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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 254, No. 1, Issue of January 10, pp. 117-126, 1979 Printed in U. a.5 A.

The Structural Specificity of Lecithin for Activation of Purified D-fl-Hydroxybutyrate Apodehydrogenase*

(Received for publication, May 4, 1978)

Yisrael A. Isaacson,+* 0 Paul W. Deroo,@ Arthur F. Rosenthal,1 Robert Bittman,s J. Oliver McIntyre,$!II Hans-Georg Bock,l( Paolo Gazzotti,ll and Sidney Fleischerll

From the Department of Chemistry, Queens College of The City University of New York, Flushing, New York 11367,s the Department of Laboratories, The Long Island Jewish Hospital, New Hyde Park, New York 11040,n and the Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 3723511

n-/3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which has an absolute requirement for lecithin for enzymic activity. We have studied the activation of the purified apodehydrogenase by a number of lecithin analogues with modifications in either the hydrophobic or polar regions of the molecule in order to map the structural specificity for the lecithin molecule. The apodehydrogenase is activated by all lecithin analogues tested which contain a phosphorylcholine moiety but with modifications in the hydrophobic or glycerol portions of the molecule: 1) D and L stereoisomers of lecithin activate the enzyme equally well; 2) octadecyleicosylphosphorylcholine, a branched alkyl- phosphorylcholine, activates the enzyme to the same extent as mitochondrial lecithin; the diacylglycerol moiety is therefore not essential to obtain activation; 3) the apodehydrogenase is activated by dialkoxyphosphatidylcholines in which the hydrocarbon chains are ether-linked rather that ester-linked to the glycerol moiety, confirming the lack of specificity of the apodehydrogenase for this part of the lecithin molecule; 4) the apodehydrogenase is activated by phosphono analogues of lecithin in which the glycerol to phosphorus oxygen is missing or replaced isosterically with a methylene group; 5) lysolecithin and sphingomyelin, both of which contain the phosphorylcholine polar moiety, activate the apodehydrogenase, although to a lesser extent than the activation by mitochondrial lecithin. Therefore, although the hydrophobic region is necessary for activation of o-fi-hydroxybutyrate apodehydrogenase, the enzyme does not exhibit specificity for this part of the lecithin molecule,

The phosphinate analogue of lecithin, in which both oxygens of the phosphate esters are missing, i.e., with glycerol and with choline, did not activate the apoenzyme. Thus, a decreased separation of the phosphoryl and quaternary ammonium groups or a specific requirement for the oxygen in the phosphorylcholine moiety prevents the activation of the apodehydrogenase by this analogue.

The specificity of o-fi-hydroxybutyrate apodehydrogenase for the polar region of the lecithin molecule has

* These studies were supported by Grants AM-14632, AM-07699, and HL-16666 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ These studies were in partial fulfillment of the requirements for the Ph.D. degree for Yisrael A. Isaacson, Queens College of The City University of New York, Flushing and for J. Oliver McIntyre, Van- derbilt University.

been further studied by synthesis of a number of analogues of octadecyleicosylphosphorylcholine in which the choline group is altered. When the -CHZCHZ- group, which links the phosphoryl and quaternary ammonium groups of the phosphorylcholine moiety, was replaced by either a substituted propyl or butyl group, the activation of the enzyme was not greatly altered; however, no activation is obtained with the compound which contains an isopropyl group in this position. Thus, the distance which separates the two charged groups is not a critical aspect of the structure of the lecithin molecule for activation of the apodehydrogenase, but there are several steric constraints for this part of the molecule. The quaternary ammonium group in lecithin is essential to obtain activation of the enzyme since neither phosphatidylethanolamine nor N,N-dimethylphosphatidylethanolamine activates the apodehydrogenase. The size of the quaternary ammonium group can be increased within defined limits; N-ethyl-N,N-dimethy- loctadecyleicosylphosphorylcholine activates the enzyme, but no activation was obtained by N,N,N-triethy- loctadecyleicosylphosphorylcholine.

From these studies, we conclude that although D-p-

hydroxybutyrate apodehydrogenase does not exhibit specificity for the hydrophobic domain, there is a high degree of specificity for the choline moiety. The quaternary ammonium group seems to be essential. The polar region can be varied within limits of steric and structural constraints.

Phospholipids are essential for the function of a number of membrane-bound enzymes (Fleischer and Fleischer, 1967; Rothfield and Romeo, 1971). For most lipid-requiring enzymes, the phospholipid requirement seems to be nonspecific. n-j?-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which specifically requires lecithin for function (Sekuzu et al., 1963; Fleischer et al., 1966). The apodehydrogenase from beef heart mitochondria has recently been purified to homogeneity, devoid of phospholipid or detergents (Bock and Fleischer, 1975). The inactive apodehydrogenase can be reactivated specifically by lecithin or phospholipid mixtures containing lecithin (Fleischer et al., 1974; Gazzotti et al., 1975). This report is concerned with mapping the structural specificity of the lecithin molecule in terms of its activation of D-p-

hydroxybutyrate apodehydrogenase. A preliminary report has appeared (Isaacson et al., 1977).

MATERIALS AND METHODS

Solutions were prepared with deionized water. Chemicals were of reagent grade unless otherwise specified. nL-P-Hydroxybutyric acid

117

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118 Activation of Hydroxybutyrate Apodehydrogenase with Lecithin Analogues

(sodium salt), bovine plasma albumin (Fraction V, powder), dithiothreitol, N,N-dimethylphosphatidylethanolamine (dipalmitoyl), L- palmitoyl-lysolecithin, nL-dipalmitoylphosphatidylcholine, and bovine brain sphingomyelin were obtained from Sigma Chemical Co. (St. Louis, MO.). Lysolecithin, prepared fom soybean phospholipids, was a gift of Dr. Hans Betzing (A. Natterman & Co., Koln, West Germany). 3-N,N-Dimethylamino-l-propanol, N,N-dimethylaminoethanol, N,N-diethylaminoethanol, I-N,N-dimethylamino-2-propanol, 4-N,N-dimethylamino-I-butanol, methyl tosylate, and ethyl tosylate were purchased from the Aldrich Chemical Co. (Milwaukee, Wis.). A solution of bovine plasma albumin, used as the protein standard, was obtained from Armour Pharmaceutical Co. (Chicago, Ill.). NAD+ was obtained from P-L Biochemicals Inc. (Milwaukee, Wis.). Naja naja siamensis venom was obtained from Miami Serpen- tsrium Laboratories (Miami, Fla.).

Assays-Protein was measured by the procedure of Lowry et al. (1951) with bovine plasma albumin as protein standard. When dithiothreitol was present in the sample, the assay was carried out as described by Ross and Schatz (1973) using iodoacetate to carboxy- methylate the dithiothreitol which would otherwise interfere with the assay for protein. Phosphorus was measured using a modification (Rouser and Fleischer, 1964) of Chen et al. (1956). For the phos- phonolipids, digestion with the 70% perchloric acid was allowed to proceed for 16 h. Control studies showed that digestion for this length of time had no effect on the quantitation of the phosphorus in potassium phosphate standards.

n-P-Hydroxybutyrate dehydrogenase activity was measured spec- trophotometrically as the rate of reduction of NAD’ with D-P-hydroxybutyrate as substrate (Bock and Fleischer, 1975). Two procedures were used to measure the activation of the enzyme by phospholipid. In the cuvette assay (1 ml tinal volume), the enzyme (usually 1 or 2 pg) was added to a l-ml cuvette (lo-mm light path) containing a final concentration of 10 mM potassium phosphate (pH 7.35), 0.5 mM EDTA, 0.4 mg/ml of bovine plasma albumin, 1.27% (v/v) ethanol, 0.3 rnM dithiothreitol, 2 InM NAD’, and variable amounts of phospholipid as a microdispersion. After incubation at 37°C for the optimal time for activation of the enzyme by the particular lipid (usually 15 min), the enzymic reaction was initiated by the addition of 0.1 ml of m-/3-hydroxybutyrate to a final concentration of 20 mru. In the complex assay, an enzyme *lipid complex was preformed for the optimum reactivation time (usually 15 min) at room temperature (-25°C) with approximately 10 pg of the apodehydrogenase and variable amounts of phospholipid microdispersion in 0.1 ml containing 20 mM Tris-HCl (pH 8.1), 1 mM EDTA, and 5 mM dithiotbreitol. The enzymic activity of the preformed complex was measured by the addition of a small aliquot (5 or 10 4) of the complex to the cuvette which contained the standard assay medium, preincubated to the desired temperature (usually 37°C). Only small differences (<lo%) were observed between results obtained using the cuvette or complex assay.

Preparation of D-/3-Hydroxybutyrate Apodehydrogenase-The apodehydrogenase was purified from beef heart mitochondria as described by Bock and Fleischer (1974, 1975). The specific activity of the preparations used in these studies varied between 80 and 90 pmol of NAD+ reduced/min/mg of protein at 37°C when activated with mitochondrial phospholipid. This is equivalent to a specific activity of 32 to 36 at 25°C.

Preparation and Analysis of Phospholipids-The purity of all phospholipid preparations was checked by thin layer chromatography on silica gel plates, containing 10% by weight magnesium silicate and developed with chloroform/methanol/water (65/35/5) (Rouser et al., 1970a). For each lipid, only one spot was observed when the plates were sprayed with Zinzadze reagent (prepared as described by Ditt- mer and Lester, 1964), which is specific for compounds that contain phosphorus. No additional spots were observed after subsequent charring (Rouser et al., 1970a).

Lipids from beef heart mitochondria were extracted as described previously (Fleischer et al., 1967). Mitochondrial diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcboline were purified from the total lipid extract by silicic acid chromatography (Rouser et al., 1967). Neutral lipids were eluted with chloroform, diphosphatidylglycerol with chloroform/methanol (9/l), phosphatidylethanolamine with chloroform/methanol (4/l), and phosphatidylcholine with chloroform/methanol (3/2). L-Dipalmitoylphosphatidyl- choline and L-dimyristoylphosphatidylcholine were synthesized using the method of Cubero-Robles and Van den Berg (1969). These two synthetic lecithins activated the enzyme to the same extent as the

synthetic lipids from commercial sources (Sigma Chemical Co.). Preparation of D-Dipalmitoylphosphatidylcholine-DL-Dipahni-

toylphosphatidylcholine, approximately 250 mg, was sonicated in 15 ml of deionized water until no further decrease in turbidity was observed. The sonic extract was diluted with buffer to a final concentration of 10 mg of lipid/ml, 25 mM glycylglycine, pH 7.4,3 mM CaCh, and 4 ml of Naja naja siamensis phospholipase A (1 mg/ml, prepared as described by Bock and Fleischer, 1974) were added. The lipid was digested by incubating the suspension at 37”C, with occasional shak- ing, for 48 h. The digested lipid was then extracted with chloroform/methanol (2/l). The organic phase was evaporated to dryness and dissolved in a small volume of chloroform. Thin layer chromatography showed the presence of two phosphorus-containing spots, one of which co-chromatographed with the starting material and the more polar spot with lysolecithin. The two spots were scraped from the plate and the phosphorus content was determined as described above, except that the silica adsorbent was removed by centrifugation prior to reading the absorbance. Equal amounts of phosphorus in the two spots indicated a complete digestion of the L isomer. The n-dipalmitoylphosphatidylcholine was then purified to homogeneity by silicic acid column chromatography as described above.

Preparation of Lecithin Analogues-The ether lipids, ditetra- decyloxyphosphatidylcholine, dihexadecyloxyphosphatidylcholine, and dioctadecyloxyphosphatidylcholine were prepared as described previously (Rosenthal, 1975). The phosphono analogues, dioctadecyloxypropylphosphonylcholine and dioctadecyloxybutylphosphonylcholine were synthesized according to Rosenthal (1966). Dioctadec- yloxypropylphosphinylcholine was prepared according to Rosenthal et al. (1969). Octadecyleicosylphosphorylcholine and the diacylpro- pylphosphonylcholines (dimyristoyl, dioleoyl, and distearoyl) were synthesized by the method of Deroo et al. (1976). Dimyristoylbutyl- phosphonylcholine (isosteric with dimyristoylphosphatidylcholine) was synthesized using an analogous procedure in which 3,4dihydrox- ybutyl bromide (generously supplied by Dr. Robert Engel, Queens Colleee of The Citv Universitv of New York. New York) was used in place”of 2,3-dihydroxypropyl iodide.

The octadecyleicosylphosphorylcholine analogues with modified phosphorylchohne moieties were synthesized following the procedures of Deroo et al. (1976) for the svnthesis of octadecvleicosvlnhos- phorylcholine. In short, this synthetic procedure involves the coupling of a quaternary ammonium alcohol tosylate salt with octadecyleico- sylphosphoric acid using trichloroacetonitrile as condensing agent. The tosylate salts used in the present work were: 3-N,N,N-trimethylamino-1-propanol tosylate, prepared by reacting 3-N,N-dimethylamino-l-propanol with methyl tosylate; N-ethyl-N,N-dimethylaminoethanol tosylate, prepared by reacting N,N-dimethylaminoethanol with ethyl tosylate; 1-N,N,N-trimethylaminoisopropyl alcohol tosylate, prepared by reacting I-N,N-dimethylaminoisopropyl alcohol with methyl tosylate; N,N,N-triethylaminoethanol tosylate, prepared reacting N,N-diethylaminoethanol with ethyl tosylate; and 4-N,N,N- trimethylamino-l-butanol tosylate, prepared by reacting 4-N,N-dimethylamino-1-butanol with methyl tosylate. All of the above salts separated as solids from the tetrahydrofuran reaction mixture with the exception of the N,N,N-triethyl compound which separated as an oil, trituration of this oil with ice-cold acetone yielded a white solid. All the tosylate salts were extremely hygroscopic and were dried under vacuum over PzO~ to render them anhydrous before use in the synthesis of the various octadecyleicosylphosphorylcholine analogues. The chemical and physical properties of the various octadecyleicosylphosphorylcholine analogues, together with their structure, are given in Table I.

Preparation of Phospholipid Microdispersions-Mitochondrial ahosnholiuid was microdisnersed in 20 mM Tris-HCl. 1 mM EDTA. pH 8.1, by the method of Fleischer and Klouwen (1961). The other phospholipids were microdispersed by sonication as follows. The various phospholipids were evaporated to dryness and resuspended in 20 mM Tris-HCl, 1 mM EDTA, pH 8.1, by mixing on a Vortex mixer using a glass bead to aid the resuspension. The resuspended phospholipids were then microdispersed, at approximately 109 pg of phosphorus/ml, by sonication using a bath sonicator (Laboratory Supplies, Inc.) at 23”-26°C for 3 to 15 min, until no further decrease in turbidity of the sample was observed visually. All unsaturated phospholipids were sonicated under a flow of nitrogen. Co-dispersions of phospholipids were prepared in a similar manner except that the components of the mixture were combined in chloroform/methanol (2/l) and dried down together, prior to the addition of the buffer and sonication.


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TABLE I

Chemical and physical properties of octadecyleicosylphosphorylcholine polar group analogues

Analogues of octadecyleicosylphosphorylcholine were synthesized as described under “Materials and Methods.” The complete structure of octadecyleicosylphosphorylcholine (Compound I) is shown in Fig. 1. The elemental analysis was calculated assuming the following hydration of the various compounds: I and II, 1.5 HsO; IV, 2.0 H20; III, V, and VI, 1.0 H20.

COIlI- pound

Melting point -

Yie1d (Decomposi- tion)

+ I -CH,-CH,--N(CH,), 71 217-219

+ II -CH2-CHZ-CHZ-N(CH& 69 222-224

+ III -CHz-CH,-CH,-CH,-N(CH& 58

+ IV -CH-CHZ-N(CH& 63 185-187

kHQ

+ V -CHi-CHZ-N(CzHs) (CH& 64 206-207

+ VI -CHs-CHZ-N(CzH& 59

0

o l-i&-0-t-R

SITE (I) (2) (3) (4)

TYPE I - PHOSPHATIDYLCHOLINE (PC)

H,C-(CH,),, CH, ?

\ , ,o/p-o--(x)

A 0-

Carbon

Calcu- Found

lated

B

Hydrogen Nitrogen

Calcu- Found

Calcu- lated lated

Found

% B

69.49 69.62 12.61 12.81 1.88 2.17 4.17 4.26

69.79 70.01 12.65 12.79 1.85 1.58 4.09 4.11

70.91 70.94 12.70 12.52 1.84 1.69 4.06 4.13

68.97 69.27 12.63 12.89 1.84 1.72 4.04 3.94

70.63

71.17

70.69 12.63 12.83 1.87

70.94 12.73 12.81 1.80

1.81

1.76

4.14 4.06

3.99 3.91

H,C-(CH,),, ii

TYPE II - OCTADECY LEICOSYL- PHOSPHORYLCHOLINE (OEPC)

FIG. 1. The structure of phosphatidylcholine and octadecyleicosylphosphorylcholine indicating structural modification of analogues. Type I compounds with modifications in different sites, as indicated, include: dialkyloxyphosphatidylcholine, lysolecithin, sphingomyelin, and octadecyleicosylphosphorylcholine altered at Site 1; diacyloxy- propylphosphonylcholine at Site 2; diacyloxypropylphosphinyl- choline without the oxygens at both Sites 2 and 3; phosphatidylethanolamine and N,N-dimethylphosphatidylethanolamine with modified structures at Site 4. The type II compounds, which are analogues of octadecyleicosylphosphorylcholine (X = choline), have modifications in the choline moiety (X) as shown in Table I.

RESULTS

Optimization of Lipid Activation Studies

The purified n-P-hydroxybutyrate apodehydrogenase used in these studies has been purified to homogeneity, is devoid of phospholipid, and is therefore inactive. The apodehydrogenase is activated specifically by lecithin or by phospholipid mixtures containing lecithin. Neither phosphatidylethanolamine nor diphosphatidylglycerol by themselves activate the apoenzyme, yet the mixture of phospholipids in mitochondria, i.e. mitochondrial phospholipids, which contain lecithin (40%) as well as phosphatidylethanolamine (37%), diphosphatidylglycerol (20%), and phosphatidylinositol (3%), activate better than the mitochondrial lecithin by itself. Thus, the

Phosphorus

Calcu- lated Found

%

highest specific activity of n-P-hydroxybutyrate dehydrogenase and the best efficiency of activation (lowest ratio of moles of lecithin/m01 of enzyme subunit) is obtained with mitochondrial phospholipids. Lecithins of varying chain lengths and unsaturation activate the apodehydrogenase, albeit to different specific activities and efficiency. The activation can gen- erally be optimized by preparing mixed dispersions of the lecithin with mitochondrial phosphatidylethanolamine alone or together with diphosphatidylglycerol, (Fleischer et al., 1974; Gazzotti et al., 1975). Therefore, in the present study, we have measured the activation of the apodehydrogenase by lecithin analogues as mixed dispersions with phosphatidylethanolamine or phosphatidylethanolamine plus diphosphatidylglycerol.

The activation of n-P-hydroxybutyrate apodehydrogenase by phospholipid is a time-dependent process. The time course of activation of the apodehydrogenase by mitochondrial phospholipid using the complex and cuvette assay methods is shown in Fig. 2. The maximal activity obtained is the same by both procedures although the time course of activation is dependent on the procedure for preincubation and on the ratio of phospholipid to apodehydrogenase. The preincubation time for optimum activation of the apoenzyme was determined for each phospholipid mixture by either the complex or cuvette assay methods, whichever procedure was used for activation of the enzyme. Lecithin or lecithin analogues, co- dispersed either with phosphatidylethanolamine or with both phosphatidylethanolamine and diphosphatidylglycerol, activate the apodehydrogenase with time courses similar to that of mitochondrial phospholipids under the same conditions (not shown).

The activation of D-/3-hydroxybutyrate apodehydrogenase by each lipid microdispersion was studied by titrating a con- stant amount of apoenzyme with variable amounts of phospholipid and measuring the enzymic activity after the optimal reactivation time. The titration curves for the activation of the enzyme by mitochondrial phospholipid and by mitochondrial lecithin are shown in Fig. 3. Two parameters are obtained from such titration curves: 1) maximal activity; this rate is usually expressed relative to the maximal activity obtained with mitochondrial phospholipid and 2) the efficiency of activation, i.e. moles of lecithin added per mol of enzyme subunit to obtain half-maximal reactivation. A larger number for efficiency of activation denotes less efficient activation by the


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120

100

0

Activation of Hydroxybutyrate Apodehydrogenase with Lecithin Analogues

I I I I J 4 8 12 16 20

INCUBATION TIME (min.) FIG. 2. Time course of activation of n-/3-hydroxybutyrate apode-

hydrogenase (BDH) by mitochondrial phospholipid using either cuvette or complex assay procedure. In the cuvette assay, the enzyme (approximately 1 pg) was preincubated at 37°C in the cuvette with mitochondrial phospholipid, either 60 ag of phosphorus/mg of D-p-

hydroxybutyrate apodehydrogenase (C - -U) or 6 pg of phosphorus/mg of n-/3-hydroxybutyrate apodehydrogenase (a- - -0) in the standard assay medium: 10 mM potassium phosphate, pH 7.35, 0.5 rnM EDTA, 0.4 mg/ml of bovine serum albumin, 1.27% (v/v) ethanol, 0.3 rnM dithiothreitol, and 2 IIIM NAD’. The enzyme reaction was initiated at the times indicated by the addition of on-P-hydroxybutyrate to a final concentration of 20 mM. The specific activity (micromoles of NAD’ reduced/min/mg of n-,B-hydroxybutyrate apodehydrogenase) was calculated from the initial rate of reduction of NAD+ measured at 340 nm. In the complex assay, a complex was preformed by incubating the enzyme (10 pg), at 25”C, with mitochondrial phospholipid, either 60 ,ag of phosphorus/mg of n-P-hydroxybutyrate apodehydrogenase (,W) or 6 pg of phosphorus/mg of D-/?-hydroxybutyrate apodehydrogenase (M) in 0.1 ml of 20 mM Tris- Cl, pH 8.1, 1 mM EDTA, and 5 mM dithiothreitol. After preincubation for the indicated times, the enzyme reaction was initiated by the addition of a 5- or 10-p aliquot of the preformed complex to the cuvette at 37°C and containing the standard assay medium with substrate. The specific activity of D-P-hydroxybutyrate apodehydrogenase was calculated as above. A shorter preincubation time is required for the complex assay as compared with the cuvette assay, although the same specific activity is obtained with longer preincubation in the cuvette assay.

lipid. The apodehydrogenase preparations used in these studies had a maximum specific activity, at 37”C, of 80 to 90 pmol of NAD+ reduced/min/mg of enzyme, when reactivated with mitochondrial phospholipid (Fig. 3). The efficiency of activation of the apodehydrogenase by mitochondrial phospholipid varied between 6 and 8 mol of lecithin/m01 of enzyme subunit

for half-maximal activity. A dispersion of mitochondrial lecithin activated apodehydrogenase to 90% of the maximum activity obtained with mitochondrial phospholipid but with

poor efficiency, requiring approximately 80 mol of lecithin/m01 of enzyme subunit to obtain half-maximal activation of the enzyme (Fig. 3). The time course for activation by lecithin (Fig. 5) is slower than by mitochondrial phospholipid (Fig. 2). Optimum activation of the apodehydrogenase requires approximately 8 and 90 min of preincubation with mitochondrial phospholipid and lecithin, respectively. The more rapid and efficient activation of the apoenzyme by lecithin in mitochondrial phospholipid than by lecithin alone is due to the presence, in mitochondrial phospholipid, of both phosphatidylethanolamine and diphosphatidylglycerol since mixtures of lecithin supplemented with phosphatidylethanol-

amine and diphosphatidylglycerol behave similarly to mitochondrial phospholipid.

When n-P-hydroxybutyrate apodehydrogenase is activated with lecithin (either mitochondrial, soybean, or dioleoyl) microdispersed together with mitochondrial phosphatidylethanolamine, the efficiency of activation and the maximum activity obtained are dependent on the diphosphatidylglycerol content of the liposome (Fig. 4). The activation of the enzyme by lecithin becomes more efficient as the diphosphatidylglycerol content of the lecithin/phosphatidylethanolamine/diphosphatidylglycerol co-dispersion is increased. Con- comitant with this increase in efficiency, there is a decrease in maximal activity. This decrease in activity is small as the diphosphatidylglycerol content is increased from 0 to 16% (by phosphorus), i.e. to a phosphorus ratio of 1.0/0.8/0.35 for lecithin / phosphatidylethanolamine / diphosphatidylglycerol, respectively. The enzyme becomes more strongly inhibited when the diphosphatidylglycerol content of the co-dispersion exceeds 16% (Fig. 4). Each lecithin analogue was co-dispersed together with mitochondrial phosphatidylethanolamine and diphosphatidylglycerol at a lecithin analogue/phosphatidylethanolamine/diphosphatidylglycerol phosphorus ratio of either 1.0/0.8/0.2 or 1.0/0.8/0.4. Two amounts of diphosphatidylglycerol were usually tested to optimize the activation of the apodehydrogenase by the different lecithin analogues.

The Activation of D-/?-Hydroxybutyrate Apodehydrogenase with Lecithin Analogues Modified in the

Acylglycerylphosphoryl Portion of the Lecithin Molecule

D- and L-Dipalmitoylphosphatidylcholine-The stereo- specificity of lecithin for the activation of the apodehydrogenase was studied using the D and L stereoisomers of dipalmitoylphosphatidylcholine, as well as the racemic mixture. The D and L stereoisomers activate the same within experimental

(MOLES LECITHIN / MOLE EDH)

0 y I

j

I 1 I 1 0 10 20 30 40

LECITHIN (~JG P/IO~JG BDH)

FIG. 3. Titration curves for the activation of n-P-hydroxybutyrate apodehydrogenase (BDH) by mitochondrial phospholipid and by mitochondrial lecithin using the complex assay procedure. The complex was formed by preincubation as described in Fig. 2; the enzyme (10 pg) was admixed with variable amounts of lecithin, either microdispersed alone (O- - -0) or as mitochondrial phospholipid (M) as indicated and preincubated for 15 min and 2 h, respectively, times required for optimum activation by mitochondrial phospholipid and lecithin dispersions, respectively ( I$ Figs. 2 and 5). The activity of each complex was measured as described in Fig. 2. The amount of lecithin required for half-maximal activity of D-P-hydroxybutyrate apodehydrogenase is indicated by the dashed vertical lines and is 7 and 80 mol of lecithin/m01 of o-P-hydroxybutyrate apodehydrogenase subunit for mitochondrial phospholipid or lecithin, respectively.


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Activation of Hydroxybutyrate Apodehydrogenase with Lecithin Analogues 121

IOC a

a

\

x t

\ \

10 b A \O

‘.8

2 OL I I I I I L

0 5 IO 15 20 25 30 35" DPG IN MIXED CODISPERSION

( % OF PHOSPHORUS )

FIG. 4. The effect of the diphosphatidylglycerol (DPG) content of lecithin/phosphatidylethanolamine/diphosphatidylglycerol co-dispersions on the activation of n-P-hydroxybutyrate apodehydrogenase (BDH) by lecithin. Co-dispersions of either mitochondrial lecithin (phosphatidylcholine (PC)) (m, 0) or dioleoylphosphatidylcholine (A, A), or soybean phosphatidylcholine (0, 0) with mitochondrial phosphatidylethanolamine (at a phosphatidylcholine/phosphatidylethanolamine phosphorus ratio of 1.0/0.8) and with variable amounts of mitochondrial diphosphatidylglycerol, as indicated, were prepared by drying down together, from organic solvent, aliquots of the purified lipids followed by sonication in 20 mM Tris-Cl, pH 8.1, 1 mM EDTA as described under “Materials and Methods.” D-,&Hy- droxybutyrate apodehydrogenase was titrated with each phospholipid dispersion using the complex assay procedure, as described in Fig. 3, to obtain the maximum n-P-hydroxybutyrate apodehydrogenase specific activity (micromoles of NAD+ reduced/min/mg of D-P-hydroxybutyrate apodehydrogenase) (solid symbols) and the efficiency of activation of the enzyme (moles of phosphatidylcholine/mol of D-p- hydroxybutyrate apodehydrogenase subunit for half-maximal activation) (open symbols). The activation of the apodehydrogenase by mitochondrial phospholipid at 37’C is shown for comparison; the maximum specific activity was 85 (x) and the efficiency of activation was 7 mol of lecithin/m01 of o-P-hydroxybutyrate apodehydrogenase subunit (B(I).

error. A double reciprocal plot of enzymic activity uersus the amount of lecithin added (not shown) gives an apparent K,,, of 16 /AM-' and a V,,, o f 84 for both the D and L stereoisomers. The titration curve (direct plot) gives somewhat lower maximal activity (Table II, Subsection A) than the V,,,,, value, due to slight inhibition with excess lipid; similar inhibition is observed occasionally in excess mitochondrial phospholipid (Fig. 3). The activation of the enzyme with a racemic mixture of the two isomers was similar to that obtained with either of the separate species (Table II, Subsection A). Thus, D-p-

hydroxybutyrate apodehydrogenase exhibits no specificity to- ward the D and L configuration of the diacylglycerol moiety of lecithin.

Octadecyleicosylphosphorylcholine - Octadecyleicosyl- phosphorylcholine is an analogue of lecithin in which a saturated branch chain alcohol replaces the diacylglyceryl moiety of lecithin (Fig. 1, type II compound with X = choline). This analogue activates the apodehydrogenase. The time for preincubation of the apoenzyme with octadecyleicosylphosphoryl-

choline to form an active complex is appreciably longer than for activation with lecithin (Fig. 5). The preincubation time required for optimal activity is also dependent on the concentration of lipid. When octadecyleicosylphosphorylcholine is co-dispersed with either phosphatidylethanolamine or with both phosphatidylethanolamine and diphosphatidylglycerol,

the time course of activation of the apoenzyme is similar to that observed for activation with mitochondrial phospholipid (Fig. 2); the activity of the n-P-hydroxybutyrate dehydrogenase . octadecyleicosylphosphorylcholine complexes remains stable for 150 min, the longest preincubation time studied. The titration curves for activation of the apodehydrogenase by octadecyleicosylphosphorylcholine microdispersed either alone or with mitochondrial phosphatidylethanolamine or with both phosphatidylethanolamine and diphosphatidylglycerol are shown in Fig. 6. The activation of the enzyme with mitochondrial lecithin under the same conditions (complex assay) are shown for comparison. The maximum activation obtained with octadecyleicosylphosphorylcholine is appreciably lower than that for lecithin (Fig. 6A). However, when each is co-dispersed with phosphatidylethanolamine plus diphosphatidylglycerol (lecithin or octadecyleicosylphosphorylcholine/phosphatidylethanolamine/diphosphatidylglycerol phosphorus ratio of 1.0/0.&l/0.4) (cf Fig. 6D and Table II, Subsection A) the percentage of maximal activation becomes more similar, as does the efficiency of activation; half-maximal activity is obtained at an octadecyleicosylphosphorylcholine enzyme to subunit molar ratio of 6.5 (by complex assay, Fig. 6) or 5.2 (by cuvette assay, Table II, Subsection A) which compares favorably with that obtained with lecithin (5.5 mol of lecithin/m01 of enzyme subunit). The variation in activity of n-P-hydroxybutyrate dehydrogenase observed between octadecyleicosylphosphorylcholine and lecithin with the different co-dispersions tested may arise from differences in the physical properties of the liposomes. In this regard, octadecyleicosylphosphorylcholine alone has a tendency to form suds, which is decreased by forming the mixed co-dispersions with phosphatidylethanolamine or phosphatidylethanolamine plus diphosphatidylglycerol. Despite these varia- tions between octadecyleicosylphosphorylcholine and lecithin, the apodehydrogenase can be activated maximally with octadecyleicosylphosphorylcholine and with good efficiency of activation. We conclude from the data in Fig. 6 that the diacylglyceryl moiety of lecithin, which is not present in octadecyleicosylphosphorylcholine, is not essential for the activation of n-P-hydroxybutyrate apodehydrogenase.

Lysolecithin and Sphingomyelin-Lysolecithin and sphingomyelin, which have the same phosphorylcholine polar moiety as lecithin, each activate the apodehydrogenase (Table II, Subsection B). The titration curves for the activation of the apoenzyme with lysolecithin were biphasic; there was diminished activity with amounts of lysolecithin above that which gives optimal activity. The activity was also lower at 37°C than at 25’C and even at this lower temperature, the highest rate is obtained initially and without preincubation, i.e. when the apoenzyme is added to the cuvette containing the lysolecithin. The activity decreases with time of preincubation and seems to be due to denaturation of the enzyme, since it is not restorable with mitochondrial phospholipid. The poor maximal activity obtained with lysolecithin seems to be due, at least in part, to inactivation by this detergent.

Bovine brain sphingomyelin co-dispersed with phosphatidylethanolamine or with phosphatidylethanolamine and diphosphatidylglycerol activated the apodehydrogenase to only 12% and lo%, respectively, of the maximal specific activity obtained with mitochondrial phospholipid (Table II, Sub- section B). This low activation is not improved when the sphingomyelin to phosphatidylethanolamine ratio was varied from half to double nor when the diphosphatidylglycerol content of the liposome was increased to a phosphorus ratio of I.O/O.8/0.4 (sphingomyelin/phosphatidylethanolamine/diphosphatidylglycerol).

Dialkoxyphosphatidylcholines-These differ from lecithin


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TABLE II

Activation of D-P-hydroxybutyrate apodehydrogenase with lecithin analogues modified in the acylglycerylphosphoryl moiety

The lecithin analogues, with modifications at different sites as droxybutyrate apodehydrogenase subunit), the less efficient is the indicated in Fig. 1, were co-dispersed either with mitochondrial phos- activation by the lipid. The percentage of maximal activity is ex- phatidylethanolamine (analogue/phosphatidylethanolamine phos- pressed relative to the maximal specific activity obtained with mito- phorus ratio of 1.0/0.8) or with mitochondrial phosphatidylethanola- chondrial phospholipids (see Footnote a below) as 100%. The specific mine and diphosphatidylglycerol (analogue/phosphatidylethanol- activity of [email protected] apodehydrogenase varied between 80 amine/diphosphatidylglycerol phosphorus ratio of l.O/O.S/O.Z). D-b- and 90 pm01 of NAD+ reduced/min/mg of D-@-hydroxybutyrate apo- Hydroxybutyrate apodehydrogenase was titrated with each lipid mi- dehydrogenase when assayed at 37°C. The maximal activity obtained crodispersion and activity was measured at 37°C (unless otherwise with the analogues, assayed at 25’C, is expressed as the percentage of indicated) using either the cuvette or complex assay. From the the maximal specific activity obtained with mitochondrial phospho- titration curve obtained with each lipid dispersion, the maximal lipids, at this temperature (32 to 36 pmol of NAD+ reduced/min/mg activation and efficiency of activation [mole ratio of lecithin (phos- of n-P-hydroxybutyrate apodehydrogenase). With mitochondrial phatidylcholine) or analogue/D-P-hydroxybutyrate apodehydrogen- phospholipids, phosphatidylcholine, and octadecyleicosylphosphor- ase subunit which gives half-maximal activity] were obtained (c/I Fig. ylcholine, the titration curves obtained by using the complex and 3). According to this representation, the greater the number for cuvette assay methods were compared; no significant differences were efficiency of activation (mole ratio of phosphatidylcholine/@-hy- observed between the results obtained by the two methods.

+Phosphatidyl- +[Phosphatidylethanolamine + di- ethanolamine phosphatidylglycerol]

Phospholipid Modification site Maximum activ- Maximum activ- Effici;;;q~)(mole

ity ity - % %

Mitochondrial phospholipid” 100 7.0 Subsection A

Mitochondrial lecithin (phosphatidylcholine) 105 93 7.2 69’ 5.5*

L-Dipalmitoylphosphatidylcholine” 58 D-Dipalmitoylphosphatidylcholine’ (1) 68 DL-Dipalmitoylphosphatidylcholine’ (1) 65 27* 4.3b Octadecyleicosylphosphorylcholine (1) 108 74 12.5

72’ 5.2b

Subsection B L-Palmitoyl-lysolecithin” Soybean lysolecithin” Sphingomyelin’

Subsection C Distearyloxyphosphatidylcholine’ Dipalmityloxyphosphatidylcholine’ Dimyristyloxyphosphatidylcholine’

Subsection D Distearoylpropylphosphonophosphatidylcholine analogud Dioleoylpropylphosphonophosphatidylcholine analogueX Dimyristoylpropylphosphonophosphatidylcholine analog& Dimyristoylbutylphosphonophosphatidylcholine analogud Distearyloxypropylphosphonophosphatidylcholine analogug Distearyloxybutylphosphonophosphatidylcholine analogud

Subsection E Distearyloxypropylphosphinophosphatidylcholineh

(1) 15 (1) 23

(1) 12

(1) 25 30 17 (1) 71 50 12

(1) 75 61 29

(2) 8.3 (2) 50

(2) 73 (2) 75

(1 & 2) 4 (1 & 2) 10

(1. 2 & 3)

28 300 10 8.0

42’ 3.8’ 346 3.7*

4 8

0 0

n The aqueous microdispersion of mitochondrial phospholipids in 20 mM Tris-HCl, 1 mM EDTA, pH 8.1, was prepared from the beef heart mitochondria. A lipid extract was prepared from which the neutral lipids were removed by silicic acid chromatography as described previously (Fleischer et al., 1967). Mitochondrial phospholipid is composed of phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol, and minor components (mainly phosphatidylinositol), which constitute 40%. 3770, 20%, and 3% respectively, of the total phosphorus. Mitochondrial phospholipid contains phosphatidylethanolamine and diphosphatidylglycerol, and as such serves as the control value for the activation of D-/&hydroxybutyrate apodehydrogenase by mitochondrial phosphatidylcholine in the presence of phosphatidylethanolamine plus diphosphatidylglycerol (Subsection A).

* These data were obtained by co-dispersing the phosphatidylcholine or analogue together with both phosphatidylethanolamine and diphosphatidylglycerol at a phosphorus ratio of 1.0/0.8/0.4 instead of 1.0/0.8/0.2.

’ D-Dipalmitoylphosphatidylcholine was prepared from DL-dipalmitoylphosphatidylcholine by prolonged digestion with phospholipase AP followed by purification of the undigested D isomer by silicic acid chromatography as described in the text (see “Materials and Methods”). The titration curves were obtained using the cuvette assay procedure.

“With lysolecithin, the reaction was initiated bv the addition of the enzyme since, when the enzyme was preincubaied with lysolecithin, the activity diminished with time. Therefore, the usual complex or cuvette assay methods give lower enzymic activity than that which is obtained. The activity at 37°C was lower than that observed at 25”C, so that the activation with lysolecithin was measured at 25°C and is expressed relative to the maximal specific activity of D-p-

hydroxybutyrate apodehydrogenase-mitochondrial phospholipid at this temperature (32 to 36 pmol of NAD’ reduced/min/mg of D-p- hydroxybutyrate apodehydrogenase).

e With sphingomyelin (from bovine brain), the activity was measured by cuvette assay at 25°C following preincubation of the lipid with the enzyme for 30 min, the optimum preincubation time. Maxi- mum activity is expressed relative to the activity of D-P-hydroxybutyrate apodehydrogenase-mitochondrial phospholipid at 25°C (see Footnote d).

‘The three dialkyloxyphosphatidylcholines (Subsection C) are racemic mixtures of the D and L isomers. The alkyl groups are ether- linked rather than ester-linked to the glycerol moietv. These lipids were slightly turbid when co-dispersed either with pgosphatidykth- anolamine or with both phosphatidylet,hanolamine and dinhosnhati- dylglycerol. The titration curves were obtained using thk cokplex assay procedure.

g The diacyloxypropyl- and diacyloxybutyl phosphonylcholine


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Activation of Hydroxybutyrate Apodehydrogenase with Lecithin Analogues

in that the alkyl chain is linked to the glycerol moiety through an ether rather than an ester linkage. The three dialkoxyphosphatidylcholines (dioctadecyl, dihexadecyl, and ditetra- decyl) which were tested, reactivate n-P-hydroxybutyrate apodehydrogenase with similar optimal activity, when either co-dispersed with phosphatidylethanolamine or with both phosphatidylethanolamine and diphosphatidylglycerol (Table II, Subsection C), to that reported previously with the analogous synthetic lecithins (Gazzotti et al., 1975). Although the activation by the ether analogues is less efficient, the ester linkage in lecithin is not essential for activation of the apodehydrogenase. This conclusion was predictable from the results obtained with octadecyleicosylphosphorylcholine (see above).

Phosphonylcholine Analogues of Lecithin-Phosphonyl- cholines lack an oxygen atom on either side of the phosphate ester. The ones we tested lacked the oxygen between the glyceryl and phosphoryl moiety (Site 2, Fig. 1). The lecithin analogues of this type activate n-/?-hydroxybutyrate apodehydrogenase (Table II, Subsection D). In particular, dimyris- toyloxypropylphosphonylcholine and dimyristoyloxybutyl- phosphonylcholine (isosteric with dimyristoyl-lecithin, the Site 2 oxygen being replaced by a methylene group) were compared (Table II, Subsection D); the activation of the apodehydrogenase by these two analogues, when co-dispersed with phosphatidylethanolamine, is the same within experimental error. The dioctadecyloxypropylphosphonylcholine and dioctadecyloxybutylphosphonylcholine activate the apoenzyme much more poorly than the dioleoyl- and dimyris- toyloxypropylphosphonylcholines.

Phosphinylcholine Analogues of Lecithin-These analogues lack oxygen atoms at both Sites 2 and 3 (Fig. 1). Dioctadecyloxypropylphosphinylcholine does not activate the apodehydrogenase (Table II, Subsection E), although some activation is obtained with the analogous dioctadecyloxypropylphosphonylcholine and dioctadecyloxybutylphosphonylcholine compounds (Table II, Subsection D) both of which contain the Site 3 oxygen atom. The oxygen atom at Site 3 in the lecithin molecule is, therefore, essential for activation of n-P-hydroxybutyrate apodehydrogenase.

Phosphatidylethanolamine and N,N-Dimethylphosphati- dylethanolamine-When these are microdispersed alone or together with diphosphatidylglycerol they do not activate the apodehydrogenase (data not shown), although phosphatidylethanolamine, in the form of liposomes, is 90% zwitterionic at pH 8 (Green and Fleischer, 1964). The quaternary ammonium group of lecithin therefore seems to be essential for activation of the enzyme.

The Activation of D-/?-Hydroxybutyrate Apodehydrogenase with Octadecyleicosylphosphorylcholine Analogues

Modified in the Choline Moiety

Since the apodehydrogenase is activated with octadecyleicosylphosphorylcholine (Fig. 6 and Table II, Subsection A), analogues of this lipid were synthesized with modified polar moieties and tested for their ability to activate the apoenzyme

analogues are missing an oxygen atom at Site 2 (cfi Fig. 1) and are racemic mixtures of the D and L isomers (Subsection D). Dimyristoyl- oxybutylphosphonylcholine analogue is isosteric with dimyristoyl- lecithin, since the Site 2 oxygen is replaced by a methylene (CH2) group. The titration curves were obtained by the cuvette assay method.

’ The dioctadecyloxypropylphosphinylcholine (Subsection E) is a racemic mixture of the D and L isomers and is missing oxygen atoms at both Sites 2 and 3 (cf Fig. 1). Activation was tested by both the complex and cuvette assay methods.

123

1 I I

e”tT---

io INCUBATION TIME (min)

FIG. 5. The time course of activation of n-/3-hydroxybutyrate apodehydrogenase (BDH) by lecithin and by octadecyleicosylphosphorylcholine using the complex assay procedure. n-/3-Hydroxybutyrate apodehydrogenase (IO pg) was incubated at 25’C to form a complex (cfi Fig. 2) either with mitochondrial phosphatidylcholine alone (400 pg of phosphorus/mg of n-,G-hydroxybutyrate apodehydrogenase, o---O, or 140 pg of phosphorus/mg of n-/s’-hydroxybutyrate apodehydrogenase, W- - -W) or with octadecyleicosylphosphorylcholine alone (500 pg of phosphorus/mg of n-P-hydroxybutyrate apodehydrogenase, M, or 140 pg of phosphorus/mg of D-/3-hydroxybutyrate apodehydrogenase, l - - -0). The n-P-hydroxybutyrate apodehydrogenase activity of each complex was measured, after the preincubation times indicated, using the complex assay method as described in Fig. 2.

TABLE III

Activation of D-,l-hydroxybutyrate upodehydrogenase with octadecyleicosylphosphorylcholine analogues modified in the

choline moiety

The octadecyleicosylphosphorylcholine analogues have the basic structure of octadecyleicosylphosphorylcholine (cf Fig. 1, type II) but with different polar structures (X) as indicated. These analogues were each co-dispersed with mitochondrial phosphatidylethanolamine (octadecyleicosylphosphorylcholine analogues/phosphatidylethanolamine, phosphorus ratio of 1.0/0.8) or with both phosphatidylethanolamine and diphosphatidylglycerol (octadecyleicosylphosphorylcholine analogues/phosphatidylethanolamine/diphosphatidylglycerol, phosphorus ratio of 1.0/0.8/0.4).

+Phos- pha- +[Phosphati-

tidyl- dylethanolam- etha- ine + diphos-

nolam- phatidylgly- COIlI- pound structure (X) ine cerol]

Maxi- Maxi- Effl- lll”lIl mum ciency” activity”

a;$- (mole ratm)

B %

+ I -CHz-CHz--N(CHs)$ 105 72 5.2

II -CHz-CHz-CHz-+N(CH& 101 70 4.8

+ III -CHz-CHz-CHz-CHz-N(CH3)3 81 45 4.0

+ IV ---CH-CHP-N(CHZ)~ 0 0

I CH,

+ V -CHz-CHa-N(CzH5) (CH,)z 93 64 3.9

+ VI -CHz-CHz-N(CzHz)3 0 0

“The percentage reactivation and efficiency of activation of the apodehydrogenase by each octadecyleicosylphosphorylcholine analogue, were obtained from titration curves such as those shown in Fig. 3 and expressed as described in Table II.


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100

= 80

5 p 60

s 40 2 2 20

x 2 100 0

% 80

5 LLI 6o

E iit 40

20

PC OR OEPC CONCENTRATION UJG P/ML)

0 20 40 0 20 40 60 1 I I I 1 I

0 A

40’ /o- - -0

c

/- T; 1

4 0 2 4 6

OEPC CONCENTRATION

UJG P/ML)

PC OR

(Table III). When the ethyl group, connecting the phosphate and quaternary ammonium groups of phosphorylcholine is replaced by a propyl moiety (Compound II) or by a butyl moiety (Compound III), the activation of the apodehydrogenase is unaffected and decreased only 20%, respectively. How- ever, when an isopropyl moiety (Compound IV) is substituted in this position, the apodehydrogenase is not activated at all. Thus, the precise distance which separates the quaternary ammonium group and the phosphoryl group is not a critical aspect of the structure of lecithin for activation of D-/?-hydroxybutyrate apodehydrogenase although the isopropyl group is sterically not suitable. Substitution on the quaternary ammonium group of one of the methyl groups by an ethyl group (Compound V) does not appreciably alter the ability of the lipid t,o activate the enzyme. However, the N,N,N-triethyl derivative (Compound VI) does not activate the apodehydrogenase. Thus, the substituents on the quaternary ammonium group can be modified within limits.

DISCUSSION

n-/$Hydroxybutyrate dehydrogenase is one of the best documented and most intensively studied lipid-requiring en-

FIG. 6. The titration curves for the activation of n-P-hydroxybutyrate apodehydrogenase (BDH) by mitochondrial lecithin (PC) or by octadecyleicosylphosphorylcholine ( OEPC) added alone, or co-dispersed together with phosphatidylethanolamine or with phosphatidylethanolamine plus diphosphatidylglycerol. The complex assay procedure was used, as described in Fig. 2; D-p- hydroxybutyrate apodehydrogenase (10 pg) was preincubated with varying amounts of either mitochondrial phosphatidylcholine (O- - -0) or octadecyleicosylphosphorylcholine o--a The phosphatidylcholine or octadecyleicosylphosphorylcholine was either (A) microdispersed alone or (B), co-dispersed with mitochondrial phosphatidylethanolamine (phosphatidylcholine or octadecyleicosylphosphorylcholine/ phosphatidylethanolamine phosphorus ratio of 1.0/0.8) or (0, co-dispersed with mitochondrial phosphatidylethanolamine and diphosphatidylglycerol (phosphatidylcholine or octadecyleicosylphosphorylcholine / phosphatidylethanolamine/diphosphatidylglycerol phosphorus ratio of 1.0/0.8/0.2) or (D) as (C) but with a phosphorus ratio of 1.0/0.8/0.4. The complex was formed by preincubation for 2 h for A and 15 min for B, C, and D (the time which is required for optimum activation) and then a 5- or lo- gl aliquot was assayed for n-P-hydroxybutyrate apodehydrogenase activity in the standard assay medium at 37°C as described in Fig. 2. The percentage of maximal activity is expressed relative to the activation with mitochondrial phospholipid (85 pmol of NAD’ reduced/min/mg of n-/3-hydroxybutyrate apodehydrogenase) which is taken as 100%. Similar results were obtained by cuvette assay.

zymes. It has a specific and absolute requirement for lecithin for enzymic activity which was first shown with impure preparations of the enzyme (Sekuzu et al., 1963; Fleischer et al., 1966; Grover et aZ., 1975). Only recently has the specificity for lecithin been documented for the purified apodehydrogenase (Fleischer et al., 1974; Gazzotti et al., 1975; Vidal et al., 1977). With the purified apodehydrogenase, we could demonstrate that the active form of the enzyme is the enzyme - phospholipid complex (Fleischer et al., 1974; Gazzotti et al., 1975). This study represents the first detailed investigation of the structural specificity of lecithin for activation of n-P-hydroxybutyrate apodehydrogenase.

One of the problems of measuring the activation of the apodehydrogenase by different lipids is that there is variation in part due to different properties of the microdispersion. This problem was faced by us previously when we studied the activation of the apodehydrogenase by lecithins of varying chain length and unsaturation (Fleischer et al., 1974; Gazzotti et al., 1975). The large difference in activation observed with the different lecithins was, in part, overcome by forming mixed microdispersions of the lecithin with phosphatidylethanolamine or phosphatidylethanolamine plus diphosphatidyl-


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Activation of Hydroxybutyrate Apodehydrogenase with Lecithin Analogues 125

glycerol, the other two major phospholipids in the mitochondrial inner membrane. We have therefore studied the activation of D-P-hydroxybutyrate apodehydrogenase by lecithin analogues as co-dispersions with phosphatidylethanolamine or phosphatidylethanolamine plus diphosphatidylglycerol to minimize the differences in the properties of microdispersions of these phospholipids.

We conclude that the apodehydrogenase does not require a specific structure in the hydrophobic region of the lecithin molecule since: 1) The D and L stereoisomers of lecithin activate the enzyme equally. 2) The activation of the apoenzyme by the dialkoxyphosphatidylcholines is similar to that obtained with the analogous synthetic diacyl (ester) lecithins. We have previously found that the activation of the apodehydrogenase by beef heart mitochondrial lecithin, which contains 50% plasmalogen (1-alkenyl-2-acylphosphatidylcholine) (Fleischer et al., 1967), is comparable with the activation by rat liver mitochondrial lecithin which is devoid of plasmalogen. 3) The enzyme is activated by several diacyloxypropyl- and diacyloxybutylphosphonylcholines, indicating that the oxygen at Site 2 (Fig. 1) is not essential for activation of the apoenzyme. 4) The apodehydrogenase is activated by octadecyleicosylphosphorylcholine, in which a branched alkyl chain is linked directly to the phosphorylcholine group.

Although the diacylglycerylphosphoryl moiety is not essential for activation of n-/?-hydroxybutyrate apodehydrogenase, the hydrophobic region is required for efficient binding of the lecithin to the apoenzyme. Our previous studies (Gazzotti et al., 1975) demonstrated that the efficiency of activation of the apodehydrogenase increases as the chain length of the acyl moieties is increased from dihexanoyl- to dioctanoyl- to didecanoyl-lecithin. In this regard, the weak association of short-chain lecithins with the apodehydrogenase could be confirmed by passage of the enzyme e dioctanoyl-lecithin complex through a gel exclusion column; complexes of the enzyme with longer chain lecithins are not readily dissociated under comparable conditions. Grover et al. (1975), using a crude preparation of the apodehydrogenase, reported that L-CC-

glycerylphosphorylcholine and L-a-diethanoylphosphatidyl- choline, do not activate the apoenzyme at all. The reason is undoubtedly referable to lack of complex formation due to negligible hydrophobic interaction. Thus, the hydrophobic moiety is essential for efficient binding to n-P-hydroxybutyrate apodehydrogenase, but there is no specificity for the diacylglycerylphosphoryl moiety of the lecithin.

The apodehydrogenase is partially activated by lysolecithin although the activity of the enzyme. lysolecithin complex decreases with time. This inactivation cannot be reversed by the subsequent addition of mitochondrial phospholipid so that it seems likely that the denaturation of the enzyme results from the detergent nature of lysolecithin. Grover et cil. (1975) reported that stearylphosphorylcholine activates a crude preparation of the apodehydrogenase and that this activity is unstable. The activation by this compound is similar to the activation with lysolecithin in terms of extent of hydrophobic- ity. Octadecyleicosylphosphorylcholine, which has twice the size of the hydrophobic moiety, forms an active stable complex with n-P-hydroxybutyrate apodehydrogenase.

Sphingomyelin from bovine brain only minimally activates the apodehydrogenase. The poor activation obtained may arise from either the high saturated fatty acid content of this lipid (Rouser et al., 1970b; Schmidt et al., 1977) or the presence of intramolecular and intermolecular hydrogen bonding in sphingomyelin liposomes (Schmidt et al., 1977). Such hydrogen bonding would likely compete with the sphingomyelin- apodehydrogenase interaction and thus limit the activation of the apoenzyme by this lipid.

In contrast with the lack of specificity of the enzyme for the structure of the diacylglycerylphosphoryl moiety, the enzyme exhibits a high degree of specificity for the choline group. Yet, some change in the structure of the choline is permissible. Increasing the distance by substituting a butyl or propyl for the ethyl group between the two charged groups of phosphorylcholine does not appreciably alter the activation of the enzyme. Substituting an isopropyl group for the ethyl group results in complete loss of activation, undoubtedly due to steric constraints. Some modification of the quaternary ammonium group is also tolerated, i.e. substitution of one of the methyl groups by an ethyl group. However, N,N,N-triethyloc- tadecyleicosylphosphorylcholine does not activate the apodehydrogenase at all. The quaternary ammonium moiety is essential since neither phosphatidylethanolamine nor N,N- dimethylphosphatidylethanolamine activate n-P-hydroxybutyrate dehydrogenase. The specific requirements of the structure of the choline moiety for activation of the apodehydrogenase are further indicated by the finding that the enzyme is not activated by dioctadecyloxypropylphosphinylcholine. The oxygen atom at Site 3 (cf Fig. l), between the phosphorus and choline, is necessary for the activation of the enzyme. Whether this oxygen atom can be replaced isosterically with a methylene group at this position was not determined.

We have previously shown that the lecithin confers on the enzyme the ability to bind its coenzyme, NADH (Gazzotti et al., 1974). We have also previously characterized the interaction of the purified enzyme with lecithins containing defined fatty acyl groups (Gazzotti et al., 1975). This is the first detailed mapping of the structural specificity of a lipid-requiring enzyme. The preparation of structural analogues of substrates is an important approach to mapping the active site of an enzyme. One of the best examples is the substrate analogue studies of acetylcholinesterase (Cohen and Oosterbaan, 1963; Nachmonsohn and Neumann, 1975). These mapping studies defined the way in which substrates and inhibitors interacted with the active site of acetylcholinesterase and led to a detailed understanding of the structure of the active site and its reactivity. Studies to map the structure of the acetylcholine receptor is another important example (Beers and Reich, 1970). Our studies of the activation of n-/3-hydroxybutyrate apodehydrogenase by lecithin analogues demonstrate that (a)

the hydrophobic moiety of lecithin is essential for effective binding but is not specific with respect to structure; (b) the quaternary ammonium seems to be required; and (c) the choline moiety exhibits high structural specificity but can be varied somewhat, within limits of steric constraints.

Acknowledgments-We thank Dr. Robert Engel for the gift of the 3,4-dihydroxybutyl bromide used in the synthesis of dimyristoyloxy- butylphosphonylcholine and Dr. Hans Betzing of the Natterman Co., Koln, West Germany, for the gift of soybean lysolecithin. We also thank Mr. R. Vicevich for capable technical assistance.

REFERENCES

Beers, W. H., and Reich, E. (1970) Nature 228,917-922 Bock, H.-G., and Fleischer, S. (1974) Methods Enzymol. 31, 374-391 Bock, H.-G., and Fleischer, S. (1975) J. Biol. Chem. 250,5774-5781 Chen, P. S., Toribara, T., and Warner, H. (1956) Anal. Chem. 28,

1756-1758 Cohen, J. A., and Oosterbaan, R. A. (1963) in Hundbuch der Expe-

rimentellen Pharmakologie, Vol. 15, 299-373, Springer-Verlag, Berlin

Cubero-Robles, E., and Van den Berg, D. (1969) Biochim. Biophys. Acta 187,520-526

Deroo, P. W., Rosenthal, A. F., Isaacson, Y. A., Vargas, L. A., and Bittman, R. (1976) Chem. Phys. Lipids 16,60-70

Dittmer, J. C., and Lester, R. L. (1964) J. Lipid Res. 5, 126-127 Fleischer, B., Casu, A., and Fleischer, S. (1966) Biochem. Biophys.

Res. Commun 24, 189-194


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Fleischer, S., Bock, H.-G., and Gazzotti, P. (1974) in Membrane Basis of Nerve Activity, pp. 105-118, Academic Press, New York Proteins in Transport and Phosphorylation (Klingenberg, M., and Rosenthal, A. F. (1966) J. Lipid Res. 7,779-785 Azzone, G. F., eds) pp. 125-136, North-Holland Publishing Co., Rosenthal, A. F. (1975) Methods Enzymol. 35,429-529 Amsterdam Rosenthal, A. F., Chodsky, S. V., and Han, S. C. H. (1969) Biochim.

Fleischer, S., and Fleischer, B. (1967) Methods Enzymol. 10,406-433 Fleishcer. S.. and Kloruven. H. (1961) Biochem. Biowhys. Res. Corn.

man. 5; 378-383 - -

Fleischer, S., Rouser, G., Fleischer, B., Casu, A., and Kritchevsky, G. (1967) J. Liwid Res. 8, 170-180

Gazzotti, P., Bock, H.-G:, and Fleischer, S. (1974) Biochem. Biophys. Res. Commun. 58,309-315

Gazzotti, P., Bock, H.-G., and Fleischer, S. (1975) J. Biol. Chem. 250, 5782-5790

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Gazzotti and S FleischerY A Isaacson, P W Deroo, A F Rosenthal, R Bittman, J O McIntyre, H G Bock, P

D-beta-hydroxybutyrate apodehydrogenase.The structural specificity of lecithin for activation of purified

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