Bioscience Reports, Vol. 6, No. 5, 1986

Uridine Sugar Nucleotides Modified in Their Nucleoside Moieties and Their Effect on

Lipid-Linked Saccharide Formation in vitro

William McDowell , 1'3 Gisbert Weckbecker 2 and Ralph T. Schwarz 1

Received March 7, I986

KEY WORDS: periodate; sugar nucleotide: 5-fluorouridine; dolichol saccharide biosynthesis.

Uridine diphospho glucose (UDP-Glc) and uridine diphospho N-acetylglucosamine (UDP-GlcNAc), modified in the uridine moiety by either periodate oxidation of the ribose ring or substitution at position 5 of the uracil ring with fluorine, have been tested as potential inhibitors of glucosyl monophosphoryl dolichol (Glc-P-Dol) or N,N- diacetylchitobiosyl pyrophosphoryl dolichol ((GlcNAc)2-PP-Dol) assembly in chick embryo cell membranes. The periodate oxidised sugar nucleotides inhibited glycosyl transfer from their respective natural counterparts by 50%0 at 230#m periodate oxidised UDP-Glc and 70/~rn periodate oxidised UDP-GlcNAc respectively. Inhibition in both cases was irreversible and addition of exogenous Dol-P stimulated only the residual non-inhibited reaction. Periodate oxidised UDP-GlcNAc preferentially inhibited the transfer of GlcNAc to GlcNac-PP-Dol.

The sugar nucleotides containing 5-fluorouridine were, on the other hand, alternative substrates for Glc-P-Dol or (GlcNAc)2-PP-Dol synthesis. FUDP-Glc was a good substrate for Glc-P-Dol formation; having Km and Vmax values equal to those of UDP-Glc, whereas FUDP-GIcNAc was a less efficient substrate for the formation of (GlcNAc)2-PP-Dol; having Km and Vmax values one half and one third respectively of those of UDP-GlcNAc.

1 lnstitut fiir Virologie, Justus Liebig Universit/it Giessen, Frankfurter Strasse 107, D-6300 Giessen, F.R.G. 2 Biochemisches Institut, Albert Ludwigs Universitfit Freiburg, Hermann Herder Strasse 7, D-7800 Freiburg

im Breisgau, F.R.G. a To whom correspondence should be addressed.

435 014448463/86/0500-0435505.00/0 �9 1986 Plenum Publishing Corporation

436 McDowell, Weckbecker, and Schwarz

INTRODUCTION

N-Glycosylation of glycoproteins is a complex process involving sugar nucleotides, lipid intermediates, glycosyltransferases and processing glycosidases (Hubbard and Ivatt, 1981 ; Kornfeld, 1982; Schwarz and Datema, 1982a; Kornfeld and Kornfeld, 1985). Sugar analogues have been shown to interfere (Schwarz and Datema, 1982a) with the assembly of GlcaMan9(GlcNAc)2 PP-Dol, the lipid-linked oligosaccharide precursor of N-linked protein-bound carbohydrate (Li et al., 1978). Specifically, in the cases of 2-deoxy glucose, 2-fluoro 2-deoxy glucose and 2-fluoro 2- deoxy mannose, the interference has been attributed to the nucleotide esters of the sugar analogues (Schwarz and Datema, 1982a; Datema and Schwarz, 1984; McDowell et al., 1985).

The effects of modifications in the nucleoside moiety of sugar nucleotides on the activity of glycosyltransferases are not as well documented as those of modification in the sugar moiety. However, in a study of the substrate specificities of glycosyltransferases involved in bacterial polysaccharide assembly (Kochetkov and Shibaev, 1974; Shibaev, 1978) it was found that for uridine diphospho glucose (UDP- Glc) only the NH group of uracil together with the 3-hydroxyl of glucose were necessary for the interaction with glucosyltransferase (Kochetkov and Shibaev, 1974; Shibaev, 1978).

A recent report by Prehm (1985) showed that UDP-GlcNAc and UDP-glucuronic acid modified by periodate oxidation of the ribose ring in their uridine moieties were effective inhibitors of hyaluronic acid synthesis. It has also been shown that the 5- fluorouracil-containing analogue of UD P-N-acetylmuramyl pentapeptide functions as a poor substrate and effective competitive inhibitor in the translocase catalysed transfer of phospho-N-acetylmuramyl pentapeptide from its UDP-donor to lipid acceptor during bacterial ceil wall biosynthesis (Stickgold and Neuhaus, 1967).

We were interested to know if 5-fluorouridine- or periodate oxidised-sugar nucleotides would similarly affect lipid-linked oligosaccharide synthesis via the dolichol pathway. In this report it is shown that periodate oxidised UDP-Glc or U D P - GlcNAc inhibit the synthesis of glucosyl monophosphoryl dolichol (Glc-P Dol) or N,N-diacetylchitobiosyl pyrophosphoryl dolichol ((GlcNAc)2-PP Dol) respectively whereas the corresponding 5-fluorouridine derivatives are alternative substrates.

MATERIALS A N D M E T H O D S

Materials

UDP-[U14C]Glc (292 Ci/mol) and UDP [6-3H]GIc (3.1 Ci/mmol) were obtained from Amersham Biichler (Braunschweig, FRG). UDP-[U~4C]GIcNAc (235 Ci/mol) and [~4C]Glc-P Dol (6.5 Ci/mmol) were from NEN (Dreieich, FRG). UDP-GIcNAc and UDP-Glc were bought from Boehringer Mannheim (Mannheim, FRG). [14C](GlcNAc)2-PP-Dol and [t4C]GlcNAc-PP-Dol were prepared as described (Schwarz and Datema, 1982b). Dol-P was from Sigma (Miinchen, FRG). All other chemical reagents were of the highest purity available.

Modified-Uridine Containing Sugar Nucieotides 437

Periodate Oxidation of UDP-GIc and UDP-GIcNAc

Sugar nucleotide (50 #mol) was dissolved in 1 ml 40 mM sodium phosphate buffer, pH 6.8 and cooled in ice. Sodium metaperiodate (50 ~tmol) was added and the mixture incubated on ice ~'o," 60 min. Oxidation was terminated by addition of 10/it (134//moll glycerol. The periodate oxidised sugar nucleotide was then purified by gel filtration through a column (1 x 17 cm) of Sephadex G-10 using water as eluent. Fractions exhibiting absorbance at 260 nm were pooled and freeze-dried. The concentration of the periodate oxidised sugar nucleotide was determined by measurement of the absorbance at 260nm assuming a molar absorption coefficient of 10,000 litre mol-1 cm i (Prehm, 1985).

Peroxidate oxidation of UDP-[X4C]GIc and UDP-l-liC]GtcNAc was carried out as above using 2/xCi (6.8 nmol) of labelled sugar nucteotide and 6.8 nmol sodium periodate in a total volume of 200 ~tl sodium phosphate buffer pH 6.8. On cellulose TLC (solvent 4), the periodate oxidised sugar nucleotides migrated as single spots with mobilities differing significantly from those of the parent compounds. The sugar moieties, released by digestion with phosphodiesterase and alkaline phosphatase (Schwarz and Schmidt, 1976), were analysed by paper chromatography (Whatmann 3MM, solvent 3) and found to comigrate with authentic glucose or N- acetylglucosamine. This confirms that the cis hydroxyls of ribose were cleaved under the conditions used and that the hexose units remained intact. Vicinal hydroxyls in a cis configuration are known to be oxidised faster than those in a transconfiguration (Bobbit, 1956; Guthrie, 1961).

Preparation of 5-Fluorouridine Diphospho (FUDP) Sugars

The procedures used to prepare and purify FUDP-GIc and FUDP-GlcNAc in unlabelled and labelled forms have already been described in detail (Weckbecker and Keppler, 1984). The specific activities of the labelled compounds were as follows: FUDP [6-3H]GIc (600 Ci/mol) and FUDP-GIcN[~4C]Ac (57 Ci/mol).

Thin Layer and Paper Chromatography

Chromatograms were developed using the following systems (all solvent ratios are by volume): (1) Chloroform-methanol-ammonia-water (65:35:4:4); (2) Chloroform-methanol-water (60:39:6); (3) Ethylacetate-pyridine-acetic acid-water (5:5:1:3); (4) Ethanol-lM ammonium acetate pH 7.5 (13:7).

Radioactivity on thin layer plates was located with a Berthold LB 2842 automatic scanning system. Unlabelled sugars were located by spraying with aniline citrate (a 1 :l mixture of 2% aniline in ethanol and 0.2 M citric acid) followed by heating at I t0~ for 10-15 min.

Other Procedures

The preparation of crude microsomaI membranes from primary chick embryo cells has been described (Schwarz and Datema, 1982b), as has the in vitro synthesis of

438

Glc-P-Dol and (GlcNAc)2-PP-Dol from labelled respectively (Schwarz and Datema, 1982b).

McDoweiI, Weckbecker, and Schwarz

UDP-GIc and UDP-GlcNAc

RESULTS

Periodate Oxidixed UDP-GIc and UDP-GIcNAc as lnhibitors of GIc-P-Dol and (GIeNAc)2-PP-Dol Biosynthesis

The transfer of [14C]Glc or [-14C]GlcNAc from their respective UDP-sugar nucleotides to material soluble in chloroform-methanol (2:1 v/v; CM) was inhibited by periodate oxidised UDP Glc (o-UDP-Glc) and periodate oxidised UDP-GIcNAc (o-UDP-GIcNAc) respectively (Fig. 1). In both cases the inhibition was concentration- dependent with 50 % inhibition of [14C] GlcNAc or [14C] Glc transfer caused by 70 #m o-UDP-GlcNAc or 230/~M o-UDP-Glc respectively. It was possible to totally inhibit [14C]Glc transfer by using 1 mM o-UDP-Glc. Analysis of CM extracts from the incubations containing 200 #M periodate oxidised sugar nucleotides by TLC showed that o-UDP-Glc was inhibiting GIc-P-Dol formation (data not shown), whereas o- UDP-GlcNAc specifically blocked the transfer of GlcNAc to GlcNAc-PP-Dol since only GlcNAc-PP-Dol was present in the CM extract (Fig. 2a,b).

Addition of exogenous DoI-P (0.3 mg/ml) did not relieve the inhibitory effects of the periodate oxidised sugar nucleotides, but it stimulated only what is assumed to be the residual non-inhibited enzyme activity (Table 1). In the case of o-UDP-GlcNAc inhibited GlcNAc transfer, only G!cNAc-PP-Dol formation was observed in the presence of exogenous Dol-P when the CM extract was examined by TLC (Fig. 2c).

r c-

>

ID O

"O

O C'6

100 a)

60 1,0 2 0 %

100[ b) 0iv" 60 ~,0 20 ~a

' 160' 260=q'5~

Inhibitor Concentration(tJM)

Fig. 1. Inhibition of Glc P-Dol and (GIcNAc) 2 PP-Dol syhthesis by modified uridine sugar nucleotides. Microsomal membranes (0.8 or 1,6 mg protein) from chick embryo cells were incubated in 60~tl of 120/4 of a solution comprising 13raM Tris HCI, pH 7.5, 100raM NaC1, 0.3raM MgC1 z, 0.3raM MnC12 and (a) 0.05#Ci UDP-[14C]Glc or (b) 0.05#Ci UDP-[t4"C]GIcNAc in the presence of the corresponding periodate oxidised sugar nucleotide (open symbols) or FUDP-sugar (closed symbols). The incorporation of radioactivity into the CM extract was determined as described by Schwarz and Datema (1982b).

Modified-Uridine Containing Sugar Nucleotides 439

2 Oh

C.. 0

L)

-'d

>.,

o qo E~

90l

50

110

1 N

b)

k cl

a/

0 5 10 15 20

Distance Migrated (cm)

Fig.2. Inhibition ofthetransfer of GlcNAc to GIcNAc PP- Dol by periodate oxidised UDP-GIcNAc. The CM extracts from incubation mixtures synthesizing (GtcNAc)z PP-Dol from UDP-[ I~C] GIcNAc and containing (a) no additions (b) o UDP GlcNAc (200/~M) or (c) o-UDP-GlcNAc (200#M) and Dol-P (0.3 mg/ml) were subjected to TLC on Silica G-60 using solvent system 2 and scanned for radioactivity. The arrows denote the positions of the following: N neutral glycotipid; l GlcNAc-PP Dol; 2 (GlcNAc)z-PP Dol.

Table 1. Effect of exogenous Dol-P on the inhibitory effects of the base-modified sugars

Sugar nucleotide

Periodate oxidised FUDP-containing

Substrate Radioactivity Radioactivity Addition (dpm) in CM Addition (dpm) in CM

UDP-( 14C)GIc

UDP-( 14C)GIcNAc

None 5910 None 2120 Dol P 27637 Dol-P 12270 o UDP-GIc 3417 FUDP-GIc 630 DoI-P + o-UDP-GIc 13100 DoI-P + FUDP-Glc 6108

None 4107 None 4698 Dol-P 15768 DoI-P 12908 o UDP-GIcNAc 1166 FUDP-GIcNAc 2507 Dol-P + o-UDP-Glc 2804 Dol P + FUDP-Glc 12485

Assay mixtures (see legend to Fig. l ) catalysing Glc-P-Dol or (GlcNAc)2-PP-Dol synthesis were incubated with Dol-P (0.3mg/mI), 200r inhibitor, of Dol-P and inhibitor together and incorporation of radioactivity into the CM extract determined after TLC.

4411 McDowell, Weckbecker, and Schwarz

Mechanism of Inhibition by Periodate Oxidised Sugar Nucleotides

It would appear from the above results that both inhibitors irreversibly block the activity of a specific glycosyltransferase. To test this possibility, the microsomal membrane preparation was preincubated with 200~m~ of either o - U D P - G l c or o - U D P - G l c N A c for various times up to 30rain. The appropriate labelled sugar nucleotide was then added and glycosyltransfer monitored. Routinely 5rain preincubation was used, and inhibitions of 47% and 60~ ~ for o - U D P G I c and o - UDPGlcNAc respectively were obtained. However, increasing the preincubation time to 10rain increased the degree of inhibition to 58% and 68% respectively and inhibitions of 77% and 85% respectively were obtained on increasing the preincubation time to 30 rain, Another approach was to incubate the membrane preparation and o - UDP-sugar (200/zm) for 15 rain and then reisolate the membranes and assay either [14C]Glc or [~4C]GlcNAc transfer in the treated membranes in the absence of further added inhibitors. The results (Table 2) show that the treated membranes have a lower capacity for transferring [I~C]Glc or [~4C]GlcNAc. These results support the idea that the periodate oxidised sugar nucleotides become covalently bound to their respective glycosyltransferases as has been observed with other systems (Colman, 1983).

Table 2. Capacity of membranes pretreated with o UDP Glc or o-UDP GIcNAc to catalyse gIucosyl transfer or N-acetylglucosaminyl transfer

Membrane Radioactivity (dpm) Substrate source incorporated into CM

UDP-(14C)G1 c Untreated 10493 o- UDP-Glc treated 7342

UDP (14C)GIcNAc Untreated 6463 o UDP GlcNAc treated 2163

Membranes (1.6 mg protein) were incubated in 120~1 of a solution comprising 13 mM Tris HCI pH 7.5, i00 mM NaCI, 0.3 mM MgCI~ and 0.3 mM McCI z, containing no inhibitor of 200 ~LM periodate oxidised sugar nucleotide, for 15 rain at 37'. The membranes were then reisolated by ultracentrifugation at 100,000 g for 60rain at #' and assayed for Glc P-Dol or (GlcNAc)z PP Dol synthesis.

Incubation of o - U D P [14C]Glc or o -UDP[14C]GlcNAc with the membrane preparation did not result in either [I~C]Glc or [14C]GlcNAc transfer to material soluble in CM, confirming that the periodate oxidised sugar nucleotides are not acting as alternative substrates.

5-Fluorouridine Diphospho Sugars in Reactions of the Dolichol Pathway

Incubation of F U D P - G l c or F U D P GIcNAc with membranes catalysing the formation of GIc -P-Dol from UDP-[14C]Glc or (GIcNAc)2-PP-Dol from U D P - [14C]GlcNAc resulted in a concentration-dependent reduction in the label incorporated into the CM-soluble extract in both cases (Fig. 1). The F U D P sugar nudeotide concentration for 50% inhibition was 8 ~M and 60 pM for F U D P - G I c and FUDP-GlcNAc , respectively. Unlike the situation with the periodate oxidised sugar nucleotides, addition of exogenous D o I - P relieved the inhibition of lipid saccharide

Modified-Uridine Containing Sugar Nucleotides 441

synthesis (Table 1). This suggests that the FUDP-sugars are competing with their labelled natural counterparts in monosaccharide transfer and are therefore alternative substrates for Glc P-Dol or (GlcNAc)2-PP-Dol synthesis.

F U D P - G l c and F U D P - G I c N A c as Substrates for Saccharide P(P) Dol Synthesis

The substrate properties of the two FUDP-sugars were tested by incubating F UDP [aH]Glc and FUDP-GlcN[14C]Ac with microsomal membranes. In both cases label was incorporated into the CM soluble extract. Subsequent analysis of the product formed from FUDP- [~H]Glc by TLC (Silica G-60 with solvent 1) showed a single product that co-migrated with authentic Glc P-Dol. Incorporation of label into Glc- P Dol was stimulated by exogenous Dol-P. The kinetic parameters (Kin, Vmax) for F U D P ~3HJGtc were equal to those of UDP-Glc for GIc-P-Dol synthesis (Table 3), indicating that the presence of a 5-fluoro group on the uridine base does not affect either recognition of the sugar nucleotide by the enzyme or the transferase reaction.

Table 3. Comparison between the kinetic parameters for FUDP-sugars and those of the natural sugar nucleotides

Sugar nucleotide Km (/~M) Vmax pmoles sugar transferred

min - 1 (rag protein)-

UDP Glc 1.0 1.0 FUDP-GIc 0.9 0.8 UDP GIcNAc 5.0 1.6 FUDP-GIcNAc t0.0 0.5

With FUDP-GlcN[I4C]Ac as substrate, TLC analysis (Silica G-60, solvent 2) showed the presence of three products. The major product (representing 61% of the label) migrated as a neutral glycolipid. The other two components comigrated with authentic GlcNAc PP-Dol and (GlcNAc)2-PP-Dol and represented 199/o and 20% of the label respectively. Addition of exogenous Do l -P stimulated incorporation into the two dolichol derivatives, which were still present in the same ratio, but no neutral glycolipid was formed under these latter conditions. In contrast to F U D P - G l c the kinetic parameters of FUDP-GIcN[14C]Ac differed from those of UDP-GlcNAc (Table 3). Thus in the case of FUDP-GtcNAc a 5-fluoro substituent on uridine lowers the affinity of the sugar nucleotide for the enzymes synthesizing (GlcNAcJ2-PP Dol and consequently glycosyltransfer is reduced.

These properties of FUD P-Glc and FUDP-GlcNAc are in line with the view that they are alternative substrates for, rather than inhibitors of, GIc-P Doi and (GlcNAc)2-PP-Dol synthesis, respectively.

DISCUSSION

Modification of the uridine moiety of UDP-sugar nucleotides, either by periodate oxidation of the vicinal hydroxyl groups of the ribose ring or by substitution with

442 McDowell, Weckbecker, and Schwarz

fluorine at position 5 of uracil, produces different effects on the glycosyltransferases catalysing the formation of lipid-linked saccharides. Periodate oxidation results in a molecule that is a suicide substrate which becomes covalently bound to enzyme active site (Colman, 1983). Periodate oxidised nucleotides have found use as inhibitors and affinity labels of enzymes utilizing ATP, G D P or NADP (Colman, 1983). Enzyme inactivation has been attributed either to the formation of a Schiff base with the E- amino group of lysine or to the formation of a dihydroxymorpholino derivative and/~- elimination of the 5' phosphate group (Colman, 1983).

It is interesting that o -UDP-GlcNAc preferentially inhibits the transfer of GIcNAc to GlcNAc-PP Dol. In this respect it resembles the antibiotic diumycin which strongly inhibits the same reaction (Villemez and Carlo, 1980), This specificity of o - UDP-GlcNAc for the second reaction in the dolichol pathway is most probably due to the fact that the first reaction entails the transfer of GlcNAc-l-phosphate to dolichol monophosphate to form GlcNAc-PP Dol. Thus the binding of the sugar nucleotide to the active site of the first enzyme will differ to that to the second enzyme in the pathway, since a pyrophosphate bond in the substrate is broken in the first reaction and a sugar- phosphate bond is broken in the second. Periodate oxidation of sugar nucleotides under mild conditions offers the possibility of easily preparing potential inhibitors of glycosyltransferases.

In contrast to the periodate oxidised sugar nucleotides, the 5-fluorouridine- containing derivatives are not inhibitors of Glc-P-Dol and (GIcNAc)2 PP Dol synthesis, but are alternative substrates. However, UDP-Glc : Dol -P glycosyltransferase tolerated the presence of the 5-fluoro substituent on UDP-Glc whereas UDPGlcNAc: Dol -P N-acetylglucosaminyl-l-phosphotransferase and UDP-GlcNAc: GlcNAc PP-Dol : N-acetylglucosaminyltransferase did so to a lesser degree. FUDP-GlcNAc was an inefficient substrate for (GlcNAc)2-PP Dol synthesis. An investigation of the substrate properties of 5-fluorouridine sugar nucleotides detected in hepatoma cells treated with the chemotherapeutic agent 5-fluorouridine (Weckbecker and Keppler, 1984) revealed that they could serve as substrates for enzymes of UDP-sugar metabolism including UDP-Glc pyrophosphorylase, U D P - Glc dehydrogenase, UDP-Glc-4'-epimerase, glycogen synthase, galactose-1- phosphate uridylyltransferase and UDP-GlcNAc 2-epimerase. However, with the exception of the latter enzyme, decreased Vmax values were observed when FUDP- sugars were used as substrates. Thus a wide range of enzymes utilizing UDP-sugars as substrates are also able to act on the fluorinated derivatives.

Substrate analogues modified in only one specific site can be used to define recognition sites on the substrate molecule necessary for interaction with the active site of the enzyme. Such an approach has been used (Shibaev, 1978) to evaluate the structural features o f U D P Glc for interacting with glucosyltransferases. Only the NH group of uracil and the 3-hydroxyl group of glucose were found to be necessary. Positions 4, 5 and 6 of the uracil ring as well as the 2-hydroxyl ofribose were not part of the recognition sites (Shibaev, 1978). Therefore the present results with 5-fluorouridine sugar nucleotides appear to fit this pattern. However, the inhibition of protein glycosylation in herpes virus infected cells, treated with the anti-viral drug (E)-5-(2- bromovinyl)-2r-deoxyuridine (BVdU) (Olofsson et al., 1985), by BVdU sugar

Modified-Uridine Containing Sugar Nncleotides 443

nucleot ides indicates that modi f ica t ion in the uraci l moie ty can p roduce inh ib i tors of

g lycosyla t ion reactions.

A C K N O W L E D G E M E N T S

This work was suppor t ed by the Deutsche Forschungsgemeinschaf t (SFB 47; F o r s c h u n g s g r u p p e Leber and S F B 154, F re ibu rg ; and a Heisenberg Fe l lowsh ip to R.T.S.) and F o n d s der Chemischen Indust r ie . The technical assis tance of Kirs t in

Salser and Ulr ike Klein was much apprec ia ted . The advice and encouragement of Dr D. Kepp l e r is also acknowledged .

REFERENCES

Bobbit, J. M. (1956). Adv. Carbohydr. Chem. 11:1~41. Colman, R. F. (1983). Ann. Rev. Biochem. 52:67-91. Datema, R. and Schwarz, R. T. (1984). Biosci. Rep. 4:213 221. Guthrie, R. D. (1961). Adv. Carbohydr. Chem. 16:105-158. Hubbard, S. C. and Ivatt, R. J. (1981). Ann. Rev. Biochem. 50:555-583. Kochetkov, N. K., and Shibaev, V. N. (1974). lzv. Acad. Nauk SSSR, Set. Khim. 1169-1189 (cited in Shibaev

(1978)). Kornfeld, R., and Kornfeld, S. (1985). Ann. Rev. Biochem. 54:631-664. KornMd, S. (1982). The Glycoconjugates (Horowitz, M. I., Ed.), Vol. III, pp. 3-22, Academic Press, New

York. Li, E., Tabas, I., and Kornfeld, S. (1978). J. Biol. Chem. 253:7762-7770. McDowell, W., Datema, R., Romero, P. A., and Schwarz, R. T. (1985). Biochemistry 24:8145 8152. Olofsson, S., Lundstrom, M., and Datevna, R. (1985). Virology 147:201 205. Prehm, P. (1985). Biochem. d. 225:699-705. Schwarz, R. T., and Datema, R. (1982a). Adv. Carbohydr. Chem. Biochem. 40:287-379. Schwarz, R. T., and Datema, R. (1982b). Meth. Enzymol. 83:432-443. Schwarz, R. T., and Schmidt, M. F. G. (1976). Eur. J. Biochem. 62:181-187. Shibaev, V. N. (1978). Pure Appl. Chem. 50:1421-1436. Stickgold, R. A., and Neubaus, F. C. (1967). J. Biol. Chem. 242:1331-1337. Villemez, C. L., and Carlo, P. L. (1980). J. Biol. Chem. 255:8174-8178. Weckbecker, G., and Keppler, D. O. R. (1984). Biochem. Pharmacol. 33:2291-2298.