plant phospholipase d · plant phospholipase d i. studies on cottonseed and cabbage phospholipase...

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PLANT PHOSPHOLIPASE D I. STUDIES ON COTTONSEED AND CABBAGE PHOSPHOLIPASE D* BY HARVEY L. TOOKEY AND A. K. BALLS (From the I)epartment of Biochemistry, Purdue University, Lafayette, Indiana) (Received for publication, June 13, 1955) Phospholipases are of current interest in two rather distinct lines of research: the general interest in phospholipide metabolism, and the special effects of such enzymes on terminal oxidase systems. The present paper deals with certain descriptive observations on the relatively little studied phospholipase D1 obtained from two plant sources. A subsequent paper is concerned with its inactivating effect on succinic oxidase. Phospholipase D is a widely distributed enzyme, occurring in such un- related plants as cabbage (5), Hevea latex (6), soy beans, maple trees (7), etc. The phospholipide of seeds disappears during germination, reappear- ing as water-soluble choline and other compounds (7), thus suggesting a general r&e for the enzyme in the utilization of reserve phospholipide. Nearly all of the work on phospholipase D has been done by measuring only the choline released from lecithin. However, Rose (8) found that the cabbage enzyme released either serine or ethanolamine from “cepha- lin.” Kates (9, 10) has recently shown that in certain leaves, notably spinach and cabbage, the enzyme is confined to the chloroplast fraction, but no soluble preparations of phospholipase D have been reported. It now appears, however, t,hat the enzyme from a new source, cottonseed, can exist in an apparently soluble form. It attacks phosphatidyl ethanol- amine as well as lecithin. The principal distinguishing properties for the cottonseed enzyme are its apparent solubilit,y in water and its lack of act,ivation by diethyl ether. * Journal Paper No. 873 of the Purdue University Agricultural Experiment Sta- tion, Lafayette, Indiana. 1 Phospholipase D hydrolyzes phospholipide to release free nitrogen base and phosphatidic acid. Since these enzymes also cleave substrates other than lecithin, they are termed phospholipases. Some authors refer to phospholipase D as phos- pholipase C or lecithinase C, following the prophecies of Contardi and Ercoli (1); we and others (2) follow the names assigned as the enzymes were discovered: leci- thinase C (Clostridium welchii a-toxin) was named in 1941 (3) before phospholipase D was discovered (4). 213 by guest on June 25, 2020 http://www.jbc.org/ Downloaded from

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Page 1: PLANT PHOSPHOLIPASE D · PLANT PHOSPHOLIPASE D I. STUDIES ON COTTONSEED AND CABBAGE PHOSPHOLIPASE D* BY HARVEY L. TOOKEY AND A. K. BALLS (From the I)epartment of Biochemistry, Purdue

PLANT PHOSPHOLIPASE D

I. STUDIES ON COTTONSEED AND CABBAGE PHOSPHOLIPASE D*

BY HARVEY L. TOOKEY AND A. K. BALLS

(From the I)epartment of Biochemistry, Purdue University, Lafayette, Indiana)

(Received for publication, June 13, 1955)

Phospholipases are of current interest in two rather distinct lines of research: the general interest in phospholipide metabolism, and the special effects of such enzymes on terminal oxidase systems. The present paper deals with certain descriptive observations on the relatively little studied phospholipase D1 obtained from two plant sources. A subsequent paper is concerned with its inactivating effect on succinic oxidase.

Phospholipase D is a widely distributed enzyme, occurring in such un- related plants as cabbage (5), Hevea latex (6), soy beans, maple trees (7), etc. The phospholipide of seeds disappears during germination, reappear- ing as water-soluble choline and other compounds (7), thus suggesting a general r&e for the enzyme in the utilization of reserve phospholipide.

Nearly all of the work on phospholipase D has been done by measuring only the choline released from lecithin. However, Rose (8) found that the cabbage enzyme released either serine or ethanolamine from “cepha- lin.” Kates (9, 10) has recently shown that in certain leaves, notably spinach and cabbage, the enzyme is confined to the chloroplast fraction, but no soluble preparations of phospholipase D have been reported. It now appears, however, t,hat the enzyme from a new source, cottonseed, can exist in an apparently soluble form. It attacks phosphatidyl ethanol- amine as well as lecithin. The principal distinguishing properties for the cottonseed enzyme are its apparent solubilit,y in water and its lack of act,ivation by diethyl ether.

* Journal Paper No. 873 of the Purdue University Agricultural Experiment Sta- tion, Lafayette, Indiana.

1 Phospholipase D hydrolyzes phospholipide to release free nitrogen base and phosphatidic acid. Since these enzymes also cleave substrates other than lecithin, they are termed phospholipases. Some authors refer to phospholipase D as phos- pholipase C or lecithinase C, following the prophecies of Contardi and Ercoli (1); we and others (2) follow the names assigned as the enzymes were discovered: leci- thinase C (Clostridium welchii a-toxin) was named in 1941 (3) before phospholipase D was discovered (4).

213

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Page 2: PLANT PHOSPHOLIPASE D · PLANT PHOSPHOLIPASE D I. STUDIES ON COTTONSEED AND CABBAGE PHOSPHOLIPASE D* BY HARVEY L. TOOKEY AND A. K. BALLS (From the I)epartment of Biochemistry, Purdue

214 COTTONSEED AND CABBAGE PHOSPHOLIPASE D

Materials and Methods

Enzymatic Assay-The assay for cephalin hydrolysis was adapted from Rose (8). In many instances it was convenient to use smaller quantities of enzyme. This was accomplished by conducting the reaction in the microdiffusion apparatus of Kirk and Tompkins (11). 1 ml. of reaction mixture usually contained 4 mg. of enzyme (dry weight), water, and 40 mg. of substrate emulsified in phosphate buffer to give a final concentration of 0.1 M phosphate. After incubation, the reaction was stopped with 0.4 ml. of saturated potassium metaborate (12). 0.1 ml. of 2 M periodic acid (HJO,J was placed in the larger bulb of the vessel (not in contact with the digest), 0.05 ml. of 20 per cent sulfuric acid was placed in the spoon, the cell evacuated, and the periodic acid then mixed with the digest. The ammonia was allowed to diffuse for 2 hours at room temperature, after which the sulfuric acid was made up to 2.00 ml. and nesslerized (13). A Beckman model B spectrophotometer at 490 rnp was used to measure the color developed. Controls were run with all assays either by using boiled enzyme or by adding the enzyme after the addition of the meta- borate. Only free ethanolamine and serine2 are measured by the method. Release of choline from lecithin was measured separately by the aqueous Reinecke salt method of Snell and Snell (14).

Purified soy bean “lecithin RG” was used as a substrate throughout. This is a mixture of phospholipides having an approximate percentile analysis as follows:3 cephalin, 29.2; lecithin, 29.2; inositol phosphatides, 31.4; glycerides, 4.0; sugars, glucosides, 5.2; insoluble materials, moisture, 1.0.

Chemical Methods-Inorganic phosphate was determined by the method of Allen (16). Turbidity in the solutions was avoided by coagulation of lecithin RG with HCIO1, followed by centrifugation. Enzymatic diges- tions were carried out in 0.1 M acetate buffer at pH 5.9 when phosphate was to be measured subsequently.

Calcium phosphate gel was prepared according to Kunitz (17). Free fatty acids were determined by the method of Fairbairn (18). Chromatography-Whatman No. 1 paper “for chromatography” was

employed throughout. Lutidine-ethanol and phenol-water developing solvents are described by Block (19). Butanol-acetic acid (Solvent I) was made by shaking together 49 ml. of n-butanol, 49 ml. of distilled H20,

s Threonine would also be measured, but this is not known to occur in phospho- lipides. The results are reported as ethanolamine N, since no serine is liberated by the enzyme, as shown by paper chromatograms.

3 Obtained through the courtesy of Mr. John Lathe of The Glidden Company. The analytical data were also furnished by The Glidden Company and are in quite good agreement with those of Scholfield et al. (15).

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H. L. TOOKEY AND A. K. BALLS 215

and 2 ml. of glacial acetic acid, then using the upper phase. Butanol- acetic acid (Solvent II) consisted of 4 volumes of n-butanol, 1 volume of glacial acetic acid, and 1 volume of water. 0.2 per cent ninhydrin in water- saturated n-butanol was used as a color reagent.

Digests for chromatography were prepared in a manner designed to concentrate t)he end-products. For example, 10 mg. of the cottonseed enzyme were incubated for 6 hours with 100 mg. of lecithin RG in the presence of 0.4 M NaCl and 0.1 M phosphate buffer at pH 5.9. (The total volume was 2.5 ml.) Boiled enzyme was used for the control. The digest was acidified, extracted with ether, and 2 ml. of the aqueous phase were evaporated to 0.2 ml. 5 ~1. of the concentrate were then applied to the paper. The digests contained appreciable amounts of NaCl, but this did not interfere in the preparation of chromatograms suitable for showing ethanolamine and serine.

RESULTS AND DISCUSSION

Enzyme Preparations

Cabbage Enzyme-The enzyme from cabbage leaves was prepared by homogenizing fresh leaves, from which the center ribs had been removed, with an equal weight of water, saturating the press juice therefrom with NaCl, and dialyzing the resulting precipitate against distilled water as in Step B below. The suspension was then lyophilized to yield a stable pale green powder.

Cottonseed Enzyme. Step A; Crude Extract-Cottonseed meal4 was extracted with hexane and air-dried. The meal was next disintegrated with water (8 to 12 ml. per gm.) in a colloid mill, and the pH was adjusted to 8.2 to 8.4 with NaOH. After standing in the cold 2 hours, the mixture was centrifuged at 800 X g to remove the larger particles.

Step B; NaCl Precipitate-The crude extract was saturated with NaCl and allowed to stand 4 hours in the cold. The precipitate was collected by 40 minutes centrifugation at 26,000 X g (average centrifugal force) and dialyzed overnight against 400 volumes of cold distilled water. The precipitate does not dissolve appreciably.

Alternative Step B; El&ion from Calcium Phosphate Gel-The crude ex- tract from cottonseed was treated with twice its volume of calcium phos- phate gel at pH 6.1, and the gel eluted with 0.07 M phosphate buffer at pH 8.2. The eluate was dialyzed overnight against 50 volumes of cold distilled water and lyophilized. The preparation does not precipitate on dialysis.

4 Cottonseed “meats” specially prepared without heating were obtained through the courtesy of Dr. Willy Lange of The Procter and Gamble Company.

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216 COTTONSEED AND CABBAGE PHOSPHOLIPASE D

Xtep C; Second E&act---The contents of the dialysis bag (from Step B) were extracted with distilled water at a slightly alkaline pH and centri- fuged 20 minutes at 26,360 X g. The clear supernatant liquid was ad- justed to pH 8.0 and lyophilized. While wet, the enzyme was unstable, but the dry preparations retained their activity completely in cold storage for at least 4 months.

The degree of enzyme purification achieved is summarized in Table I. All values are averages of at least two determinations.

TABLE I

Steps in Purification of Cottonseed Phospholipase D

Enzyme preparation Specific activity Yield

Defatted meali ................................... Step A;crude extract .............................

“ B; NaCl ppt., lyophilized .................... “ C;2nd extract ...............................

Alternative Step B; calcium phosphate gel ........

5.9$ 19.0 9.1

units* per gm. meal

220 344 384 357 157

* For brevity, the activity has been expressed in units defined as follows: 1 unit corresponds to the release of 1 y of ethanolamine nitrogen in 15 hours at 25” in 0.10 M phosphate buffer at pH 5.9. The system contained 60 mg. of enzyme preparation and 200 mg. of lecithin RG in a total volume of 4.0 ml.

t Prepared by mixing the meal with 0.10 M phosphate, pH 5.9. Increasing yields might be attributed to a pH difference between this and the other preparations, al- though the presence of an inhibitor is not excluded.

$ The usual preparation from cabbage leaves corresponding to this step had a specific activity of 5.0 units per mg.

Cottonseed phospholipase D has thus been obtained in an aqueous fraction which is not sedimented by 20 minutes centrifugation at 26,360 X g, in contrast t,o cabbage phospholipase D which is in the chloroplast fraction (10). Since various authors give values from 10,000 to 20,000 X g as sufficient for the removal of plastids and mitochondria (20, 21), it is felt that the cottonseed phospholipase D is soluble, or at least a very small particle.

By way of a further distinction between the two enzymes, the enzyme from cabbage leaves has been found to be potently activated by saturation of the aqueous medium with diethyl ether (10). Under corresponding conditions, we found the cottonseed enzyme to exhibit only 70 per cent of its normal activity. It is plausible that the particulate nature of the cabbage preparation is responsible for this difference in properties.

pH Optimum-The pH observed as optimal was essentially the same with preparations from both cottonseed and cabbage, as shown in Fig. 1.

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H. L. TOOKEY AND A. K. BALLS 217

Calibration of Assay-The relation between the ethanolamine liberated at pH 5.9 and the amount of enzyme present is shown in Fig. 2; that be- tween observed activity and amount of substrate in Fig. 3. These findings for the release of ethanolamine are similar to Dhe results of Kates (10)

P 0

3 4 5 6 7 8 120 160

PH MILLIGRAMS ENZYME

FIN. 1 FIG. 2

FIG. 1. pH dependence of phospholipase D activity. One preparation from cab- bage leaves (X), and two preparations (Step B, lyophilised) from cottonseed (0, 0). The ethanolamine measured was that released in 15 hours at 25” by 60 mg. of enzyme preparation from 200 mg. of substrate in 0.07 to 0.10 M phosphate buffer. Total volume 4.0 ml.

FIG. 2. Dependence of activity on concentration of enzyme. Liberation of etha- nolamine by cottonseed preparation (Step C) (0) was measured after 2 hours at 30” (400 mg. of lecithin RG, total volume 10.0 ml.); by cabbage leaf preparation (X) after 15 hours at 25” (150 mg. of lecithin RG, total volume 4.0 ml.). 0.10 M phos- phate buffer at pH 5.9 was employed.

for the release of choline by the cabbage enzyme. With the Lineweaver- Burk equation (22), an apparent Michaelis constant of 2.1 X 1w2 M may be calculated for cabbage phospholipase D by using the data of Fig. 3.

Stability Studies-The thermal stability of the cottonseed enzyme was determined by assays after 5 minute exposures to various temperature levels in 0.07 M phosphate buffer at pH 5.9. The results (Fig. 4) indicate that the enzyme possesses no unusual heat stability.5 The thermal stability of the cabbage enzyme has been extensively studied elsewhere (5, 10) ; it was not studied on these preparations, but it was noted that, their enzymatic activity was destroyed by brief boiling.

The pH range of optimal stability of the cottonseed enzyme was tested by exposure at room temperature for 4.5 hours. It proved to be rather

6 This observation does not necessarily apply to the stability in the whole seed, where lipides are high and water relatively low. The question has been of some in- dustrial importance.

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218 COTTONSEED AND CABBAGE PHOSPHOLIPASE D

broad, there being less than 10 per cent destruction of the enzyme between pH 6.40 and 8.75. Despite its wide range, the pH of optimal stability does not coincide with the pH of optimal activity.

Specificity-Compared with the amount of nitrogenous base liberated by our cottonseed preparations, the liberation of inorganic phosphate was quite small. The concomitant release of choline, ethanolamine, and in- organic phosphate is reported in Table II. The preparations sre thus

3

)c “0 25 50 20 40 60 80 100

MG SUBSTRATE / ML TEMPERATURE IN “C

FIG. 3 FIG.~

FIG. 3. Dependence of activity on concentration of lecithin RG with cottonseed preparation (0) and cabbage leaf preparation (X). The cottonseed preparation (20 mg., Step C) was incubated for 2 hours at 30” and contained 0.10 M phosphate, 0.5 M NaCl, in a volume of 4.0 ml. at pH 5.9. The cabbage enzyme (equivalent of 33 gm. of leaves) was incubated for 9 hours at 25” in 0.07 M phosphate, in a volume of 4.0 ml. at pH 5.9.

FIG. 4. Thermal inactivation of cottonseed phospholipase D. Per cent activity remaining after 5 minute exposures at the indicated temperatures. Ethanolamine released in 15 hours at 25” from 200 mg. of lecithin RG by 60 mg. of cottonseed phos- pholipase D (Step B), in 0.07 M phosphate, in a volume of 4.0 ml. at pH 5.9.

shown to give an effect typical of phospholipase D, with very little sub- sidiary phosphatase action.6 In like manner, there is little free fatty acid released (Table II), indicating that the preparation is relatively free from lipase or phospholipase A action.

An interesting but still open question about phospholipase D concerns its substrates among the phospholipides. The cabbage and carrot prep-

6 For similar data on cabbage preparations, see Hanahan and Chaikoff (5) and Kates (lo), where the results are quite similar to those reported here for the cotton- seed enzyme.

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H. L. TOOKEY AND A. K. BALLS 219

arations used by Hanahan and Chaikoff (4, 5) liberated choline from a mixture of phospholipides; similar (but not identical) preparations were observed to liberate either ethanolamine or serine by a method in which both are measured ((8), also this paper). It follows from Table II that the enzyme from cottonseed hydrolyzes both lecithin and phosphatidyl ethanolamine. The cabbage enzyme also acts on both of these substrates.

Chromatograms prepared from the substrate after acid hydrolysis (6 hours boiling in 2.5 N HCl) indicated that little or no serine was present (Fig. 5). The existence of phosphatidyl serine in “soy bean lecithin” has

TABLE II

Action of Cottonseed Phospholipase D on Soy Bean Lecithin RG

Release of phospholipide hydrolysis products

Experiment A I

Experiment B

Inorganic P ............. 0.26 0.05 Free fatty acid*. ........ 1.4t 0.13 Ethanolamine N ......... 1.7 0.35 3.3 0.31 Choline N. .............. 3.2 0.65 7.4 0.69

Experiment A, 4 mg. of cottonseed phospholipase D (Step C) incubated 2 hours at 30” with 40 mg. of lecithin RG in 0.10 M acetate buffer at pH 5.9. Total volume 1.0 ml. Experiment B, same as Experiment A except in 0.10 M phosphate buffer, pH 5.9, and in the presence of 0.4 M NaCl as an activator. All values are an aver- age of at least two determinations.

* Calculated as linoleic acid. t The experimental results were 5 times larger than shown. All ingredients were

increased 5-fold because of the limits of the method.

been postulated on occasion, but seems to be dubious. Scholfield’s (15) separat,ion of soy bean phospholipides did not rule out the presence of serine, because his method of analysis did not distinguish between ethanolamine and serine. In order to identify the nitrogenous base re- leased by the enzymes, soy bean lecithin RG, the usual substrate, was digested with enzyme preparations from both sources. The resulting material was then examined by paper chromatography. Paper chromato- grams developed with a variety of solvents always showed spots corre- sponding to ethanolamine, but never to serine, in the digests of both cottonseed and cabbage phospholipase D. These were developed with several solvents as one-dimensional chromatograms, and also as two- dimensional chromatograms with n-butanol-acetic acid (Solvent I), fol-

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220 COTTONSEED AND CABBAGE PHOSPHOLIPASE D

lowed by either of t’he other solvents described. However, cleavage of phosphatidyl serine cannot be eliminated as a property of the enzymes, but it is not a factor in this case.

FIG. 5. Chromatogram of an acid hydrolysate of lecithin RG. E, known ethanol- amine; S, known nL-serine; A, 5 ~1. of hydrolysate; A’, 10 ~1. of hydrolysate. De- veloped with n-butanol-acetic acid-water (4: 1: 1) ; sprayed with ninhydrin. The lowest (third) spot on E + S appears to be a contaminant of redistilled ethanol- amine.

E$ect of NaC&--A curious effect of NaCl was observed on cottonseed phospholipase D. With the routine preparation, the presence of 0.4 M

NaCl gave 60 to 75 per cent activation and tended to give greater activa- tion with increasing time of digestion. On the other hand, with a prep- aration made with calcium phosphate gel (alternative Step B), 0.4 M

NaCl gave 118 per cent activation during a 15 hour incubation (Table III), but during a 2 hour incubation it inhibited the phospholipase D, the degree

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II. L. TOOKEY AND A. K. BALLS 221

of inhibition varying with the salt concentration. This reversal of effect might be due to some protective action of the salt during the longer in-

TABLE III

Effect of XaCl on Cottonseed Phospholipase D

Enzyme preparation

Step C

Alternative Step B

-

--

-

Length of incubation

- I

hrs.

2 2 3

2 2 2 2 2

15

_-

-

N&l

M

0.5 0.4 0.4

0.001 0.01 0.10 0.5 1.0 0.4

Per cent change in activity

+60 +64 +74

0 0

-39 -31

0 +11s

The complete system contained 20 mg. of enzyme, 200 mg. of lecithin RG, and NaCl in 0.10 M phosphate, pH 5.9, in 4.0 ml. total volume. The incubation tempera- ture was 30” except in the 15 hour assay, which was at 25”.

0 20 40 60 80

GRAMS CABBAGE LEAF PROVIDING ENZYME

FIG. 6. Relation of activity to amount of cabbage enzyme used. Ethanolamine released in 15 hours at 25” from 150 mg. of lecithin RG in 0.07 M phosphate buffer at pH 5.9. Total volume 4.0 ml. Undialysed NaCl precipitate (0) ; dialyzed NaCl precipitate (0). For details see the text.

cubation. Our preparations of cabbage phospholipase D were also ac- tivated by NaCl, 1.0 M NaCl giving 22 per cent activation during a 15 hour incubation, but inhibition by NaCl was not observed under the conditions employed.

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222 COTTONSEED AND CABBAGE PHOSPHOLIPASE D

Naturally Occurring Inhibitor-The enzyme in cabbage leaves appears to be accompanied by a material inhibitory t,hereto. In the case of the precipitate obtained from cabbage leaf juice with NaCl, large doses of

TABLE IV

Effect of Inhibitor from Cabbage Leaves

Enzyme preparation

12 mg. cabbage preparation

2 mg. cottonseed (Step C)

Correspond. ng weight o

cabbage leaves

k-m.

2.6 2.0 2.0 2.0 2.0 2.0 2.0

2.0 2.0

If

-

Heat treatment Weight

Boiled 2 min.

Boiled 3 min. Ashed

I‘

-

,PZg.

50 50

50 4.4 4.4

50 50

T

Per cent change in activity

-62 -72 -34 -45 -24

+30 +33

+15 +29

The complete system contained enzyme, inhibitor, and 40 mg. of lecithin RG in 0.10 M phosphate buffer, pH 5.9, in a total volume of 1.0 ml. The ethanolamine re- leased was measured after 15 hours incubation at 25”.

TABLE V

Comparison of Phospholipase D from Cottonseed and Cabbage

Physical state pH optimum Digestion of lecithin

“ “ phosphatidyl ethanolamine

;lelease of inorganic P and free fatty acids

pH stability Thermal stability

Activation by NaCl Occurs Inhibitor in cabbage Not inhibited Activation by diethyl ether “ activated

Cottonseed Cabbage*

Withstands 26,360 X g Particulate (10) pH 5.CL6.0 pH 5.F6.0 Occurs Occurs (5, 10)

‘< I‘

Low

pH 6.4-8.75 Destroyed at 70”

-

Low (5)

Destroyed by boiling, but see (5) for con- trary finding

Occurs Inhibited Activated (10)

* The figures in parentheses refer to the bibliography.

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II. I,. TOOKEY AND A. 6. BALLS 223

material prior to dialysis had less effect than smaller doses, as shown in Fig. 6. After dialysis this effect disappeared, and the inhibitory material’ could be recovered from the diffusate. Thus when cabbage press juice was brought to pH 2 to 3 and dialyzed against distilled water, which was then evaporated to dryness at low temperature, a solid was obtained. When dissolved in water and adjusted to pH 5 t’o 6, the solid exhibited inhibitory power, as shown in Table IV.

The inhibitory material is slowly destroyed by boiling and complet,ely destroyed by ashing; the inorganic ash produces instead a small activation (Table IV). It is plausible that the inhibitory material from cabbage contains activating salts and an organic inhibitor which overcomes them. It. should be noted t,hat the inhibitory effects seen are small, considering the amounts necessary to produce inhibition.

The material from cabbage did not inhibit the cottonseed enzyme (Table IV). The cottonseed activation may well be due to the salts present in the “inhibitory” material.

The discussion of a purely descriptive study must necessarily concern itself with details. The similarity of phospholipase D in cottonseed to that in cabbage leaves is striking. The principal differences observed between the behavior of the enzyme in the two preparations can well be attributed t’o particle size (or solubility). A comparison of these prepara- tions is presented in Table V. Whether the similarity bespeaks a common function must obviously await further study.

SUMMARY

An enzyme which releases ethanolamine and choline from phospholipides is reported in cottonseed. It is obtained as a stable dry product with a specific activity against phosphatidyl ethanolamine 86 times t,hat of defatted cottonseed meal. An aqueous solution of the enzyme withstands a centrifugal force of 26,360 X g for 20 minutes. This is the first report of an apparently soluble phospholipase D.

The similar enzyme from cabbage is shown to release ethanolamine from phospholipide. An inhibitor in cabbage juice is described.

The pH optimum, thermal stability, and the effects of diethyl ether and of NaCl on the cottonseed enzyme are described and compared to those of the cabbage enzyme.

BIBLIOGRAPHY

1. Contardi, A., and Ercoli, A., Riochem. Z., 261, 275 (1933). 2. Sumner, J. B., and Myrbiick, K., The enzymes, New York, 996 (1952).

3. MacFarlane, M. G., and Knight, B. C. J. G., Biochevz. J., 36,884 (1941).

7 Kates (10) has also reported an inhibitory substance in cabbage.

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224 COTTONSEED AND CABBAGE PHOSPHOLIPASE D

4. Hanahan, D. J., and Chaikoff, I. L., J. Biol. Chem., 169, 699 (1947). 5. Hanahan, D. J., and Chaikoff, I. L., J. Biol. Chem., 172, 191 (1948). 6. Smith, R. H., Biochem. J., 66,240 (1954). 7. Ducet, G., Ann. Agron., 19, 184 (1949). 8. Rose, W. G., Food Tech., 4, 230 (1950). 9. Kates, M., Nature, 172, 814 (1953).

10. Kates, M., Cunao!. J. Biochem. and Physiol., 32, 571 (1954). 11. Kirk, P. L., Quantitative ultramicroanalysis, New York, 179 (1950). 12. Burmaster, C. F., J. Biol. Chem., 166, 1 (1946). 13. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and

tissue metabolism, Minneapolis, 161 (1949). 14. Snell, F. D., and Snell, C. T., Calorimetric methods of analysis, New York,

3rd edition, 4, 69 (1954). 15. Scholfield, C. R., Dutton, H. J., Tanner, F. W., and Cowan, J. C., .J. Am. Oil

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Page 13: PLANT PHOSPHOLIPASE D · PLANT PHOSPHOLIPASE D I. STUDIES ON COTTONSEED AND CABBAGE PHOSPHOLIPASE D* BY HARVEY L. TOOKEY AND A. K. BALLS (From the I)epartment of Biochemistry, Purdue

Harvey L. Tookey and A. K. BallsPHOSPHOLIPASE D

ON COTTONSEED AND CABBAGE PLANT PHOSPHOLIPASE D: I. STUDIES

1956, 218:213-224.J. Biol. Chem. 

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