4 Phospholipases

Phospholipases DONALD J. HANAHAN I. Introduction

. . . . . . . . . . . . .

. . . . . . . . . . . . A. Sources . . . . . . . . . . . . . €3. Substrates and Mode of Attack . . . . . . .

11. Phospholipasc A2

C. Isolation and Purification of Enzyme

. . . . . .

D. Physical and Chemical Characteristics

111. Phospholipase C . . . A. Sources . . . . €3. Isolation and Purification

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. . . . . .

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.

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. . . . . . . . .

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71 73 74 74 74 78 82 82 83

I. Introduction

The phospholipases, which are classified as hydrolases, are of quite widespread occurrence in nature. There are four well-documented established types which can attack a typical phosphoglyceride, phosphatidylcholine, illustrated below at the indicated positions :

Thus, using the above molecule as a model substrate, phospholipase A, would catalyze release of a fatty acid from the 1 position, phospholipase 71

72

DONALD J . HANAHAN

A? the fatty acid from the 2 position, phospholipase C the phosphorylated base, and phospholipase D the nitrogenous base only. Other compounds may serve as substrates for these enzymes, as shall be discussed below. In addition to the above-mentioned there is another recognized activity, phospholipase B, which has as its primary substrate a monoacylphosphoglyceride (a “lyso” compound). The products of this reaction are a fatty acid and the corresponding glycerylphosphoryl base. There appears to be some slight activity of this enzyme toward the diacylphosphoglycerides. Evidence has also been presented for the occurrence of a phosphatidate phosphatase (phosphohydrolase) which has as its substrate, diacylglycerylphosphoric acid (phosphatidic acid). The products of the reaction are diglyceride and inorganic phosphate. Certain historical facets related to isolation and identification of the phospholipases may be obtained by reference to reviews by Hanahan ( 1 ) and by Kates ( 2 ) .A description of progress in this field as of 1965 was provided by van Deenen and deHaas (3). Recently, a detailed consideration of the approaches used for purification and assay of the phospholipases was published and essentially covered the field through 1967 ( 4 ) . On the basis of information presented in this latter review, it was decided to focus attention here primarily on those phospholipases which have been purified to a significant degree and/or on which detailed physical and chemical constants are available, together with any new information regarding their mode of action. Thus, this presentation essentially represents a status report on two enzymes, namely, phospholipase A, and phospholipase C. Phospholipases A, and C have been the most frequently studied and widely used enzymes in this class of hydrolases. Phospholipase A,, with its specific attack on the 2 position of phosphoglycerides, such as phosphatidylcholine and phosphatidylethanolamine, has provided a wealth of information regarding not only the specific positioning of fatty acids but also the relative metabolic behavior of these acids. I n many instances phospholipase C, which releases diglyceride and phosphorylcholine from phosphatidylcholine, has allowed further examination of the structure of the phosphoglycerides and a convenient route to preparation of diglycerides. Currently, considerable interest has been 1 . D. J. Hanahan, Frog. Chem. Fats Lipids 4, 141 (1957). 2. M. Kates, in “Lipid Metabolism” (K. Bloch, ed.), p. 165. Wiley, New York,

1960. 3. I,. L. M. van Deenen and G. H. deHaas, Ann. Rev. Biochem. 35, 157 (1966). 4. J . M. Lowenstein, ed., “Methods in Enzymology,” pp. 131-213, 1969.

4.

PHOSPHOLIPASES

73

manifested in the use of these phospholipases to establish a role of phosphoglycerides in the structure and behavior of membranes. All too often, though, investigators have employed quite impure enzyme preparations in such studies and any resulting alterations to a membrane may provide little or no information of import as to the role of phosphoglycerides in membrane structure. Furthermore, the action of a phospholipase on a phosphoglyceride can cause a considerable change in the physical state of the lipids, which then undoubtedly influences the physical state of contiguous proteins and other components. Artifacts, which are not unknown to the lipid or membrane field, can arise and conclusions regarding the role of lipids would be of little value. There is little doubt that these enzymes could prove useful in certain types of membrane studies, yet the problems alluded to above should cause some pause for reflection on the meaning of experimental results using these enzymes. With the availability of highly purified and crystalline phospholipases, the opportunity to investigate the three-dimensional structure of these enzymes by X-ray crystallographic analysis (coupled with sequencing of their amino acids) provides an excellent opportunity to explore and explain certain of their unusual properties (heat stability, activity in solvents such as diethyl ether, etc.). Of equal importance these enzymes provide a fine opportunity for study of lipid-protein interactions. The availability of a wide range of stereochemically pure phosphoglycerides provides the necessary components for a highly varied experimental attack. II. Phospholipase A,

Phospholipase A, (phosphatide acyl-hydrolase, EC 3.1.1.4j catalyzes the specific removal of the acyl group from the 2 position of an sn-3 phosphoglyceride (5) which represents the predominant stereochemical form of these phospholipids in nature. I t has been the most widely studied of all the phospholipases and, as might be expected, has been obtained in the highest state of purity. Two sources, snake venom and pancreas, have provided the best preparations of this enzyme, and a description of the results derived from these studies is given below. 5. IUPAC-IUB Commission on Biochemical Nomenclature, Biochemistry 6, 3287 (1967).

74

DONALD J . HANAHAK

Phospholipase A, activity has been reported in rat liver mitochondria ( 6 ) , in many different tissues of the rat ( 7 ) , and in the venoms of seven different species of snakes (8).

B. SUBSTRATES AND MODEOF ATTACK Under proper conditions, this enzyme can attack a wide variety of phosphoglyceridcs, provided their stereochemical configuration is that of an sn-glycero-3-phosphate. Phosphatidylcholine, phosphatidyletlianolamine, phosphatidylglycerol, and diphosphatidylglycerol are examples of compounds attacked by phospholipase A,. The attack in each instance is the same in that it catalyzes release of one fatty acid per glycerol chain and in the sn-3 series, only from the 2 position (9-11). It is of interest to note that a “P-lecithin,” an sn-2 phosphoglyceride, could be attacked stereochemically with release of only one fatty acid (12). I t is also of interest to note that the phospholipase A, isolated from the venom of Crotalus atroz (Western diamondback rattlesnake) attacks the acyl ester (in the 2 position) of the vinyl ether (OCH=CHR in the 1 position) containing phosphoglycerides (plasmalogen) much inore slowly than the diacyl acyl ester derivative (13). Similarly, the ncyl cstcr of the saturated ether (0-CH,CH,R in 1 position) phosphoglycerides appears to be no re slowly attacked by phospholipase A? from C. adanianteus than the diacyl ester compound ( 1 4 ) . Preliminary evi- glyceride may be quite dence suggests that the phosphono (-C-0-P-C) resistant to attack by this enzyme (14). C. ISOLATION A N D PURIFICATION OF ENZYME Recently, three different laboratories have isolated and purified phospholipase A,, using make venom and pig pancreas as the source of en6. G. I,. Scherphof and L. L. M. van Deenen, BBA 98, 204 (1965). 7. J. J. Gallai-Hatchard and R. H. S. Thompson, BBA 98, 128 (1965). 8. L. J. Nutter and 0. S. Privett, Lipids 1, 258 (1966). 9. N. H. Tattrie and J. R. Bennett, Can. J . Biochem. Physiol. 41, 1983 (1963). 10. D. J. Hanahan, H. Brockerhof, and E. J. Barron, JBC 235, 1917 (1960). 11. I,. I,. M. vnn Deencn and G. H. deHass, BBA 70, 211 (1963). 12. G. H. deHaas and L. L. M. van Deenen, BBA 84, 569 (1964). 13. E. I,. Oottfried iind M. M. Rapport, JBC 237, 329 (1962). 14.

D. J . Hanahan, unpublished obscrvations.

4.

PHOSPHOLIPASES

75

zyme. The salient features of each of these isolation procedures is outlined below. 1. Crotalus adamanteus Venom

Wells and Hanahan (16)employed commercially available lyophilized venom of Crotalus adamanteus (Eastern diamondback rattlesnake) as starting material. The venom was suspended in tris-ethylenediaminetetraacetate (EDTA) buffer and then subjected to gel filtration on Sephadex G-100. The activity was eluted as a single peak and was further chromatographed on BioRex 70 and subsequently on DEAE-cellulose (Whatman DE52) from which two well-separated peaks of activity were eluted (16).Final purification could be achieved on SE-Sephadex. These two activities represented 2-476 of the starting protein and approximately 30% of the original activity in each peak. The two enzymic activities could be crystallized in the cold by slow addition of ammonium sulfate. The assay procedure for phospholipase A, activity was based on an earlier observation (17) that this enzyme was active, in diethyl ether, on highly purified phosphatidylcholine. The assay method involves titration of the liberated fatty acid. The increase in specific activity of the enzyme over the starting material was found to be 14-fold. The final specific activity was 3150 and 3250 units (microequivalents of fatty acid liberated in 1 min) per milligram protein, respectively, for each fraction. The turnover number of both enzymes is 1600 moles of substrate hydrolyzed per mole of enzyme per second. The presence of two forms of this enzyme appear unique to this venom. The same two forms were obtained whether fresh or lyophilized venom was used as the starting material, and they did not appear to be equilibrium forms since one could not be converted to the other. No phosphodiesterase, protease, or amino acid oxidase activity could be detected. These two forms of phospholipase A, are very stable and can be maintained for several months a t 4" without loss in activity. Each enzyme showed a single, but of different mobility, band on gel electrophoresis. A combination of the two forms could be resolved on gel electrophoresis. Sedimentation velocity patterns of the two enzymes indicated no detectable asymmetry. An interesting facet of the assay for this enzymic activity is the influence of water. In the usual procedure, diethyl ether, which contains a small amount of added methanol, is used as solvent for the substrate, 15. M. A. Wells and D. J. Hanahan, Biochemktw 8, 414 (1969). 16. K. Saito and D. J. Hanahan, Biochemistry 1, 521 (1962). 17. D. J. Hanahan, JBC 195, 199 (1952).

76

DONALD J . HANAHAN

and the enzyme in an aqueous solution is added to it. The amount of water required for maximal activity of the enzyme is 100 times that consumed in the reaction. Thus, far more water than necessary for hydrolysis was nccded before thc reaction was initiated. The relationship of the water to the physical structure of the substrate is complex and is under current investigation (18).I n addition to the water requirement, both sodium chloride and a divalent cation such as Ca2+ are required for maximal activity (18). I n this latter study, other cations such as Mg2+, Mn2+, and Cd2+ also activate to varying degrees, whereas Zn2+, Co'+, Fez+, Al.", and Ba2+ arc potcnt inhibitors. The mode of action of calcium, for example, in activation is not simply as a cofactor per se or in the removal of liberated fatty acids but appears to be involved possibly in a con formational change in the enzyme. Furthermore, zinc, in inhibiting the enzymic action, may affect regions or groups on this protein quite removed from the calcium active sitc(s) . 2. Pig Pancreas DeHaas et al. (19) used freshly prepared homogenates of pig pancreas for the source of enzyme. The homogenate is allowed to autolyze for 1224 hr at room temperature and then subjected (after adjustment of pH and removal of precipitate) to ammonium sulfate fractionation. The material insoluble a t 0.6 saturation was dissolved in 0.75M NaCl and chromatographed on Sephadex G-50. The eluate containing enzymic activity was then passed through a DEAE-cellulose column and the single peak of activity then chromatographed on CAT-cellulose. The activity from the lattcr chromatographic procedure moved as a single band on disc gel electrophoresis. The enzyme was obtained in a 31% yield. In this fractionation procedure, an egg yolk assay system was employed and phospholipasc A, activity was reported as alkali uptake in microequivalents per minute per milligram protein. Apparently calcium was necessary for optimal activity in this system, but zinc, cadmium, and lead, as well as EDTA, strongly inhibited. The purified enzyme was not inhibited by diisopropylpliosphofluoridate (DFP). It showed no change in activity aftcr 5 min a t 98" a t pH 4.0, and 45% of its activity was recovered after heating for 1 hr a t 98" a t p H 4.0. Furthermore, storage of the enzyme in 8 M urea a t 20" for 22 hr caused no decrease in activity. The mode of action of thc purified enzyme on several synthetic mixed 18. M. A. Wells. rinpuhlishcd ohscrwtions.

19. G . H. deHans, N. M.Postrma, W. Nieuwenhuiaen, and L. L. M . van Derncn, BBA 159, 103 (1968).

4. PHOSPHOLIPASES

77

acid phosphoglycerides (in a deoxycholate-diethyl ether medium) was reported in this same study. This enzyme preparation had no activity toward an sn-glycerol-1-phosphatidylcholine and, as expected then, only 50% activity towards a racemic phosphatidylcholine. However, the reactivity toward sn-glycero-3-phosphatidylcholine (s) and ethanolamine(s) was maximal with no preference shown for chain length or unsaturation of the fatty acyl group in the 2 position. No specific activity value for this enzyme on these synthetic substrates was provided. Of considerable interest, this enzyme exhibited very high activity toward anionic substrates such as phosphatidic acid, phosphatidylglycerol, and diphosphatidylglycerol (cardiolipin) . This latter action is in contradistinction to that found for the enzyme obtained from snake venom. DeHaas et al. (19) further noted that freshly prepared homogenates of pig pancreatic tissue contained a low level of phospholipase A, activity, but during autolysis a considerable rise in enzymic activity was noted. It was reasoned that the increase in activity in pancreas homogenates was related to proteolytic activation. This activation process was increased by trypsin and decreased by DFP. Subsequent investigation (20) led to the isolation of a pre-phospholipase A,, a zymogen of phospholipase A,, through use of the same procedure as noted above for (pancreas) phospholipase A, preparation. Upon treatment with trypsin, this material yielded phospholipase A,. Diisopropylphosphofluoridate was found to inhibit activation during isolation. Further information on this zymogen is provided later.

3. Crotalus atrox Venom Wu and Tinker (21) used this venom for their starting material. The venom was subjected to ammonium sulfate fractionation and then Sephadex G-75 column chromatography. Active protein appeared as a single band on polyacrylamide gel disc chromatography (at pH values from 4.5 to 8.4) and was eluted from Sephadex G-75 with a constant specific activity some 35 times that of the crude venom. The final yield of material was 0.48% of the starting protein and 16.8% of the initial activity. It exhibited exclusive action a t the 2 position of (liver) lecithin. No detectable proteasc, phosphodiesterase, or monoesterase activity was found. The assay system was a titrimetric one employing purified lecithin as substrate (in diethyl ether, chloroform, or as a sonicated lecithin dispersion in water) and cresol red as the indicator. 20. G . H. deHans, N. W. Postema, W. Nieuwcnhuieen, and L. L. M. van Deenen, BBA 159, 118 (1968). 21. T. W. Wu and D. 0. Tinker, Biochemistry 8, 1558 (1969).

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DONALD J . HANAHAX

Contrary to the results with phospholipases from pancreas and Crotalus adamanteus venom as described above, Wu and Tinker reported that their preparation was inactivated by DFP. It did not show any isozymic forms. However, there was a drastic loss of enzymic activity in the initial ammonium sulfate fractionation and though it is doubtful that any isozyme would be lost specifically a t this point, it is worthy of note. The purified phospholipase A? withstands heating to 80" for 30 min and pH 3.0, but similar treatment a t pH 7 immediately inactivated it. Lyophilization caused inactivation of the enzyme, but activity was restored to a considerable extent in an aqueous medium over a 24 hr period. Michaelis-Menten kinetics apparently are applicable to this enzyme in ether and in chloroform, K,,, 8.3 and 8.5 mM, respectively. The enzyme was activated to varying degrees by Ca2+ and other divalent cations. However, certain divalent cations such as Zn'+ inhibited its action. Trace amounts of inhibitory cations required that EDTA be present in the assay mixture.

D. PHYSICAL AND CHEMICAL CHARACTERISTICS

A considerable amount of data have now become available on the physical and chemical characteristics of the phospholipase A, from Crotalus adamanteus venom and pig pancreas. Inasmuch as there are many aspects of these data that 'may prove of importance in explaining certain of their unusual properties, e.g., reactivity in solvents such as diethyl ether and chloroform, it was considered of import to collate these results a t this point. 1. Molecular Weight Molecular weights have been obtained on phospholipase A, preparations from C. adamanteus venom, C . atrox venom, and pig pancreas. In addition, data on the molecular weight of pre-phospholipase A, (zymogen) isolated from pig pancreas are available. The figures obtained from sedimentation equilibrium, Sephadex G-75 chromatography, and amino acid assays are recorded in Table I. It is of considerable interest to note that the molecular weight of the isozyme forms of this enzyme from C. adamanteus venom is approximately twice that of the enzyme isolated from C . atros venom and pig pancreas. No explanation for this difference can be provided a t this time. The lower molecular weight of the phospholipase A, of pig pancreas, as compared to its zymogen form, can be attributed to the loss of a hepta-

TABLE I MOLECULAR WEIGHTVALUES Source of enzyme

Pig pancreasc

Crohlus adamanteusa venom

Technique Sephadex G-75 chromatography Amino acid content (calculated) Sedimentation equilibrium (&Is,D ) From Wells and Hanahan (16). From Wu and Tinker (21). c From deHaaa et al. (19, 20). 6

b

Q

B

Not run 29,864 29,800

Not run 29,864 29,800

Crotalvs atro9

Zymogen

0%)

Active form

14,500 f 500 Not run Not run

14,800 f 500 14,749 Not run

13,900 f 4.50 14,150 13,500 5%

venom

80

DONALD J. HANAHAN

peptide during the enzymic conversion of the pre-phospholipase A, (nonactive) to the active form. 2. Amino Acid Content

The amino acid content of the phospholipase A, obtained from C. adamanteus venom and of the zymogen and active form of this enzyme isolated from pig pancreas is presented in Table 11. Certain aspects of these results and comments on other applicable observations are presented below. a. Phospholipase A, of Crotalus adamanteus Venom. Both forms of this enzyme showed identical amino acid content. One is immediately impressed by the high content of cystine residues, and since no free sulfhydryl groups could be detected in the intact protein, this indicatcs the presence of nearly 15 disulfide bonds per mole. This value is confirmed by results of treatment of the protein with mercaptoethanol. This TABLE I1

AMINOACID COMPOSITION

Pig pancreas"

Amino acid ASP Thr Ser Glu Pro GlY Ala Val CYS Met Ile Leu TYr Phe Lya His A% TrP NH3 a

b

Pre-phospholipase Phospholipase A¶ Ai (nearest integer/14,000 g) 23

7

12 9 6

7

8 2 14 2 6

7

8 5 9 3 5 2 -

From Maroux el al. (22). From Wells and Hanahan (16).

23 7 10 7 6 6 8 2 14 2 5

7

8 5 9 3 4 2 -

C. adamanteus venomb

Phospholipase AZ (nearest integer/ 29,864 g) 30 13 13 24 16 24 15 11 30 2 11 11 16 10 16 5 12

7

16

4.

PHOSPHOLIPASES

81

latter procedure, which eliminated all the enzymic activity, produced 29 residues of free sulfhydryl groups per mole of protein. These data agreed well with results on cysteic acid content after performic acid oxidation. Of considerable interest, no N-terminal groups on either native or modified protein could be detected. b. Pig Pancreas. The amino acid content of the pre and active forms of phospholipase A,, as might be expected, are quite comparable. However, close inspection of the data in Table I1 shows that the active form contains approximately &7 less amino acids. This difference is attributable to the cleavage (by trypsin) of a heptapeptide from the prephospholipase A,. It is now believed (22) that pre-phospholipase A, contains only one tryptophan residue instead of two, and 12 half-cystine residues instead of the 14 listed in Table 11. Pre-phospholipase A, contains a single polypeptide chain, with 7 disulfide bonds and no N-terminal amino acid, but it does have a cystine carboxyl terminal group. On treatment with trypsin, cleavage of the seventh bond (Arg-Ala) in the chain occurs. The released heptapeptide was shown by mass spectrometry to have the following structure: PyroGlu-Glu-Gly-Ile-Ser-Ser-Arg. The origin of the N-terminal pyroglutamic acid is unknown, i.e., whether it was present in the original molecule or whether it derived from a cyclization of a glutamate or glutamine residue during the isolation period. The resulting major peptide fragment, phospholipase A?, had full enzymic activity and was indistinguishable from that found in autolyzates of pancreas. It contained an N-terminal alanine and a C-terminal cystine. On disc gel electrophoresis it migrated as a single band which was distinct from phospholipase A, but could be converted to the phospholipase A, band by treatment with trypsin. 3 . Amino Acid Sequence

The sequence of amino acids in the active phospholipase A, obtained by tryptic attack on the pre-phospholipase A, from pig pancreas has been reported by Maroux et al. ( 2 2 ) . These results are presented in Fig. 1 and illustrate the unique structural characteristics of this protein. The peptide released from the pre-phospholipase A? was attached through its arginine residue to the alanine (N-terminal) present in the active form of this enzyme. Four of the six disulfide bridges have been identified and are connected between residues 18 and 83, 34 and 129, 68 and 98, and 91 22. S. Marous. A . Puigscrver, V. Dlouha, P. Desnuelle, G . D. deanas, A. J. Slotboom, P. P. M. Bonsen, N. Nieunenhuizen, and L. L. M. van Deenen, BBA 188, 351 (1969).

82

DONALD J. HANAHAX

8 9 10 11 12 13 14 15 16 17 18 19 Ala -Leu-Trp-Gln -Phe-ArgSer -Met- Ile-Lys-Cys-Ala 20 21 22 23 24 25 26 27 28 29 30 31 Ile -Pro -GlySer -His -Pro -Leu-Met-Asp-Phe-Asri-Asn32 33 34 35 36 37 38 39 40 41 42 43 Tyr-Gly-Cys-Tyr-Cys-Gly-Leu-Gly -GlySer -Gly -Thr44 45 46 47 48 49 50 51 52 53 54 55 Pro -Val -Am-Glu-Leu-Asn- Arg-Cys -Glu-His -Thr-Asp56 57 58 59 60 61 62 63 64 65 66 67 Asii-Cys-Tyr-Arg-Asp-Ala -Lys-Asn -Leu-Asn-Asp-Ser 68 69 70 71 72 73 74 75 76 77 78 79 Cys-Lys-Phe-Leu-Val -Asp-Asn-Pro -Tyr-Thr-Glu-Ser 80 81 82 83 84 85 86 87 88 89 90 91 Tyr-Ser -Tyr-Cys-Ser -Ser -Am-Thr -Glx -1le -Thr-Cys92 93 94 95 96 97 98 99 100 101 102 103 Asii-Ser -Lys-Asii-Asii-Ala -Cys-Glu -Ala -Phe-Ile -Cys104 105 106 107 108 109 110 111 I12 113 114 115 Am-Arg//Asii-Ala -Ala -1le -Cys-Phe -Ser -Lys-Ala -Pro116 117 118 ll!) 120 121 122 123 124 125 126 127 Tyr-.hi-Lys-Glu-His-Lys-Asn-Leu -Asn-Thr-Lys-Lys128 129 Tyr-Cys

1;tr;. 1. .\mino ncid srrliicnrc of rrduced phospliolipnse A,. Ala8 represents the S-trriiiind nniino acid in the active enzyme, which was derived by tryptic cleavage of a heptapeptide from the zymogen form. The source of this material was pig pancreas. Data from the work of Maroux et al. (22). Reproduced from BBA 188, 351

(1969).

and 103. Undoubtedly, the high cross-linking via the disulfide bonds contributes to the unusual stability of this enzyme. Ill. Phospholipase C

The enzymic activity of pliospholipabe C (phosphatidylcholine:Cholinep l i o ~ ~ ~ l i o l i y d r o lEC a ~ ~ 3.1.4.3 , catalyzca tlie hydrolytic cleavage of diacyl-sn-glycero-3-phosphorylcholine,as an example, to a 1,a-diacylglycerol (diglyceride) and phosphorylcholine.

A. SOURCES This enzyme is found mainly in bacteria, with Clostridiwu welchii and Bacillus cereus being the most frequently used sources. It has been reported to be present in the marine planktonic chrysomonad Monochrysis

4. PHOSPHOLIPASES

83

lutheri (25). No evidence for its presence in mammalian tissues has been noted to date. B. ISOLATION AND PURIFICATION Two procedures have been reported for the isolation and partial purification of this enzyme from bacterial sources and are outlined below. 1. Bacillus cereus

Kleiman and Lands (24) used a late-log phase (20-24 hr) static culture of Bacillus cereus, strain 7004, as starting material. This was centrifuged to remove cells and the supernatant fluid brought to 70% saturation with ammonium sulfate. The precipitated material contained all the phospholipase C activity. Subsequent dialysis and lyophilization yielded material for further purification by two different approaches. The first procedure involved chromatography of the crude enzyme preparation, in tris-buffer pH 7.4 a t 0", directly on a polyethyleneimine cellulose column with elution of enzyme activity in the solvent front. This resulted in a 22fold purification. The second technique involved careful addition of protamine to the crude enzyme preparation with formation of a precipitate. The enzyme was recovered in the soluble fraction and a 2-3-fold increase in specific activity was obtained. The latter fraction was then chromatographed on DEAE-cellulose columns using a gradient of salt and buffer. This latter approach allowed a good recovery of activity and a 23-fold purification. Two different systems were used for assay of this enzymic activity, one of which utilized an ether-containing mixture of the substrate (lecithin) to which the enzyme was added. At the end of the incubation time, extraction with chloroform-methanol and washing with water yielded one product of the reaction, namely, watersoluble organic phosphorus or phosphorylcholine, which was then assayed for total phosphorus. An alternative method involved use of sonically dispersed phospholipid substrate, to which the enzyme was added. Subsequent to incubation, the extraction and remainder of the procedure was the same as described above. The activity of this purified phospholipase C preparation toward various phosphoglyceride substrates was undertaken and activities were evaluated. It was evident that this enzyme was different in its specificity as compared t o the phospholipase C from Clostridiu?n perfringens toxin in that it was active toward a 23. E. Bilinski, N. J. Antia, and Y. C. Lan, BBA 159, 496 (1968). 24. J. H. Kleiman and W. E. M. Lands, BEA 187, 477 (1969).

84

DONALD J. HANAHAN

diacylglyceryl~~hoqdiorylcholine(phosphatidylcholine), diacylglycerylpliosphorylethanolnlnine (phosphatidylethanolaniinc) and diacylglycerylphosphoryl monomethylethanolamine. It was inactive toward diacylglycerylphosphate (phosphatidic acid). No evidence on the presence of other enzymic activity, e.g., proteases, in this phospholipase C preparation was presented by these investigators. This enzyme preparation had relatively little or no activity toward sphingomyelin.

Activrrtors ( i n d Inhibitors. 1)itliiotlircitol bhowed an inliibitory effect at :i i-angc of 1 to 10 Inill. p-Chloromercuribenzoate was not inhibitory tit concentrations near 0.1 m.11; similarly, 5,5’-dithiobis (2nitrobenzoic acid) was not inhibitory in the range of 5000-4 p M . Zinc ions showed little effect on this reaction. There was some question as to the importance of calcium ion in this reaction system. No chemical or I)hysical parameters biicli as amino arid content, sedimentation equilibrium v:iluc~s,or molecular weights were presented.

2. Clostiidizim perfr ingen s Pastan et al. (25) have outlined a procedure wherein a phospholipase

C specific for bphingomyelin and one specific for phosplioglycerides (for phosphatidylcholine) can be isolated in reasonable purity from cultures of C. perfringens. C’lostritliuw perfringens (ATCC 10543 was grown in liquid medium ant1 at tlic proper time the cell culture was centrifuged and the cells discarded. The supernatant was then subjected to ammoiiium sulfate fractionation and later to chromatography on Sephades G-100. I n the latter instance, interestingly, two peaks of activity were obtained, one of which was active primarily toward sphingomyelin and the other of which was active primarily toward lecithin. Subsequent chromatography of these two fractions on DEAE-cellulose columns (0.01 M trisHCl, pH 6) showed that the lecithin hydrolyzing activity was not adsorbed, whereas the sphingomyelin hydrolyzing activity was adsorbed and could be eluted later with sodium chloride. However, as noted by these authors, the sphingomyelinase activity was not present in all preparations. Phospholipase C activity was assayed by the release of water-soluble organic phosphorus (phosphorylcholine) from a soiiically dispersed subbtrate. The unreacted substrate and tlie other product ceramide (acyl sphingosine) were removed by solvent extraction. Specific comments on tlie individual hydrolyases are as follows. 25. J. Pastan, V. Macchia, and

R. Kntzen, JBC 243, 3750 (1968).

4.

PHOSPHOLIPASES

85

a. Sphingoniyelin Hydrolyase Activity. The optimal pH for this hydrolysis was 7.8-8.8 and the apparent K,, was 8 X M . The enzyme was completely inhibited by preincubation with EDTA and was partially inhibited by preincubation with mercaptoethanol. Iodoacetic acid, iodoacetamide, DFP, and N-ethylmaleimide did not decrease this enzymic activity. There was apparently no product inhibition in this reaction. Diethyl ether added to the reaction mixture increased the enzymic activity twofold. This latter observation would be in accord with an earlier report that a crude preparation of phospholipase C from C. perfringens toxin would attack lecithin in a diethyl ether solution (26). Interestingly, Mg*+ activated the sphingomyelin hydrolyzing activity whereas Ca'+ inhibited it. Zinc, copper, and iron were also inhibitory to varying degrees. This preparation did exhibit some activity toward lysolecithin and lecithin, but the major activity (on a rate basis, a tenfold difference) was toward sphingomyelin. It showed no activity toward phosphatidylserine or (dipalmitoyl) phosphatidylethanolamine. No activity toward an impure phosphatidylinositol was observed. b. Lecithin Hydrolyase Activity. This enzyme fraction showed high activity toward lecithin with some reactivity toward sphingomyelin (approximately 20% of the rate) : Ca2+activated this enzyme system which was only 10% as active in its absence. Interestingly, most previous assays for phospholipase C on sphingomyelin included calcium in the medium, which would inhibit sphingomyelinase. Thus, this latter activity would not have been noted. Addition of Mg2+to a calcium-inhibited preparation of sphingomyelinase restored its activity toward sphingomyelin. Phosphatidylethanolamine and phosphatidylserine were not hydrolyzed by this lecithin hydrolyase.

26. D. J. Hanahan and R. Vercamer? JACS 76, 1804 (19&4).