An acid phospholipase C from Tetrahymena culture medium

Comp. Biochem. Physiol. Vol. 85B, No. 1, pp. 143--148, 1986 Printed in Great Britain

0305-0491/86 $3.00+ 0.00 Pergamon Journals Ltd

AN ACID PHOSPHOLIPASE C FROM TETRAHYMENA CULTURE MEDIUM J. FLORIN-CHRISTENSEN*~, M. FLORIN-CHRISTENSEN*§, L. RASMUSSEN* and J. KNUDSEN~ *Institute of Anatomy and Cytology and tInstitute of Biochemistry, Odense University, Campusvej 55, DK-5230 Odense M, Denmark

(Received 29 October 1985) Al~traet--1. A phospholipase C in Tetrahymena thermophila culture medium was assayed with 1,2-di[1-J4C]stearoyl-sn-glycero-3-phosphorylcholine as substrate and purified by ammonium sulphate precipitation of the extracellular growth medium, DEAE cellulose ion exchange chromatography and gel filtration on Sepharose 6B. 2. The enzyme is associated with high tool. wt complexes, probably containing lipids. Phospholipase C activity has a pH optimum of 5 and is not affected by addition of CaC!2 or EDTA. It does not hydrolyse p-nitrophenylphosphorylcholine. 3. Phospholipase C copurifies with haemolytic activity. The chromatographic patterns of both activities are coincident. Haemolysis is inhibited by phosphatidylcholine, has an acid pH optimum and is unaffected by CaC12 or EDTA. Phospholipase C is thermolabile and trypsin-sensitive and its destruction is accompanied by loss of haemolytic activity. These results suggest that phospholipase C is a lytic factor in Tetrahymena culture medium. INTRODUCTION We found recently that Tetrahymena thermophila (Ciliata) releases haemolytic proteins to the culture medium (Florin-Christensen et aL, 1985). In the same study we showed that haemolysis was inhibited by phospholipids and that the medium contained phospholipase A and C activities. A possible involvement of these enzymes in haemolysis was therefore proposed. Of all the phospholipases studied only phospholipase C from Clostridium perfringens has been shown to haemolyse h u m a n erythrocytes. Other enzymes, like phospholipase A2 from Naja naja venom, can attack m e m b r a n e phospholipids, but do not lyse them, whereas others like phospholipase C from Bacillus cereus are unable to hydrolyse phospholipids from intact cell membranes (Colley et aL, 1973; Zwaal et aL, 1973). In the present work we have isolated phospholipase C activity from T. thermophila extracellular medium. We have studied the properties of this enzyme and shown that it copurifies with haemolytic activity. This, together with other results, indicates that Tetrahymena phospholipase C attacks intact red cell membranes causing haemolysis. MATERIALS AND METHODS Materials T. thermophila strain DIII (Nanney and McCoy, 1976) was used throughout this study. Proteose peptone and yeast ~:Correspondenee to be addressed to: Dr J. FlorinChristensen, C~itedra de Biologla Celular, Facultad de Ciencias Exaetas y Naturales, Ciudad Universitaria, Pab. II 4°-P, 1428 Buenos Aires, Argentina. §Present address: C/ttedra de Biologla Celular, Faeultad de Ciencias Exactas y Naturales, Ciudad Universitaria, Pab. II, 4°-P, 1428 Buenos Aires, Argentina.

extract were obtained from Difco Laboratories (Detroit, MI, USA); 1,2-di[1-'4C]stearoyl-sn-glycero-3-phosphorylcholine and 1-palmitoyl-2-[1-t4C]oleoyl-sn-glycero-3phosphorylcholine, from The Radiochemical Centre (Amersham, UK); soy bean and chicken egg phosphatidylcholine and trypsin, from Sigma Chemical Co. (St. Louis, MO, USA); Bacillus cereus phosphotipase C, from Boehringer (Manrdaeim, FRG); Silica Gel HR 60, from Merck (Darmstadt, FRG); Hydrocount from J. T. Baker Chemicals, B.V. (Deventer, Holland); DEAE cellulose, from Whatman (H. Reeve Angel and Co., London, UK); Sepharose 6B and Blue Dextran 2000, from Pharmacia (Uppsala, Sweden).

Methods Cultures. Axenie cultures of T. thermophila DIII were grown at 37°C for 70-80 hr in a medium composed of 0.75% (w/v) proteose peptone, 0.25% (w/v) yeast extract and salts supplemented with 0.15M NaCI (FlorinChristensen et al., 1985). The cultures were grown in 2 and 0.51 Fernbach flasks with 300 and 75 ml of medium, respectively, without stirring. The generation time under these conditions was around 5 hr. The cell-free supernatant was harvested by centrifugation at 10°C for 3 min at 2500 rpm in a fixed angle rotor (PR6000 IEC centrifuge). Phospholipase C assay. The e ~ e was routinely assayed by incubation with 1,2-di[1-14C]stearoyl-sn-glycero-3phosphorylcholine, which was diluted with soy bean phosphatidylcholine to a specific activity of 0.06 Ci/mol. Before use, the substrate was dispersed at a concentration of 3.2#mol/ml by sonication in a Branson sonifier (50W, 3 min, room temp.). The assay mixture contained an aliquot of 100 #1 of the sample to be tested, 50 #1 of the substrate suspension and 50 #1 of 0.25 M sodium acetate, adjusted to pH 5 with 0.25 M acetic acid, containing 4% Triton X-100 (v/v). The detergent stimulated phospholipase C activity 2-fold. After incubation at 37°C for 1 to 3 hr, the reactions were stopped by addition of 0.75 ml chloroform/methanol (I :2, v/v) and lipids were extracted according to Bligh and Dyer (1959). Carrier 1,2-diacylglycerol, obtained by enzyme hydrolysis of chicken egg phosphatidylcholine by Bacillus cereus phospholipase C, was added to each sample and the

143

144

J. FLORIN-CHRISTENSEN et al.

extracted lipids were separated by thin layer chromatography on Silica Gel HR plates using hexane/diethyl ether/acetic acid/methanol (90:20:2:3, v/v) as developing solvent mixture. The lipids were made visible by brief exposure to iodine vapour and the 1,2-diacylglycerol spots were scraped off into a counting vial and counted in an LS 8000 Beckman liquid scintillation counter after addition of 0.5 ml of water and 5 ml of Hydrocount. Assay of hydrolysis of 1-palmitoyl-2-[ 1-14C]oleoyl-sn-glycero-3-phosphorylcholine by phospholipase C was carried out as described above for 1,2-di[l-14C]stearoyl-sn-glycero-3-phosphorylcholine. Haemolytic assays. Haemolytic activity was routinely assayed with sheep erythrocytes. Aseptic sheep blood was kept in sterile Alsever solution. Prior to use the erythrocytes were washed three times in 0.15 M NaC1/0.02 M Tris-HC1 (pH 7.0) and resuspended in the same buffer at a concentration yielding an absorbance of 0.3 at 540nm upon complete haemolysis by 20-fold dilution in distilled water. The samples to be tested were made 0.15 M with NaCI and 0.03 M with sodium acetate (pH 5.5) by addition of a 10-fold concentrated stock solution. Serial dilutions were prepared in the same buffer. Aliquots of 900 #1 of each dilution were mixed with 100 #1 of the red cell suspension. After 1 hr of incubation at 37°C, the samples were centrifuged (30 sec in a Dich high speed microcentrifuge), the supernatants were collected and the amount of released haemoglobin was determined speetrophotometrically at 540 nm. The dilution at which 50% of the haemoglobin became released was determined by linear interpolation. Such dilution contained, by definition, one haemolytic unit (HU) per ml. The number of HU per ml present in the undiluted sample was calculated as the reciprocal of the corresponding dilution factor. Unless otherwise indicated, haemolytic activity was measured in this way. Kinetics of haemolysis. Study of the time course of haemolysis was used as a sensitive method to investigate the action of different factors affecting this activity. Changes in the apparent absorbance of haemolysing red cell suspensions at 37°C were followed spectrophotometrically at 500 nm as described previously (Florin-Christensen et al., 1985). Purification ofphospholipase C. All steps were carried out at 0-5°C. Cell-free extracellular medium (1.21) was taken to 243 g/l of ammonium sulphate with solid salt. After I hr of standing in the cold, the precipitated protein was collected by centrifugation at 16,000 g for 30 min. The precipitate was dissolved in 0.075 M NaCI/0.01 M Tris-HCl (pH 7.1), dialysed for 3 h r against this buffer and loaded onto a 7.5 x 2.5 cm DEAE cellulose column, equilibrated with the same buffer. The column was washed with 125 ml of this buffer and phospholipase C was eluted with a linear gradient of NaCI, obtained by mixing 2 bed volumes of each 0.075 M NaCI/0.01 M Tris-HC1 (pH 7.1) and 1 M NaCI/0.01M

Tris-HCl (pH 7.1). Flow rate was 90ml/hr and 10ml fractions were collected. The fractions containing the peak of phospholipase C activity were pooled, concentrated by dialysis against solid sucrose, loaded on a 35 x 2.5cm Sepharose CL 6B column equilibrated with 0.01M Tris-HCl (pH 7.1). Elution of phospholipase C was carried out with the same buffer at a flow rate of 24 ml/hr and fractions of 6.5 ml were collected.

Assay of hydrolysis of p-nitrophenylphosphorylcholine. The incubation mixtures contained 0.1 ml of sample and 0.1 ml of 40 mM p-nitropbenylphosphorylcholine in 75% (v/v) glycerol/0.25 M sodium acetate, pH 5.0. After incubation at 37°C for up to 60 min, the reactions were stopped by addition of 1 ml of 0.4 M glycine adjusted to pH 10.4 with NaOH. Release of p-nitrophenol was measured spectrophotometrically at 410 nm. Blank samples without enzyme were incubated simultaneously. Trypsin treatment. Samples of purified phospholipase C were incubated in 0.01 M Tris-HCl (pH 8.1) with 5 mg/ml of trypsin for 1 hr at 37°C. Trypsin was added as a 10 mg/ml sterile filtered solution in 0.01 M Tris-HCl (pH 8.1). Controls received the same treatment, except that trypsin was omitted. Assays of phospholipase C and haemolytic activities in treated and control samples were carried out at pH 5 and 5.5, respectively, in 0.03 M sodium acetate buffer. For haemolysis, samples were supplemented with 0.15 M NaCI (final concentration). Heat treatment. This was carried out in 0.15M NaC1/0.02 M sodium acetate (pH 5.5) by immersion of the samples for the indicated times in a water bath set at 60°C or in boiling water.

Influence of pH on phospholipase C and haemolytic activities. Assays of phospholipase C activity were carried out in 0.2M sodium citrate/0.2 M "Iris adjusted to the different pH values. Haemolytic activity was tested adjusting the pH of the enzyme solution with either 0.2 M Trizma base or 0.2 M acetic acid as required.

RESULTS

Purification o f p h o s p h o l i p a s e C is outlined in Table 1. Preliminary experiments in which the g r o w t h m e d i u m was f r a c t i o n a t e d with different concentrations o f a m m o n i u m sulphate showed t h a t the highest specific activity for b o t h p h o s p h o l i p a s e C a n d haemolysis was o b t a i n e d at 243 g/l a m m o n i u m sulphate. This c o n c e n t r a t i o n was selected for the initial purification step. The chromatographic patterns of phospholipase C a n d haemolytic activities eluting from D E A E cellulose a n d Sepharose 6B are s h o w n in Figs 1 a n d 2. In b o t h columns, closely parallel elution o f phos-

Table 1. Partial purification of phospholipase C and haemolytic activity from the extracellular fluid of Tetrahymena thermophila

Fraction Ammonium sulphate precipitate DEAE cellulose Sepharose 6B

Total protein (rag) 39.6 2.89 0.34

Total activity (nmol diacylglycerol/hr) 1720 350 230 Total activity (HU)

Ammonium sulphate precipitate DEAE cellulose Sepharose 6B

39.6 2.89 0.34

3890 315 205

Phospholipase C activity Spec. act. (nmol diacylglycerol/ Recovery hr/mg) (%) 43 121 676 Hemolytic activity Spec. act. (HU/mg) 98.3 109 600

100 20 13.5 Recovery (%) 100 8.1 5.3

Purification factor 1 2.8 15.5 Purification factor 1 1.1 6.1

Acid phospholipase C

145

f-

"T e=.

O

I

O

t-

E •

"a-

15 .1

10

C~

-~10-l

5

E G$ "-r-

~

l

.~

, l, . . . . . ......

T T 7 "T

2

6

•

I

"~118~-1~ ~ 02~.

/~;~\/~ . . . .

11"l/Ill l

•

---ho .~.-[ 0.10

~ : ~~

0 "0S

"I .

I-T

10

"T T

1L,

T

"T "T ~

I

18 22 26 Fraction number

l

l

30

I

l

3/+

Fig. 1. D E A E cellulose ion exchange chromatography o f phospholipase C and haemolytic activities. Start of gradient is indicated by arrow. For experimental details see the Materials and Methods section. DO: diacylglycerol.

pholipase C and haemolytic activities is observed. A constant ratio between haemolytic and phospholipase C activities of 0.9 HU/nmol diacylglycerol formed/hr is found in the active fractions, as well as in the pools resulting from these two steps. The crude ammonium sulphate precipitate contains a second haemolytic factor, different from phospholipase C. This can be shown when the DEAE cellulose step is omitted and the ammonium sulphate precipitated protein is directly chromatographed on Sepharose 6B. In this case, two separate haemolytic peaks are observed, one coinciding with phospholipase C, and the other not associated with it. The existence of this second haemolytic factor, which was not further investigated, can account for the higher ratio between haemolytic and phospholipase C activities observed for the ammonium sulphate precipitate (2.26 HU/nmol diacylglycerol). Phospholipase C is present in high mol. wt complexes (>4.106). This is indicated by the low concen-

tration of ammonium sulphate required to precipitate the activity and by its elution pattern on Sepharose 6B, where it elutes in the void volume (Fig. 2). The detergent Triton X-100 drastically alters the elution volume of the enzyme from Sepharose 6B. Figure 3 compares the chromatographic patterns observed in the absence and presence of this detergent. In the first case, phospholipase C from DEAE cellulose was directly chromatographed on Sepharose 6B, equilibrated and eluted with l0 mM Tris--HCl, pH 7.1. In the second, Triton X-100 (final concentration of 2%) was added to phospholipase C from the same source. After standing in the cold for 12 hr, the sample was loaded onto the Sepharose 6B column, which was equilibrated this time with 0.5 % Triton X-100/10mM Tris-HCl, pH 7.1, and elutecl with the same buffer. In this case, phospholipase C is considerably retained and elutes together with Triton X-100 micelles. If lower Triton X-100 concentrations were used, the peak at the void volume was only

T 0

I

0

i

E -1-

,m

-~

w

~$

~6

5

?k •

>,15

~10

0.15

-

4

-

-0,0

o N

0.05 <

I/b O

g. 2

13 12 16 20 2Z~ 28 32

36

Fraction number Fig. 2. Sepharose 6B gel filtration of phospholipase C. For experimental details see the Materials and Methods section. Void volume determined with Blue Dextran 2000 is indicated by arrow. DO: diacylglycerol. C.B.P. 8 5 / I B - - - J

146

J. FLORIN-CHRISTENSENet al.

p-= 10

t-~

,

a

~=~

2 11

15

17

23

27

31

35

Fraction humor

Fig. 3. Effect of Triton X-100 on the chromatographic pattern of phospholipase C on Sepharose 6B. In both cases 7 ml of an enzyme preparation eluted from DEAE cellulose, containing 0.64 mg of protein were loaded on a 80 x 2.5 ern column. Flow rate was 25 ml/hr and 9 ml fractions were collected. 1"7:without Triton X-100; I1: sample dissolved in 2% Triton X-100 eluted in the presence of 0.5% of this detergent. partially solubilized and, therefore, two peaks of phospholipase C activity were observed. Delipidation of phospholipase C preparations by the procedures of van den Bosch et al. (1973) and Williams and J u t (1976) resulted in loss of enzyme activity. This inactivation could not be reversed by addition of Triton X-100 or soy bean phospholipids. Phospholipase C hydrolyses 1,2-di[1-~4C]stearoylsn-glycero-3-phosphorylcholine and l-palmitoyl-2[1.~C]oleoyl-sn-glycero-3-phosphorylcholine at similar rates. The final preparation of enzyme had no detectable activity against p-nitrophenylphosphorylcholine, a substrate reported to be useful for phospholipase C assay (Kurioka and Matsuda, 1976). A phosphodiesterase active against this substrate is present in the ammonium sulphate precipitate. This enzyme elutes in the wash-out of the D E A E cellulose step and is thus separated from phospholipase C. Our results agree with what van den Bosch (1982) has indicated for this substrate. The variation of phospholipase C with pH is shown in Fig. 4. In this experiment purified phospholipase C was used as source of enzyme. No increase in free fatty acids was detected at any pH, indicating that no deacylation of either phosphatidylcholine or diacylglycerols took place in the preparations. Phospholipase C showed optimum activity at pH 5.0. Portions of the purified preparation were used to study the variation of the rate of haemolysis with the

!

0.02

~ 7

5

6

7

8

9 pH

Fig. 4. Effect of the pH on phospholipase C activity and rate of haemolysis. Purified phospholipase C (0.013 mg/ml of protein) was used in the assays which were carded out as described in the Materials and Methods section.

Table 2. Effects of CaCI2, EDTA, heat treatment and trypsin on phospholipasaC and hacmolyticactivities Phospholipas¢ Haemolytic Additions or C activity activity treatments % of controls % of controls 5 mM 104 100 CaCl2 10 mM 99 96 1 mM 98 96 EDTA 2.5 mM 101 97 60°C 10rain 60 55 20 min 34 35 30 min 19 14 100°C 10rain 0.1 0 trypsin 0.5 0 Assays of phospholipaseC were carried out as described in the Materials and Methods section. The assay mixtures contained 3.2/~g/mlof purifiedenzymepreparation.Data are expressedas percentages of the respectivecontrol values. pH. Figure 4 shows that the rate of haemolysis is maximal at acidic pH values. Controls which showed no haemolysis were performed for each pH value with buffer without enzyme protein. Below pH 5.5, erythrocytes become progressively unstable, precluding determinations under more acidic conditions. The results of the assay of phospholipase C and haemolysis in the presence of 5 and 10 mM CaCI2 and 1 and 2.5 m M EDTA are shown in Table 2. These substances did not affect either .phospholipase C activity or haemolytic activity, indicating that Ca 2+ has no inhibitory effect and that this or other divalent cations are not required for the activities. Phospholipase C and haemolytic activities are thermolabile. Exposure to 60°C causes progressive and parallel degradation of both activities (Table 2). Incubation at 100°C for 10 rain abolishes both activities. Incubation with trypsin, as described in Materials and Methods, resulted in destruction of phospholipase C and haemolytic activity (Table 2). It was found that 25 gg/ml of chicken egg phosphatidylcholine completely prevented the haemolysis caused by purified phospholipase C preparations containing 4 HU. DISCUSSION Several mammalian cells have been shown to possess phospholipase C activity with acidic optimum (Heller and Shapiro, 1966; Weinreb et al., 1968; Beaudet et al., 1980). A lysosomal origin for this enzyme has been demonstrated in rat liver hepatocytes (Matsuzawa and Hostetler, 1980; Kunze et al., 1982), and it has been suggested that the acid phospholipase C originates from the lysosomal enzyme system in other ceils (Matsuzawa and Hostetler, 1980). This enzyme has recently attracted attention because it may be identical to the lysosomal sphingomyelinase. The deficiency of the latter is believed to be the cause of Niemann-Pick's disease, a serious human disorder where lysosomes accumulate large amounts of phospholipids (Beaudet et al., 1980; Wherrett and Huterer, 1983; Huterer et aL, 1983). The phospholipase C we have found to be released by Tetrahymena has an acidic pH optimum, is not inhibited by EDTA and does not require Ca2+; feature,s which are characteristic of the lysosomal phospholipase C from rat hepatocytes (Matzuwa and

Acid phospholipase C Hostetler, 1980; Kunz¢ et al., 1982). It is well established that Tetrahymena cells release large amounts of lysosomal enzymes to the medium (Mfiller, 1972; Blum and Rothstein, 1975). We therefore suggest that the acidic phospholipase C partly purified in this study is of lysosomal origin. In the present work we show that phospholipase C copurifies with haemolytic activity against sheep erythrocytes. This result, together with the fact that haemolysis is inhibited by egg phosphatidylcholine and the close correlation between phospholipase C and haemolytic activity observed in different experiments, indicates that Tetrahymena phospholipase C is able to lyse sheep erythrocytes. The final preparation is also active on human red cells (data not shown). As already mentioned, phospholipases with haemolytic activity on human red cells have, so far, only been found to be of type C and from bacterial origin (van Deenen et al., 1976). Tetrahymena is therefore the first eukaryotic cell in which an enzyme with such properties is detected. Some predator ciliates such a Dendrocometes paradoxus and Lacrymaria olor have been reported to possess cytolytic factors (Faurt-Fremiet, 1962), but their nature and the molecular basis of their action are unknown. A phospholipase C like that found in the ciliate Tetrahymena could participate in the cytoiytic actions of the secretions of these predator ciliates. Tetrahymena phospholipase C was found to be associated to high mol. wt micelles. The drastic effect of Triton X-100 on the gel filtration chromatographic behaviour of the enzyme suggests that the micelles contain lipids which are replaced by the detergent in the experiment illustrated in Fig. 3. The natural lipids associated with phospholipase C may play an important role in the interaction of the enzyme with intact cell membranes. As pointed out above, lytic phospholipases have, so far, only been reported to be produced by bacteria, organisms that bear protective cell walls. In contrast, Tetrahymena lacks cell wall, exposing an almost naked plasma membrane to the medium. We have suggested (Florin-Christensen et aL, 1985) that the tolerance of this organism to its own released phospholipases may be due to the inclusion in its surface membrane of a peculiar kind of phospholipids, the phosphonolipids (Kennedy and Thompson, 1970), which are highly resistant to enzymatic hydrolysis by phospholipases (Rosenthal and Pousada, 1968; Thompson, 1969; Rosenthal and Han, 1970). The release of a cytolytic phospholipase which can be tolerated by these cells may result in selective destruction of competitors and protection against predators, thus conferring an obvious evolutionary advantage to the species. Acknowledgements--We gratefully acknowledge support from the Danish Ministry of Foreign Affairs (to Jorge and Monica Fiorin-Christensen) and from the Danish Science Research Council. REFERENCES Beaudet A. L., Hampton M. S., Patel K. and Sparrow J. T. (1980) Acidic phospholipascs in cultured human

147

fibroblasts: deficiency of phospholipase C in Niemann-Pick disease. Clinica chim. acta 108, 403-414. Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Phys/oL 37, 911-917. Blum J. J. and Rothstein T. L. (1975) Lysosomes in Tetrahymena. In Lysosomes in Biology and Pathology (Edited by Hawthorne J. N. and Ansell G. B.), Chap. 9, pp. 313-357. Elsevier Biomedical Press, Amsterdam. (Edited by Dingle J. T. and Dean R. T.), Vol. 4, pp. 33-45. Elsevier/North-Holland, Amsterdam. Colley C. M., Zwaal R. F. A., Roeiofsen B. and van Deenen L. L. M. (1973) Lytic and non-lytic degradation of phospholipids in mammalian erythrocytes by pure phospholipases. Biochim. biophys. Acta 307, 74--82. Faur~-Fremiet E. (1963) Pouvoir lytique et phosphatase acid chez les cili~s. C.r. hebd. s$anc. Acad. Sci., Paris 254, 2691-2693. Florin-Christensen J,, Florin-Christensen M., Knudsen J. and Rasmussen L. (1985) Cytolytic activity released from Tetrahymena. J. Protozool., 32, 657-660. Heller M. and Shapiro B. (1966) Enzymic hydrolysis of sphingomyelin by rat liver. Biochem. J. 98, 763-769. Huterer S., Wherrett J. R., Poulos A. and Callahan J. W. 0983) Deficiency of phospholipase C acting on phosphatidylglycerol in Niemann-Pick disease. Neurology (NY) 33, 67-73. Kennedy K. E. and Thompson G. A., Jr. (1970) Phosphonolipids: localization in surface membranes of Tetrahymena. Science, Wash. 168, 989-991. Kunze H., Hesse B. and Bohn E. (1982) Hydrolytic degradation of phosphatidylethanolamine and phosphatidylcholine by isolated rat liver lysosomes. Biochim. biophys. Acta 711, 10-18. Kurioka S. and Matsuda M. (1976) Phospholipase C assay using p-nitrophenylphosphorylcholine together with sorbitol and its application to studying the metal and detergent requirement of the enzyme. Analyt. Biochem. 75, 281-289. Matsuzawa Y. and Hostetler K. Y. (1980) Properties of phospholipase C isolated from rat liver lysosomes. J. biol. Chem. 255, 646-652. Mfiller M. (1972) Secretion of acid hydrolases and its intracellular source in Tetrahymena pyriformis. J. Cell Biol. 52, 478-587. Nanney D. L. and McCoy J. W. (1976) Characterization of the species of the Tetrahymena pyriformis complex. Trans. Am. Microsc. Soc. 95, 664-682. Plesner P., Rasmussen L. and Zeuthen E. (1964) Techniques used in the study of synchronous Tetrahymena. In Synchrony in Cell Division and Growth (Edited by Zeuthen E.), pp. 543-563. Interscience Publishers, New York. Rosenthal A. F. and Pousada M. (1968) Inhibition of phospholipase C by phosphate analogs of glycerophosphatides. Biochim. biophys, dcta 164, 226-237. Rosenthal A. F. and Han S. C.-H. (1979) A study of phospholipase A inhibition by glycerophosphatide analogs in various systems. Biochim. biophys. Acta 218, 213-220. Takahashi T., Suyahara T. and Ohsaka A. (1974) Purification of CIostridium perfringens phospholipase C (~-toxin) by affinity chromatography on agarose-linked egg yolk lipoprotein. Biochim. biophys. Acta 351, 155-171.

Thompson G. A., Jr. (1969) The metabolism of 2-aminoethylphosphonate lipids in Tetrahymena pyriformis. Biochim. biophys. Acta 176, 330-338. van Deenen L. L. M., Demel R. A., Geurts Van Kessel W. S. M., Kamp H. H., Roclofsen B., Verkleij A. J., Wirtz K. W. A. and Zwaal R. F. A. (1976) Phospholipases and monolayers as tools in studies on membrane structure. In The Structural Basis of Membrane Function (Edited by

148

J. FLORIN-CHRISTENSENet al.

Hatefi Y. and Djavadi-Ohaniance L.), pp. 21-38. Academic Press, Inc., New York. van den Bosch H., Aarsman A. J., de Jong J. G. N. and van Deenen L. L. M. (1973) Studies on lysophospholipases I. Purification and some properties of a lysophospholipase from beef pancreas. Biochim. biophys. Acta 296, 94-104. van den Bosch H. (1982) Phospholipases. In Phospholipids Weinreb N. J., Brady R. O. and Tappel A, L. (1968) The lysosomal localization of sphingolipid hydrolases. Biochim. biophys. Acta 159, 141-146.

Wherrett J. R. and Huterer S. (1983) Deficiency of taurocholate-dependent phospholipase C acting on phosphatidylcholine in Niemann-Pick disease. Neurochem. Res. 8(1), 87-96. Williams J. T. and Juo P.-S. (1976) Release and activation of a particulate bound acid phosphatase from Tetrahymena pyriformis. Biochim. biophys. Acta 422, 120-126. Zwaal R. F. A., Roelofsen B. and CoUey C. M. (1973) Localization of red cell membrane constituents. Biochim. biophys. Acta 300, 159-182.