A pancreatic-type phospholipase A2 in rat gastric mucosa

A phospholipase A2, which is immuno-crossreactive with the anti-rat pancreatic phospholipase A2 antibody, is present in rat gastric mucosa. The content of the enzyme in the gastric mucosa was comparable to that in the pancreas, but the specific activity in the gastric mucosa homogenate (60.7 +/- 19.5 nmol/min/mg) was higher than that in the pancreas homogenate (3.16 +/- 0.77 nmol/min/mg). A greater proportion of the enzyme was found in the particulate fraction. The gastric enzyme and its proenzyme were purified from the supernatant. The amino acid sequence of the N-terminal 15 residues of the gastric enzyme was determined and found to be identical with that of rat pancreatic phospholipase A2. Like the pancreatic proenzyme, the gastric proenzyme was activated on trypsin treatment.     

Subcellular localization of rat gastric phospholipase A2

In the present study, we have performed experiments to gain some insight into the subcellular localization and biochemical properties of gastric mucosal phospholipase A2. After classical subcellular fractionation of whole glandular stomach mucosa, we found that gastric phospholipase A2 was essentially enriched in the 105,000 x g pellet that contains microsomes and plasma membranes. Except for the cytosol, all the subcellular fractions exhibited similar phospholipase A2 activity (i.e., optimum of pH, calcium dependence, apparent Km and positional specificity). The high-speed pellet was further characterized by ultracentrifugation on a sucrose gradient. Data showed that the sedimentation profile of phospholipase A2 was quite similar to those of plasma membrane markers and more specifically to an apical membrane marker. These results, taken together, showed that a gastric phospholipase A2 is distributed among the various subcellular fractions (as a result of cross-contamination) together with the membrane fraction on which it is associated. It is proposed that this fraction is the apical plasma membrane which would be the main site of phospholipase A2 action for arachidonic acid release. Lysophospholipase showed the same sedimentation profile as phospholipase A2, whereas acyl CoA-lysophosphatidylcholine: acyltransferase mainly sedimented with heavy microsomes. The substrate specificity of the enzyme was assessed by endogenous hydrolysis of gastric mucosal phospholipids. We were able to show that the enzyme acts at nearly the same rate on two major gastric membrane phospholipids, namely phosphatidylcholine and phosphatidylethanolamine.     

Phospholipid-deacylating enzymes of rat stomach mucosa

1. Rat stomach mucosa exhibited three distinguishable phospholipid-deacylating enzyme activities: lysophospholipase, phospholipase A1 and phospholipase A2. 2. The lysophospholipase hydrolyzed 1-palmitoyl lysophosphatidylcholine to free fatty acid and glycerophosphorylcholine. This enzyme had an optimum pH of 8.0, was heat labile, did not require Ca2+ for maximum activity and was not inhibited by bile salts or buffers of high ionic strength. 3. Phospholipase A2 and phospholipase A1 deacylated dipalmitoyl phophatidylcholine to the corresponding lyso compound and free fatty acid. The specific activity of phospholipase A2 was 2--4-fold higher than that of phospholipase A1 under all the conditions tested. Both activities were enhanced 4--7.5-fold in the presence of bile salts at alkaline pH and 11-18-fold at acidic pH. 4. In the absence of bile salts, phospholipase A1 exhibited pH optima at 6.5 and 9.5 and phospholipase A2 at pH 6.5, 8.0 and 9.5. The pH optima for phospholipase A1 were shifted to pH 3.0, 6.0 and 9.0 in presence of sodium taurocholate; the activity was detected only at a single pH of 9.5 in the presence of sodium deoxycholate and at pH 10.0 in the presence of sodium glycocholate. Phospholipase A2 optimum activity was displayed at pH 3.0, 6.0 and 8.0 in presence of taurocholage, pH 7.5 and 9.0, in presence of glycocholate and only at pH 9.0 in presence of deoxycholate. 5. Ca2+ was essential for optimum activity of phospholipases A1 and A2. But phospholipase A1 lost complete activity in presence of 0.5 mM ethyleneglycolbis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) at pH 6.0, whereas phospholipase A2 lost only 50%. 6. Phospholipases A1 and A2 retained about 50% of their activities by heating at 75 degrees for 10 min. At 100 degrees, phospholipase A1 retained 22% of its activity, whereas phospholipase A2 retained only 7%.     

Phospholipid-deacylating enzymes of rat small intestinal mucosa.

1. Two phospholipase activities, provisionally designated as phospholipase activity I and phospholipase activity II, were found to be present in the mucosal homogenates of rat small intestine. These phospholipase activities were present in the membraneous particle fraction and were characterized in this study without further purification, using phosphatidylcholine as a substrate. Phospholipase activity I was assayed at pH 5.9 in the absence of deoxycholate, whereas phospholipase activity II was assayed at pH 9.4 in the presence of deoxycholate. Phospholipase activity I was more easily inactivated by heat treatment and trypsin digestion than phospholipase activity II. Both phospholipase activities were inhibited by diisopropyl-fluorophosphate but not by SH-binding reagents. 2. Phospholipase activity I had a pH optimum at 5.9. A sigmoid curve was obtained when the amount of the enzyme preparation was plotted against the phospholipase activity I. The unusually low activity found at low enzyme concentrations was enhanced by addition of the heat-inactivated enzyme preparation to a level where a linear relationship was found between the amount of enzyme and the activity. The effector present in the enzyme preparation was tentatively identified as fatty acid(s). The addition of oleic acid or linoleic acid to the incubation mixture enhanced the phospholipase activity I. At 1 mM levels of these fatty acids the highest activity was obtained when 1.5 mM phosphatidylcholine was used as a substrate. 3. The phospholipase activity II increased on addition of deoxycholate. In the presence of 5 mM deoxycholate, a pH optimum was found at 9.6. It was found that the maximal extent of hydrolysis of phosphatidylcholine in the incubation mixture was dependent on the concentration of deoxycholate. This indicates that deoxycholate facilitates the action of phospholipase activity II, presumably by forming deoxycholate-phosphatidylcholine mixed micelles. Phospholipase activity II was found to deacylate specifically the 2-acyl moiety of phospholipids.     

Phospholipase of rat intestine: mode of action]

Phospholipase activities of rat intestinal mucosa homogenate have been determined from lysophosphatidylcholines [14C] and phosphatidylcholines [-3H-14C]. In the presence of phosphatidylcholines, at pH 6.5, the homogenate has a phospholipase B activity. At pH 8.5, a phospholipase A2 activity was shown. In the presence of lysophospatidylcholines, at pH 6.5, we notice a lysophospholipase A1 activity. A kinetic study of the reactions allows us to separate the activity B into a phospholipase A2 activity and a lysophospholipase A1 activity. Thus, it appears that the total phospholipase activity of rat intestinal mucosa would results from a phospholipase A2 activity and a lysophospholipase A1 activity.     

Studies on phospholipase A inhibitor in blood plasma. II. Interaction of phospholipase A inhibitor with phospholipase A and its specificity

Non-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed unless the inhibitor was preincubated with the enzyme. The inhibition seemed to be due to the formation of the enzyme-inhibitor complex, which was identified by immunoelectrophoresis. The enzyme-inhibitor interaction was observed maximally on incubation at physiological pH, but not below pH 5. The inhibitor was inactivated by trypsin digestion and heat treatment. It suppressed the phospholipase A2 activities of rat blood plasma as well as of the snake venom and porcine pancreas, but not the enzyme activities such as those of phospholipase C of Bacillus cereus, lipase of porcine pancreas, trypsin, and papain. The inhibitor also showed the ability to decrease membrane-bound phospholipase A1 and A2 activities in intracellular organelles such as plasma membranes, mitochondria, lysosomes, and microsomes. In view of these facts, it was concluded that the plasma inhibitor is specific for phospholipase A.     

Presence of pancreatic-type phospholipase A2 mRNA in rat gastric mucosa and lung

The content of mRNA for a pancreatic-type phospholipase A2 present in rat gastric mucosa was much greater than that in pancreas. In lung the mRNA for this pancreatic-type phospholipase A2 was also detected, but less than in pancreas. Nucleotide sequence analysis showed that these pancreatic-type phospholipase A2 cDNAs derived from rat gastric mucosa and lung were completely identical to that from rat pancreas (Ohara et al. (1986) J. Biochem. 99, 733-739). This demonstrates that the pancreatic-type phospholipase A2 present in gastric mucosa and lung does not originate from pancreas.     

Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C

The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.     

Characterization and Localization of Phospholipase A2 Activity in Sympathetic Ganglia

Superior cervical ganglion phospholipase A2 activity was characterized using 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine as a substrate. The enzyme activity exhibited linearity with interval of incubation and tissue concentration; there appeared to be two pH optima of the enzyme, at pH 6.0 and 9.0. A Lineweaver-Burk plot of the reciprocal of activity versus substrate concentration yielded an apparent Km of 0.53 mM and a Vmax of 5.3 nmol/h/mg of protein. The enzyme exhibited a partial Ca2+ dependence; in the absence of Ca2+ and presence of EGTA, activity was reduced by 40%. The phospholipase A2 activity was heat sensitive and was completely inactivated after treatment at 100 degrees C for 30 min. For determination of whether the enzyme had a preference for hydrolysis of specific fatty acid substituents in the 2 position of phosphatidylcholine, several different substrates were tested. The order of preference for hydrolysis by the ganglionic enzyme was 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine = 1-palmitoyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-2-[1-14C]palmitoyl-sn-glycero-3-phosphocholine. For determination of the localization of the phospholipase A2 enzyme in sympathetic ganglia, two approaches were used. Guanethidine, which results in destruction of adrenergic cell bodies in sympathetic ganglia, was administered to rats; an approximately 50% decline in phospholipase A2 activity was observed after this treatment. In other experiments, the preganglionic nerve to the ganglion was sectioned in rats; after 2 weeks of denervation, no significant change in ganglionic phospholipase A2 activity was seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible regulation of thiamine diphosphatase activity in rat brain microsomes by lipids.

The effects of various treatments, which affect membrane structure, on microsomal thiamine diphosphatase and thiamine triphosphatase activities of rat brain, were examined. The treatment of micorosomes at alkaline pH caused a 2-fold activation of the thiamine diphosphatase, this being related to a change in membrane structure which was evidenced by a decrease of the turbidity of the microsomal suspension. Repeated freezing and thawing after hypo-osmotic treatment also increased the activity of microsomal thiamine diphosphatase. In addition, the thiamine diphosphatase activity was enhanced by treatment of the microsomes with phospholipase C or acetone. This lipid depletion resulted in a marked reduction in the apparent Km value of the thiamine diphosphatase with a corresponding loss in heat stability of the enzyme. We found further that brain thiamine diphosphatase was solubilized by Triton X-100. This decreased the phospholipid content in the preparation, but did not affect the apparent Km value and heat stability of the enzyme. In contrast with thiamine diphosphatase, thiamine triphosphatase was inactivated by treatment at alkaline pH or with acetone. However, treatment with phospholipase C did not affect the activity of thiamine triphosphatase.     

Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex

Phospholipase A has been isolated from a crude lysosomal fraction from rat kidney cortex and purified 7600-fold with a recovery of 9.8% of the starting activity. The purified enzyme is a glycoprotein having an isoelectric point of pH 5.4 and an apparent molecular weight of 30,000 by high-pressure liquid chromatography gel permeation. Naturally occurring inhibitors of lysosomal phospholipase A are present in two of the lysosomal-soluble protein fractions obtained in the purification. They inhibit hydrolysis of 1,2-di[1-14C]oleoylphosphatidylcholine by purified phospholipase A1 with IC50 values of 7-11 micrograms. The inhibition is abolished by preincubation with trypsin at 37 degrees C, but preincubation with trypsin at 4 degrees C has no effect, providing evidence that the inhibitors are proteins. The results suggest that the activity of lysosomal phospholipase A may be regulated in part by inhibitory proteins. Lysosomal phospholipase A from rat kidney hydrolyzes the sn-1 acyl group of phosphatidylcholine, does not require divalent cations for full activity, and is not inhibited by ethylenediaminetetraacetic acid. It has an acid pH optimum of 3.6-3.8. Neither p-bromophenacyl bromide, diisopropyl fluorophosphate, nor mercuric ion inhibits phospholipase A1. In contrast to rat liver, which has two major isoenzymes of acid phospholipase A1, kidney cortex has only one isoenzyme of lysosomal phospholipase A1.     

Purification and characterization of a membrane-associated phospholipase A2 from rat spleen. Its comparison with a cytosolic phospholipase A2 S-1

A membrane-associated phospholipase A2 was purified from rat spleen. The phospholipase A2 was solubilized from the 108,000 x g pellet fraction with 0.3% lithium dodecyl sulfate and then purified to homogeneity by successive DEAE-Cellulofine AM, octyl-Sepharose, Cellulofine GCL 300-m, S-Sepharose, and Bio-Gel P-30 chromatographies in the presence of 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate. The apparent Mr of the enzyme, estimated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, was about 13,600. The purified enzyme had a pH optimum in the range of pH 8.0-9.5 and required the presence of Ca2+ (4 mM) for its maximal activity. The enzyme preferentially hydrolyzed the 2-acyl ester bonds of phosphatidylglycerol in the presence and absence of sodium cholate or sodium deoxycholate. Unlike the phospholipase A2 of rat spleen supernatant, no immunocross-reactivity was observed between the purified enzyme and anti-rat pancreatic phospholipase A2 antibody. The N-terminal amino acid sequence of the enzyme was determined and found to be homologous to that of viperid and crotalid venom phospholipases A2. The results in this and the preceding report (Tojo, H., Ono, T., Kuramitsu, S., Kagamiyama, H., and Okamoto, M. (1988) J. Biol. Chem. 263, 5724-5731) demonstrate that rat spleen contains two genetically distinct phospholipase A2 isoenzymes.     

Enzymatic sulfation of cholesterol by rat gastric mucosa

A sulfotransferase which catalyzes transfer of the sulfate group from 3'-phosphoadenosine-5'phosphosulfate to cholesterol has been demonstrated in the rat gastric mucosa. The product of the reaction was characterized as cholesterol sulfate by two-dimensional thin-layer Chromatographic behavior, and gas-liquid chromatography of cholesterol after acid solvolysis. The bulk of enzyme activity was found in the cytosol fraction. Sulfation of cholesterol did not require added Mg⁺², Mn⁺², or Ca⁺², and was unaffected by ethylenedia-minetetraacetate. Triton X-100 moderately enhanced the enzyme activity. A broad pH optimum from pH 6.0–9.0 was exhibited with a maximum at pH 7.0–7.5. The apparent Km for PAPS was 0.8 × 10^?6M. The possible function of cholesterol sulfate in gastric mucosa is discussed.     

Mechanisms in regulating the release of serotonin from the perfused rat stomach

The mechanisms regulating the release of serotonin into the portal circulation as well as into the gastric lumen were studied in the isolated vascularly and luminally perfused rat stomach. Immunohistochemical study of the rat stomach showed that serotonin-containing enterochromaffin (EC) cells were densely packed in the antral mucosa, sparsely scattered in the corpus, and not found in the fundus. Such morphological findings suggest that serotonin detected in this study may have originated from antral EC cells. Luminal acidification stimulated the vascular release of serotonin but did not affect the luminal release of serotonin. The basal release of serotonin into the vasculature was 10 times higher than that into the gastric lumen at intragastric pH 2. The vascular release of serotonin is regulated by stimulation from cholinergic nicotinic mechanisms, whereas inhibitory neurotransmitters such as vasoactive intestinal peptide and NO are probably not involved. Somatostatin and peptide YY originating from endocrine cells may exert direct inhibitory effects, possibly via somatostatin and peptide YY receptors on the EC cells, and a cholinergic muscarinic mechanism may exert indirect effects on the vascular release of serotonin via the muscarinic receptor on the endocrine cells.     

Characterization of ATP and ADP hydrolysis activity in rat gastric mucosa

The degradation of nucleotides is catalyzed by the family of enzymes called nucleoside triphosphate diphosphohydrolases (NTPDases). The aim of this work was to demonstrate the presence of NTPDase in the rat gastric mucosa. The enzyme was found to hydrolyze ATP and ADP at an optimum pH of 8.0 in the presence of Mg2+ and Ca2+. The inhibitors ouabain (0.01-1 mM), N-ethylmaleimide (0.01-4 mM), levamisole (0.10-0.2 mM) and Ap5A (0.03 mM) had no effect on NTPDase 1 activity. Sodium azide (0.03-30 mM), at high concentrations (>0.1 mM), caused a parallel hydrolysis inhibition of ATP and ADP. Suramin (50-300 microM) inhibited ATP and ADP hydrolysis at all concentrations tested. Orthovanadate slightly inhibited (15%) Mg2+ and Ca2+ ATP/ADPase at 100 microM. Lanthanum decreased Mg2+ and Ca2+ ATP/ADPase activities. The presence of NTPDase as ecto-enzyme in the gastric mucosa may have an important role in the extracellular metabolism of nucleotides, suggesting that this enzyme plays a role in the control of acid and pepsin secretion, mucus production, and contractility of the stomach.     

Studies on the phospholipases of rat intestinal mucosa

1. Subcellular distribution and characteristics of different phospholipases of rat intestinal mucosa were studied. 2. The presence of free fatty acid was necessary for the maximal hydrolysis of lecithin (phosphatidylcholine), but there was no accumulation of lysolecithin (1 or 2-acylglycerophosphorylcholine);lysolecithin accumulated when the reaction was carried out in the presence of sodium deoxycholate and at or above pH8.0. 3. The fatty acid-activated phospholipase B as well as lysolecithinase showed optimum activity at pH6.5, whereas for the phospholipase A it was about pH8.6. 4. The bulk of the phospholipase A was present in the microsomal fraction, whereas the phospholipase B and lysolecithinase activities were distributed between the microsomal and soluble fractions of the mucosal homogenate. 5. Phospholipase A was equally distributed between the brush border and brush-border-free particulate fraction, with the brush border having highest specific activity, whereas the other two activities were distributed between the brush-border-free particulate and soluble fractions. 6. Various treatments showed marked differences between the phospholipase A and phospholipase B activities, but not between phospholipase B and lysolecithinase activities. 7. By using (beta[1-(14)C]-oleoyl) lecithin it was shown that the mucosal phospholipase A was specific for the beta-ester linkage of the lecithin molecule.     

Effect of diacarb on the carbonic anhydrase activity in the blood and gastric mucosa and pepsinogen concentration in the gastric mucosa of the rat

It was established that the activity of blood and gastric mucosa carboanhydrase increased after the introduction of food irritant (milk) into the stomach, as well as after the subcutaneous injection of histamine. This was accompanied by the increase of pepsinogen content in the gastric mucosa. When introduced into the stomach before the food irritant or histamine, acetazolamide inhibited blood and gastric mucosa carboanhydrase and reduced the content of pepsinogen in the gastric mucosa. Oral administration of acetazolamide for 5 days resulted in a more remarkable inhibition of blood and gastric mucosa carboanhydrase and in a drastically reduced content of pepsinogen in the gastric mucosa. The rate of pepsinogen biosynthesis by the gastric mucosa seems to depend on the activity of carboanhydrase in blood and in the gastric mucosa.     

Effect of ethanol on mucus glycoprotein fatty acyltransferase from gastric mucosa

The enzyme activity that catalyzes the transfer of palmitic acid from palmitoyl coenzyme A to the deacylated intact or deglycosylated gastric mucus glycoprotein was demonstrated in the detergent extracts of the microsomal fraction of antral and body mucosa of the rat stomach. Both types of mucosa exhibited similar acyltransferase activities and acceptor specificities. A 10-14% decrease in the fatty acyltransferase activity was observed with the reduced and S-carboxymethylated mucus glycoprotein, but the proteolytically degraded glycoprotein showed no acceptor capacity. This indicated that the acylation of mucus glycoprotein with fatty acids occurs at its nonglycosylated polypeptide regions and that some of the fatty acids may be linked via thiol esters. Optimum enzyme activity was obtained at pH 7.4 with the detergent Triton X-100, NaF, and dithiothreitol. The apparent Km values for the intact and deglycosylated mucus glycoproteins were 0.45 and 0.89 microM, respectively. The acyltransferase activity of the microsomal enzyme was inhibited by ethanol. With both intact and deglycosylated glycoprotein substrates, the rate of inhibition was proportional to the ethanol concentration up to 0.4 M and was of the competitive type. The K1 values were 0.80 microM for the intact mucus glycoprotein and 1.82 microM for the deglycosylated glycoprotein. Preincubation of the microsomal enzyme with low concentrations of ethanol (up to 0.5 M) did not seem to exert any additional deterrent effect on acyltransferase activity. Higher concentrations of ethanol (1.0 M and above), however, caused substantial reduction in the transferase activity due to denaturation of the enzyme.     

The phospholipase activity of sheep pancreatic juice.

Sheep pancreatic juice was found to contain at least two enzymes which hydrolysed biliary lecithin. One enzyme was heat and acid labile and hydrolysed the fatty acid from position 1 (phospholipase A1); the other was heat and acid stable hydrolysing the fatty acid at position 2 (phospholipase A2). Lysophospholipase activity was also present. The phospholipases were active at pH values greater than 4.2, and would therefore function in the acid conditions (pH 3-6) of the sheep small intestine. The activity of the pancreatic phospholipases, and A2 in particular, was dramatically stimulated by the presence of the secretions of Brunner's glands which could be important in accelerating the hydrolysis of biliary lecithin in the lumen of the intestine. Phospholipase A1 was sensitive to acid in the range pH 2.5-3.5 and could therefore be partially inactivated by abomasal digesta; but phospholipase A2 was resistent to acid treatment.     

Lysosomal phospholipase A activities of rat ovarian tissue.

1.1. Lysosome-enriched fractions were prepared by differential centrifugation of homogenates of luteinized rats ovaries. Acid phospholipase A activities were characterized with [U-14C]diacyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-[9,10-3H]- or [1-14C]oleoyl-sn-glycero-3-phosphocholine as substrates. Acid phospholipase A1 activity had properties similar to other hydrolases of lysosomal origin; subcellular distribution, latency and acidic pH optimum. Acid phospholipase A2 activity with similar characteristics was also tentatively identified. We were unable to exclude the possibility that the combined action of phospholipase A1 and lysophospholipase contributed to the release of acyl moieties from the 2-position of the synthetic substrates. 2. Lysophospholipase activity was present in the lysosome-enriched fractions. This activity had an alkaline pH optimum. 3. Phospholipase A1 and A2 activities solubilized from lysosome fractions by freeze-thawing were inhibited by Ca2+ and slightly activated by EDTA. A Ca2+- stimulated phospholipase A2 activity, with an alkaline pH optimum, remained in the particulate residue of freeze-thawed lysosome preparations. This activity is believed to represent mitochondrial contamination. 4. Activities of acid phospholipase A, as well as other acid hydrolases, increased approx. 1.5-fold between 1 and 4 days following induction of luteinizatin, suggesting a hormonal influence on lysosomal enzyme activities.