Purification and Characterization of Membrane-Bound Inositol Phospholipid-Specific Phospholipase C from Suspension-Cultured Rice (Oryza sativa L.) Cells (Identification of a Regulatory Factor)

A membrane-bound inositol phospholipid-specific phospholipase C was solubilized from rice (Oryza sativa L.) microsomal membranes and purified to apparent homogeneity using a series of chromatographic separations. The apparent molecular mass of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000 D, and the isoelectric point was 5.1. The optimum pH for the enzyme activity was approximately 6.5, and the enzyme was activated by both Ca2+ and Sr2+. The chemical and catalytic properties of the purified membrane-bound phospholipase C differed from those of the soluble enzyme reported previously (K. Yotsushima, K. Nakamura, T. Mitsui, I. Igaue [1992] Biosci Biotech Biochem 56: 1247-1251). In addition, we found a regulatory factor for the phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolyzing activity of phospholipase C from rice cells. The regulatory factor was dissociated from the catalytic subunit of phospholipase C during the purification. The regulatory factor was necessary to induce PIP2-hydrolyzing activity of both membrane-bound and -soluble phospholipase C; these purified enzymes had no activity alone. Because the plasma membranes isolated from rice cells could also act as a regulatory factor, the regulatory factor seems to be localized in the plasma membranes. Regulation of inositol phospholipid turnover in rice cells is discussed.     

Subcellular localization of the phospholipases A of rat heart: evidence for a cytosolic phospholipase A1

During myocardial ischemia increased levels of lysoglycerophospholipids have been reported which may be deleterious to myocardial function. Phospholipases are presumed to be important in the regulation of this process. To further quantify and characterize the activity of heart phospholipases, we carried out a systematic analysis of phospholipase A activity in rat heart subcellular fractions isolated by the method of Palmer et al. (J. Biol. Chem. 1972. 262: 8731-8739). Neutral phospholipase A was recovered predominately in the cytosolic (soluble) fraction which represented 46% of recovered activity, while the microsomal and subsarcolemmal mitochondrial fractions represented 15% and 12% of the total recovered activity, respectively. Cytosolic phospholipase A differed from the two principal membrane-bound phospholipases A in its pH dependence and apparent Km for substrate. The cytosolic enzyme had a Km (apparent) for dioleoylphosphatidylcholine of 0.07 mM versus 0.28-0.33 mM for the membrane-associated phospholipases A. Acid phospholipase A activity had a subcellular distribution consistent with a lysosomal localization. Lysophospholipase was found principally in the cytosolic, microsomal, and the subsarcolemmal and interfibrillar mitochondrial fractions where it represented 46, 17, 6.3, and 6.9% of the recovered activity, respectively. The positional specificity of the respective phospholipases was assessed. This analysis was complicated by the fact that in heart, lysophospholipase has an observed Vmax 3.6- to 4.5-fold greater than that of phospholipase A in the various subcellular fractions. Equations were derived to obtain corrected values for the activity of phospholipases A1 and A2. Using this method we found that the cytosolic and lysosomal fractions contained phospholipase A1, while the mitochondrial fractions contained primarily phospholipase A2. In heart microsomes, the positional specificity of phospholipase A could not be determined because lysophospholipase activity was very high and lysophosphatidylcholine did not accumulate.     

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by incubation of rat hepatocytes with phospholipase A2

The effect of phospholipase A2 treatment of rat hepatocytes on CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis was investigated. Cytidylyltransferase is recovered from the cytosol and in a membrane-bound form with the microsomes. Digitonin treatment of cells causes rapid release into the medium of the cytosolic, but not the microsomal form of the cytidylyltransferase. Incubation of hepatocytes for 10 min with phospholipase A2 (0.9 units/dish) in the medium, resulted in a 33% decrease in the cytidylyltransferase activity released by digitonin treatment (2.5 +/- 0.15 nmol/min per mg compared to 3.9 +/- 0.10 nmol/min per mg in the control). In agreement with the digitonin experiments, incubation with 0.9 units/dish of phospholipase A2 resulted in a decrease in the cytidylyltransferase activity in the cytosol (from 4.3 +/- 0.10 nmol/min per mg to 2.6 +/- 0.14 nmol/min per mg) and a corresponding increase in the microsomal fraction (from 0.9 +/- 0.16 nmol/min per mg to 1.8 +/- 0.20 nmol/min per mg). The effect of phospholipase A2 on cytidylyltransferase translocation was concentration- and time-dependent. Incubation of hepatocytes in the presence of phospholipase A2 (0.9 units/dish) for 10 min prior to pulse-chase experiments resulted in an increase in radiolabel incorporation into phosphatidylcholine (from 2.4 +/- 0.02.10(-5) dpm/dish to 3.1 +/- 0.1.10(-5) dpm/dish) and a corresponding decrease in radiolabel associated with the choline (from 2.5 +/- 0.05.10(-5) to 1.4 +/- 0.03.10(-5) dpm) and phosphocholine fractions (from 8.5 +/- 0.07.10(-5) to 6.9 +/- 0.05.10(-5) dpm). We conclude that phospholipase A2 can cause a stimulation of CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in cultured rat hepatocytes.     

The action of purified lysophospholipases on microsomal membrane-bound lysophosphatidylcholine

Rat liver membranes were labelled by intraperitoneal injection of [Me-14C]choline chloride. Isolated microsomal membranes were briefly treated with pancreatic phospholipase A2 to produce different levels of membrane-bound lysophosphatidylcholine. The hydrolysis of this lysophosphatidylcholine by two purified lysophospholipases from beef liver was studied. The specific activity of enzyme I at saturating membrane concentrations appeared to increase linearly with the lysophosphatidylcholine level in the membranes until the lysoderivative represented 15% of the original phosphatidylcholine. In contrast, the specific activity of enzyme II was independent of the lysophosphatidylcholine level, at least in the range of 4.9-34.0% tested. These different kinetics are discussed in terms of the possible functions of both enzymes in liver.     

Enzymatic degradation and partial biosynthetic reconstitution of microsomal and mitochondrial membranes.

Guinea pig liver microsomal and mitochondrial membranes were degraded with phospholipase C and D followed by partial biosynthetic reconstitution. Activities of phosphatidylinositol synthetase in microsomal membranes and NADPH-cytochrome c reductase were almost completely lost after phospholipase C and D treatment; almost complete restoration of the original activity was achieved after biosynthesis of phosphatidylcholine in degraded microsomes, but was not reparable after biosynthesis of cytidinediphosphodiglycerides (CDP-diglycerides). The mitochondrial biosynthesis of polyglycerophosphatides was completely retained after degradation of these membranes with phospholipase C, but after similar treatment with phospholipase D, only about one-quarter of the original activity remained, the relative composition of polyglycerophosphatides being significantly different. The activity of NADPH-cytochrome c reductase of microsomes represented about 76% of the original activity after phospholipase C treatment, but only approximately 1% after treatment with phospholipase D. Although this activity could not be restored with CDP-diglyceride synthesis, it was restored to about 75% of the original activity after the biosynthesis of phosphatidylcholine in these fragments. These and additional experimental findings are discussed in terms of the relation between structural organization of lipids and proteins and enzymatic activities of membrane-bound phospholipid-synthesizing enzymes in microsomal and mitochondrial membranes isolated from guinea pig liver.     

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.     

Purification and characterization of a phospholipase A2 associated with rabbit lung microsomes: some evidence for its mitochondrial origin

Phospholipase A2 (EC 3.1.1.4) activity appeared to be unevenly distributed among the subcellular fractions of rabbit lung homogenates. The mitochondrial/lysosomal fraction, which possessed the highest specific activity, was the second most abundant source of enzyme, following the 1000 x g pellet. Crude microsomes, which were the poorest source of enzyme, had a specific activity intermediate between that of crude mitochondria and of cytosol. Despite these observations, in view of the putative role of microsomal phospholipase A2 in remodelling phosphatidylcholines for pulmonary surfactant biosynthesis, the purification of phospholipase A2 from microsomal membranes was investigated. The activity was solubilized from rabbit lung microsomes with 1 M KCl and resolved into two distinct peaks by ion-exchange chromatography. The larger peak (95% of the recovered activity) was subjected to a combination of hydroxyapatite and gel-filtration chromatography, resulting in a purification factor in excess of 70,000 relative to the microsomal membranes. There was no indication for the removal of endogenous inhibitor(s) during the purification. Application of the same purification protocol to a 1 M KCl extract of lung mitochondria resulted in phospholipase A2 profiles in each of the four columns employed that had exactly the same elution characteristics as those generated by the microsomal extracts. The purified enzyme is specific for the sn-2 ester bond of phosphatidylcholine, requires Ca2+ for activity and has an alkaline pH optimum. It is heat-labile and susceptible to treatment by p-bromophenacyl bromide and by 2-mercaptoethanol but remains unaffected by NaF, diisopropylfluorophosphate and thiol reagents.     

Effect of Phospholipase Digestion and Lysophosphatidylcholine on Dopamine Receptor Binding

[3H]Spiperone specific binding by microsomal membranes isolated from sheep caudate nucleus is decreased by trypsin and phospholipase A2 (Vipera russeli), but is insensitive to neuraminidase. The inhibitory effect of phospholipase A2 is correlated with phospholipid hydrolysis. After 15 min of phospholipase (5 micrograms/mg protein) treatment, a maximal effect is observed; the maximal lipid hydrolysis is about 56% and produces 82% reduction in [3H]spiperone binding. Equilibrium binding studies in nontreated and treated membranes showed a reduction in Bmax from a value of 388 +/- 9.2 fmol/mg protein before phospholipase treatment to a value of 52 +/- 7.8 fmol/mg protein after treatment, but no change in affinity (KD = 0.24 +/- 0.042 nM) was observed. Albumin washing of treated membranes removes 47% of lysophosphatidylcholine produced by phospholipid hydrolysis without recovering [3H]spiperone binding activity. However, the presence of 2.5% albumin during phospholipase A2 action (1.5 micrograms/mg protein) prevents the inhibitory effect of phospholipase on [3H]spiperone binding to the membranes, although 28% of the total membrane phospholipid is hydrolysed. Lysophosphatidylcholine, a product of phospholipid hydrolysis, mimics the phospholipase A2 effect on receptor activity, but the [3H]spiperone binding inhibition can be reversed by washing with 2.5% defatted serum albumin. Addition of microsomal lipids to microsomal membranes pretreated with phospholipase does not restore [3H]spiperone stereospecific binding. It is concluded that the phospholipase-mediated inhibition of [3H]spiperone binding activity results not only from hydrolysis of membrane phospholipids, but also from an alteration of the lipid environment by the end products of phospholipid hydrolysis.     

Effect of phospholipase A2 on Ca2+-stimulated adenosine triphosphatase activity in microsomal fraction of rat submandibular gland

The effect of phospholipase A2 on the Ca2+-ATPase (EC 3.6.1.3) activity in the microsomal fraction of rat submandibular gland was kinetically studied in vitro. The Ca2+-ATPase activity was significantly increased by the treatment with phospholipase A2 in the presence of bovine serum albumin as a scavenger for hydrolyzed products. When the microsomal fraction was incubated with phospholipase A2 in the absence of bovine serum albumin, the Ca2+-ATPase activity was not altered. The Vmax and Km values for both ATP and Ca2+ were increased by the phospholipase A2 treatment, respectively. These results indicated that the activation of Ca2+-ATPase by the phospholipase A2 treatment is due to the increase of Vmax.     

Influence of Carbicron (O-[(2-butenoic acid)-N,N-dimethylamide-3-YL] O,O-dimethylphosphate) on some biochemical and biophysical parameters of rat liver membranes

• 1.1. Treatment of rats with carbicron induced a reduction of the phospholipids in both microsomal and plasma membranes.
• 2.2. A decrease of the structural order parameter (S_DPH) and an increase of the pyrene excimer-to-monomer fluorescence ratio (I_E/I_M) was also observed, indicating membrane fluidization.
• 3.3. The specific activity of membrane-bound phospholipase A₂ and phospholipase C were decreased in both types of membranes, whereas acyl-CoA:lysophosphatidylcholine acyltransferase activity was augmented due to carbicron treatment.

     

Pulmonary phosphatidic acid phosphatase. A comparative study of the aqueously dispersed phosphatidate-dependent and membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activities of rat lung.

1. The properties of the aqueously dispersed phosphatidate-dependent phosphatidic acid phosphatase (EC 3.1.3.4) activities of rat lung have been studied in microsomal and cytosol preparations and compared with the properties of the membrane-bound phosphatidate-dependent activities. 2. The microsomal phosphatidic acid phosphatase displayed a prominent pH optimum at 6.5 with a minor peak which varied between 7.5--8 in different experiments. With the cytosol, the major activity was at the higher pH (7.5--8.0) but a distinct optimum was also observed at pH 6.0--6.5. With the membrane-bound substrate, a single broad optimum was observed between pH 7.4 and 8.0 with the cytosol and 6.5--7.5 with the microsomal fraction. 3. Subcellular fractionation studies revealed that the microsomal fraction possessed the greatest proportion of the total phosphatidic acid phosphatase activity and the highest relative specific activity. However, studies with marker enzymes indicated that the aqueously dispersed phosphatidate-dependent activity could be present in plasma membrane, lysosomes and osmiophilic lamellar bodies as well as in the endoplasmic reticulum. 4. The aqueously dispersed phosphatidic acid-dependent activities present in the microsomal and supernatant fractions were inhibited by Ca2+, Mn2+, F- and by high concentrations of Mg2+. In contrast to the membrane-bound phosphatidate-dependent activities, there was little Mg2+ stimulation and only a very slight inhibitory effect was noted with EDTA. A small EDTA-dependent Mg2+ stimulation could be observed with the microsomal fraction but only at the lower pH optimum (6.5). 5. The presence of a number of phosphate esters tended to stimulate rather than inhibit the microsomal activity, indicating that the hydrolase is relatively specific for lipid substrates. Marked inhibitions were noted with lysophosphatidic acid and phosphatidylglycerol phosphate. Phosphatidylcholine produced a slight inhibition. 6. The results indicate that the bulk of the aqueously dispersed phosphatidate-dependent phosphatidic acid phosphatase activities of rat lung microsomes and cytosol is not related to the activities observed with membrane-bound phosphatidate. The Mg2+-dependent hydrolase activities may be synonymous. However, unequivocal conclusions will only be possible when the polypeptide or polypeptides responsible for these activities can be purified.     

The influence of exogenous and of membrane-bound phosphatidate concentration on the activity of CTP: phosphatidate cytidylyltransferase and phosphatidate phosphohydrolase.

Rat liver microsomes were treated with phospholipase D to obtain microsomal membranes with varying amounts of membrane-bound phosphatidate. This treatment did not impair the activity of two microsomal-bound enzymes acting with phosphatidate as substrate, i.e. CTP: phosphatidate cytidylyltransferase and phosphatidate phosphohydrolase. The dependency of the activity of these enzymes on the concentration of membrane-bound phosphatidate was determined. Both enzymes showed a linear increase in activity with membrane-bound phosphatidate concentrations up to at least 100 nmol phosphatidate/mg microsomal protein. These results indicate that both enzymes have a large reserve capacity and suggest that the enzymes are operating intracellularly, i.e. at phosphatidate concentrations of 5-10 nmol/mg endoplasmic reticulum protein, far below their maximal capacity. The ratio of phosphatidate conversion into CDP-diglyceride and 1,2-diglyceride seems to be constant for a large range of membrane-bound phosphatidate concentrations. The membrane-bound enzymes cannot utilize phosphatidate substrate present in heat-denatured membranes, but are active on phosphatidate incorporated into membranes of phospholipid vesicles.     

4-Hydroxy-2,3-trans-nonenal stimulates microsomal lipid peroxidation by reducing the glutathione-dependent protection

Glutathione (GSH) protects liver microsomes against lipid peroxidation. This is probably due to the reduction of vitamin E radicals by GSH, a reaction catalyzed by a membrane-bound protein. Pretreatment of liver microsomes with 0.1 or 1mM 4-hydroxy-2,3-trans-nonenal (HNE), a major product of lipid peroxidation, reduces the GSH-dependent protection. GSH and vitamin E concentrations are not affected by this pretreatment. Pretreatment with 0.1 mM N-ethyl maleimide (NEM), a synthetic sulfhydryl reagent, resulted in a reduction similar to that with HNE of the GSH-dependent protection against lipid peroxidation. The reduction of the GSH-dependent protection by HNE and NEM is probably the result of inactivation of the membrane-bound protein by covalent binding to an essential SH group on the protein. If the GSH-dependent protection would proceed via the microsomal GSH transferase, pretreatment with NEM, which activates the microsomal GSH transferase, should enhance the GSH-dependent protection. Actually a decrease in the GSH-dependent protection is found. Apparently the GSH-dependent protection does not proceed via the microsomal GSH transferase. Also the microsomal phospholipase A2 is not involved, since addition of 0.1 mM mepacrine, an inhibitor of phospholipase A2, did not preclude the GSH-dependent protection. Once the process of lipid peroxidation, either in vivo or in vitro, has started, the protection of liver microsomes by GSH is less effective. This might be the result of formed HNE. In this way an endproduct of lipid peroxidation stimulates the process that generates this product.     

Effect of liver plasma membrane fluidity on endogenous phospholipase A2 activity

Investigations have been carried out on the influence of the phospholipid composition and the physicochemical properties of rat liver plasma membranes on the endogenous activity of membrane-bound phospholipase A2. The membrane phospholipid composition was modified by the incorporation of different phospholipids in the lipid bilayer by the aid of lipid transfer proteins. The results indicate that the endogenous activity of phospholipase A2 in liver plasma membranes depends upon membrane fluidity and not upon the presence of a specific phospholipid in the enzyme's microenvironment.     

Characterization and distribution of a phospholipase A2 activity from adult rabbit lung

A phospholipase A2 activity was characterized in adult rabbit lung. This activity was calcium- and deoxycholate-dependent and displayed an alkaline pH optimum. Km and Vmax were 0.176 mM and 256.8 pmoles/min./mg protein respectively. The microsomal fraction displayed the highest enzymatic specific activity; the lowest activity was present in the cytosol. Yet this latter fraction accounted for the majority of the total activity. Although the specific activity was high within the lamellar body fraction this compartment contained only approximately 2% of the total activity. Phospholipase A2 activity was inhibited by bromophenacyl bromide, chlorpromazine and mepacrine in decreasing order of effectiveness. Treatment of the microsomes with increasing concentrations of NaC1 indicated that the lung phospholipase A2 activity was relatively loosely bound to the microsomal membranes and was maximally removed with salt at a concentration only slightly higher than physiological. Addition of calmodulin to the enzyme assay did not significantly alter hydrolysis of labelled phosphatidylcholine.     

Association of changes in lysophosphatidylcholine metabolism and in microsomal membrane lipid composition to the pulmonary injury induced by oleic acid

Alterations in the lipid composition of lung microsomal membranes occur in oleic acid-induced respiratory distress. The marked decrease in the phosphatidylcholine/lysophosphatidylcholine molar ratio could be related with an altered metabolism of lysophosphatidylcholine in these membranes. Results revealed that the activity of phospholipase A increased whereas that of acyl-CoA:lysophosphatidylcholine acyltransferase decreased. Microsomal lysophospholipase activity remained unchanged. On the other hand, the microsomal enzyme system involved in the de novo synthesis of diacylglycerol was impaired, and cholinephosphotransferase activity was lowered. These changes in the activity of some membrane-bound enzymes were not caused by changes in the membrane lipid fluidity since lipid structural order parameter (SDPH) did not change and neither did the major factors on which the fluidity depends. The possible significance of microsomal lipid alterations in the pathogenesis of respiratory distress induced by oleic acid is discussed.     

Membrane phospholipid composition, fluidity and phospholipase A2 activity of human hepatoma cell line HepG2

1. Investigations have been carried out on the phospholipid composition, physical state and phospholipase A2 activity of plasma and microsomal membranes from HepG2 cells. 2. The results showed a great similarity in the physico-chemical properties of plasma and microsomal membranes from HepG2 cells. 3. The activity of phospholipase A2 was found to depend on the membrane physical state in both types of membranes.     

Defining the role of protein kinase c in calcium-ionophore-(A23187)-mediated activation of phospholipase A2 in pulmonary endothelium.

We sought to investigate the mechanisms by which the calcium ionophore A23187 triggers arachidonic acid release in bovine pulmonary endothelial cells and to test the hypothesis that protein kinase C is involved in this process. Our results indicate that the mechanism by which A23187 increases phospholipase A2 activity and arachidonic acid release in bovine pulmonary arterial endothelial cells depends upon the concentration studied. At concentrations of 1 microM and 2.5 microM, A23187 increases phospholipase A2 activity and arachidonic acid release without stimulating protein kinase C. At concentrations of 5-12.5 microM, A23187 increases arachidonic acid release and phospholipase A2 activity in conjunction with a dose-dependent activation of membrane-bound protein kinase C. To test the hypothesis that these doses of A23187 increase phospholipase A2 activity by stimulating protein kinase C, we studied the effect of prior treatment with the protein kinase C inhibitor sphingosine. Sphingosine inhibits the increase in phospholipase A2 activity and arachidonic acid release caused by A23187 over the range 5-12.5 microM. To investigate further the potential role of protein kinase C, we studied the effects of the inactive phorbol ester 4 alpha-phorbol 12 beta-myristate 13 alpha-acetate (4 alpha-PMA) and an active phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (4 beta PMA). Neither 4 alpha-PMA nor 4 beta-PMA affected basal arachidonic acid release. 4 alpha-PMA also did not augment the effects of A23187. In contrast, 4 beta-PMA significantly augments the increase in phospholipase A2 activity and arachidonic acid release caused by lower doses of A23187. Under these conditions, sphingosine completely inhibits the stimulatory effects of 4 beta-PMA on protein kinase C translocation, phospholipase A2 and arachidonic acid release. Thus, at low doses (1 microM and 2.5 microM) A23187 increases phospholipase A2 activity and arachidonic acid release by a mechanism that does not involve protein kinase C. At these A23187 doses, activating membrane-bound protein kinase C with 4 beta-PMA causes a synergistic increase in phospholipase A2 activity and arachidonic acid release. At higher doses (5-12.5 microM), A23187 acts in large part by stimulating protein kinase C translocation. Overall, our results indicate that activating membrane-bound protein kinase C by itself is an insufficient stimulus to increase phospholipase A2 activity and arachidonic acid release in pulmonary endothelial cells, but activating protein kinase C can substantially augment the increase in phospholipase A2 activity and arachidonic acid caused by a small increase in intracellular calcium.     

Purification of 5'-nucleotidase from human placenta after release from plasma membranes by phosphatidylinositol-specific phospholipase C

5'-Nucleotidase was purified greater than 1000-fold from human placenta by treatment of plasma membranes with S. aureus phosphatidylinositol-specific phospholipase C and affinity chromatography on Con A Sepharose and AMP-Sepharose. The resulting enzyme had a specific activity of greater than 5000 mumol/hr/mg protein and a subunit molecular weight of 73,000. Goat antibodies against 5'-nucleotidase inhibited enzyme activity and detected 5'-nucleotidase after Western blotting. These antibodies also recognized a soluble form of 5'-nucleotidase and residual membrane-bound 5'-nucleotidase which could not be released by phosphatidylinositol-specific phospholipase C treatment, suggesting that the three forms of the enzyme are structurally related. The soluble 5'-nucleotidase may be derived from the membrane-bound form by the action of an endogenous phospholipase C. The structural basis for the inability of some of the membrane-bound 5'-nucleotidase to be released by phosphatidylinositol-specific phospholipase C is unknown.     

Increased phospholipase A2 activity in the kidney of spontaneously hypertensive rats

Phospholipase A2 activity was studied in the renal cortex and medulla of stroke-prone spontaneously hypertensive rat (SHRSP) and normotensive rat (WKY), and the subcellular localization of its activity was determined. Enhanced activity was found in both the cortical and medullary microsomes in SHRSP kidneys. In SHRSP, but not in WKY, phospholipase A2 activity progressively increased with age. This phospholipase A2 had substrate specificity toward phosphatidylethanolamine. There were no differences in optimal pH, substrate specificity, heat lability, and responses to Triton X-100 and deoxycholate between SHRSP and WKY. Ca2+ stimulated phospholipase A2 activity in both animals. The maximal activation was achieved at 5 mM Ca2+, and EDTA strongly inhibited the activity. But the response to Ca2+ was different in each. Ca2+ enhanced this activity in SHRSP markedly compared with WKY. It seems that Ca2+ is specifically required for phospholipase A2 activity in SHRSP. Though the influx of Ca2+ into microsomal membranes was not enhanced, the Ca2+ efflux of microsomal membranes decreased in SHRSP. This results in increases of intramicrosomal Ca2+, which may cause the subsequent activation of phospholipase A2. The Ca2+ permeability may be one of the factors in the increased phospholipase A2 activity in SHRSP.