Ectoenzyme release from rat liver and kidney by phosphatidylinositol-specific phospholipase C

Ectoenzyme release from rat liver and kidney by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis was studied. Alkaline phosphatase and 5'-nucleotidase were released from rat kidney slices to extents of up to 60% and 30%, respectively. Release of alkaline phosphatase was observed at lower amounts of PI-specific phospholipase C than that of 5'-nucleotidase. Both enzymes were more easily released from microsomal fractions or free cells. From kidney cells, alkaline phosphatase was released without cell lysis, and more than 80% release of alkaline phosphatase was observed at 3.8% hydrolysis of PI. Isoelectric focusing profiles of alkaline phosphatase released by PI-specific phospholipase C were significantly different from the control in the cases of both rat liver and kidney. Lubrol-solubilized alkaline phosphatase was eluted at the void volume of a Toyopearl HW-55 column, while the enzyme obtained by further treatment with PI-specific phospholipase C was eluted in the lower-molecular-weight region corresponding to 100,000-110,000 daltons. Furthermore, Lubrol-solubilized phosphatase became more thermostable on treatment with PI-specific phospholipase C.     

Release of alkaline phosphodiesterase I from rat kidney plasma membrane produced by the phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP.     

Alkaline phosphodiesterase I release from eucaryotic plasma membranes by phosphatidylinositol-specific phospholipase C. I. The release from rat organs

From various rat organs, alkaline phosphodiesterase I was liberated by the action of phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis. Especially, a large amount of alkaline phosphodiesterase I was released from slices of small intestine, testis, lung, and kidney, but not from pancreas and liver. The release of the enzyme induced by phospholipase C was dependent on, or proportional to, the reaction time and the concentrations of the phospholipase C and the weight of the slices of small intestine or testis. Furthermore, little enzyme was released from the homogenate of pancreas. These results suggest an important role of phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membranes of rat small intestine and pancreas. The alkaline phosphodiesterase I released from slices of rat small intestine and testis had a molecular weight of about 240,000, and was activated by Mg2+ and Ca2+ but inhibited by EDTA. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9, having the Km values of 0.36 mM (small intestine) and 0.25 mM (testis). The intestinal enzyme differed from the testis enzyme in pI values, thermostability, and Arrhenius plot having a single breakpoint.     

Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins.

The ability of the phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes to hydrolyze glycosyl phosphatidylinositol (GPI)-anchored membrane proteins was compared with the ability of the PI-PLC from Bacillus thuringiensis to hydrolyze such proteins. The L. monocytogenes enzyme produced no detectable release of acetylcholinesterase from bovine, sheep, and human erythrocytes. The cleavage of the GPI anchors of alkaline phosphatase from rat and rabbit kidney slices was less than 10% of the cleavage seen with the PI-PLC from B. thuringiensis. Activity for release of Fc gamma receptor IIIB (CD16) on human granulocytes was also low. Variations in pH and salt concentration had little effect on the release of GPI-anchored proteins. Our data show that L. monocytogenes PI-PLC has low activity on GPI-anchored proteins.     

Electrophoretic characterization of hepatic alkaline phosphatase released by phosphatidylinositol-specific phospholipase C. A comparison with liver membrane and serum-soluble forms.

Alkaline phosphatase was solubilized from plasma membrane of rat liver with butanol-ol, bile acids or sodium deoxycholate, and electrophoretically compared with a soluble form in serum which was derived from the liver. The three enzyme preparations from the plasma membrane migrated at the same position on polyacrylamide-gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulphate. The mobility of them, however, was distinctly different from that of the serum-soluble form of the liver-derived alkaline phosphatase. On the other hand, phosphatidylinositol-specific phospholipase C isolated from Bacillus cereus was used to release alkaline phosphatase from plasma membrane. The released alkaline phosphatase was demonstrated to have the same mobility as the serum-soluble form on polyacrylamide-gel electrophoresis in the presence or absence of detergents. The phospholipase C also converted the butan-1-ol-extracted membrane form into the serum-soluble form. The results suggest that release of alkaline phosphatase from the liver into serum is not simply caused by a detergent effect of bile salts, but involves an enzymic hydrolysis of phosphatidylinositol, with which alkaline phosphatase may strongly interact in the membrane.     

Hydrophobic interactions of brush border alkaline phosphatases: the role of phosphatidyl inositol

Tissue-specific (intestinal) and tissue-nonspecific (kidney) rat alkaline phosphatases are released from their respective brush border membranes by different enzymes. To elucidate the mechanism underlying their membrane attachment, we tested the ability of these enzymes to partition into lipid or aqueous phases both before and after treatment with phospholipases and proteases. Interaction with Triton X-114 micelles was eliminated or decreased by treatment of intestinal enzyme with phospholipase A2 or papain, while only phosphatidylinositol (PI)-specific phospholipase C (PIPLC) and subtilisin were effective with the kidney enzyme. Binding to octyl Sepharose for the intestinal enzyme was decreased by phospholipase A2 more than by PIPLC, whereas the reverse was true for the kidney enzyme. Treatment with phospholipases decreased the apparent mass of the phosphatases by 50-80 kDa, presumably due to loss of bound lipid and detergent. PIPLC treatment of the kidney, but not the intestinal enzyme, prevented binding of the phosphatase to phospholipid vesicles. These results show that both enzymes are bound to respective membranes by hydrophobic anchor peptides to which phospholipids are bound. However, their sensitivity to phospholipases is different. The data are consistent with the hypothesis that, in the kidney enzyme, the PI is bound covalently, while with the intestinal enzyme, binding of PI appears to be tight but not covalent.     

The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.     

Alkaline phosphatase in the lactating bovine mammary gland and the milk fat globule membrane. Release by phosphatidylinositol-specific phospholipase C.

1. Alkaline phosphatase is covalently bound to bovine mammary microsomal membranes and milk fat globule membranes through linkage to phosphatidylinositol as demonstrated by the release of alkaline phosphatase following treatment with phosphatidylinositol-specific phospholipase C. 2. The release of alkaline phosphatase from the pellet to the supernatant was demonstrated by enzyme assays and electrophoresis. 3. Electrophoresis of the solubilized enzymes showed that the alkaline phosphatase of the microsomal membranes contained several isozymes, while only one band with alkaline phosphatase activity was seen in the fat globule membrane. 4. Levamisole and homoarginine were potent inhibitors of the alkaline phosphatase activities in both membrane preparations and in bovine liver alkaline phosphatase, but not in calf intestinal alkaline phosphatase.     

The hydrolysis of phosphatidylinositol by lysosomal enzymes of rat liver and brain.

1. Lysosomes from rat liver contain two enzymic systems for hydrolysing phosphatidyl-inositol: a deacylation via lysophosphatidylinositol producing glycerophosphoinositol and non-esterified fatty acid, and a phospholipase C-like cleavage into inositol 1-phosphate and diaclygycerol. 2. The separate enzyme systems involved can be distinguished by gel filtration, differential temperature-stability and the inhibitory action of detergents. 3. The enzyme systems both have pH optima at 4.8 and their attack on a pure phosphatidylinositol substrate is inhibited by many bivalent metals including Ca2+ and Mg2+, and cationic drugs. 4. Whereas the deacylation system will attack other glycerophospholipids, the phospholipase C shows a marked specificity towards phosphatidylinositol, although it will also slowly attach phosphatidylcholine with the liberation of phosphocholine. 5. Gel filtration and temperature-stability distinguish the phospholipase C from lysosomal phosphatidic acid phosphatase, but not from sphingomyelinase. 6. Evidence is presented that an EDTA-insensitive phospholipase C degrading phosphatidylinositol is present in rat brain.     

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 x slower in liposomes and 100 x slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.     

Purification and partial characterization of alkaline phosphatase of matrix vesicles from fetal bovine epiphyseal cartilage. Purification by monoclonal antibody affinity chromatography

Alkaline phosphatase of matrix vesicles isolated from fetal bovine epiphyseal cartilage was purified to apparent homogeneity using monoclonal antibody affinity chromatography. The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Tween 20 whereas the major portion of nonspecific protein was removed by this single step. Of various agents tested, 0.6 M 2-amino-2-methyl-1-propanol, pH 10.2, was the most effective in eluting 80-100% of the enzyme initially applied. Both Tween 20 and 2-amino-2-methyl-1-propanol associated with the eluted enzyme were effectively removed by the sequential application of DEAE-cellulose and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 81,000. This molecular weight was nearer reported values for rat liver (Ohkubo, A., Langerman, N., and Kaplan, M. M. (1974) J. Biol Chem. 249, 7174-7180) and porcine kidney (Cathala, G., Brunel, C., Chapplet-Tordo, D., and Lazdunski, M. (1975) J. Biol. Chem. 250, 6040-6045) alkaline phosphatase, than to previously reported values for chicken (Cyboron, G. W., and Wuthier, R. E. (1981) J. Biol. Chem. 256, 7262-7268) and fetal calf (Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1980) Calcif. Tissue Int. 30, 217-225) cartilage matrix vesicle alkaline phosphatase. The purified alkaline phosphatase was activated by micromolar Mg2+. The amino acid composition of cartilage alkaline phosphatase was found to be similar to that previously described for porcine kidney (Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141, 273-282). Double immunoprecipitation data indicated that monoclonal antibody against cartilage alkaline phosphatase cross-reacted with fetal bovine liver or kidney enzyme but failed to react with calf intestinal or rat cartilage enzyme. Thus these observations suggest that alkaline phosphatase of matrix vesicles from calcifying epiphyseal cartilage is a liver-kidney-bone isozyme.     

The action of phosphatidylinositol-specific phospholipases C on membranes.

A phospholipase C prepared from lymphocytes readily hydrolysed pure phosphatidyl-inositol but was relatively ineffective against phosphatidylinositol in erythrocyte "ghosts" and rat liver microsomal fraction and also against sonicated lipid extracts from these membranes. In contrast, a phospholipase C prepared from Staphylcoccus aureus readily hydrolysed phosphatidylinositol in sonicated lipid extracts but had only low activity against purified phosphatidylinositol. Unlike the enzyme from lymphocytes, the S. aureus phospholipase C did not require Ca2+ for its activity and was inhibited by cations. The previously reported specificity of this enzyme was confirmed by our observation of hydrolysis of approx. 75% of the phosphatidylinositol in ox, sheep and cat erythrocyte "ghosts" together with no detectable effect on the major erythrocyte membrane phospholipids. The phosphatidylinositol of rat liver microsomal fraction was hydrolysed only to a maximum of 15%. Some preliminary experiments showed that approx. 60% of the phosphatidylinositol of ox or sheep erythrocytes could be hydrolysed without causing substantial haemolysis.     

Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding

Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.     

Axolotl pronephric duct cell migration is sensitive to phosphatidylinositol-specific phospholipase C

On the basis of its distribution pattern in embryos of the axolotl (Ambystoma mexicanum), we recently identified alkaline phosphatase as a molecule potentially involved in guiding the migration of the pronephric duct. Alkaline phosphatase is a cell surface protein anchored to cell membranes via a covalent linkage to a phosphatidylinositol glycan (PI-G). The enzyme phosphatidylinositol-specific phospholipase C (PIPLC) specifically releases from cell surfaces molecules anchored by the PI-G linkage. In order to test the possibility that a PI-G anchored protein is involved in directing pronephric duct cell migration, PIPLC was applied to axolotl embryos. The enzyme was introduced into embryos through the use of a novel slow-release bead material, hydrolysed polyacrylamide. PIPLC blocked pronephric duct cell migration without interfering with somite fissure formation, a concurrent, neighbouring morphogenetic cell rearrangement which occurs with little if any alkaline phosphatase present. In addition, alkaline phosphatase activity was markedly diminished in the vicinity of the implanted beads. These observations suggest that at least one protein anchored to the cell membrane by a PI-G linkage, possibly alkaline phosphatase, is involved in guiding or promoting pronephric duct cell migration.     

Membrane interactions of rat intestinal alkaline phosphatase: role of polar head groups

Lipid-protein interactions with purified membranous intestinal alkaline phosphatase have been studied by using rat intestine. The enzyme was incorporated equally well into neutral lecithin and anionic liposomes, including those made from phosphatidic acid alone. It could not be solubilized with chaotropic salts nor by phospholipases C and D from either native membranes or phospholipid vesicles. Detergents effected nearly complete release of enzyme from the vesicles. Phosphatase activity was lost upon treatment with phospholipase D alone. The activity was restored with free choline, or choline containing phospholipids, but not by the addition of other phospholipids or amines. The catalytic activity was also lower when the enzyme was bound to a phosphatidylcholine vesicle containing additional phosphatidic acid. Neither phosphatidylserine nor phosphatidylinositol addition altered enzyme activity. These results show that the enzyme binds to the membrane by a primary hydrophobic interaction with membrane phospholipids without requiring the polar head group and that the enzyme activity is affected via a secondary interaction with choline. We suggest that choline protects the active site of brush border alkaline phosphatase from inhibition by endogenous membrane phosphate groups.     

Conversion of human placental alkaline phosphatase from a high Mr form to a low Mr form during butanol extraction. An investigation of the role of endogenous phosphoinositide-specific phospholipases.

Alkaline phosphatase in a wide range of tissues has been shown to be anchored in the membrane by a specific interaction with the polar head group of phosphatidylinositol. It has previously been suggested that the production of low Mr alkaline phosphatase during the commonly used butanol extraction procedure may result from the activation of an endogenous phosphoinositide-specific phospholipase C which removes the 1,2-diacylglycerol responsible for membrane anchoring. This conversion process was investigated in greater detail with human placenta used as the source of alkaline phosphatase. Mr and hydrophobicity of the alkaline phosphatase were determined by gel filtration on TSK-250 and partitioning in Triton X-114, respectively. Alkaline phosphatase extracted from human placental particulate fraction with butanol at pH 5.4 or released by incubation with Staphylococcus aureus phosphatidylinositol-specific phospholipase C produced a form of alkaline phosphatase of Mr approx. 170,000 and relatively low hydrophobicity. By contrast, the butanol extract prepared at pH 8.3 was an aggregated form of Mr approx. 600,000 and was relatively hydrophobic. The effect of a variety of inhibitors and activators on the amount of low Mr alkaline phosphatase produced during butanol extraction revealed that it was a Ca2+- and thiol-dependent process. Proteinase inhibitors had no effect. [3H]Phosphatidylinositol hydrolysis by the particulate fraction, unlike low Mr alkaline phosphatase production, was relatively sensitive to heat inactivation, indicating that the phosphoinositide-specific phospholipases C from cytosol and lysosomes were unlikely to be responsible for conversion. A butanol-stimulated activity which removed the [3H]myristic acid from the variant surface glycoprotein ( [3H]mfVSG) of Trypanosoma brucei was detectable in the human placental particulate fraction. Since this activity was acid active, Ca2+- and thiol-dependent and relatively heat stable, it may be the same as that responsible for production of low Mr alkaline phosphatase. The only 3H-labelled product identified was phosphatidic acid, suggesting that the [3H]mfVSG-cleaving activity is a phospholipase D. These data strongly support the proposal that production of low Mr alkaline phosphatase during butanol extraction is an autolytic process occurring as the result of an endogenous phospholipase. However, they also suggest that the lysosomal and cytosolic phosphoinositide-specific phospholipases C that have previously been described in many mammalian tissues are not responsible for this process.     

The distribution of some hydrolases in glomeruli and tubular fragments prepared from rat kidney by zonal centrifugation

1. A collagenase digest of rat kidney cortex was separated into four bands by zonal centrifugation. 2. Two of these bands were shown by light-microscopy to contain glomeruli and tubular fragments, which were free from each other and well separated from other renal material. 3. Protein, N-acetyl-beta-glucosaminidase, 5'-nucleotidase, l-leucine beta-naphthylamidase, leucine aminopeptidase, acid phosphatase and alkaline phosphatase were assayed across the gradient. 4. The greater proportion of these enzyme activities was recovered in the tubular fragments and acid phosphatase was the only enzyme detected in significant amounts in the glomeruli. 5. Tubular fragments and glomeruli were sedimented and multiple forms of beta-naphthylamidase, N-acetyl-beta-glucosaminidase, acid phosphatase and alkaline phosphatase were investigated by starch-gel electrophoresis.     

The catabolism of phosphatidylinisitol by an EDTA-insensitive phospholipase A1 and calcium-dependent phosphatidylinositol phosphodiesterase in rat brain

1. A rat brain supernatant and microsomal fraction contained a phospholipase A1 enzyme which hydrolysed phosphatidylinositol at pH 8 in the absence of calcium. Triolein and phosphatidylcholine were not attacked under the same incubation conditions. 2. No evidence could be obtained for a phospholipase A2 in the microsomal preparation, and in the presence of Ca2+ the release of fatty acid observed was due to phosphatidylinositol phosphodiesterase followed by diacylglycerol lipase action. 3. Brain phosphatidylinositol phosphodiesterase showed extensive activity in the alkaline range (7-8.5) as well as at pH 5-5.5. The activity at higher pH values required higher calcium concentrations and disappeared on purification of the soluble enzyme by ammonium sulphate fractionation. 4. In general the ratio between inositol 1,2-(cyclic)phosphate and inositol 1-phosphate produced by phosphodiesterase action decreased with increasing pH.     

Molecular aspects of the regulation of rat kidney alkaline phosphatase

The mechanisms by which phosphate regulates the activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) in rat kidney were investigated. Measurements of incorporation of [(14)C]leucine into kidney alkaline phosphatase in rats fed on complete or phosphate-free diet provide evidence of a twofold increase in the rate of synthesis of the enzyme in diet-treated animals. Cycloheximide experiments indicated that control and diet-adapted enzyme decreases in activity according to first-order kinetics with a calculated half-life of 10.3 and 6.5h after complete and phosphate-free diet administration respectively. Basal and diet-adapted enzymes exhibit similar K(m) values for several phosphomonoesters and an identical degree of inhibition is produced by cysteine. In addition, the enzyme from both sources is the same with regard to heat inactivation at 45, 56 or 64 degrees C, to the profile of elution from Sephadex and to electrophoretic properties on polyacrylamide gel. A failure of rat kidney alkaline phosphatase to respond to cortisol (hydrocortisone) was also observed.     

A histochemical study about the influence of lytic enzymes on plasma membrane enzyme activities in rat liver and kidney.

1. The effect of lipolytic, glycolytic and proteolytic enzymes on the activities of plasma membrane enzyme activities in rat liver and kidney has been investigated by a pretreatment of tissue sections with the lytic enzymes. 2. The action of the proteolytic enzymes causes a very strong decrease of leucyl-beta-naphthylamidase activity, whereas the activities of ATP-ase, 5'-nucleotidase and alkaline phosphatase show a lesser decrease. This indicates a different membrane anchorage of leucyl-beta-naphthylamidase as compared to that of the phosphatases. 3. Treatment with glycolytic enzymes results in a decrease of 5'-nucleotidase and ATP-ase activity, whereas liver alkaline phosphatase and leucyl-beta-naphthylamidase show an increase in activity. 4. Treatment with phospholipase C gives about the same results. The very strong decrease of 5'-nucleotidase activity indicates a great dependence on phospholipids.