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.     

Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1

The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of alkaline phosphatase from human osteosarcoma cells by phosphatidylinositol phospholipase C: effect of tunicamycin

Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase [alkaline optimum], EC 3.1.3.1) expressed in two human osteosarcoma cell lines (Saos-2 and KTOO5) in culture was the tissue nonspecific type and was released from the plasma membrane by phosphatidylinositol (PI) phospholipase C. Despite a difference of 10-fold between the two cell lines in the amount of alkaline phosphatase expressed, the phospholipase solubilized nearly all of the phosphatase from resuspended cells of the two lines. Alkaline phosphatase released with Nonidet-P40 from Saos-2 cells had a Mr of 445,000 by gradient gel electrophoresis in the absence of detergent; that released by PI-phospholipase C was 200,000. The subunit Mr of both solubilized forms was 86,000. Thus, tetrameric alkaline phosphatase in the membrane is attached by a PI-glycan moiety and is converted to dimers when released by PI-phospholipase C. Tunicamycin treatment of Saos-2 cells in culture affected the release of alkaline phosphatase by a high concentration of PI-phospholipase C, but not by a low concentration; both the rate and extent of release were lower from treated cells. However, the enzyme released from the treated cells was in two forms with different molecular weights; it seems that both glycosylated and nonglycosylated dimers were transported to the cell surface and incorporated into the plasma membrane. Glycosylation does not appear to be necessary for alkaline phosphatase to be anchored in the membrane via PI.     

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.     

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.     

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.     

Differential release of proteins from bovine fat globule membrane

Alkaline phosphatase and 5'-nucleotidase are covalently linked to phosphatidylinositol in bovine fat globule membrane, as demonstrated by their release following treatment with phospholipase C specific for phosphatidylinositol. The failure of this treatment to liberate phosphodiesterase I may indicate that it has a variant linkage resistant to release. In a test of exposure at the membrane surface, alkaline phosphatase and phosphodiesterase I, but not 5'-nucleotidase, were released from fat globule membrane by treatment with proteinase K. These apparent differences in accessibilities of membrane surface proteins suggest that attachment to phosphatidylinositol does not necessarily impart greater exposure to proteins with which it is linked.     

Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity.

A phosphatidylinositol phosphodiesterase from the culture broth of Bacillus cereus, was purified to a homogeneous state as indicated by polyacrylamide gel electrophoresis, by ammonium sulfate precipitation and chromatography with DEAE-cellulose and CM-Sephadex. The enzyme (molecular weight: 29000 +/- 1000) was maximally active at pH 7.2-7.5, AND NOT INFLUENCED BY EDTA, ophenanthroline, monoiodoacetate, p-chloromercuribenzoate or reduced glutathione. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, under the conditions examined. The products from phosphatidylinositol of enzyme reaction were diacylglycerols and a mixture of myoinositol 1- and 1, 2-cyclic phosphates, suggesting that the enzyme was a phosphatidylinositol-specific phospholipase C. The enzyme released alkaline phosphatase quantitatively from rat kidney slices. A kinetic analysis was made on the release of alkaline phosphatase. The results suggest that phosphatidylinositol-specific phospholipase C can specifically act on plasma membrane of rat kidney slices.     

Decay-accelerating factor (DAF) shares a common carbohydrate determinant with the variant surface glycoprotein (VSG) of the African Trypanosoma brucei

Decay-accelerating factor (DAF) is an integral membrane protein that inhibits amplification of the complement cascade on the cell surface. We and other investigators have shown that DAF is part of a newly characterized family of proteins that are anchored to the cell membrane by phosphatidylinositol (PI). The group includes the variant surface glycoprotein (VSG) of African trypanosomes, the p63 protein of Leishmania, acetylcholinesterase (AChE), alkaline phosphatase, Thy-1, 5'-nucleotidase, and RT6.2--an alloantigen from rat T cells. The structure of the membrane anchor has been best characterized for VSG, but chemical studies of the membrane anchors of AChE and Thy-1 suggest that similar glycolipid moieties anchor these proteins to the cell surface. In the VSG, the membrane anchor consists of an ethanolamine linked covalently to an oligosaccharide and glucosamine; the entire complex is anchored to the cell membrane by PI. Immunologically, this glycolipid defines an epitope, the cross-reacting determinant (CRD), that is only revealed after removal of the diacyl glycerol anchor by a phospholipase C. By Western blotting, we show here that DAF-S (DAF released from the membrane by PI-specific phospholipase C [PIPLC]) also contains CRD. Using a newly developed immunoradiometric assay (IRMA) in which the solid-phase capturing antibody is a monoclonal antibody to DAF and the second antibody is anti-CRD, we have been able to quantitate DAF-S. By IRMA, we show that the reaction between anti-CRD and DAF-S is specific, since the binding is competitively inhibited only by the soluble form of the VSG. These observations further support the concept that the glycolipid anchors of this new family of proteins have similar structures. DAF is also found as a soluble protein in various tissue fluids as well as in Hela cell supernatants. No evidence for the presence of the CRD epitope was found on these proteins, suggesting that these forms of DAF are not released from the surface of cells by endogenous phospholipases.     

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.     

Selective release of plasma-membrane enzymes from rat hepatocytes by a phosphatidylinositol-specific phospholipase C.

When isolated hepatocytes are incubated with phosphatidylinositol-specific phospholipase C, three cell-surface enzymes show markedly different behaviour. Most of the alkaline phosphatase is released at very low values of phosphatidylinositol hydrolysis, whereas further phosphatidylinositol hydrolysis releases only a maximum of about one-third of the 5'-nucleotidase. Alkaline phosphodiesterase I is not released. If cells containing phosphatidyl[3H]inositol are similarly treated, then the released [3H]inositol is in the form of inositol phosphate: no evidence has been obtained for any covalent association between released [3H]inositol and alkaline phosphatase.     

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.     

5'-Nucleotidase in rat liver lysosomal membranes is anchored via glycosyl-phosphatidylinositol

We have previously demonstrated that 5'-nucleotidase, known as a plasma membrane enzyme, is also distributed both in rat liver tritosomal membranes and contents (J. Biochem. 101, 1077-1085, 1987). When the lysosomal membranes isolated from rat livers were incubated with phosphatidylinositol-specific phospholipase C purified from B. thuringiensis, about 70% of 5'-nucleotidase activity was released from the membranes. Judging from the result by phase separation with Triton X-114, the enzyme solubilized by the phospholipase C digestion showed a hydrophilic nature such as that of the tritosomal contents. Immunoblot analysis showed that the molecular weight of 5'-nucleotidase released from the lysosomal membranes by the phospholipase C digestion was almost identical with that of the enzymes from the Tritosomal contents. The above results showed that the phosphatidylinositol-specific phospholipase C-like enzyme in the lysosomes may be responsible for the conversion of the lysosomal membrane-bound 5'-nucleotidase to the soluble form present in the lysosomal matrix.     

ACTH stimulates the release of alkaline phosphatase through Gi-mediated activation of a phospholipase C and the release of inositolphosphoglycan

We have previously reported that ACTH activates a phospholipase C that hydrolyzes glycosylphosphatidylinositol (GPI), which would release inositolphosphoglycan (IPG) to the extracellular medium, and that an IPG purified from Trypanosoma cruzi is able to inhibit ACTH-mediated steroid production in adrenocortical cells. In the present paper, it was found that anti-inositolphosphoglycan antibodies (anti-CRD) increased ACTH-mediated corticosterone production, which indicates that an endogenous IPG is a physiological inhibitor of ACTH response. On the other hand, we investigated the release to the extracellular medium of the GPI-anchored enzyme, alkaline phosphatase, by ACTH. We found that: (a) the released enzyme appeared in the aqueous phase after Triton X-114 partitioning, consistent with loss of the GPI, (b) the phospholipase C inhibitor, U73122, impaired the release of the enzyme by the hormone and (c) two inhibitors of IPG uptake, inositol 2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium. These results suggest that ACTH releases alkaline phosphatase by activation of a phospholipase C. Dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) was able to increase the release of alkaline phosphatase from adrenocortical cells and this effect was inhibited by U73122, suggesting that cAMP is involved in the activation of phospholipase C. In addition, it was found that a pertussis-toxin sensitive G-protein is required for ACTH- and db-cAMP-mediated release of alkaline phosphatase and that incorporation of anti-Gi antibodies in adrenocortical cells inhibited the release of alkaline phosphatase by ACTH. Our results suggest that ACTH increases the release of alkaline phosphatase by activation of a phospholipase C through cAMP and Gi which would contribute to produce IPG It was also found that the two inhibitors of IPG uptake, inositol-2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium of ACTH-treated cells more than in control cells, indicating that ACTH also stimulates the uptake of IPG These data support a role of GPI and the involvement of Gi in ACTH action.     

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.     

Differential Expression of Phospholipase C Specific for Inositol Phospholipids at the Cell Surface of Rat Glial Cells and REF52 Rat Embryo Fibroblasts

Abstract: Phosphatidylinositol(PI)-specific phospholipase C activity was detected on the surface of rat astrocytes, rat C6 glioma cells, and rat embryo (REF52) fibroblasts. The cell surface phospholipase C (ecto-PLC) activity was calcium-dependent, did not result from secreted phopholipase C, and was not released from the cell surface by bacterial PI-specific phospholipase C. Agents known to stimulate intracellular PI turnover, including carbachol, L-glutamic acid, acetylcholine, and orthovanadate, did not induce measurable alterations in the activity of the ecto-PLC. The expression of ecto-PLC activity by REF52 fibroblasts was density-dependent: subconfluent cultures of REF52 exhibited low levels of activity (less than 80 pmol of inositol phosphate formed/min/10⁶ cells), whereas in confluent cultures ecto-PLC activity increased to approximately 300 pmol/min/10⁶ cells. In contrast to this behavior and that exhibited by previously reported ecto-PLC-positive cell types, the ecto-PLC activity exhibited by astrocytes (approximately 1,000 pmol/min/10⁶ cells) and by C6 glioma cells (approximately 100 pmol/min/10⁶ cells) was independent of cell culture density up to confluence. The constitutive expression of ecto-PLC activity of astroglial cells may be related to their function as accessory cells in close association with neurons.     

Differences in the release of 5'-nucleotidase and alkaline phosphatase from plasma membrane of several cell types by PI-PLC

1. We have compared the effect of phosphatidyl inositol specific phospholipase C (PI-PLC) on the attachment of both 5'-nucleotidase and alkaline phosphatase to the liver plasma membrane from different species. 2. Our results demonstrate differences in the susceptibilities of both enzymes to PI-PLC treatment in relation to their origin. 3. These results were confirmed by immunoblotting using polyclonal anti-5'-nucleotidase antibodies. 4. In addition, in a single animal, susceptibility of both enzymes to PI-PLC treatment is different from one tissue to another. 5. The different percentages of released enzymes could be explained either by a polymorphism in the anchoring of these proteins at the cell surface membrane, or by a different steric hindrance or environment at the cleavage site itself.     

Action of phosphatidylinositol specific phospholipase C on platelets: nonlytic release of acetylcholinesterase, effect on thrombin and PAF induced aggregation

Phosphatidylinositol (PI) specific phospholipase C treatment of rabbit platelets caused 95% release of acetylcholinesterase in the supernatant and 4 to 6% hydrolysis of membrane PI in 2 min. Under these conditions there was no cell lysis as monitored by lack of lactate dehydrogenase activity in the medium. The phospholipase C had no activity towards phosphatidylinositol-4- phosphate and phosphatidylinositol-4,5-bis phosphate. Platelets pretreated with the phospholipase C responded normally to thrombin and platelet activating factor. It is concluded that acetylcholinesterase exists in specific interaction with PI in platelet membranes. Further, the membrane protein release phenomenon caused by the PI-specific phospholipase C did not effect the physiological responsiveness of platelets. Possible implications of these findings to the linkage between PI and membrane enzyme are also discussed.