Characterization of phosphoinositide-specific phospholipase C from human platelets.

Phosphoinositide-specific phospholipase C (PI-PLC) from human platelet cytosol was purified 190-fold to a specific activity of 0.68 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein. It hydrolyses PI and phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. The enzyme exhibits an acid pH optimum of 5.5 and has a molecular mass of 98 kDa as determined by Sephacryl S-200 gel filtration. It required millimolar concentrations of Ca2+ for PI hydrolysis, whereas micromolar concentrations are optimal for PIP2 hydrolysis. Mg2+ could substitute for Ca2+ when PIP2, but not PI, was used as the substrate. EDTA was more effective than EGTA in inhibiting the basal PI-PLC activity towards PIP2. Sodium deoxycholate strongly inhibits the purified PI-PLC activity with either PI or PIP2 as substrate. Ras proteins, either alone or in the form of liposomes, have no effect on PI-PLC activity.     

Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate

A soluble phospholipase C from rat liver was purified to homogeneity using phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. After ammonium sulfate fractionation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose CL-6B, hydroxylapatite, Reactive Blue 2 dye-linked agarose, and Mono S cation exchanger. Under the conditions of the assay, the pure enzyme had a specific activity of 407 mumol/mg protein/min. It migrated as a single band with a molecular mass of 87 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The water-soluble product formed during the hydrolysis of PIP2 by the purified enzyme was inositol 1,4,5-trisphosphate. The enzyme shows one-half of maximum velocity at 2 microM Ca2+ with PIP2 as substrate. Between 0 and 100 microM Ca2+, the enzyme shows approximately the same activity with phosphatidylinositol 4-phosphate (PIP) as it does with PIP2, and very low activity with phosphatidylinositol. The enzyme is activated by low concentrations of basic proteins; for example, with PIP2 as substrate, 1 microgram/ml histone activates the enzyme 3.6-fold. The enzyme shows an almost absolute requirement for monovalent salts which can be met by different alkali metal halides. A second, minor peak of PIP2-hydrolyzing phospholipase C activity was resolved during chromatography of the enzyme on hydroxylapatite. The substrate specificity suggests that PIP and PIP2 are normal substrates of this enzyme. Under physiological conditions of activation, the enzyme may therefore generate inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in amounts determined by the ratio of PIP and PIP2 present in the cellular membranes.     

Ca2+ regulation of phosphatidylinositol turnover in the plasma membrane of tobacco suspension culture cells.

The biochemical properties of the enzymes involved in phosphatidylinositol (PI) turnover in higher plants were investigated using the plasma membrane isolated from tobacco suspension culture cells by aqueous two-phase partitioning. Submicromolar concentrations of Ca2+ inhibited PI kinase and phosphatidylinositol 4-phosphate (PIP) kinase and stimulated phospholipase C. Diacylglycerol (DG) kinase was inhibited by Ca2+, but required a higher concentration than the physiological level. From the above results we postulate the following scheme: signal coupled activation of phospholipase C produces IP3 which induces Ca2+ release from the intracellular Ca2+ compartment, the increased cytoplasmic Ca2+ in turn activates phospholipase C and causes a further increase of the cytoplasmic Ca2+ level. This inhibits PI kinase and PIP kinase and brings about a limited supply of PIP2, the substrate of phospholipase C. Consequently, IP3 production decreases and Ca2+ mobilization ceases. Then cytosolic Ca2+ returns to the stationary level by the Ca2+ pump at the plasma membrane and at the endoplasmic reticulum and Ca2+/H+ antiporter at the plasma membrane and at the tonoplast.     

Beta-lactamase inhibitors. The inhibition of serine beta-lactamases by specific boronic acids.

Two kinds of phosphoinositide-specific phospholipase C (PLC) were purified from rat liver by acid precipitation and several steps of column chromatography. About 50% of the activity could be precipitated when the pH of the liver homogenate was lowered to pH 4.7. The redissolved precipitate yielded two peaks, PLC I and PLC II, in an Affi-gel Blue column, and each was further purified to homogeneity by three sequential h.p.l.c. steps, which were different for the two enzymes. The purified PLC I and PLC II had estimated Mr values of 140,000 and 71,000 respectively on SDS/polyacrylamide-gel electrophoresis. Both enzymes hydrolysed phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in a Ca2+- and pH-dependent manner. PLC I was most active at 10 microM- and 0.1 mM-Ca2+ for hydrolysis of PI and PIP2 respectively, whereas PLC II showed the highest activity at 5 mM- and 10 microM-Ca2+ for that of PI and PIP2 respectively. The optimal pH of the two enzymes also differed with substrates or Ca2+ concentration, in the range pH 5.0-6.0. Hydrolysis of phosphoinositides by these enzymes was completely inhibited by Hg2+ and was affected by other bivalent cations. From data obtained by peptide mapping and partial amino acid sequencing, it was clarified that PLC I and PLC II had distinct structures. Moreover, partial amino acid sequences of three proteolytic fragments of PLC I completely coincided with those of PLC-148 [Stahl, Ferenz, Kelleher, Kriz & Knopf (1988) Nature (London) 332, 269-272].     

Identification of Ca2+-stimulated polyphosphoinositide phospholipase C in isolated plant plasma membranes

A polyphosphoinositide phospholipase C has been identified in highly purified plasma membranes from shoots and roots of wheat seedlings. The enzyme preferentially hydrolysed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate and had a different phosphoinositide substrate profile from soluble phospholipase C. The enzyme activity was lower in plasma membranes isolated from light-grown shoots than from dark-grown ones, whereas no differences in activity between plasma membranes from light- and dark-grown roots were seen. Maximum activity of the membrane-bound enzyme was observed around pH 6. It was activated by micromolar concentrations of Ca2+, but not by GTP or GTP analogues. The enzyme may participate in signal transduction over the plant plasma membrane.     

Resolution of the phosphoinositide-specific phospholipase C isolated from porcine lymphocytes into multiple species. Partial purification of two isoenzymes.

Phospholipase C isolated from porcine mesenteric lymph node lymphocytes was distributed between the soluble and particulate fractions. Enzyme activity was found predominantly in the soluble fraction with optimal activity at pH 5.5. Gel filtration chromatography of the soluble phospholipase C revealed that it was composed of multiple species of enzyme activity. The activity associated with the particulate fraction had optimal activity at pH 7.0, as also did one of the species of soluble phospholipase C. Cellulose phosphate chromatography resolved the major soluble form into two species designated PLC-A and PLC-B. Both phenyl-Sepharose chromatography and hydroxyapatite chromatography purified these species still further. PLC-A and PLC-B demonstrated similar activities against phosphatidylinositol with a pH optimum near 5.5. The phospholipase C activities were abolished against this substrate by the addition of 1 mM-EDTA. When assayed in the presence of Ca2+-EDTA buffers providing a range of Ca2+ free concentrations, both enzymes exhibited optimal activity near 10(-3) M free Ca2+, but PLC-B was inhibited above this concentration more than PLC-A. PLC-B exhibited markedly lower activity against phosphatidylinositol 4,5-bisphosphate, suspended as liposomes of the pure phospholipid, than did PLC-A.     

Purification and characterization of bovine heart phosphoinositide-specific phospholipase C: kinetic analysis of the Ca2+ requirement and La3+ inhibition

Bovine heart contains multiple phosphoinositide-specific phospholipase C (PIC) activities separable by ion-exchange chromatography. One PIC activity was purified to apparent homogeneity and migrated as a single band of Mr 85,000 on SDS-PAGE. The purified PIC was characterized with sonicated suspensions of either pure phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol (PI) as substrates. At pH 7, apparent Vmax and Km values were higher for PIP2 than for PI, but the value of Vmax/Km was similar for the two substrates. PIC required Ca2+ for the hydrolysis of either PI or PIP2, and increasing free Ca2+ concentrations from 20 to 300 nM saturated PIC activity. The requirement of Ca2+ for PIC activity and the sensitivity of PIC to Ca2+ concentrations in the physiological range suggested the ion may be a cofactor. The PIC reaction mechanism was determined by two-substrate kinetic analysis; the data fit a model in which PIC contained single sites for Ca2+ and phosphoinositide, and utilized a rapid-equilibrium, random-order ternary mechanism for phosphoinositide hydrolysis. The KCa value for either PI or PIP2 hydrolysis was approximately 30 nM, suggesting resting intracellular free Ca2+ concentrations are sufficient to saturate the Ca2+ site of PIC. La3+ was used as a calcium analogue to modulate PIC activity. Low concentrations of LaCl3 (0.01-0.3 microM) inhibited PIC activity competitively with respect to calcium, consistent with a Ca2+ binding site on the enzyme.     

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.     

Studies on PIP2-phosphomonoesterase activity in human platelets

An occurrence of phosphatidylinositol 4,5-bisphosphate (PIP2) phosphomonoesterase in human platelets was demonstrated by analyzing phosphoinositides metabolism. The activity of the enzyme was maximum at pH 7.0. It was active even in the absence of Ca2+ or Mg2+ but it was enhanced in the presence of Mg2+ or NaF. The activity was inhibited by pyrophosphate. The activity was not altered in the presence of Ca2+. Thereby, besides phosphodiesteric cleavage by phospholipase C, the amount of PIP2 in activated platelets may be reduced by the combined effect of PIP2-phosphomonoesterase and suppressed activity of PI-kinase by Ca2+.     

A 57-kDa phosphatidylinositol-specific phospholipase C from bovine brain.

A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.     

Existence of phosphoinositide-specific phospholipase C in rat liver nuclei and its change during liver regeneration.

We found phosphoinositide-specific phospholipase C (PtdIns-PLC) activity in nuclei isolated from rat liver. The enzyme hydrolyzed phosphatidylinositol, phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate in a Ca(2+)-dependent manner, and produced inositol mono-, bis-, and triphosphate, respectively. Neither phosphatidylcholine, phosphatidylethanolamine, nor phosphatidylserine was utilized as a substrate. After partial hepatectomy, the PtdIns-PLC activity in isolated nuclei increased transiently in the S phase (20-22 h post-hepatectomy), to 2.5-fold higher than in the control, when measured with PIP. This result suggests a close relationship between the nuclear PtdIns-PLC, especially its PIP-hydrolyzing activity, and cell proliferation.     

A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. I. Purification and properties

Eighty-three percent of polyphosphoinositide-specific phospholipase C activity was recovered in a cytosolic fraction after nitrogen cavitation of turkey erythrocytes. This activity has been purified approximately 50,000-fold when compared to the starting cytosol with a yield of 1.7-5.0%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phospholipase C preparation revealed a major polypeptide of 150 kDa. The specific activity of the purified enzyme was 6.7-14.0 mumol/min/mg of protein with phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 4-phosphate as substrate. Phospholipase C activity was markedly dependent on the presence of Ca2+. The phospholipase C showed an acidic pH optimum (pH 4.0). At neutral pH, noncyclic inositol phosphates were the major products formed by the phospholipase C, while at pH 4.0, substantial formation of inositol 1:2-cyclic phosphate derivatives occurred. Properties of the purified 150-kDa turkey erythrocyte phospholipase C were compared with the approximately 150-kDa phospholipase C-beta and -gamma isoenzymes previously purified from bovine brain (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1987) J. Biol. Chem. 262, 12511-12518). The turkey erythrocyte phospholipase C differed from the two mammalian phospholipases with respect to the effect of sodium cholate on the rate of polyphosphoinositide hydrolysis observed. Moreover, when presented with dispersions of pure inositol lipids, phospholipases C-beta and -gamma displayed comparable maximal rates of polyphosphoinositide and phosphatidylinositol hydrolysis. By contrast, the turkey erythrocyte phospholipase C displays a marked preference for polyphosphoinositide substrates.     

Characterization of partially purified phospholipase C from human platelet membranes.

A phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-hydrolytic activity was found to be present in the human platelet membrane fraction, with 20% of the total activity of the homogenate. The membrane-associated phospholipase C activity was extracted with 1% deoxycholate (DOC). The DOC-extractable phospholipase C was partially purified approx. 126-fold to a specific activity of 0.58 mumol of PtdIns-(4,5)P2 cleaved/min per mg of protein, by Q-Sepharose, heparin-Sepharose and Ultrogel AcA-44 column chromatographies. This purified DOC-extractable phospholipase C had an Mr of approx. 110,000, as determined by Ultrogel AcA-44 gel filtration. The enzyme exhibits a maximal hydrolysis for PtdIns-(4,5)P2 at pH 6.5 in the presence of 0.1% DOC. The addition of 0.1% DOC caused a marked activation of both PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) hydrolyses by the enzyme. The enzyme hydrolysed PtdIns(4,5)P2 and PtdIns in a different Ca2+-dependent manner; the maximal hydrolyses for PtdIns(4,5)P2 and PtdIns were obtained at 4 microM- and 0.5 mM-Ca2+ respectively. In the presence of 1 mM-Mg2+, PtdIns(4,5)P2-hydrolytic activity was decreased at all Ca2+ concentrations examined, but PtdIns-hydrolytic activity was not affected.     

Phosphatidyl inositol 4,5-bisphosphate-specific phospholipase C in bovine rod outer segment membranes.

Polyphosphoinositide-specific phosphodiesterase (phospholipase C) activity against phosphatidylinositol 4,5-bisphosphate has been examined in disrupted bovine retinal rod outer segments. The enzyme was strictly modulated by free calcium ion concentration and maximally activated at 10(-5) M Ca2+ (91 +/- 4 nmoles phosphatidylinositol 4,5-bisphosphate hydrolyzed/min/mg of protein). Guanine nucleotides did not affect in vitro phospholipase C activity either in the presence or absence of light, carbachol or epinephrine. The pH optimum at 10(-5) M Ca2+ in the presence of sodium deoxycholate was 6.5. The enzyme of bovine rod outer segments was concluded to be indirectly regulated by the phototransduction events.     

Partial purification and properties of a phosphatidylinositol 4,5-bisphosphate hydrolyzing phospholipase C from the soluble fraction of soybean sprouts

Three soluble enzyme fractions (F-I, F-II, and F-III) that hydrolyze phophoinositides were separated from soybean sprouts by using Matrex green gel column chromatography. Among the three phosphatidylinositol (PI)-specific phopholipsase C (PLC) enzymes, only the third fraction (F-III) was able to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) as substrates. The F-I and F-II fractions only showed enzymatic activities for PI and PIP. The PIP2-hydrolyzing PLC protein, F-III, was partially purified using the chromatographic steps of the Matrex green gel, phenyl Toyopearl, Matrex orange gel, Mono S cation exchange, and superose 6 gel filtration columns. The molecular weight of the F-III protein was estimated to be about 64 kDa on SDS-PAGE. The protein showed immunocross-reactivity with a polyclonal antibody that was prepared against the X and Y motifs of animal PLC enzymes, the conserved catalytic domains. Ca2+ ion critically affected the PIP2-hydrolyzing PLC activity of the F-III protein, representing maximal activity at 10 microM Ca2+ concentration. The PIP2-hydrolyzing PLC activity of the protein was also significantly increased by sodium deoxycholate (SDC) from 0.05 to 0.08%. However, the activity was greatly reduced above the concentration, and no activity was detected at 0.3% SDC. In addition, the protein exhibited maximal PIP2-hydrolyzing PLC activity at pH, in the range of 6.5-7.5.     

A novel phospholipase A2 associated with nuclear matrix: stimulation of the activity and modulation of the Ca2+ dependency by polyphosphoinositides

Neutral phospholipase A2 activity, which hydrolyzed phosphatidylcholine and phosphatidylethanolamine with the same efficiency, was identified in the nuclear matrix prepared from purified nuclei of rat ascites hepatoma cells (AH 7974). The enzyme activity was optimal at pH 7.0 and required Ca2+ absolutely. Concentrations of Ca2+ for a maximal and a half-maximal activation were 1.10(-2) and 1.10(-3) M, respectively, and little activity was detected at Ca2+ concentrations lower than 1.10(-5) M. Addition of acidic phospholipids markedly stimulated the enzyme activity, and further, lowered the minimum Ca2+ concentration required for activation. In particular, the polyphosphoionositides phosphatidylinositol 4-monophosphate and 4,5-diphosphate were most effective. These two polyphosphoinositides lowered the Ca2+ concentration required for half-maximal activation to 10(-5) M and dramatically stimulated the activity at that Ca2+ concentration (greater than 30-fold). The neutral phospholipase A2 activity such as characterized in the present study was very low in the other subcellular fractions including mitochondria, microsome, plasma membrane and cytosol.     

Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein

Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (EC 2.7.1.67), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112), alkaline phosphatase (EC 3.1.3.1), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.     

Interaction of protein kinase C isozymes with phosphatidylinositol 4,5-bisphosphate.

Interaction of protein kinase C (PKC) isozymes with phosphatidylinositol 4,5-bisphosphate (PIP2) was investigated by monitoring the changes in the intrinsic fluorescence of the enzyme, the kinase activity, and phorbol ester binding. Incubation of PKC I, II, and III with PIP2 resulted in different rates of quenching of PKC fluorescence and different degrees of inactivation of these enzymes. Other inositol-containing phospholipids such as phosphatidylinositol and phosphatidylinositol 4-phosphate also caused differential rates of quenching of the intrinsic fluorescence of these enzymes. These latter two phospholipids were, however, less potent in the inactivation of PKCs than PIP2. The IC50 of PIP2 were 2, 4, and 11 microM for PKC I, II, and III, respectively. Inactivation of PKCs by PIP2 cannot be reversed by extensive dilution of PIP2 with Nonidet P-40 nor by digestion of PIP2 with phospholipase C. Interaction of PIP2 with the various PKC isozymes was greatly facilitated in the presence of Mg2+ or Ca2+ as evidenced by the accelerated quenching of the PKC fluorescence, however, these divalent metal ions protected PKC from the PIP2-induced inactivation. Binding of PIP2 to PKC in the absence of divalent metal ion also caused a reduction of [3H]phorbol 12,13-dibutyrate binding as a result of reducing the affinity of the enzyme for phorbol ester. Based on gel filtration chromatography, it was estimated that one molecule of PKC interacted with one PIP2 micelle with an aggregation number of 80-90. The PIP2-bound PKC could further interact with phosphatidylserine in the presence of Ca2+ to form a larger complex. Binding of PKC to both PIP2 and phosphatidylserine in the presence of Ca2+ was also evident by changes in the intrinsic fluorescence of PKC. As the interaction of PKC with PIP2, but not with phosphatidylserine, could be enhanced by millimolar concentrations of Mg2+, we propose that PIP2 may be a component of the membrane anchor for PKC under basal physiological conditions when [Ca2+]i is low and Mg2+ is plentiful. Under the in vitro assay conditions, PIP2 could stimulate PKC activity to a level approximately 10-20% of that by diacylglycerol. The stimulatory effect of PIP2 on PKC apparently is not due to binding to the same site recognized by diacylglycerol or phorbol ester, because PIP2 cannot effectively compete with phorbol 12,13-dibutyrate in the binding assay.     

Partial purification and characterization of membrane-bound and cytosolic phosphatidylinositol-specific phospholipases C from murine thymocytes

Membrane-bound and cytosolic phosphatidylinositol (PI)-specific phospholipases C in murine thymocytes have been partially purified and characterized. The membrane-bound enzyme was extracted from microsomes with sodium cholate and purified by sequential column chromatographies on Sephadex G-100, heparin-Sepharose CL-6B, and Sephadex G-100. The cytosolic enzyme was purified from the cytosol by sequential column chromatographies on Sephadex G-100 and FPLC-Mono S. Specific activities of the membrane-bound enzyme and the cytosolic enzyme increased more than 1,800- and 1,400-fold, respectively, compared with those of microsomes and the cytosol. The molecular weights of the both enzymes were estimated to be about 70,000 by gel filtration. These purified enzymes also hydrolyzed phosphatidylinositol 4,5-bisphosphate (PIP2). At neutral pH and low Ca2+ concentrations, the membrane-bound enzyme hydrolyzed PIP2 in preference to PI and showed higher activity than the cytosolic enzyme. These activities were also affected differently by various lipids. For PIP2 hydrolysis, all lipids investigated except lysophosphatidylcholine enhanced the activity of the membrane-bound enzyme, while phosphatidylcholine (PC) and phosphatidylserine (PS) did not significantly affect the activity of the cytosolic enzyme. PC, PE, and PS inhibited the activities of the membrane-bound and cytosolic enzymes for PI hydrolysis. The physiological implications of these results are discussed.     

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) : Orientation of Active Site and Activation by Ca and Mg

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP₂ phospholipase C was dependent on Ca²⁺ with maximum activity at 10 to 100 μm free Ca²⁺ and half-maximal activation at 0.1 to 1 μm free Ca²⁺. In the presence of 10 μm Ca²⁺, 1 to 2 mm MgCl₂ or MgSO₄ further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) inhibited the enzyme activity. The stimulatory effect by Mg²⁺ was observed also when 35 mm NaCl was included. Thus, the PIP and PIP₂ phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP₂ as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca²⁺, with maximum activity at 1 mm CaCl₂, and could not be further stimulated by Mg²⁺.