Effect of neomycin on phosphoinositide labelling and calcium binding in guinea-pig inner ear tissues in vivo and in vitro.

—Phospholipids of guinea-pig inner ear tissues were labelled in vivo by perilymphatic perfusion of the cochlea with [³²P]orthophosphate or myo-[³H]inositol. After 20-40 min the most highly labelled ³²P-lipids were phosphatidylinositol phosphate and diphosphate. Incorporation of [³H]inositol proceeded in the order phosphatidylinositol > phosphatidylinositol phosphate > phosphatidylinositol diphosphate. After treatment of animals with neomycin for 3 weeks, ³²P-incorporation into phosphatidylinositol diphosphate, but not into other lipids, was significantly decreased in the preparations of the organ of Corti and stria vascularis. In homogenates of inner ear tissues, the labelling of the polyphosphoinositides by [γ-³²P]ATP was increased and the hydrolysis of these lipids was blocked in the presence of 10^?4m -neomycin. Neomycin also competitively inhibited the binding of ⁴⁵Ca²⁺ to homogenates of these tissues.     

Inhibition by neomycin of polyphosphoinositide turnover in subcellular fractions of guinea-pig cerebral cortex in vitro

The addition of 10(-5) M to 10(-3) M neomycin to incubations of subcellular fractions of guineapig cerebral cortex increased the labelling of phosphatidylinositol phosphate and decreased the labelling of phosphatidylinositol diphosphate by [gamma-32P]ATP. The effect was observed in all subcellular fractions tested and depended on the cationic form of the antibiotic. Similar effects on lipid labelling were exerted by related aminoglycosidic antibiotics, by neamine, spermine and poly-L-lysine. Other neomycin fragments, antibiotics, local anesthetics or small polyamines were ineffective. Neomycin also inhibited the enzymatic hydrolysis of 32P-polyphosphoinositides. The addition of the drug to aqueous dispersions of these lipids increased the turbidity and lowered the pH of the suspensions. It is suggested that the effects of neomycin on polyphosphoinositide metabolism result from the formation of an ionic complex between the lipids and the antibiotic.     

Inositol Phospholipid Hydrolysis by Rat Sciatic Nerve Phospholipase C

Rat sciatic nerve cytosol contains a phosphodiesterase of the phospholipase C type that catalyzes the hydrolysis of inositol phospholipids, with preferences of phosphatidylinositol 4'-phosphate (PIP) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol 4',5'-bisphosphate (PIP2), at a pH optimum of 5.5-6.0 and at maximum rates of 55, 13, and 0.7 nmol/min/mg protein, respectively. Analysis of reaction products by TLC and formate exchange chromatography shows that inositol 1,2-cyclic phosphate (83%) and diacylglycerol are the major products of PI hydrolysis. [32P]-PIP hydrolysis yields inositol bisphosphate, inositol phosphate, and inorganic phosphate, indicating the presence of phosphodiesterase, phosphomonoesterase, and/or inositol phosphate phosphatase activities in nerve cytosol. Phosphodiesterase activity is Ca2+-dependent and completely inhibited by EGTA, but phosphomonoesterase activity is independent of divalent cations or chelating agents. Phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) inhibit PI hydrolysis. They stimulate PIP and PIP2 hydrolysis up to equimolar concentrations, but are inhibitory at higher concentrations. Both diacylglycerols and free fatty acids stimulate PI hydrolysis and counteract its inhibition by PC and lysoPC. PIP2 is a poor substrate for the cytosolic phospholipase C and strongly inhibits hydrolysis of PI. However, it enhances PIP hydrolysis up to an equimolar concentration.     

Purification and properties of a phosphohydrolase from Enterobacter aerogenes.

A phosphohydrolase from Enterobacter aerogenes which hydrolyzes phosphate mono- and diesters has been purified approximately 50-fold to apparent homoeneity and crystallized. The enzyme is produced when the bacteria utilize phosphate diesters as sole phosphorus source. From sedimentation equilibrium experiments the molecular weight of the native enzyme is 173,000; from sodium dodecyl sulfate polyacrylamide gel electrophoresis the subunit molecular weight is 29,000, indicating that the enzyme is hexameric. The hydrolytic activity of the enzyme using both mono- and diesters is maximal at pH 5; THE Km of the enzyme for bis-p-nitrophenyl phosphate is constant from pH 5 to 8.5 whereas that for p-nitrophenyl phosphate increases about 40-fold as the pH increases over the same range. The phosphodiesterase activity is not inhibited by chelating agents but is inhibited by several divalent metal ions. 31-P NMR spectroscopy was used to identify the hydrolysis products of glycoside cyclic phosphates. The enzyme-catalyzed hydrolysis of methyl beta-D-ribofuranoside cyclic 3:5-phosphate yields exclusively the 5-phosphate whereas that of adenosine 3:5-monophosphate yields a 4:1 mixture of 3- and 5- AMP.     

P-nitrophenyl phosphate as substrate for rabbit liver fructose diphosphatase

Purified rabbit liver fructose diphosphatase has been found to catalyze the hydrolysis of p-nitrophenyl phosphate, PNPP. It has been established that the hydrolysis of p-nitrophenyl phosphate is due to fructose diphosphatase through studies of the chromatographic properties of the enzyme, its temperature sensitivity, dependence on divalent cations and its inhibition by fructose diphosphate. The K_m for PNPP is 6 × 10⁻³M at pH 9.2, 5 × 10⁻⁴M at pH 7.5. This substrate should facilitate studies of the kinetics and mechanism of action of fructose diphosphatase and the comparison of this enzyme with other alkaline phosphatases.     

A calcium-activated polyphosphoinositide phosphodiesterase in the plasma membrane of human and rabbit erythrocytes.

Haemoglobin-free human erythrocyte ghosts that were prepared in the presence of EDTA and were then exposed to Ca2+ showed a substantial loss of phosphatidylinositol phosphate and phosphatidylinositol diphosphate, measured either chemically or by loss of 32P from the lipids of prelabelled membranes. At the same time there was, as reported previously (Allan, D. and Michell, R.H., (1976) Biochim. Biophys. Acta 455, 824--830), and approximately equivalent rise in the diacylglycerol content of the membranes. Analysis of the 32P-labelled water-soluble material released during this process showed that the major products were inositol diphosphate and inositol triphosphate. No change was seen in the phosphatidylinositol or phosphatidate content of the membranes, and there was no Ca2+-activated loss of 32P from the phosphatidate of prelabelled membranes: this suggests that Ca2+ did not activate phosphoinositide phosphomonoesterases or phosphatidate phosphomonoesterase in human erythrocyte membranes. It is concluded that human erythrocyte membranes contain at their cytoplasmic surface a Ca2+-activated phosphodiesterase that is active against both phosphatidylinositol phosphate and phosphatidylinositol diphosphate. Rabbit erythrocytes also contained this enzyme, but in these cells there was also evidence for the presence of a Ca2+-activated phosphatidate phosphomonoesterase.     

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.     

Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain

Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract of rat brain. The purification procedures consisted of chromatography of both enzymes on Affi-Gel blue and cellulose phosphate, followed by three sequential high performance liquid chromatography steps, which were different for the two enzymes. The resultant preparations each contained homogeneous enzyme with a Mr of 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these enzymes (PLC-II) was found to hydrolyze phosphatidyl-inositol 4,5-bisphosphate (PIP2) at a rate of 15.3 mumol/min/mg of protein and also phosphatidylinositol 4-monophosphate and phosphatidylinositol (PI) at slower rates. For hydrolysis of PI, this enzyme was activated by an acidic pH and a high concentration of Ca2+ and showed a Vmax value of 19.2 mumol/min/mg of protein. The other enzyme (PLC-III) catalyzed hydrolysis of PIP2 preferentially at a Vmax rate of 12.9 mumol/min/mg of protein and catalyzed that of phosphatidylinositol 4-monophosphate slightly. The rate of PIP2 hydrolysis by this enzyme exceeded that of PI under all conditions tested. Neither of these enzymes had any activity on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid. These two enzymes showed not only biochemical but also structural differences. Western blotting showed that antibodies directed against PLC-II did not react with PLC-III. Furthermore, the two enzymes gave different peptide maps after digestion with alpha-chymotrypsin or Staphylococcus aureus V8 protease. These results suggest that these two forms of PLC belong to different families of PLC.     

Synthesis of ATP and free radicals in an aqueous solution, containing NADH, riboflavin, ADP and inorganic phosphate

pH-dependence of initial (admixture) adenosine triphosphate (ATP) changes (ATP synthesis and hydrolysis) was studied for aerated and deaerated aqueous solutions during the incubation of adenosine diphosphate (ADP) and inorganic phosphate (Pi) in the presence of reduced nicotinamide adenine dinucleotide (NADH) and riboflavin (Rf). The preferential pH regions were indicated both for ATP synthesis and ATP hydrolysis (pH less than pH 5.4, 5.9 divided by pH 6.5 and pH greater than 6.6, pH 5.5 divided by pH 5.8 respectively). 'Free radical content measurements were paralleled by ESR technique. On the basis of the obtained results it was assumed that a part of ESP signal attributed to ADP radicals was increased during ATP synthesis.     

Hydrolysis and isomerization of trans,trans-farnesyl diphosphate by Andrographis tissue-culture enzymes.

Incubation of (3R,5S)-[5-3H1]mevalonate + (3RS)-[2-14C]mevalonate with Andrographis cell-free extract leads to trans,trans-farnesol and cis,trans-farnesol which both totally retain tritium. 2. This conflicts with our previous results which predict one third tritium loss in the cis,trans-farnesol. Inversion at C-1 during hydrolysis of trans,trans-farnesyl diphosphate to trans,trans-farnesol could explain this anomaly. 3. (1s)-trans,trans-[1-3H1]Farnesyl diphosphate and phosphate and (1R)-trans,trans-[1-3H1]-farnesyl diphosphate and phosphate, all prepared chemically, were hydrolysed with Andrographis phosphatase, and alkaline phosphatase and hydrogenolysed with lithium aluminium hydride and the product alcohols exchanged with liver alcohol hydrogenase. 4. Both Andrographis phosphatase and alkaline phosphatase hydrolyse trans,trans-farnesyl diphosphate and trans,trans-farnesyl phosphate with retention. 5. Hydrolysis of trans,trans-[1-18O]farnesyl diphosphate in H2(18O with both phosphatases supports P-O fission. 6. The C-1 configuration in (1S)-TRANS,TRANS-[1-3H1]farnesyl diphosphate and phosphate and (1R)-trans,trans-[1-3H1]farnesyl diphosphate and phosphate is progressively racemised in 0.01 M NH4OH/MeOH (1/9) AT - 20 degrees C.     

A calcium-activated polyphosphoinositide phosphodiesterase in the plasma membrane of human and rabbit erythrocytes

Haemoglobin-free human erythrocyte ghosts that were prepared in the presence of EDTA and were then exposed to Ca²⁺ showed a substantial loss of phosphatidylinositol phosphate and phosphatidylinositol diphosphate, measured either chemically or by loss of ³²P from the lipids of prelabelled membranes. At the same time there was, as reported previously (Allan, D. and Michell, R.H., (1976) Biochim. Biophys. Acta 455, 824–830), an approximately equivalent rise in the diacylglycerol content of the membranes. Analysis of the ³²P-labelled water-soluble material released during this process showed that the major products were inositol diphosphate and inositol triphosphate. No change was seen in the phosphatidylinositol or phosphatidate content of the membranes, and there was no Ca²⁺-activated loss of ³²P from the phosphatidate of prelabelled membranes: this suggests that Ca²⁺ did not activate phosphoinositide phosphomonoesterases or phosphatidate phosphomonoesterase in human erythrocyte membranes. It is concluded that human erythrocyte membranes contain at their cytoplasmic surface a Ca²⁺-activated phosphodiesterase that is active against both phosphatidylinositol phosphate and phosphatidylinositol diphosphate. Rabbit erythrocytes also contained this enzyme, but in these cells there was also evidence for the presence of a Ca²⁺-activated phosphatidate phosphomonoesterase.     

Purification, Properties, and Phosphorylation by Protein Kinase C of Two Phosphoinositidase C Isozymes from Rat Brain

Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl-Sepharose, and Mono Q. Gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC-beta-1 and PLC-gamma of bovine brain. With both enzymes phosphatidylinositol 4,5-bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2-cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC-beta-1 and PLC-gamma on Mono Q. Purified rat brain protein kinase C phosphorylated PLC-gamma but not PLC-beta-1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphorylation of another protein in the preparations.     

d-myoInositol 1:2-cyclic phosphate 2-phosphohydrolase

1. An enzyme in extracts of mammalian tissues catalyses the hydrolysis of d-myoinositol 1:2-cyclic phosphate (an intermediary in the enzymic degradation of phosphatidylinositol) to produce d-myoinositol 1-phosphate. 2. The enantiomorph of the substrate is not attacked. 3. The pH optimum is about 8.1-8.3 and the reaction is stimulated by Mg(2+) ions. 4. Extracts from rat kidney cortex and medulla are very rich sources of the enzyme; brain, testis and small intestine contain intermediary activities, and other tissues contain very small amounts.     

Characterization of phospholipase C-mediated phosphatidylinositol degradation in rat heart ventricle

Phosphoinositide-specific phospholipase C (PI-PLC) activity was investigated in the rat heart ventricle. Incubation of ventricle homogenate or 100,000g supernatant fraction with [3H]myoinositol or [3H]arachidonate-labeled phosphatidylinositol in the presence of Ca2+ resulted in a decrease in phosphatidylinositol with a concomitant increase in water-soluble [3H]inositol phosphate or [3H]diglyceride, respectively. Total overt homogenate PI-PLC activity could be accounted for in the supernatant fraction. Neutral, zwitterionic, cationic, or anionic detergents did not unmask membrane-associated activity. While cytosolic phospholipase C was active against a pure phosphatidylinositol substrate in the presence of Ca2+, no hydrolytic activity was detected when phosphatidylinositol was presented as a component (4-5%) of a mixture of phospholipids. However, addition of deoxycholate to the incubation mixture (pH 6.5, Ca2+ 10(-3) M) containing mixed phospholipids resulted in the exclusive hydrolysis of inositol phospholipids. Ventricular supernatant phospholipase C-mediated phosphatidylinositol degradation has a sharp pH optimum at 5.5 and a specific requirement for Ca2+. Activity is maximal at 1 to 2 X 10(-3) M Ca2+, with inhibition occurring at higher levels. Under optimized conditions phosphatidylinositol is hydrolyzed at a rate of 20-25 nmol/min/mg protein. Multivalent cations inhibit Ca2+-dependent PI-PLC activity while monovalent cations and anions have no effect. There is no apparent selectivity for specific fatty acid moieties on phosphatidylinositol. Soluble PI-PLC is inhibited by sulfhydryl reagents, neomycin, mepacrine, trifluoperazine, and propranolol. Chlorpromazine, dibucaine, and tetracaine exert a biphasic influence, stimulating at lower and inhibiting at higher concentrations.     

Thrombin-induced phosphodiesteratic cleavage of phosphatidylinositol bisphosphate in human platelets

The addition of thrombin to human platelets prelabeled with 32Pi led to significant loss of radioactivity in phosphatidylinositol 4,5-bisphosphate within 5 s, followed by recovery or even increase by 2 min. Loss of label from phosphatidylinositol phosphate was much less marked. Stimulated loss of label from phosphatidylinositol was not seen, while labeled phosphatidate increased severalfold. The principal labeled water-soluble phosphates observed, in addition to 32Pi and [32P] ATP, co-migrated with inositol diphosphate and inositol triphosphate. This suggests that a pool of polyphosphoinositides is constantly undergoing phosphodiesteratic cleavage and resynthesis. Thrombin addition led to rapid increase in radioactivity in inositol triphosphate, but not in inositol diphosphate. We conclude that this early consequence of the thrombin-platelet interaction is the result of an increase in the phosphodiesteratic cleavage of phosphatidylinositol bisphosphate.     

Fructose 1,6-diphosphate aldolase from rabbit muscle. Effect of pH on the rate of formation and on the equilibrium concentration of the carbanion intermediate.

The rate of oxidation of ferricyanide of the aldolase-dihydroxyacetone phosphate complex was measured under different conditions. The following conclusions are drawn. 1. In the cleavage of fructose diphosphate, catalysed by native aldolase, the steady-state concentration of the enzyme-dihydroxyacetone phosphate carbanion intermediate represents less than 6% of the total enzyme-substrate intermediates. 2. Fructose diphosphate and dihydroxyacetone phosphate compete for the four catalytic sites on aldolase, the binding of fructose diphosphate being about twice as tight. 3. The equilibrium concentration of the carbanion intermediate formed by reaction of carboxypeptidase-treated aldolase with dihydroxyacetone phosphate is independent of pH between 5.0 and 9.0. The rates of fromation of the carbanion intermediate and of the reverse reaction are, however, concomitantly increased by increasing pH between 5.0 and 6.5.     

The metal ion catalyzed decomposition of nucleoside diphosphate sugars.

The metal ion catalysed decomposition of the nucleotide diphosphate sugars, uridine diphosphate glucose, uriding diphosphate galactose, uridine diphosphate N-acetylglucosamine, guanosine diphosphate mannose, and guanosine diphosphate fucose (UDPGlc, UDPGal, UDPGlc-NAc, GDPMan, and GDPFuc, respectively), has been studies as a function of pH. UDPDlc and UDPGal decompose readily to the a,2-cycle phosphate derivative of the sugar and uridine 5'-phosphoric acid (UMP) in the presence of Mn2+. Under all conditions tested, UDPGal decomposes two to three times more rapidly than does UDPGlc. GDPFuc is slowly degraded to free fucose under similar conditions; the other nucleotide diphosphate sugars are stable. The rate of reaction increases with increasing hydroxide ion concentration from pH 6.5 to 7.9 and with metal ion concentration from 10 to 200 mm. Several metal ions are effective catalysts; at pH 7.5 WITH 20 mM UDPGal and 20 mM metal ion, the following apparent first-order rate constants (min-1 x 10(4)) were obtained: Eu3+ 700; Mn2+, 70; Co2+ 27; Zn2+, 22; Ca2+, 3.0; Cu2+, 2.4; and Mg2+, 0. It appears that Mn2+ concentrations that have been used in studies with nucleotide diphosphate sugars at neutral pH can catalyze significant decomposition leading to erroneous interpretation of kinetic and incorporation experiments.     

Cytosolic and particulate phosphatidylinositol phospholipase C activities in pollen tubes of Lilium longiflorum

Pollen tubes of Lilium longiflorum Thunb. cv. White Europe contain three distinguishable phosphatidylinositol phospholipase C activities (EC 3.1.4.10). Two of these are particulate and have optima at pH 5.2 and 7.0, respectively. The third one, a cytosolic activity, has an optimum at pH 6.0. The distribution of radioactivity in reaction products from phosphatidylinositol, labeled in either the inositol, glycerol or phosphate moiety, indicates that the three phospholipase activities cleave only the bond between glycerol and phosphate. The dependence on divalent cations slightly differs, though Ca²⁺ is the most stimulatory ion species for all the three enzyme activities. Activity is not observed in the presence of EDTA. When anionic phospholipids are mixed with phosphatidylinositol substrate an increase in phosphatidylinositol phospholipase C activities is observed, except for the particulate activity with an optimum at pH 5.2. Phosphatidylcholine and phosphatidylethanolamine are inhibitory.     

Hydrolysis of the tumor-inhibiting ruthenium(III) complexes HIm trans-[RuCl4(im)2] and HInd trans-[RuCl4(ind)2] investigated by means of HPCE and HPLC-MS

High performance capillary electrophoresis (HPCE) as well as high performance liquid chromatography-mass spectrometry (HPLC-MS) have been applied to the separation, identification and quantification of the tumor-inhibiting ruthenium compounds HIm trans-[RuCl4(im)2] (im = imidazole) and HInd trans-[RuCl4(ind)2] (ind = indazole) and their hydrolysis products. The half-lives for the hydrolytic decomposition of the Ru(III) compounds were determined by monitoring the relative decrease of the original complex anion under different conditions by means of capillary electrophoresis. The decomposition follows pseudo-first-order kinetics. The rate constants in water at 25 degrees C are 1.102 +/- 0.091 x 10(-5) s-1 for HIm trans-[RuCl4(im)2] and 0.395 +/- 0.014 x 10(-5) s-1 for HInd trans-[RuCl4(ind)2]. About 8% of HIm trans-[RuCl4(im)2] but only about 2% of HInd trans-[RuCl4(ind)2] were hydrolyzed after 1 h at room temperature. Whereas the hydrolysis rate of the imidazole complex is independent of the pH value, the indazole complex hydrolyzes much faster at higher pH. The half-life of HInd trans-[RuCl4(ind)2] in phosphate buffer at pH 6.0 and 37 degrees C is 5.4 h, whereas it is less than 0.5 h at pH 7.4. In contrast to the imidazole complex, where no dependence on the buffer system was observed, hydrolysis of the indazole complex is even faster if a buffer containing hydrogen carbonate is used. The formation of [RuCl2(H2O)2(im)2]+ could be demonstrated by HPLC-MS measurements. In the case of the indazole complex, a release of the indazole ligands results in the formation of [RuCl4(H2O)2]-.     

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.