Affinity-Purified Anti-B-50 Protein Antibody: Interference with the Function of the Phosphoprotein B-50 in Synaptic Plasma Membranes

Abstract: Affinity-purified anti-B-50 protein antibodies were used to study the previously proposed relationship of the phosphorylation state of B-50 protein and polyphosphoinositide metabolism in synaptic plasma membranes. Antibodies were raised against a membrane extract enriched in the B-50 protein and its adrenocorticotropin-sensitive protein kinase, obtained from rat brain. Anti-B-50 protein immunoglobulins were purified by affinity chromatography on a solid immunosorbent prepared from B-50 protein isolated by an improved procedure. The purified antibodies reacted only with the B-50 and B-60 protein, a proteolysis derivative (of B-50), as assessed by the sodium dodecyl sulfate-gel immunoperoxidase method. These antibodies inhibited specifically the endogenous phosphorylation of B-50 protein in synaptic plasma membranes, without affecting notably the phosphorylation of other membrane proteins. This inhibition was accompanied by changes of the formation of phosphatidylinositol 4,5-diphosphate and phosphatidic acid in synaptic plasma membranes, whereas formation of phosphatidylinositol 4-phosphate was not altered. Inhibition by ACTH _1–24 of the endogenous phosphorylation of B-50 protein in membranes was associated only with an enhancement of the phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-diphosphate. These data support our hypothesis on the functional interaction of B-50 protein and phosphatidylinositol 4-phosphate kinase in rat brain membranes. The evidence shows that purified anti-B-50 protein antibodies can be used to probe specifically the function of B-50 protein in membranes.     

Subcellular localization and enzymatic properties of rat liver phosphatidylinositol-4-phosphate kinase

The phosphatidylinositol-4-phosphate kinase activity in rat liver showed a subcellular distribution different from that of phosphatidylinositol kinase. It was preferentially associated with plasma membrane-rich subcellular fractions, while no or minimal activity could be ascribed to mitochondria, lysosomes, Golgi membranes or the endoplasmic reticulum. The plasma membrane enzyme phosphorylated endogenous and exogenously added phosphatidylinositol 4-phosphate at comparable initial rates. The phosphorylation of endogenous substrate was strongly inhibited by Triton X-100, while the phosphorylation of added substrate was enhanced, suggesting that endogenous phosphatidylinositol 4-phosphate was readily available to the enzyme in unperturbed plasma membranes. The total activity of phosphatidylinositol-4-phosphate kinase in rat liver was only 1/20 that of phosphatidylinositol kinase. The enzyme activity showed an unusually broad pH-optimum in the neutral range. Mg2+ was the preferred divalent cation and Km towards ATP was about 3-fold higher than the corresponding value for phosphatidylinositol kinase.     

Dioctanoylglycerol and phorbol diesters enhance phosphorylation of phosphoprotein B-50 in native synaptic plasma membranes

The short chain diacylglycerol, 1,2-dioctanoylglycerol, at concentrations of 100-300 microM stimulated phosphorylation of the nervous system-specific membrane protein B-50 (Mr 48 kDa, IEP 4.5) in isolated synaptic plasma membranes both in the presence and absence of exogenous protein kinase C. Comparable enhancement of histone phosphorylation by purified protein kinase C was achieved with 1 microM neutral lipid. Phorbol dibutyrate was 100 times more potent than the diacylglycerol in stimulating endogenous B-50 kinase in the membranes, whereas 4-alpha-phorbol was without effect. These results further confirm that B-50 is phosphorylated physiologically by a C kinase. Our data are consistent with a negative feedback mechanism in which generation of 1,2-diacylglycerol by enhanced phosphatidylinositol-4,5-bisphosphate hydrolysis could stimulate B-50 phosphorylation, thereby diminishing phosphatidylinositol-4-phosphate kinase activity and decreasing phosphatidylinositol-4,5-bisphosphate biosynthesis.     

ACTH-SENSITIVE PROTEIN KINASE FROM RAT BRAIN MEMBRANES

—The protein kinase which in rat brain synaptosomal plasma membranes is responsible for the phosphorylation of a protein band B-50 (MW 48, 000) was inhibited by the behaviorally active peptide ACTH_1–24 and not stimulated by cAMP. Treatment with 0.5% Triton X-100 in 75 mM-KCl solubilized 15% of the total B-50 protein kinase activity and preserved the sensitivity of the enzyme to ACTH_1–24. The rate of endogenous phosphorylation of protein band B-50 was different in intact SPM, solubilized fraction and residue. cAMP stimulated the endogenous phosphorylation of the solubilized fraction in a rather general manner. The solubilized membrane material also phosphorylated B-50 proteins which were previously extracted from membranes. Column chromatography of the solubilized material over DEAE-cellulose pointed to the presence of multiple protein kinase activities from rat brain synaptosomal plasma membranes, one of which was the ACTH-sensitive B-50 protein kinase.     

A synaptosomal protein kinase is regulated by phosphatidylinositol 4-phosphate and Mg2+

Triton X-100 extracts of purified rat brain synaptosomes exhibited marked phosphorylation of an endogenous Mr 87,000 polypeptide following chromatography on DEAE-cellulose. The protein kinase catalyzing this reaction was insensitive to cyclic AMP, Ca2+, calmodulin, and phorbol esters. However, phosphatidylinositol 4-phosphate (PIP) proved to be a potent inhibitor of the Mr 87,000 polypeptide phosphorylation at submicromolar concentrations, whereas phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol were less potent inhibitors. Unsaturated fatty acids could also mimic the effects of PIP at levels above 4 micrograms/ml. The inhibitory effect of PIP largely reflected a profound increase in the apparent Km for Mg2+ such that increasing Mg2+ levels could partially offset the action of PIP. The PIP-sensitive protein kinase was enriched in hypotonic lysates of synaptosomes from which it was partially purified by DEAE-cellulose, hydroxylapatite, and gel permeation chromatography. This purification separated the enzyme from its Mr 87,000 substrate; however, the presence of this polypeptide in heat-inactivated alkali extracts of rat brain provided an exogenous source of substrate which could be used to assay enzyme activity. The relevance of these data to a possible role for PIP and Mg2+ in cellular signaling is discussed.     

Purification of a Phosphatidylinositol 4-Phosphate Kinase from Bovine Brain Membranes

A phosphatidylinositol 4-phosphate (PIP) kinase (EC 2.7.1.68) was purified from bovine brain membranes in a six-step procedure involving solubilization of the enzyme with 170 mM NaCl followed by chromatography on diethylaminoethyl-cellulose, phosphocellulose, Ultrogel AcA44, hydroxylapatite, and ATP-agarose. The enzyme preparation was nearly homogeneous and was purified 5,600-fold with a final specific activity of 85 nmol/min/mg of protein and a yield of 20%. Its molecular mass was 110 kilodaltons, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was specific for PIP; phosphorylation of phosphatidylinositol and diacylglycerol was not observed.     

Immunochemical characterization of phosphatidylinositol 4-phosphate kinase from rat brain.

Affinity-purified antibodies were used to identify a protein of molecular mass 45 kDa (45 kDa protein) in rat brain cytosol as phosphatidylinositol 4-phosphate (PtdIns4P) kinase. Antibodies were raised in rabbits by immunization with the purified 45 kDa protein. Anti-(45 kDa protein) immunoglobulins were isolated by affinity chromatography of the antiserum on a solid immunosorbent, which was prepared by coupling a soluble rat brain fraction, the DEAE-cellulose pool containing 10-15% 45 kDa protein, to CNBr-activated Sepharose 4B. The purified IgGs were specific for the 45 kDa protein as judged by immunoblot and by immunoprecipitation. The purified anti-(45 kDa protein) IgGs inhibited the enzyme activity of partially purified PtdIns4P kinase, whereas preimmune IgGs were ineffective. Immunoprecipitation of the 45 kDa protein from the partially purified enzyme preparation with the purified IgGs resulted in a concomitant decrease in the amount of 45 kDa protein and in PtdIns4P kinase activity. The amount of 45 kDa protein remaining in the supernatant and the activity of PtdIns4P kinase correlated with a coefficient of r = 0.87. The evidence presented lends further support for the notion that the catalytic activity of PtdIns4P kinase in rat brain cytosol resides in a 45 kDa protein.     

Brain protein phosphorylation in vitro: selective substrate action of insulin

Insulin action on [32P]-phosphate incorporation into brain membranes was determined. Hippocampal homogenate tissue was phosphorylated with [32P]-ATP, and insulin was introduced at various times before or after ATP addition. With 50 microM Mg++ in the medium, insulin selectively stimulated the phosphorylation of a 47kD phosphoprotein, Protein F1. This effect required the prior presence of ATP. No effect of insulin on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material, was observed under these conditions. At 1 mM Mg++, insulin selectively decreased the phosphorylation of the alpha-subunit of pyruvate dehydrogenase. Insulin had no effect on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material under these conditions. The present study suggests a role for insulin in the modulation of brain protein phosphorylation. Since Protein F1 is phosphorylated by exogenous C kinase, and is likely the CNS-specific B-50 protein, these data also indicate a brain-specific function for insulin, possibly by action on a Ca++/phospholipid protein kinase.     

Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour.

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

Catalytic properties of a purified phosphatidylinositol-4-phosphate kinase from rat brain.

A phosphatidylinositol-4-phosphate (PIP) kinase activity was purified from rat brain extract through several chromatographic steps to yield an active preparation (specific activity 1 mumol of 32P incorporated into phosphatidylinositol 4,5-bisphosphate/min per mg of protein) with an apparent molecular size of 100-110 kDa in the native form. The isolated PIP kinase required Mg2+ (optimally 20-30 mM) for its activity and was not influenced by Ca2+. The enzyme used ATP (Km 25 microM) and GTP (Km 133 microM) as phosphate sources and appeared specific for PIP (Km 3.3 micrograms/ml) as the lipid substrate. The PIP-phosphorylation reaction was inhibited by micromolar concentrations of heparin [ID50 (concn. giving 50% inhibition) 2 micrograms/ml] and the flavonoid quercetin (ID50 0.2 microM). Whereas heparin behaves as a competitive inhibitor to PIP, quercetin was competitive towards ATP (or GTP). Phosphorylation of the preparation by a highly active purified protein kinase C did not detectably alter PIP kinase activity. Whereas 12-O-tetradecanoylphorbol acetate and various phospholipids had no effect, phosphatidylserine elicited a dose-dependent activation of PIP activity. This suggests that a phosphatidylserine-PIP kinase interaction may be considered as a possible regulatory process at the cell-membrane level.     

Polylysine and polyamine stimulation of the phosphatidylinositol kinases of amphibian oocyte membranes

Phosphatidylinositol kinase present in Xenopus laevis oocyte membranes catalyzes the formation of phosphatidylinositol 4-phosphate using phosphatidylinositol and ATP as substrates while the activity of a second enzyme, phosphatidylinositol-4-phosphate kinase, results in the synthesis of phosphatidylinositol 4,5-bisphosphate. Large (Mr greater than 20,000) homopolymers of L-lysine or L-ornithine can stimulate the activity of both of these enzymes by at least 2-fold at 10-20 microM concentrations. Under similar conditions poly-L-arginine fails to stimulate the reaction causing a partial inhibition. Smaller polylysine (25 lysines) or lysine-rich oligopeptides such as one corresponding to the last 14 amino acids of the carboxyl end of c-Ki-ras 2 protein produce appreciable stimulation of phosphatidylinositol but at concentrations of 300-500 microM. Spermine and spermidine at millimolar concentrations also stimulate exogenous phosphatidylinositol phosphorylation. The amino-glycoside antibiotic neomycin has a biphasic effect, stimulating the phosphatidylinositol kinase at concentrations below 0.5 mM and strongly inhibiting at higher concentrations. Polylysine also moderately stimulates the loss of radioactivity of phosphatidylinositol-4-[32P] phosphate observed in oocyte membranes. Polylysine and polyornithine do not change the apparent Km for ATP of the phosphatidylinositol kinase but increase the Vmax of the reaction.     

Biochemical basis for the specificity of alloxan inactivation of calmodulin-dependent protein kinase II.

The specificity and biochemical basis of inactivation of calmodulin-dependent protein kinase II by alloxan was studied in dispersed rat brain cells and a partially purified kinase preparation from an insulin-secreting tumor-cell line, RINm5f. When mechanically dispersed rat brain cells were incubated with [32P]-phosphate to label endogenous ATP, depolarization with 44 mM KCl produced a significant (P = 0.03) increase in phosphorylation of endogenous synapsin (132 +/- 8% of basal). Pre-treatment of the brain cells with 1.5 mM alloxan reduced depolarization-sensitive synapsin phosphorylation (109 +/- 5%). Phosphopeptide mapping of depolarization-phosphorylated synapsin showed that alloxan pre-treatment reduced phosphorylation specifically at synapsin sites phosphorylated by calmodulin-dependent protein kinase II. The results demonstrate selective inactivation of calmodulin-dependent protein kinase II activity by alloxan in an intact cell system, which may be useful in the study of the Type II kinase in cells and tissues. Using a partially purified kinase preparation from RINm5f cells, alloxan (100 microM) inactivated 76 +/- 1% calmodulin-dependent protein kinase II activity in 5 min at 37 degrees C. Subsequent incubation with dithiothreitol restored most of the activity. 5,5'-Dithiobis (2-nitrobenzoic acid) (I50 = 2.5 microM) also inactivated the kinase. These results suggested that a sulfhydryl group was involved at the inactivation site. Iodoacetamide (1.0 mM) had no inhibitory effect; however, preincubation with iodoacetamide protected the kinase activity from subsequent inactivation by alloxan. Covalent binding of [14C]-alloxan to calmodulin-dependent protein kinase was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of phosphatidylinositol kinase from bovine brain myelin.

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.     

Characterization and purification of membrane-associated phosphatidylinositol-4-phosphate kinase from human red blood cells

The membrane-bound form of phosphatidylinositol-4-phosphate (PtdInsP) kinase was purified 4,300-fold from human red blood cells to a specific activity of 117 nmol min-1 mg-1. Although this enzyme copurified with red blood cell membranes, it was solubilized by high salt extraction in the absence of detergent indicating that it is a peripheral membrane protein. The major protein seen in the most purified preparation migrated at 53,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major PtdInsP kinase activity in this preparation was also coincident with this 53,000-dalton band upon renaturation of activity from SDS-PAGE. To test further whether the 53,000-dalton protein contained PtdInsP kinase activity, antibodies were prepared against the gel-purified 53,000-dalton protein. This antiserum was able to precipitate both the 53,000-dalton peptide and PtdInsP kinase activity from red blood cell membranes. The apparent size of the native enzyme in the most purified preparation was determined to be 150,000 +/- 25,000 daltons by gel filtration. This PtdInsP kinase activity was at least 100-fold more active in phosphorylating PtdInsP than phosphatidylinositol and was easily separated from the red cell membrane phosphatidylinositol kinase by salt extraction. Analysis of the reaction product, phosphatidylinositol 4,5-bisphosphate, indicates that the enzyme phosphorylates phosphatidylinositol 4-phosphate specifically at the 5'-hydroxyl of the inositol ring. The apparent Km for ATP was 2 microM, and the concentrations of Mg2+ and Mn2+ giving half-maximal activity were 2 and 0.2 mM, respectively. Mg2+ supported 3-fold higher activity than Mn2+ at optimal concentrations. The enzymatic activity was inhibited by its product, phosphatidylinositol 4,5-bisphosphate and enhanced by phosphatidylserine.     

Protein phosphorylation and synaptic transmission: receptor mediated modulation of protein kinase C in a rat brain fraction enriched in synaptosomes

Aspects of protein phosphorylation related to events occurring during synaptic transmission were briefly reviewed. High resolution two-dimensional electrophoresis was used to study protein phosphorylation catalysed by protein kinase C in a fraction from rat brain enriched in synaptosomes. Incubation of 32P-labelled synaptosomes with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate resulted in an increase in the phosphorylation of a 45 K polypeptide (generally known as B-50) and an 82 K polypeptide; other major phosphoproteins in the preparation were unaffected by this treatment. It appears therefore that the 45 K and 82 K polypeptides are the only significant substrates for protein kinase C in synaptosomes. Depolarisation of labelled synaptosomes by high K+ increased the phosphorylation of the 82 K polypeptide, synapsin I and several unknown phosphoproteins. Incubation of labelled synaptosomes with the cholinergic agonist carbachol resulted in a modest, but statistically significant, increase in the phosphorylation of the 45 K (B-50) and 82 K polypeptides. This effect was blocked by atropine. The results are discussed in relation to a possible role for the B-50 phosphoprotein in regulating the resynthesis of polyphosphoinositides following cholinergic stimulation.     

Inhibition of cyclic AMP-dependent protein kinase by the polar head group of an insulin-sensitive glycophospholipid

A glycophospholipid has been purified from rat liver membranes and shown to copurify with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid is a phospho-oligosaccharide generated by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. It has been proposed that this phospho-oligosaccharide, which is also generated in response to insulin, may play a role in insulin action. Incubation of the catalytic subunit of cyclic AMP-dependent protein kinase with this phospho-oligosaccharide inhibited the activity of the kinase to phosphorylate histone IIA, a purified preparation of phospholipid methyltransferase and kemptide, a phosphate-accepting peptide. Inhibition of kinase activity was dose-dependent and 50% inhibition of histone phosphorylation was demonstrated with a concentration of phospho-oligosaccharide of around 2 microM. This effect was demonstrated in the presence of ATP at concentrations up to 1 mM, indicating that the phospho-oligosaccharide acts at physiological concentrations of ATP and that it does not compete with this nucleotide for the same binding site in the kinase. Inhibition by the phospho-oligosaccharide of kinase activity could be reversed by dilution or dialysis and was not reproduced by up to 50 microM myo-inositol, glucosamine, galactose, myo-inositol 1-phosphate, glucosamine 1-phosphate, galactose 1-phosphate or phosphorylcholine. The inhibitory activity was resistant to mild acid treatment but was labile to treatment with alkali, exposure to nitrous acid or incubation with sodium periodate. The phospho-oligosaccharide had no effect on the phosphorylation of lysine-rich histone by rat brain protein kinase C and on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase. In conclusion, the data in this study suggested that a phospho-oligosaccharide generated from an insulin-sensitive glycophospholipid may play a role in insulin action by modulating cyclic AMP-dependent protein kinase activity.     

Phosphatidic acid is a specific activator of phosphatidylinositol-4-phosphate kinase.

The lipid dependence of phosphatidylinositol-4-phosphate (PIP) kinase purified from bovine brain membranes was investigated. In the assay used, PIP-Triton X-100 micelles containing the lipid to be tested were presented to the enzyme. Under these conditions, phosphatidic acid (PA) stimulated the enzyme activity in a concentration-dependent manner up to 20-fold when an equal molar ratio of PA to PIP was attained. Stimulation by PA was highly specific; other lipids including lyso-PA and dicetylphosphate had a relatively small effect. The activation by PA was completely suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). To investigate the effect of PA on PIP kinase activity in natural membranes, endogenous PA was generated in rat brain synaptosomal plasma membranes by incubation with phospholipase D. Subsequent phosphorylation with [gamma-32P]ATP yielded an enhanced labeling of PIP2 but not of PIP in these membranes. These results suggest that PIP kinase activity may be under control of PA levels in membranes. This may have important implications for the regulation of cellular responses by agonist-induced phosphoinositide turnover.     

Purification and Some Characteristics of an ACTH-Sensitive Protein Kinase and Its Substrate Protein in Rat Brain Membranes

Abstract: ACTH inhibits the phosphorylation of a rat brain membrane-bound protein (B-50). Both the protein kinase and the substrate protein could be extracted from the membranes by means of treatment with Triton X-100 in 75 mM-KCl. Using column chromatography over DEAE-cellulose and ammonium sulphate precipitation a protein fraction (ASP 55–80) enriched in endogenous B-50 phosphorylating activity was obtained. The time course of the endogenous phosphorylation of B-50 in this fraction showed a linear incorporation with time for at least 10 min and reached an estimated maximal incorporation of 0.65 mol P/mol B-50 after 60 min. The inhibition by ACTH_{1_24} of the B-50 protein kinase in ASP 55–80 was dose-dependent; the half-maximal effective concentration was 5 × 10⁻⁶ M, being 10 to 50 times lower as compared with intact synaptic plasma membranes (SPM). cAMP, cGMP and various endor-phins had no effect on the B-50 protein kinase. The B-50 protein kinase required both magnesium and calcium for optimal activity. Using two-dimensional electrophoresis on polyacrylamide slab gels the B-50 protein kinase and the B-50 protein could be identified and purified. The isoelectric point (IEP) of the kinase is 5.5 and the apparent molecular weight 70,000, whereas the IEP of the substrate protein B-50 is 4.5 and the apparent molecular weight 48,000. Amino acid analysis on microgram quantities of purified kinase and B-50 protein revealed basic/acidic amino acid ratios in agreement with the respective lEP's. It is speculated that the inhibition of B-50 protein kinase may be related to known modulatory effects of ACTH and related peptides on certain types of neurotransmission and behaviour.     

Phosphatidylinositol Phosphodiesterase (Phospholipase C) Activity in the Pineal Gland: Characterization and Photoneural Regulation

Phosphatidylinositol phosphodiesterase (PL-C) appears to be a key element in the adrenergic regulation of pineal cyclic AMP levels. In the present study, the rat pineal enzyme was characterized using exogenous [3H]phosphatidylinositol (0.5 mM) as substrate. Half the enzyme activity was found in the cytosolic fraction, but the highest specific concentration was associated with the membrane fraction. Two pH optima (5.5 and 7.5) of enzyme activity were observed for the membrane fraction but only one in the cytosol fraction (pH 5.5). Enzyme activity in both fractions was Ca2+ dependent. In the case of the membrane protein in pH 7.5, the enzyme activity was sensitive to changes in Ca2+ in the 10-100 nM range. Addition of an equimolar concentration of phosphatidylinositol 4-phosphate nearly completely inhibited the hydrolysis of [3H]phosphatidylinositol; other phospholipids (1.0 mM) were less potent. This may reflect our present finding that [3H]phosphatidylinositol 4-phosphate is a better substrate than [3H]phosphatidylinositol for the enzyme. Stimulus deprivation (2 weeks of constant light or superior cervical ganglionectomy) reduced the cytosolic activity by 30% and had no effect on the membrane-associated enzyme.