Lipoxygenase activities of human platelets and their subcellular fractions: Comparison between lipoxygenase-deficient platelets and normal platelets

Lipoxygenase activities were estimated in washed platelets (intact platelets) and their subcellular fractions obtained from 7 patients with deficient platelet lipoxygenase activities and 9 normal subjects. From sonicated platelet preparations, 12,000 g supernatant (F-I), cytosol (F-II) and microsomal fractions (F-III) were prepared by differential centrifugation. When 12-hydroxyeicosatetraenoic acid (12-HETE) produced by the incubation of arachidonic acid with intact platelets or each of their subcellular fractions from normal subjects was measured by reversed-phase high-performance liquid chromatography analysis, the lipoxygenase activities of F-I, F-II and F-III were 87%, 31% and 17%, respectively, of the enzyme activity of intact platelets. One of the patients showed no detectable lipoxygenase activity in any preparation, while the other patients showed reduced enzyme activities in all preparations. The addition of CaCl2 significantly increased 12-HETE synthesis solely by F-I from these patients. In most of these patients, contrary to normal subjects, it appeared that the lipoxygenase activity was not fully expressed in intact platelets, since the F-I produced more 12-HETE than the intact platelets.     

A cytosolic protein activator of cardiac sarcolemmal phosphoinositide phospholipase C

Ca2+ dependent polyphosphoinositide phospholipase C (PLC) activity in cardiac sarcolemma hydrolyzed both endogenous and exogenous phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with an associated increase in inositol bisphosphate (IP2). Dialyzed cytosol and certain fractions of cytosol isolated by anion exchange or gel filtration chromatography activated sarcolemmal PLC activity by approx. 100%. The PLC activator eluted with an apparent molecular weight of 160 Kdal on a Sephacryl 300 column and was destroyed by heat or trypsin treatment. Exogenous 3H-PIP2 was not hydrolyzed by cytosolic fractions containing sarcolemmal PLC activator. These studies demonstrate that the polyphosphoinositide PLC in cardiac sarcolemma is regulated by a cytosolic protein.     

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.     

Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets

Two peaks (mPLC-I and mPLC-II) of phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity were resolved when 1% sodium cholate extract from particulate fractions of human platelet was chromatographed on a heparin-Sepharose column. The major peak of enzyme activity (mPLC-II) was purified to homogeneity by a combination of Fast Q-Sepharose, heparin-Sepharose, Ultrogel AcA-44, Mono Q, Superose 6-12 combination column, and Superose 12 column chromatographies. The specific activity increased 2,700-fold as compared with that of the starting particulate fraction. The purified mPLC-II had an estimated molecular weight of 61,000 on sodium dodecyl sulfate-polyacrylamide gels. The minor peak of enzyme activity (mPLC-I) was partially purified to 430-fold. Both enzymes hydrolyzed PIP2 at low Ca2+ concentration (0.1-10 microM) and exhibited higher Vmax for PIP2 than for phosphatidylinositol. PIP2-hydrolyzing activities of both enzymes were enhanced by various detergents and lipids, such as deoxycholate, cholate, phosphatidylethanolamine, and dimyristoylphosphatidylcholine. The mPLC-I and mPLC-II activities were increased by Ca2+, but not by Mg2+, while Hg2+, Fe2+, Cu2+, and La3+ were inhibitory. GTP-binding proteins (Gi, Go, and Ki-ras protein) had no significant effects on the mPLC-II activity.     

Characterization of phospholipase C-mediated polyphosphoinositide hydrolysis in rat heart ventricles

Phospholipase C (PLC)-mediated degradation of polyphosphoinositides (phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP] was found to be present in rat heart ventricular soluble and total membrane fractions (100,000g supernatant and pellet). Distribution of polyphosphoinositide-specific phospholipase C activity between the membrane and soluble fraction was approximately 63 and 33% of total activity, respectively, whereas, phosphatidylinositol (PI) degradation could be detected only in the soluble fraction. Optimal PIP2-PLC activity occurred at a pCa2+ of 4.5. A similar peak in PIP-PLC activity could be demonstrated in soluble and membrane preparations; however, the rate of PIP degradation in the soluble fraction continued to increase at the highest calcium level tested (pCa2+ 3). With the exception of Sr2+, other noncalcium polycations did not support homogenate PIP2-PLC activity. In the presence of Ca2+, addition of Mg2+, La3+, or Sr2+ (10(-3) M) inhibited PIP2-PLC while Mn2+ and Gd3+ stimulated activity. In both the total membrane and soluble fractions, maximal polyphosphoinositide degradation occurs at pH 5.5 and 6.8. The detergents deoxycholate, cholate, and saponin exert a biphasic effect on PIP2-PLC activity (stimulating at lower concentrations and inhibiting at higher concentrations). The deoxycholate effect is observed in both the cytosolic and membrane fractions. Neutral and cationic detergents inhibit PIP2-PLC activity in a concentration-dependent manner. Similar to cytosolic PI-PLC activity, PIP2-PLC appears to depend on intact sulfhydryl groups. In the presence of a mixture of all three inositol phospholipids or the three phosphoinositides plus noninositol phospholipids, polyphosphoinositides are preferentially degraded.     

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.     

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].     

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.     

Control of glycogen synthase by insulin and isoproterenol in rat adipocytes. Changes in the distribution of phosphate in the synthase subunit in response to insulin and beta-adrenergic receptor activation

Rat adipocytes were incubated with [32P]phosphate to label glycogen synthase, which was rapidly immunoprecipitated from cellular extracts and cleaved using either CNBr or trypsin. All of the [32P]phosphate in synthase was recovered in two CNBr fragments, denoted CB-1 and CB-2. Isoproterenol (1 microM) rapidly decreased the synthase activity ratio (-glucose-6-P/+glucose-6-P) and stimulated the phosphorylation of both CB-1 and CB-2 by approximately 30%. Insulin opposed the decrease in activity ratio and blocked the stimulation of phosphorylation by isoproterenol. Incubating cells with insulin alone changed the 32P content of neither CB-1 nor CB-2. Trypsin fragments were separated by reverse phase liquid chromatography and divided into peak fractions, denoted F-I-F-VII in order of increasing hydrophobicity. F-V contained almost half of the [32P]phosphate and was phosphorylated when synthase was immunoprecipitated from unlabeled fat cells and incubated with [gamma-32P]ATP and the cAMP-independent protein kinase, FA/GSK-3. That F-V also had the same retention time as the skeletal muscle synthase fragment containing sites 3(a + b + c) suggests that it contains sites 3. Muscle sites 1a, 5, 1b, and 2 eluted with F-I, F-II, F-VI, and F-VII, respectively. F-V was increased approximately 25% by isoproterenol, but the largest relative increases were observed in F-I (4-fold), F-III (4-fold), and F-VI (2-fold). These results indicate that beta-adrenergic receptor activation results in increased phosphorylation of multiple sites on glycogen synthase. Insulin plus glucose decreased the overall 32P content of synthase by approximately 30%, with the largest decrease (40%) occurring in F-V. Without glucose, insulin decreased the [32P]phosphate in F-V by 17%, an effect which was balanced by increases in F-I, F-II, and F-III so that no net change in the total 32P contents of the fractions was observed. Thus, activation of glycogen synthase by the glucose transport-independent pathway seems to involve a redistribution of phosphate in the synthase subunit.     

Human neutrophil cytosolic phospholipase C: partial characterization.

The activity of neutrophil cytosolic phospholipase C on PIP2 and PI was compared employing [3H]inositol-labeled heat-inactivated membranes of differentiated HL-60 cells, into which tracer [32P]PIP2 was incorporated. Hydrolysis of PIP2 did not require Ca2+ and was stimulated when the content of PIP2 in the membrane was increased by incorporation of unlabeled inositol lipid. At equal concentrations of PI and PIP2 in the membrane, hydrolysis of PIP2 was faster and no evidence of competition between the two substrates was obtained. Incorporation of PI into PE-[32P]PIP2 vesicles, accelerated PIP2 hydrolysis also at conditions that favor hydrolysis of PI. Partial purification of neutrophil cytosolic PLC on Q Sepharose, phenyl Sepharose and heparin-Agarose columns is described. From heparin-Agarose column, two PLC activity peaks exhibiting different substrate specificities were eluted. The elution profile of the main PLC species from Superose 12 gel filtration column was compatible with an approx. 150 kDa protein.     

Mechanism of protein kinase C activation by phosphatidylinositol 4,5-bisphosphate.

The mechanism of protein kinase C (PKC) activation by phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI) was investigated by using Triton X-100 mixed micellar methods. The activation of PKC by PIP2, for which maximal activity was 60% of that elicited by sn-1,2-diacyglycerol (DAG), was similar to activation by DAG in several respects: (1) activation by PIP2 and DAG required phosphatidylserine (PS) as a phospholipid cofactor, (2) PIP2 and DAG reduced the concentration of Ca2+ and PS required for activation, (3) the concentration dependences of activation by PIP2 and DAG depended on the concentration of PS, and (4) PIP2 and DAG complemented one another to achieve maximal activation. On the other hand, PIP2 activation of PKC differed from activation by DAG in several respects. With increasing concentrations of PIP2, (1) the optimal concentration of PS required was constant at 12 mol%, (2) the maximal activity at 12 mol% PS increased, and (3) the cooperativity for PS decreased. PIP2 did not inhibit [3H]phorbol 12,13-dibutyrate (PDBu) binding of PKC at saturating levels of PS; however, at subsaturating levels of PS, PIP2 enhanced [3H]PDBu binding by acting as a phospholipid cofactor. PIP did not function as an activator but served as a phospholipid cofactor in the presence of PS. While PIP2, PIP, and PI did not support DAG-dependent PKC activation as phospholipid cofactors, their presence reduced the amount of PS required for maximal activation to as low as 2 mol% from 8 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.

The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.     

Stimulation of polyphosphoinositide hydrolysis by thrombin in membranes from human fibroblasts.

One of the earliest actions of thrombin in fibroblasts is stimulation of a phospholipase C (PLC) that hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. In membranes prepared from WI-38 human lung fibroblasts, thrombin activated an inositol-lipid-specific PLC that hydrolysed [32P]PIP2 and [32P]phosphatidylinositol 4-monophosphate (PIP) to [32P]IP3 and [32P]inositol 1,4-bisphosphate (IP2) respectively. Degradation of [32P]phosphatidylinositol was not detected. PLC activation by thrombin was dependent on GTP, and was completely inhibited by a 15-fold excess of the non-hydrolysable GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]). Neither ATP nor cytosol was required. Guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) also stimulated polyphosphoinositide hydrolysis, and this activation was inhibited by GDP[S]. Stimulation of PLC by either thrombin or p[NH]ppG was dependent on Ca2+. Activation by thrombin required Ca2+ concentrations between 1 and 100 nM, whereas stimulation of PLC activity by GTP required concentrations of Ca2+ above 100 nM. Thus the mitogen thrombin increased the sensitivity of PLC to concentrations of free Ca2+ similar to those found in quiescent fibroblasts. Under identical conditions, another mitogen, platelet-derived growth factor, did not stimulate polyphosphoinositide hydrolysis. It is concluded that an early post-receptor effect of thrombin is the activation of a Ca2+- and GTP-dependent membrane-associated PLC that specifically cleaves PIP2 and PIP. This result suggests that the cell-surface receptor for thrombin is coupled to a polyphosphoinositide-specific PLC by a GTP-binding protein that regulates PLC activity by increasing its sensitivity to Ca2+.     

蚯蚓纤溶酶新基因EfP-0的电子克隆及其编码区序列RT-PCR验证

利用生物信息学手段,以期获得蚯蚓纤溶酶F-Ⅰ-0组分的基因。根据从粉正蚓(Lumbricusrubellus)中分离的F-Ⅰ-0组分的N端氨基酸序列VVGGSDTTIGQYPHQL,利用DNAMAN软件通过电子克隆方法,从Lumbricidae的dbEST中获得该组分的核酸序列信息,设计特异引物,经过RT-PCR,成功地从赤子爱胜蚓(Eiseniafoetida)中克隆到一条蚯蚓纤溶酶新基因,命名为EfP-0。EfP-0基因全长678bp,编码225个氨基酸的成熟肽,属丝氨酸蛋白酶,胰蛋白酶家族,与F-Ⅰ-0组分的氨基酸组成非常接近。BLAST证明,EfP-0与已报道的蚯蚓纤溶酶基因之间的相似性均低于40%,因此为蚯蚓纤溶酶中的一个新基因,GenBank登录号为DQ836917。构建的pMAL-c2x-EfP-0重组质粒,在大肠杆菌TB1中获得融合蛋白MBP-EfP-0的可溶性表达,表达产物有酪蛋白平板溶解活性。     

Persistent activation of platelet membrane phospholipase C by proteolytic action of trypsin and thrombin

Trypsin causes rapid activation of intact platelets that mimics many actions of thrombin, including the stimulation of phospholipase C (PLC). We have examined the effects of thrombin and trypsin on PLC in a platelet membrane preparation using exogenous [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. Trypsin induced PIP2 breakdown, which was maximal at 20 micrograms/ml, but was reduced at higher concentrations. alpha- and gamma-Thrombins also stimulated PLC-induced hydrolysis of PIP2 in membranes. This effect was inhibited by leupeptin. Exogenous [3H]phosphatidylinositol 4-monophosphate (PIP) was hydrolyzed in response to both thrombin and trypsin in the same ratio as PIP2. Activation of membrane-bound PLC persisted after removal of thrombin and trypsin. The hydrolysis of [3H]phosphatidylinositol was not activated by alpha-thrombin and trypsin. We examined the question of whether calpain was involved in the observed PLC activation by thrombin and trypsin. Although dibucaine activated a Ca2(+)-dependent protease as judged by the hydrolysis of actin-binding protein and by the activation of phosphoprotein phosphatases, it failed to stimulate the generation of phosphatidic acid in 32P-prelabeled platelets. Moreover, when PLC was assayed in the membranes, the addition of Ca2(+)-activated neutral proteinases did not increase the rate of hydrolysis of either PIP or PIP2. Our results show that proteases such as trypsin and thrombin are able to stimulate membrane-bound PLC, but this activation does not seem to be related to calpain.     

Inositol phospholipid-induced suppression of F-actin-gelating activity of smooth muscle filamin.

Filamin, a high molecular weight actin-binding protein, cross-links actin filaments and produces a gel composed of F-actin. The effects of polyphosphoinositides on the gelating activity of smooth muscle filamin were examined by measuring the low shear viscosity of the F-actin solutions containing filamin incubated with phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), or phosphatidylinositol 4,5-bisphosphate (PIP2). Micelles of these inositol phospholipids bound to filamin inhibited the ability to form a gel of F-actin. The inhibiting activity of each phospholipid was in the following order, PIP2 greater than PIP greater than PI. The F-actin binding assay of filamin revealed that the inhibition of F-actin-gelation resulted in the loss of the F-actin-binding activity of filamin. Thus, polyphosphoinositides may play important roles in regulating the gelating activity of filamin.     

Autophosphorylation of type I phosphatidylinositol phosphate kinase regulates its lipid kinase activity

Phosphatidylinositol phosphate kinases (PIPKs) have important roles in the production of various phosphoinositides. For type I PIP5Ks (PIP5KI), a broad substrate specificity is known. They phosphorylate phosphatidylinositol 4-phosphate most effectively but also phosphorylate phosphatidylinositol (PI), phosphatidylinositol 3-phosphate, and phosphatidylinositol (3,4)-bisphosphate (PI(3, 4)P(2)), resulting in the production of phosphatidylinositol (4, 5)-bisphosphate (PI(4,5)P(2)), phosphatidylinositol 3-phosphate, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P(2)), phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P(2)), and phosphatidylinositol (3,4,5)-trisphosphate. We show here that PIP5KIs have also protein kinase activities. When each isozyme of PIP5KI (PIP5KIalpha, -beta, and -gamma) was subjected to in vitro kinase assay, autophosphorylation occurred. The lipid kinase-negative mutant of PIP5KIalpha (K138A) lost the protein kinase activity, suggesting the same catalytic mechanism for the lipid and the protein kinase activities. PIP5KIbeta expressed in Escherichia coli also retains this protein kinase activity, thus confirming that no co-immunoprecipitated protein kinase is involved. In addition, the autophosphorylation of PIP5KI is markedly enhanced by the addition of PI. No other phosphoinositides such as phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, or phosphatidylinositol trisphosphate have such an effect. We also found that the PI-dependent autophosphorylation strongly suppresses the lipid kinase activity of PIP5KI. The lipid kinase activity of PIP5KI was decreased to one-tenth upon PI-dependent autophosphorylation. All these results indicate that the lipid kinase activity of PIP5KI that acts predominantly for PI(4,5)P(2) synthesis is regulated by PI-dependent autophosphorylation in vivo.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

Stimulation of polyphosphoinositide turnover upon activation of protein kinases in human erythrocytes

Activation of protein kinase C in erythrocytes by 4-beta-phorbol 12-myristate 13-acetate (PMA) resulted in a parallel stimulation (time course and dose response) of the phosphorylation of both membrane proteins (heterodimers of 107 kDa and 97 kDa, protein 4.1 and 4.9, respectively) and of phosphatidylinositol 4-phosphate (PIP) and, to a lesser extent, of phosphatidylinositol 4,5-bisphosphate (PIP2). Evidence that the effect on lipid was mediated by protein kinase C activation and not by a direct action of PMA was provided by (1) the lack of effect of a phorbol ester that did not activate protein kinase C or of PMA addition on isolated membranes from control erythrocytes, (2) the reversal of the effect in the presence of protein kinase C inhibitors (alpha-cobrotoxin, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) or trifluoperazine). PMA treatment did not change the specific activity of ATP or the content of PIP2, but increased the content of PIP and decreased that of PI, indicating that the phosphorylation or dephosphorylation reactions linking PI and PIP were the target for the action of PMA. PMA treatment had no effect on the Ca2+-dependent PIP/PIP2 phospholipase C activity measured in isolated membranes. Mezerein, another protein kinase activator, had similar effects on both protein and lipid phosphorylation, when added with alpha-cobrotoxin. Activation of protein kinase A by cAMP also produced increases in phosphorylation, although quantitatively different from those induced by protein kinase C, in proteins and PIP. Simultaneous addition of PMA and cAMP at maximal doses resulted in only a partially additive effect on PIP labelling. These results show that inositol lipid turnover can be modulated by a protein kinase C and protein kinase A-dependent process involving the phosphorylation of a common protein. This could be PI kinase or PIP phosphatase or another protein regulating the activity of these enzymes.     

Cyclic GMP-dependent protein kinase stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol.

The effect of phosphorylation by cyclic GMP-dependent protein kinase (G-kinase) on the activity of the plasmalemmal Ca2+-transport ATPase was studied on isolated plasma membranes and on the ATPase purified from pig erythrocytes and from the smooth muscle of pig stomach and pig aorta. Incubation with G-kinase resulted, in both smooth-muscle preparations, but not in the erythrocyte ATPase, in a higher Ca2+ affinity and in an increase in the maximal rate of Ca2+ uptake. Cyclic AMP-dependent protein kinase (A-kinase) did not exert such an effect. The stimulation of the (Ca2+ + Mg2+)-dependent ATPase activity of the purified Ca2+ pump reconstituted in liposomes depended on the phospholipid used for reconstitution. The stimulation of the (Ca2+ + Mg2+)-ATPase activity by G-kinase was only observed in the presence of phosphatidylinositol (PI). G-kinase, but not A-kinase, stimulated the phosphorylation of PI to phosphatidylinositol phosphate (PIP) in a preparation of (Ca2+ + Mg2+)-ATPase obtained by calmodulin affinity chromatography from smooth muscle, but not in a similar preparation from erythrocytes. Adenosine inhibited both the phosphorylation of PI and the stimulation of the (Ca2+ + Mg2+)-ATPase by G-kinase. In the absence of G-kinase the (Ca2+ + Mg2+)-ATPase was stimulated by the addition of PIP, but not by PI. In contrast with previous results of Furukawa & Nakamura [(1987) J. Biochem (Tokyo) 101, 287-290], no convincing evidence for a phosphorylation of the (Ca2+ + Mg2+)-ATPase was found. Evidence is presented showing that the apparent phosphorylation occurs in a contaminant protein, possibly myosin light-chain kinase. It is proposed that G-kinase stimulates the plasmalemmal Ca2+ pump of smooth-muscle cells indirectly via the phosphorylation of an associated PI kinase.