Interrelationships between protein kinases and spectrin phosphorylation in human erythrocytes

Casein kinase and histone kinase(s) are solubilized from human erythrocyte membranes by buffered ionic solutions (0.1 mM EDTA and subsequent 0.8 M NaCl, pH 8) containing 0.2% Triton X-100. Casein kinase is separated from histone kinase(s) by submitting the crude extracts directly to chromatography on a phosphocellulose column, eluted with a continuous linear gradient of potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. Under these conditions, the membrane-bound casein kinase activity is almost completely recovered into a quite stable preparation, free of histone kinase activity. In contrast, it undergoes a dramatic loss of activity when the extraction and the subsequent phosphocellulose chromatography are carried out with buffers which do not contain Triton X-100. Isolated spectrin, the most abundant membrane protein, is phosphorylated, in the presence of [gamma-32P]ATP, only by casein kinase while histone kinase is ineffective. Only the smaller subunit (band II) of isolated spectrin (and not the larger one (band I) is involved in such a phosphorylation process, as in the endogenous phosphorylation occurring in intact erythrocytes.     

Regulation of polyphosphoinositide synthesis in cardiac membranes

The relative distribution of phosphatidylinositol (PI) and phosphatidylinositol-4-phosphate (PIP) kinase activities in enriched cardiac sarcolemma (SL), sarcoplasmic reticulum (SR), and mitochondrial fractions was investigated. PI and PIP kinase activities were assayed by measuring 32P incorporation into PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous and exogenous PI in the presence of [gamma-32P]ATP. PI and PIP kinase activities were present in SL, SR, and mitochondrial fractions prepared from atria and ventricles although the highest activities were found in SL. A similar membrane distribution was found for PI kinase activity measured in the presence of detergent and exogenous PI. PI and PIP kinase activities were detectable in the cytosol providing exogenous PI and PIP and Triton X-100 were present. Further studies focused on characterizing the properties and regulation of PI and PIP kinase activities in ventricular SL. Alamethacin, a membrane permeabilizing antibiotic, increased 32P incorporation into PIP and PIP2 4-fold. PI and PIP kinase activities were Mg2+ dependent and plateaued within 15-20 min at 25 degrees C. Exogenous PIP and PIP2 (0.1 mM) had no effect on PIP and PIP2 labeling in SL in the absence of Triton X-100 but inhibited PI kinase activity in the presence of exogenous PI and Triton X-100. Apparent Km's of ATP for PI and PIP kinase were 133 and 57 microM, respectively. Neomycin increased PIP kinase activity 2- to 3-fold with minor effects on PI kinase activity. Calmidazolium and trifluoperazine activated PI kinase activity 5- to 20-fold and completely inhibited PIP kinase activity. Quercetin inhibited PIP kinase 66% without affecting PI kinase activity. NaF and guanosine 5'-O-(3-thiotriphosphate) had no effect on PI and PIP kinase activities, indicating that these enzymes were not modulated by G proteins. The probability that PIP and PIP2 synthesis in cardiac sarcolemma is regulated by product inhibition and phospholipase C was discussed.     

Purification and characterization of membrane-bound phosphatidylinositol kinase from rat brain

A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with Triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad pH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak.     

Phosphatidylinositol kinase of bovine adrenal chromaffin granules: kinetic properties and inhibition by low concentrations of Ca2+

The kinetics and regulatory properties of phosphatidylinositol (PI) kinase were studied in chromaffin granule ghosts isolated from the bovine adrenal medulla. Phosphatidylinositol 4-phosphate (PIP) was the major 32P-labelled phospholipid formed when the isolated membranes were phosphorylated by [gamma-32P]ATP. The PI kinase activity was rather independent of pH, but highly dependent on Mg2+ with a maximal stimulation at 60 mM Mg2+. By contrast, KCl and NaCl had a slight inhibitory effect. The Km value for MgATP was 44 and 62 microM in the presence of 1 and 20 mM MgCl2, respectively. The PI kinase was almost fully and reversibly inhibited by free Ca2+ (calmodulin-independent) in the nanomolar and low micromolar range, depending on the concentration of Mg2+. The inhibition was not dependent on Ca2+-stimulated protein phosphorylation, and it could not be explained by a dephosphorylation of PIP.     

Protein kinases in brown adipose tissue of developing rats. II. two soluble kinase activities and their affinities for nucleotide and protein substrates.

A heat-stable, soluble component of brown adipose tissue from newborn rats was found to be readily phosphorylated by protein kinase of the same subcellular fraction. The concentration of this component in brown fat decreased with the age of the animals. A boiled crude microsomal preparation from rat liver was also phosphorylated by brown fat protein kinase. The GTP-linked phosphorylation of the endogenous heat-stable protein was not stimulated by ATP (in contrast to phosphorylation of histone). The maximum velocity of phosphorylation achieved with GTP was about 2.5 times higher than that with ATP as nucleotide substrate. This difference was not due to ATPase activity in the assay. With histone as the protein acceptor both activities were the same. The affinity of protein kinase(s) for ATP was lower with the endogenous heat-stable brown-fat protein and with boiled microsomes (Km of 0.21 mM and 0.17 mM, respectively) than with histone (Km of 0.05 M). No detectable ATPase activity was present in either acceptor protein. It is concluded that the 100 000 times g supernatant fraction from brown fat of infant rats contains two protein kinase activities. One preferentially uses ATP and histone as substrates and the other uses endogenous heat-stable soluble proteins and either ATP or GTP.     

Purification and kinetic properties of a membrane-bound phosphatidylinositol kinase of the bovine adrenal medulla

Phosphatidylinositol (PI) kinase (EC 2.7.1.67), an integral membrane protein of chromaffin granule ghosts of the bovine adrenal medulla, was found to phosphorylate PI in the 4-position of the inositol ring. The PI kinase was purified about 200-fold from a membrane fraction containing chromaffin granules and microsomes by extraction with Triton X-114, followed by phase partition (clouding) and heparin Sepharose chromatography. The PI kinase preparation (specific activity of 5.1 nmol PIP/mg protein per min) was free from other enzymatic activities that metabolize polyphosphoinositides. Km values of 55 microM and 40 microM for ATP and PI, respectively, were estimated for the purified enzyme. Concentrations of Triton X-100 above the critical micellar concentration (0.01%, w/v) were necessary to support significant enzyme activity, which was optimal at about 0.1% (w/v). Its dependence of pH was similar to that of the membrane-bound enzyme, with a broad optimum around pH 7. Mes in the millimolar concentration range was found to strongly inhibit the activity of the purified PI kinase (I50 at about 4 mM). The enzyme was almost totally inhibited by low micromolar concentrations of free calcium, and stimulated by hydrophilic cations, e.g., Mg2+ and poly(L-lysine), with the same potencies as for the membrane-bound enzyme. The amphiphilic cation trifluoperazine, however, stimulated the activity of purified PI kinase less effectively than the membrane-bound enzyme (Husebye, E.S. and Flatmark, T. (1988) Biochem. Pharmacol. 37, 449-456), whereas the inhibitory effect of near millimolar concentrations of trifluoperazine was the same for the two forms of the enzyme. It is concluded that the membrane-bound PI kinase of this tissue is of type II according to the classification of Cantley and co-workers (Whitman et al. (1987) Biochem. J. 247, 165-174).     

Distribution of protein kinase activities in subcellular fractions of rat brain.

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

Platelet membrane phosphatidylinositol kinase activity. Triton X-100 effects provide evidence for intramicellar reaction.

Phosphatidylinositol (PI) kinase activity of platelet membranes was solubilized and partially purified by anion-exchange chromatography to measure the initial enzymatic rates. Kinetic studies were performed in the presence of Triton X-100 to obtain mixed micelles. The partially purified enzyme exhibited a Michaelian behaviour towards ATP, with a Km of 58 microM. The enzymatic rates were dependent upon Triton concentrations. Upon increasing its concentration, this detergent exhibited an activating effect followed by an inhibitory one. The optimal micellar Triton concentration was proportionnal to the PI concentration used in the assay. Conversely, the behaviour of the enzyme towards PI was dependent upon the Triton concentration. However, when PI concentration was expressed as its surfacic concentration within the micelles, the activity became independent of the detergent concentration, and a Km value of 0.09 mol/mol was estimated. Therefore, in vitro phosphorylation of phosphatidylinositol by PI kinase is rate-limited by an intramicellar reaction, and provides a study model for the in vivo reaction.     

Characterization of undecaprenol kinase from Lactobacillus plantarum.

A membrane-bound undecaprenol kinase from Lactobacillus has been identified by observing the ATP-dependent phosphorylation of [14C]undercaprenol. The product of this reaction was shown to be [14C]undecaprenyl monophosphate by comparison of its chromatographic mobilities with authentic undecaprenyl monophosphate. It was shown that 32P from [gamma-32P]ATP was incorporated into undecaprenyl monophosphate. The kinase was partially solubilized by a variety of methods utilizing Triton X-100. Both the membrane-associated and solubilized enzymes required Mg2+, Triton X-100 and dimethylsulfoxide for activity. The enzyme preferentially phosphorylated the C34, C50 AND C 55 polyprenols. Geranylgeraniol (C20) and dolichol (C100), however, were utilized only 6% and 13% as well as undecaprenol, respectively. Despite the 8-fold difference in apparent V values, the apparent Km values for dolichol and undecaprenol were both 14 microM. The apparent Km for the nucleotide cosubstrate, ATP, was 2 mM. No other nucleoside triphosphate could substitute for ATP.     

PROTEIN KINASES IN MYELIN OF RAT BRAIN: SOLUBILIZATION AND CHARACTERIZATION

Myclin from rat brain contained adenosine 3′, 5′-monophosphate (cyclic AMP)-dependent protein kinase activity, which was solubilized by 0.2% Triton X-100 and required exogenous protein substrate for its activity. Also present was a protein kinase which catalysed the phosphorylation of the endogenous substrate and which was neither solubilized by Triton X-100 nor stimulated by cyclic AMP. Sodium fluoride was required to maintain the activity of the endogenous phosphorylation, probably by inhibiting ATPase activity, but had no effect on the phosphorylation of histone by the solubilized enzyme. Protamine and myelin basic protein served as well as histone as a substrate for the solubilized enzyme. A protein kinase modulator had no effect on the endogenous phosphorylation, but inhibited histone phosphorylation by the solubilized enzyme. Cyclic AMP-binding activity was observed in both the solubilized and non-solubilized preparations. The concentration of cyclic AMP required to give half-maximal binding activity of the preparations was about 2.5 nM. The results indicate that the cyclic AMP-binding site of the protein kinase in myelin may partially be accessible, whereas the catalytic site may be integrated into the membrane structure of myelin.     

Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells.

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.     

Purification and Characterization of a Soluble Phosphatidylinositol 4-Kinase from Carrot Suspension Culture Cells

Previously we reported the presence of a soluble phosphatidylinositol 4-kinase (PI 4-Kinase) in carrot (Daucus carota L.) suspension culture cells (C.M. Okpodu, W. Gross, W.F. Boss [1990] Plant Physiol 93: S-63). We have purified the enzyme over 1000-fold using Q-Sepharose ion exchange, hydroxylapatite, and G-100 gel filtration column chromatography. The Mr of the enzyme was estimated to be 83,000 by gel filtration. PI 4-kinase activity was recovered after renaturation of the 80-kD region of polyacrylamide gels, and an 80-kD peptide cross-reacted with antibodies to the yeast 55-kD membrane-associated PI 4-kinase on western blots. The isolated lipid kinase phosphorylated PI but not lysophosphatidylinositol or phosphatidylinositol monophosphate. Maximal PI kinase activity occurred when the substrate was added as Triton X-100/PI mixed micelles at pH 8. The enzyme required divalent cations. At low concentrations (1-5 mM), Mn2+ was more effective than Mg2+ in increasing enzyme activity; however, maximal activity occurred at 25 to 40 mM Mg2+. Calcium from 0.01 [mu]M to 1 mM had no effect on the enzyme activity. The Km of the enzyme for ATP was estimated to be between 400 and 463 [mu]M. The enzyme was inhibited by adenosine (100 [mu]M); however, ADP (up to 100 [mu]M) had no effect on the activity. The biochemical characteristics of the carrot soluble PI 4-kinase are compared with the previously reported PI 4-kinases from animals and yeast.     

Phosphorylation of a pancreatic zymogen granule membrane protein by endogenous calcium/phospholipid-dependent protein kinase

The occurrence of phospholipid-sensitive calcium-dependent protein kinase (referred to as C kinase) and its endogenous substrate proteins was examined in a membrane preparation from rat pancreatic zymogen granules. Using exogenous histone H1 as substrate, C kinase activity was found in the membrane fraction. The kinase was solubilized from membranes using Triton X-100 and partially purified using DEAE-cellulose chromatography. An endogenous membrane protein (Mr approximately equal to 18 000) was found to be specifically phosphorylated in the combined presence of Ca2+ and phosphatidylserine. Added diacylglycerol was effective in stimulating phosphorylation of exogenous histone by the partially purified C kinase, but had no effect upon phosphorylation of the endogenous 18 kDa protein by the membrane-associated C kinase. Phosphorylation of the 18 kDa protein was rapid (detectable within 30 s following exposure to Ca2+ and phosphatidylserine), and highly sensitive to Ca2+ (Ka = 4 microM in the presence of phosphatidylserine). These findings suggest a role for this Ca2+-dependent protein phosphorylation system in the regulation of pancreatic exocrine function.     

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.     

Hepatic phosphatidylinositol kinase activity in continuously endotoxemic rats.

The activity of phosphatidylinositol (PI) kinase and the content and fatty acid composition of inositol phospholipids (IPLs) were analyzed in the livers of rats that had been continuously infused with Escherichia coli endotoxin (ET) or saline for 30 h. Maximal enzymatic activity in total liver membrane fractions was observed in the presence of 1 mM ATP, 20 mM MgCl2, exogenously added 0.3 mM PI and Triton X-100 (0.25%). The activity of PI kinase for endogenous and exogenous PI was 43 and 79% higher respectively, in ET- as compared with saline-infused rats. The Km of the enzyme for ATP was not altered (0.175 mM), while the apparent Vmax was higher for ET- as compared with saline-infused rats (0.48 and 0.38 nmol of phosphatidylinositol 4-phosphate formed/mg protein per min, respectively). The ET-induced higher activity of PI kinase was paralleled by a 68-78% increase in the content of polyphosphoinositides (PPI), while PI content was unchanged. All IPLs from livers of endotoxemic rats had a lower content of arachidonic acid. We demonstrate for the first time that ET can directly and/or indirectly stimulate the net synthesis of PPI in liver cells. This effect could serve to modulate the PPI derived signals by increasing the availability of the substrate phosphatidylinositol 4,5-bisphosphate.     

Specific substrate for histone kinase II: a synthetic nonapeptide

Based on the previously determined intrinsic substrate specificity of histone kinase II, a nonapeptide was synthesized which was a specific substrate for this enzyme. The Vmax value of phosphorylation of the peptide (Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide) was about the same as that for H1 histone and the apparent Km for the phosphorylation of the peptide was 0.2 mM, an order of magnitude higher than that for H1 histone. H1 histone inhibited the phosphorylation of the peptide, while the peptide did not inhibit the phosphorylation of H1 histone. In the crude extracts of calf thymus, spleen and liver, histone kinase II was the only enzyme which phosphorylated the synthetic peptide. The rate of phosphorylation of this peptide was used to determine the activity of histone kinase II in the crude extracts of several tissues obtained from different species.     

Stimulation of a histone H4 protein kinase in Triton X-100 lysates of rabbit peritoneal neutrophils pretreated with chemotactic factors. Effect of fMet-Leu-Phe and partial characterization of the protein kinase

Rabbit peritoneal neutrophils were stimulated with either the chemotactic factor, fMet-Leu-Phe (10(-8) M, 10 s) or the protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), (0.1 microgram/ml, 3 min) at 37 degrees C, lysed with Triton X-100 at the indicated times and the histone H4 kinase activity of the lysate measured. The histone H4 protein kinase activity was increased severalfold by fMet-Leu-Phe but not PMA. The inclusion of the potent protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (50 microM) inhibited little if any of the histone H4 protein kinase activity. The effect of fMet-Leu-Phe was transient, maximum stimulation occurring within 10 s and decaying thereafter. The soluble fraction (extract) of the Triton X-100 lysates from control and fMet-Leu-Phe-treated cells was found to contain both histone H4 protein kinase and calcium-phospholipid-activated protein kinase (protein kinase C) activities. The histone H4 protein kinase activity obtained after fMet-Leu-Phe treatment was very little affected by calcium, phospholipid, and PMA and preferred histone H4 but not H1 or H2A as its substrate. In contrast, the calcium-phospholipid-activated protein kinase activity of the extract preferred histones H1 or H2A as substrates and was strongly inhibited by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine. The histone H4 protein kinase was partially separated from kinase C by DEAE-cellulose and phenyl-Sepharose 4B chromatography. It phosphorylated mostly serine in histone H4. The results indicate that the chemotactic factor, fMet-Leu-Phe, stimulates a protein kinase with substrate specificity and biochemical properties distinct from calcium-phospholipid-activated protein kinase C.     

Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells

We have shown previously that a nuclear phosphatidylinositol (PI) 4-kinase activity was present in intact nuclei isolated from carrot suspension culture cells (Daucus carota L.). Here, we further characterized the enzyme activity of the nuclear enzyme. We found that the pH optimum of the nuclear-associated PI kinase varied with assay conditions. The enzyme had a broad pH optimum between 6.5–7.5 in the presence of endogenous substrate. When the substrate was added in the form of phosphatidylinositol/phosphatidylserine (PI/PS) mixed micelles (1 mM PI and 400 μM PS), the enzyme had an optimum of pH 6.5. In comparison, the pH optimum was 7.0 when PI/Triton X-100 mixed micelles (1 mM PI in 0.025 %, v/v final concentration of Triton X-100) were used. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). Calcium at 10 μM inhibited 100 % of the nuclear-associated enzyme activity. The K_m for ATP was estimated to be between 36 and 40 μM. The nuclear-associated PI kinase activity was inhibited by both 50 μM ADP and 10 μM adenosine. Treatment of intact nuclei with DNase, RNase, phospholipase A₂ and Triton X-100 did not solubilize the enzyme activity. Based on sensitivity to calcium, ADP, detergent, pH optimum and the product analysis, the nuclear-associated PI 4-kinase was compared with previously reported PI kinases from plants, animals and yeast.     

Further enzymatic characteristics of a thylakoid protein kinase

The enzymatic characteristics of a protein kinase purified from thylakoids are further described. ATP (KM approximately 30 microM) and Mg2+ ion (greater than 1.0 mM) were required for activity, while ADP was a competitive inhibitor (Ki = 100 microM). Activity was 55% inhibited by the sulfhydryl inhibitor p-chloromercuribenzoate (1 mM) and was less sensitive to substituted maleimides. Lysine-rich histones (H1) were utilized as an exogenous phosphorylation substrate both by thylakoid-bound kinase and by isolated enzyme; threonine was predominantly phosphorylated by the in situ enzyme, whereas the isolated enzyme phosphorylated closely related serine residues as determined by peptide mapping. Detergents that proved useful in extracting the kinase from thylakoids markedly inhibited activity of the isolated enzyme, whereas Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid had little effect. The enzyme could be freed from detergent and behaved as an active monomer on size-exclusion chromatography. The phosphate contents of the light-harvesting chlorophyll a/b protein complex of photosystem II isolated from maximally phosphorylated thylakoid membranes of spinach and pea were equivalent to approximately 6% and approximately 19% phosphorylation, respectively. Corresponding values for nonphosphorylated membranes were approximately 3% and approximately 14.5%.     

Partial purification and characterization of phosphatidylinositol kinases from human platelets

Most of human platelet phosphatidylinositol (PI) kinase activity (approx. 80%) was associated with the membrane fraction and its majority was released by the extraction with Triton X-100 after KCl treatment. Two major activity peaks (mPIK-I and mPIK-III) were obtained by Mono Q column chromatography. They were distinct from each other with regard to Mr (76,000 and 80,000 as determined by gel-filtration chromatography), apparent Km values for ATP, effect of arachidonic acid and phosphatidylserine and detergent requirement. Triton X-100 inhibited the activity of mPIK-I but rather weakly enhanced the mPIK-III activity, and sodium cholate remarkably inhibited both mPIK-I and mPIK-III activities. Their products were identified to be phosphatidylinositol 4-phosphate. On the other hand, about 20% of PI kinase activity was recovered from the cytosolic fraction and two activity peaks (cPIK-I and cPIK-II) were resolved on Mono Q column chromatography. There were no significant differences in biochemical properties between cPIK-I and cPIK-II. Both of them had Mr approx. 550,000 as determined by gel-filtration chromatography and were activated by sodium cholate to a greater extent than by Triton X-100. The results suggest that the major PI kinases (mPIK-I and mPIK-III) are PI 4-kinase and mPIK-I is distinct from PI 4-kinases in other sources especially with regard to the effect of Triton X-100.