The phosphatidylinositol kinase of rat brain

1. The presence of a phosphatidylinositol kinase in homogenates of adult rat brain was shown by using labelled ATP or labelled phosphatidylinositol. 2. The kinase was activated by Mg(2+) or Mn(2+) and inhibited by Ca(2+), Cu(2+), K(+), Na(+) and F(-). 3. The detergents sodium deoxycholate, Cutscum and Triton X-100 markedly stimulated the reaction; sodium taurocholate, Tween-20 and cetyltrimethyl-ammonium bromide were less effective. 4. The activity of the enzyme was dependent on SH groups. 5. The subcellular distribution of the kinase in brain resembled that of Na(+)-plus-K(+)-stimulated adenosine triphosphatase and 5'-nucleotidase.     

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.     

Bovine aorta contains at least two related forms of heparan sulphate proteoglycan

• 1.1. The proteoglycan peak from anion exchange chromatography of an extract of bovine aorta was digested with chondroitinase ABC. The residual heparan sulphate proteoglycans were further purified by chromatography on Sepharose CL4B and DEAE-Sephacel to yield two species, of high and low charge density.
• 2.2. Higher molecular weight material had a higher proportion of high charge density proteoglycan, while the lower molecular weight species had a higher proportion of low charge density heparan sulphate proteoglycan.
• 3.3. The two species shared epitopes as they both reacted with an antibody to heparan sulphate proteoglycan from bovine glomerular basement membrane.
• 4.4. On electron microscopy, both high and low charge density proteoglycans were visualized as ‘tadpole-like’ molecules, which showed a tendency to aggregate via their globular heads.
• 5.5. Bovine aortic smooth muscle cells were cultured in the presence of [³⁵S]sulphate and [³H]glucosamine. Proteoglycans were isolated from medium and cell layer extract by the methods outlined above.
• 6.6. The major HSPG species isolated from medium were significantly larger than those from cell layer and displayed substantial heterogeneity in both size of HS chain after papain digestion and size of protein core after heparitinase digestion. 7. The major cell layer species yielded two HS species of widely differing mol. wt after papain digestion, and a very small protein core after heparitinase digestion. Therefore cell layer-associated HSPGs show a good deal more homogeneity than those found in the medium.
• 7.8. Further ion-exchange chromatography after digestion with chondroitinase ABC revealed HSPG species of lower charge density, possibly derived from a hybrid chondroitin sulphate-dermatan sulphate proteoglycan (CS/DSPG) after removal of the CS/DS chains.

     

Partial purification and charcterization of phosphatidylinositol kinase from bovine brain

Purification of Phosphatidylinositol (PI) kinase was attempted from bovine brain. A seven step purification protocol increased the specific activity 100×but attempts at further purification were unsuccessful. Labeling of the partially purified PI kinase with the ATP analog fluorosulfonylbenzoyl adenosine reproducibly identified three bands on polyacrylamide gel electrophoresis of 76 K, 45 K, and 29 K, one of which likely represents PI kinase. Kinetic studies showed aK _m of 17 M for ATP, 0.02 mg/ml for PI and aV _m of 1830 pmol/min/mg protein for ATP and 820 pmol/min/mg protein for PI.     

Purification and characterization of bovine brain type I phosphatidylinositol kinase

Investigation into the phosphatidylinositol kinase activities in bovine brain has revealed the presence of a type I PtdIns kinase activity. This classification is based upon potent inhibition by neutral detergent and the production of a phosphatidylinositol phosphate that can be distinguished from phosphatidyl-inositol-4-phosphate [PtdIns(4)P] by thin-layer chromatography. The enzyme has been substantially purified and the activity is associated with an 85-kDa polypeptide on SDS/polyacrylamide gel electrophoresis. Analysis of the product confirms the identification of the enzyme as a type I PtdIns kinase. The purified kinase has been characterized with respect to substrate dependence (Mg2+, ATP, PtdIns), substrate presentation (pure lipid versus mixed micelle) and specificity [PtdIns versus PtdIns(4)P and phosphatidylinositol 4,5-bisphosphate].     

Detergent solubilization and hydrophobic chromatography of rat brain phosphatidylinositol kinase

Rat brain microsomal phosphatidylinositol kinase activity was maximally activated in the presence of either 3 mM sodium deoxycholate, 2% Triton-X-100, or 30–40 mM octylglucoside. Among these detergents, 1% Triton-X-100 was most effective in solubilizing the enzyme, and after treatment with, this agent, 100% of the activity was recovered in the high speed supernatant. Octylglucoside solubilized 40% of the enzyme at concentrations below its critical micelle concentration of 25 mM and up to 80% at higher levels. Solubilized phosphatidylinositol kinase failed to adsorb to adenosine nucleotide affinity resins. However, when the Triton-X-100 extract was chromatographed on an uncharged hydrophobic resin, consisting of dodecyl chains attached to Sepharose 4B by ether bonds, nearly all the enzyme activity was retained, and from 44–85% could be eluted with 8 mM sodium deoxycholate. Solubilization followed by hydrophobic chromatography resulted in several-fold purification of phosphatidylinositol kinase and may have disrupted interactions of the enzyme with other hydrophobic proteins sufficiently to allow its substantial purification by conventional or affinity chromatography techniques.The abbreviations used are phosphatidylinositol 1,2-diacyl-sn-glycero-3-phosphoryl-1-l-myo-inositol - phosphatidylinositolphosphate 1,2-diacyl-sn-glycero-3-phosphoryl-1-l-myo-inositol-4-monophosphate - phosphatidylinositolbisphosphate 1,2-diacyl-sn-glycerol-3-phosphoryl-1-l-myo-inositol-4,5-bisphosphate - octylglucoside 1-0-n-octyl-d-glucopyranoside     

Protein kinase C contains two phorbol ester binding domains

A series of deletion and truncation mutants of protein kinase C (PKC) were expressed in the baculovirus-insect cell expression system in order to elucidate the ability of various domains of the enzyme to bind phorbol dibutyrate (PDBu). A PKC truncation mutant consisting of only the catalytic domain of the enzyme did not bind [3H]PDBu, whereas a PKC truncation mutant consisting of the regulatory domain (containing the tandem cysteine-rich putative zinc finger regions) bound [3H]PDBu. Deletion of the second conserved region (C2) of PKC did not abolish [3H]PDBu binding, whereas a deletion of the first conserved region (C1) of PKC, containing the two cysteine-rich sequences, completely abolished [3H]PDBu binding. Additional truncation and deletion mutants helped to localize the region necessary for [3H]PDBu binding; all PKC mutants that contained either one of the cysteine-rich zinc finger-like regions possessed phorbol ester binding activity. Scatchard analyses of these mutants indicated that each bound [3H]PDBu with equivalent affinity (21-41 nM); approximately 10-20-fold less than the native enzyme. In addition, a peptide of 146 amino acid residues from the first cysteine-rich region, as well as a peptide of only 86 amino acids residues from the second cysteine-rich region, both bound [3H]PDBu with high affinity (31 +/- 4 and 59 +/- 13 nM, respectively). These data establish that PKC contains two phorbol ester binding domains which may function in its regulation.     

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.     

Scrapie prion protein contains a phosphatidylinositol glycolipid

The scrapie (PrPSc) and cellular (PrPC) prion proteins are encoded by the same gene, and their different properties are thought to arise from posttranslational modifications. We have found a phosphatidylinositol glycolipid on both PrPC and PrP 27-30 (derived from PrPSc by limited proteolysis at the amino terminus). Ethanolamine, myo-inositol, phosphate, and stearic acid were identified as glycolipid components of gel-purified PrP 27-30. PrP 27-30 contains 2.8 moles of ethanolamine per mole. Incubation of PrP 27-30 with a bacterial phosphatidylinositol-specific phospholipase C (PIPLC) releases covalently bound stearic acid, and allows PrP 27-30 to react with antiserum specific for the PIPLC-digested glycolipid linked to the carboxyl terminus of the trypanosomal variant surface glycoprotein. PIPLC catalyzes the release of PrPC from cultured mammalian cells into the medium. These observations indicate that PrPC is anchored to the cell surface by the glycolipid.     

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.     

Purification and chemical modification of a phosphatidylinositol kinase from sheep brain.

A membrane-bound phosphatidylinositol (PtdIns) kinase has been purified approximately 9500-fold to apparent homogeneity from sheep brains. The purification procedure involves: solubilisation of the membrane fraction with Triton X-100, ammonium sulphate fractionation and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 1149 nmol.min-1.mg-1. The molecular mass of the enzyme was estimated to be 55 kDa by SDS/PAGE and 150 +/- 10 kDa by HPLC gel filtration in the presence of Triton X-100. Kinetic measurements have shown that the apparent Km value of PtdIns kinase for the utilisation of PtdIns is 22 microM and for ATP 67 microM. Mg2+ was the most effective divalent cation activator of PtdIns kinase, with maximal enzymatic activity reached at a concentration of 10 mM Mg2+. In addition to adenosine and ADP, the 2'(3')-O-(2,4,6-trinitrophenyl) derivative of ATP was found to be a strong competitive inhibitor of the enzyme, with a Ki of 32 microM. Enzymatic activity was found to be stimulated by Triton X-100 but inhibited by deoxycholate.     

Bovine brain phosphatidylinositol transfer protein. Selective inhibition by chlorpromazine and other amphiphilic amines

Cationic amphiphilic amines of varied pharmacological activity were evaluated as modulators of the protein-catalyzed, intermembrane transfers of phosphatidylinositol and phosphatidylcholine. The catalytic agent was brain phosphatidylinositol transfer protein; the membrane system consisted of two populations of single bilayer phospholipid vesicles. The majority of the amines tested caused decreases in phospholipid transfer activity with the relative potencies in the following order: chlorpromazine greater than dibucaine greater than propranolol much greater than tripelennamine approximately chloroquine greater than dipyridamole. Concentrations required for 50% inhibition of phosphatidylinositol transfer were 0.24 mM chlorpromazine, 0.46 mM dibucaine, and 0.78 mM propranolol. The phosphatidylcholine transfer activity of this protein was somewhat less sensitive to these compounds. Comparison of chlorpromazine and its quaternary amine analogue, methochlorpromazine, at different pH values indicated that the observed inhibition can be attributed in large part to the charged forms of the amphiphiles. Direct association of methochlorpromazine with egg phosphatidylcholine bilayers was demonstrated by molecular sieve chromatography; no such association of the amphiphile with phosphatidylinositol transfer protein was apparent. Anionic agents, such as indomethacin, phenylbutazone, and tolmetin, were without significant effect on protein-catalyzed phospholipid transfers. Electrostatic interaction between the cationic amines and anionic or zwitterionic phospholipids, forming ion pairs in the lipid bilayers, is suggested as a possible molecular mechanism for the observed inhibition.     

Cardiotoxin from cobra venom increases the level of phosphatidylinositol 4-monophosphate and phosphatidylinositol kinase activity in two cell lines

In rat basophilic leukemia-2H3 (RBL-2H3) and Madin-Darby canine kidney (MDCK) cells, cardiotoxin from cobra venom induced a marked decrease in the level of [3H] phosphatidylinositol and a corresponding increase in the level of [3H]phosphatidylinositol 4-monophosphate over the course of 20 min as demonstrated in cells that had been labeled to equilibrium with [3H]inositol. The effect was dependent on the concentration (5-30 micrograms/ml) of the toxin. In plasma membrane-enriched fractions isolated from the two cell lines, the cardiotoxin enhanced the endogenous activity of phosphatidylinositol kinase especially at temperatures above 14 degrees C. In RBL-2H3 cells, cardiotoxin also induced release of substantial amounts of histamine and lactate dehydrogenase. The release of histamine, but not of lactate dehydrogenase, was totally dependent on external calcium and this release probably represented an exocytotic response of the cells to cardiotoxin. Although, initially, treatment with the toxin did not impair antigen-induced hydrolysis of inositol phospholipids or prevent the antigen-induced rise in the concentration of cytosol Ca2+, prolonged exposure to the toxin did result in a progressive loss of responsiveness of RBL-2H3 cells to antigen.     

Molecular cloning and sequencing of cDNA encoding the phosphatidylinositol kinase from rat brain.

A complementary DNA (cDNA) clone that encodes phosphatidylinositol 4-kinase (PI 4-kinase) was isolated from a rat brain cDNA library. The deduced amino acid sequence of 697 residues revealed that the protein contains two putative transmembrane sequences and that the N-terminal part of the protein has several sequences representing potential phosphorylation sites for cAMP- and calmodulin-dependent kinase. The C-terminal region is probably a phosphotransferase domain homologous to the kinase region of protein kinase family proteins. Specific antibody against the protein expressed in Escherichia coli successfully immunoprecipitated rat brain PI 4-kinase. The messenger RNA for PI 4-kinase was found predominantly in brain and rat neural cell lines. This PI kinase may play a specific role in neural signal transduction.     

Evidence that phosphorylase kinase exhibits phosphatidylinositol kinase activity

Phosphorylase kinase phosphorylates the pure phospholipid phosphatidylinositol. Furthermore, it catalyzed phosphatidylinositol 4-phosphate formation using as substrate phosphatidylinositol that is associated with an isolated trypsin-treated Ca2+-transport adenosinetriphosphatase (ATPase) preparation from skeletal muscle sarcoplasmic reticulum. On this basis a fast and easy assay was developed that allows one to follow the phosphatidylinositol kinase activity during a standard phosphorylase kinase preparation. Both activities are enriched in parallel approximately to the same degree. Neither chromatography on DEAE-cellulose nor that on hydroxyapatite in the presence of 1 M KCl separates phosphatidylinositol kinase from phosphorylase kinase. The presence of a lipid kinase, phosphatidylinositol kinase, in phosphorylase kinase is not a general phenomenon; diacylglycerol kinase can be easily separated from phosphorylase kinase. Polyclonal anti-phosphorylase kinase antibodies as well as a monoclonal antibody directed specifically against the alpha subunit of phosphorylase kinase immunoprecipitate both phosphorylase kinase and phosphatidylinositol kinase.     

Serum of lipopolysacharide-treated mice contains two types of colony-stimulating factor,separable by affinity chromatography

Serum from mice traated with bacterial lipopolysaccharide (LPS) was fractionated by Con A-Sepharose affinity chromatography, and assayed in vitro for colony-stimulating factor (CSF) using mouse bone marrow cells. The CSF failing to bind to concanavalin A-Sepharose (pool A) had similar biological properties to the unfractionated serum, i.e., it stimulated the formation of about equal numbers of granulocytic, mixed granulocyte-macrophage and macrophage colonies. The fraction eluted from the Con A-Sepharose column with α-methyl-D-glucopyranoside (pool B) had a steeper dose-response curve than either the unfractionated serum or the pool A CSF and most of the colonies were composed of macrophages. A mixture of the pool A and pool B CSFs stimulated colonies in a similar way as unfractionated serum and pool A. The apparent molecular weights of the two types of CSF were determined by two different gel-filtration procedures. Sephacryl S-200 gel-filtration suggested an apparent molecular weight of 85,000 for pool A CSF and 180,000 for pool B CSF. Gel-filtration on Sepharose CL-6B in the presence of guanidine hydrochloride (6M) yielded an apparent molecular weight of approximately 23,000 for pool A CSF and 33,000 for pool B CSF. The colony-forming cells (CFC) responding to pool B CSF were found to have a relatively high sedimentation velocity (peak sedimentation velocity 5.6–6.2 mm/hr) compared to the CFC responding to mouse-lung conditioned medium (MLCM) whose peak sedimentation velocity was between 4.0–4.5 mm/hour. The CFC responding to pool A CSF had an intermediate sedimentation velocity (peak 4.6–5.2 mm/hour). A time-course analysis of the morphology of clones or colonies in cultures stimulated with either MLCM or pool B CSF showed that the proporation of different colony types depends significantly on the incubation period and suggested that pool B CSF induced an early commitment of CFC towards macrophage differentiation.     

Association of phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities with the cytoskeleton in human platelets

The inositol lipid kinases were investigated in the cytoskeletons of human platelets. In the absence of added lipids the kinases were only barely detectable in the Triton-soluble fractions and undetectable in cytoskeletons of resting cells. However at least 30% of the total phosphatidylinositol kinase was present in the cytoskeleton as revealed by saturation of the enzyme. Phosphatidylinositol 4-phosphate kinase was also found in significant amounts in the cytoskeletons. On the other hand, both enzymes being only recovered in the particulate fraction of the cells, we suggest that inositol lipid kinases may be present near the anchoring points of the cytoskeletons at the membranes.     

Coated vesicles contain a phosphatidylinositol kinase

When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism.     

Membrane-associated phosphatidylinositol kinase from Saccharomyces cerevisiae

Membrane-associated phosphatidylinositol kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was partially purified 93-fold from Saccharomyces cerevisiae. Activity was dependent on magnesium ions (10 mM) and the optimum pH was 8.5. The apparent Km values for ATP and phosphatidylinositol were 0.21 mM and 71 microM, respectively. Activity was stimulated by sodium cholate and inhibited by sodium, potassium, lithium, and fluoride ions.