Purification and characterization of phosphatidylinositol kinase from Saccharomyces cerevisiae

The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 8,000-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of microsomal membranes, DE-52 chromatography, hydroxylapatite chromatography, octyl-Sepharose chromatography, and two consecutive Mono Q chromatographies. The procedure resulted in the isolation of a protein with a subunit molecular weight of 35,000 that was 96% of homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidylinositol kinase activity was associated with the purified Mr 35,000 subunit. Maximum phosphatidylinositol kinase activity was dependent on magnesium ions and Triton X-100 at pH 8. The true Km values for phosphatidylinositol and MgATP were 70 microM and 0.3 mM, and the true Vmax was 4,750 nmol/min/mg. The turnover number for the enzyme was 166 min-1. Results of kinetic and isotopic exchange reactions indicated that phosphatidylinositol kinase catalyzed a sequential Bi Bi reaction mechanism. The enzyme bound to phosphatidylinositol prior to ATP and phosphatidylinositol 4-phosphate was the first product released in the reaction. The equilibrium constant for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 31.5 kcal/mol, and the enzyme was thermally labile above 30 degrees C. Phosphatidylinositol kinase activity was inhibited by calcium ions and thioreactive agents. Various nucleotides including adenosine and S-adenosylhomocysteine did not affect phosphatidylinositol kinase activity.     

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.     

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.     

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.     

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.     

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.     

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.     

Purification and reconstitution of phosphatidylinositol 4-kinase from human erythrocytes.

A membrane-bound phosphatidylinositol 4-kinase (PtdIns kinase) has been purified to apparent homogeneity from human erythrocytes. Enzyme activity was solubilized from urea-KCl-stripped, inside-out membrane vesicles by 3% Triton X-100. Purification to apparent homogeneity was accomplished by cation-exchange chromatography on phosphocellulose, followed by heparin-acrylamide chromatography. This resulted in a nearly 3900-fold purification of PtdIns kinase activity to a specific activity of 44 nmol min-1 mg-1. The purified enzyme has an Mr of 59,000 on silver-stained SDS-PAGE; however, many preparations also contain 54 kDa and 50 kDa proteins which are related to the 59 kDa protein and have PtdIns kinase activity. Kinetic analysis of the PtdIns kinase indicate apparent Km values of 40 and 35 microM for phosphatidylinositol and ATP, respectively. The purified enzyme has been reconstituted into phospholipid liposomes and shown to phosphorylate phosphatidylinositol.     

Separation of the protein-tyrosine kinase and phosphatidylinositol kinase activities of the human placental insulin receptor

On immunoprecipitation using a specific antiphosphotyrosine antibody, phosphatidylinositol kinase (EC 2.7.1.67) activity was separated from the protein-tyrosine kinase (EC 2.7.1.112) activity of the wheat germ agglutinin (WGA) -purified insulin receptor from human placenta. This clearly indicates that protein-tyrosine kinase and phosphatidylinositol kinase activity do not reside on the same polypeptide chain as previously has been suggested. Quantitatively, the fraction of phosphatidylinositol kinase that was bound to WGA sepharose and eluted together with the insulin receptor amounted to 2% of the Triton X-100 soluble phosphatidylinositol kinase. The apparent Km values of the bound and unbound phosphatidylinositol kinase with respect to PI and ATP were very similar (0.4 and 0.3 mmol/l and 8 and 7 mumol/l, respectively) suggesting that the WGA-bound phosphatidylinositol kinase is not a different enzyme, but rather represents a small portion of the bulk Triton X-100-soluble phosphatidylinositol kinase that is bound to the lectin tightly associated with the insulin receptor. The synthetic polymer (Glu80Tyr20)n, a model substrate of the insulin receptor tyrosine kinase, at 0.5 mmol/l, inhibited phosphatidylinositol kinase of WGA-purified insulin receptor by 70-90%. This inhibition was not overcome by increasing the concentrations of ATP or PI as one would expect if a functional interrelationship of the protein-tyrosine kinase and the phosphatidylinositol kinase would exist.     

Purification and characterization of a soluble phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae.

A phosphatidylinositol (PI) 4-kinase was purified 25,000-fold from the cytosolic fraction of extracts from the yeast Saccharomyces cerevisiae. The purification consisted of an ammonium sulfate fractionation followed by chromatography on sulfonated-agarose (S-Sepharose), phosphocellulose, threonine-agarose, and quaternary amino (Mono Q), and sulfonated (Mono S) beads. Major contaminants in the purification, Hsc82 and Hsp82 (yeast homologs of the mammalian heat shock protein Hsp90), were eliminated by using a combination of molecular genetics (to construct a null mutation in HSC82), altered growth conditions (to minimize expression from the inducible HSP82 gene), and high ionic strength fractionation conditions (to remove the residual Hsp82). The purified enzyme had an apparent subunit molecular weight of 125,000, much larger than any other well characterized PI-4-kinase reported previously. Like mammalian PI-4-kinases, the yeast enzyme specifically phosphorylated PI on position 4 of the inositol ring and was stimulated by Triton X-100. However, activity was not inhibited by adenosine, a potent inhibitor of certain (type II) mammalian PI-4-kinases. The enzyme displayed typical Michaelis-Menten kinetics with apparent Km values of 100 microM for ATP and 50 microM for PI. To date, this yeast enzyme is the first soluble PI-4-kinase purified from any source.     

The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes

Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.     

Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae

Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity was associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.     

Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets

The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.     

Purification, characterization, and kinetic analysis of a 55-kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae.

A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae. The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromatography with DE52, hydroxylapatite I, Q-Sepharose, Mono Q, and hydroxylapatite II. The procedure resulted in a nearly homogeneous 55-kDa phosphatidylinositol 4-kinase preparation. The 55-kDa phosphatidylinositol 4-kinase and the previously purified 45-kDa phosphatidylinositol 4-kinase differed with respect to their amino acid composition, isoelectric points, and peptide maps. Furthermore, the two forms of phosphatidylinositol 4-kinase did not show an immunological relationship. Maximum 55-kDa phosphatidylinositol 4-kinase activity was dependent on magnesium (10 mM) or manganese (0.5 mM) ions and Triton X-100 at the pH optimum of 7.0. The activation energy for the reaction was 12 kcal/mol, and the enzyme was labile above 30 degrees C. The enzyme was inhibited by thioreactive agents, MgADP, and calcium ions. A detailed kinetic analysis of the purified enzyme was performed using Triton X-100/phosphatidylinositol-mixed micelles. 55-kDa phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol and followed surface dilution kinetics. The interfacial Michaelis constant (Km) and the dissociation constant (Ks) for phosphatidylinositol in the Triton X-100 micelle surface were 1.3 mol % and 0.035 mM, respectively. The Km for MgATP was 0.36 mM. 55-kDa phosphatidylinositol 4-kinase catalyzed a sequential reaction mechanism as indicated by the results of kinetic and isotopic exchange reactions. The enzyme bound to phosphatidylinositol before ATP and released phosphatidylinositol 4-phosphate before ADP. The enzymological and kinetic properties of the 55-kDa phosphatidylinositol 4-kinase differed significantly from those of the 45-kDa phosphatidylinositol 4-kinase. This may suggest that the two forms of phosphatidylinositol 4-kinase from S. cerevisiae are regulated differentially in vivo.     

A phospholamban protein phosphatase activity associated with cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic phospholamban protein phosphatase activity, which is also effective in dephosphorylating phosphorylase a. The phosphatase associated with sarcoplasmic reticulum membranes was solubilized with Triton X-100 and subjected to chromatography on Mono Q HR 5/5 and polylysine-agarose. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. Thermal denaturation of the enzyme resulted in progressive and coincident loss of both phospholamban and phosphorylase a phosphatase activities. Enzymic activity was partially inhibited by protein phosphatase inhibitor 1. Migration of the enzyme during sucrose density gradient ultracentrifugation corresponded to a globular protein with an apparent Mr of 46,000. This enzyme preparation could dephosphorylate both the calcium-calmodulin-dependent as well as the cAMP-dependent sites on phospholamban. Thus, dephosphorylation of phospholamban by this sarcoplasmic reticulum-associated phosphatase may participate in modulating sarcoplasmic reticulum function in cardiac muscle.     

A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae. Purification and characterization

We have recently described a luminal guanosine diphosphatase activity in Golgi-like vesicles of Saccharomyces cerevisiae (Abeijon, C., Orlean, P., Robbins, P. W., and Hirschberg, C. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6935-6939). The presumed in vivo role of this enzyme is to convert GDP into GMP. GDP is a reaction product following outer-chain mannosylation of luminal proteins and a known inhibitor of mannosyltransferases. It is hypothesized that GMP then returns to the cytosol. We have purified this enzyme to apparent homogeneity. Following solubilization from a membrane pellet using a buffer containing Triton X-100, the enzyme was purified on a concanavalin A-Sepharose column followed by Mono Q fast protein liquid chromatography (FPLC) and Superose-12 FPLC columns. After treatment with endoglycosidase H, the deglycosylated active enzyme was applied to a second Mono Q FPLC column and a phenyl-Superose FPLC column. The final enzyme activity was enriched 6500-fold over that of the Triton X-100 extract. The apparant molecular mass of the deglycosylated enzyme is 47 kDa. The purified enzyme is highly specific for guanosine diphosphate, requires Ca2+ for maximal activity, and has a broad pH optimum between 7.4 and 8.2. The apparent Km for GDP is 0.1 mM; the Vmax is 4.9 mmol/min/mg of protein. An enzyme activity with similar substrate specificity has also been detected in membranes of Schizosaccharomyces pombe.     

Purification of Phosphatidylinositol Synthetase from Rat Brain by CDP-Diacylglycerol Affinity Chromatography and Properties of the Purified Enzyme

A purification procedure for rat brain phosphatidylinositol synthetase (PI synthetase; CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase; EC 2.7.8.11) is described. The enzyme was purified 200-250-fold from the homogenate by solubilization with Triton X-100 from microsomal membranes and affinity chromatography on CDP-diacylglycerol-Sepharose. Elution of enzyme activity required the presence of Triton X-100, CDP-diacylglycerol, and either phosphatidylcholine or asolectin. The product that was obtained in 5-10% yield from whole brain and in 70% yield from the microsomal fraction contained three protein bands as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation contained levels of CDP-diacylglycerol hydrolase and CDP-diacylglycerol: sn-glycero-3-phosphate 3-phosphatidyltransferase activities that were less than 1% of PI synthetase activity. The purified enzyme displayed a pH optimum of 8.5-9.0, required either Mg2+ or Mn2+ and exhibited a Km of 4.6 mM for myo-inositol.     

Purification and characterization of a phosphatidylinositol kinase from A431 cells

A phosphatidylinositol kinase from A431 cells has been purified to near homogeneity. Purification was achieved through the use of a combination of chromatography steps including affinity elution of the enzyme from a heparin-agarose column with PI. Characterization of the [32P]PIP formed by the purified PI kinase indicates that the enzyme phosphorylates the inositol on the 4-position and is therefore a phosphatidylinositol 4-kinase. The enzyme has a subunit weight of 55,000 as estimated by SDS gel electrophoresis and appears to be active as a monomer. Studies of the hydrodynamic properties of the enzyme indicate that the PI kinase binds substantial amounts of Triton X-100 and is actually present in detergent-containing solutions as a complex with a molecular weight of approximately 120,000. The Km of the enzyme for PI is 16 microM and for ATP is 74 microM. The enzyme is inhibited by adenosine with an IC50 of 100 microM. These properties are essentially identical with those of the membrane-bound PI kinase in A431 cells which is stimulated by EGF. The data therefore suggest that the EGF-stimulated PI kinase is a 55,000-Da monomer.     

Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes

Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis.     

Characterization of the somatogenic receptor in rat liver. Hydrodynamic properties and affinity cross-linking

Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H2O and D2O, and affinity cross-linking using 125I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of 125I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble 125I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the 125I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity. Furthermore, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (first dimension, nonreducing and second dimension, reducing) showed that a disulfide-linked binder at Mr 43,000 is contained within the Mr 86,000 species. As with pregnant rats, female and male rats both showed 125I-bovine growth hormone binders of Mr 95,000, 84,000, 55,000, 43,000, and additionally an Mr 35,000 binder.