A Nucleoside Diphosphate Kinase from Paramecium tetraurelia with Protein Kinase Activity

Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.     

Nucleoside triphosphate requirements for superoxide generation and phosphorylation in a cell-free system from human neutrophils. Sodium dodecyl sulfate and diacylglycerol activate independently of protein kinase C.

The NADPH-oxidase of human neutrophils can be activated in a cell-free system comprised of plasma membrane, cytosol, and an anionic amphiphile such as arachidonate or sodium dodecyl sulfate (SDS). Recently, we showed that diacylglycerol acts synergistically with SDS in the cell-free system to stimulate superoxide generation, with concurrent phosphorylation of a 47-kDa cytosolic protein which is thought to be a component of the oxidase (Burnham, D. N., Uhlinger, D. J., and Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559). We report herein that when undialyzed cytosol is used along with either SDS alone or SDS plus diacylglycerol as activators, adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) both stimulated superoxide generation several fold, yielding about the same maximal velocity. ATP and GTP showed lower levels of stimulation. Stimulation by ATP gamma S and GTP gamma S was nonadditive, and showed a 5-7-fold greater specificity for GTP gamma S. ATP gamma S stimulation was inhibited by the nucleoside diphosphate (NDP) kinase inhibitor UDP. In contrast, when extensively dialyzed cytosol was used, most of the stimulation by ATP gamma S was lost, while most of that by GTP gamma S was retained. Addition of GDP restored the ability of ATP gamma S to stimulate, consistent with NDP kinase-catalyzed formation of GTP gamma S from ATP gamma S plus GDP. This activity was demonstrated directly in both cytosol and plasma membrane. Using undialyzed cytosol, phosphorylation of p47 showed a similar nonspecificity for nucleoside triphosphates, due to NDP kinase activity, but revealed the expected ATP specificity when dialyzed cytosol was used. Neither ATP gamma S nor GTP gamma S were good substrates for protein phosphorylation. Under a variety of conditions, phosphorylation of p47 or other neutrophil proteins failed to correlate with oxidase activation. The present studies indicate that SDS and diacylglycerol stimulation of superoxide generation in the cell-free system is independent of protein kinase C or other protein kinase activity, and suggest a novel role for diacylglycerol in cell regulation.     

Purification and characterization of nucleoside diphosphate kinase from spinach leaves.

Two types of nucleoside diphosphate kinase (NDP kinase I and NDP kinase II) have been purified from spinach leaves to electrophoretic homogeneity. The enzymes were copurified with apparent [35S]GTP-gamma S-binding activities. NDP kinase I, which was not adsorbed to a hydroxyapatite column, and NDP kinase II, which was adsorbed, had molecular weights of 16,000 and 18,000, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights determined by gel filtration were 92,000 and 110,000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor differences in some amino acids between NDP kinase I and NDP kinase II were observed when both enzymes were analyzed for amino acid composition. The apparent [35S]GTP gamma S-binding activity of purified NDP kinase I and NDP kinase II was found to be due to the formation of a [35S]thiophosphorylated enzyme, which is the intermediate of the NDP kinase reaction.     

Purification, properties and specificity of a NDP kinase from Alyssum murale grown under Ni(2+) toxicity

During the growth of Alyssum murale, a nickel accumulator plant, three root peptides chains of 55, 18 and 16kDa undergo phosphorylation. The intensity of the phosphorylated bands decreased in the course of growth in nutrient solution supplied with 0.5mM Ni(2+). In the shoot only two phosphorylated peptide chains with a size of 18 and 16kDa were detected. These two shoot peptides disappeared on the 19th day of growth in Ni(2+)-exposed plants, while the root peptide of 16kDa continued to be present in less intensity. This peptide was identified as the catalytic subunit of nucleoside diphosphate kinase (NDP kinase: E.C. 2.7.4.6) and was named NDPK-B. The enzyme was purified by means of ammonium sulphate precipitation, DEAE-sepharose and hydroxyapatite column chromatography. NDPK-B was thermostable, displayed a molecular mass of 103,000 and was comprised of six catalytic subunits. The autophosphorylated enzyme displayed an isoelectric point (pI) of 6.5. The NDPK-B autophosphorylation activity was metal-dependent. With regard to the transfer reaction, NDPK-B exhibited the following properties: (a) the enzyme had an optimum pH of 7.6; (b) it was capable of using both (gamma-(32)P) ATP and (gamma-(32)P) GTP as phosphate donors and of using all the available NDPs except dCDP as phosphate acceptors; (c) its activity using NDPs as substrates was metal dependent; (d) in the presence of (gamma-(32)P) GTP as the phosphate donor, it phosphorylated exclusively ADP when a mixture of NDPs was added in the reaction mixture; and, (e) ADP had a very low K(m) value towards 8.4nM. This high affinity towards ADP suggests that the enzyme may play a crucial function in the formation of the amount of ATP necessary for Alyssum murale to survive Ni(2+) stress.     

Biochemical characteristics of bovine retina nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDP kinase; ATP: NDP phosphotransferase; EC 2.7.4.6) was purified from bovine retina. The molecular mass of the native enzyme was found to be 72 kDa, and those of its subunits were 17.5 and 18.5 kDa. Kinetic characteristics of the enzyme were determined. It was shown that NDP kinase exists in retina in both soluble and membrane-bound forms.     

Kinetics of activation of phospholipase C by P2Y purinergic receptor agonists and guanine nucleotides

Membranes prepared from [3H]inositol-labeled turkey erythrocytes express a phospholipase C that is markedly stimulated by stable analogs of GTP (Harden, T. K., Stephens, L., Hawkins, P. T., and Downes, C. P. (1987) J. Biol. Chem. 262, 9057-9061). We now report that P2-purinergic receptor-mediated regulation of the enzyme occurs in the membrane preparation. The order of potency of a series of ATP and ADP analogs for stimulation of inositol phosphate formation, i.e. 2-methylthioadenosine 5'-triphosphate (2MeSATP) greater than adenosine 5'-O-(2-thiodiphosphate) greater than adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than 5'-adenylyl imidodiphosphate approximately ADP greater than alpha, beta-methyleneadenosine 5'-triphosphate greater than beta, gamma-methyleneadenosine 5'-triphosphate, was consistent with that for the P2Y-purinergic receptor subtype. Agonist-stimulated effects were completely dependent on the presence of guanine nucleotide. Activation of phospholipase C by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) occurred with a considerable time lag. The rate of activation followed first order kinetics and was markedly increased by increasing concentrations of a P2Y receptor agonist; in contrast, the rate of activation at a fixed agonist concentration was independent of guanine nucleotide concentration. Addition of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) prior to addition of agonist and GTP, 5'-guanylyl imidodiphosphate (Gpp(NH)p), or GTP gamma S blocked in a concentration-dependent manner the stimulatory effect of guanine nucleotide. GDP beta S, added subsequent to preactivation of membranes with 2MeSATP and GTP gamma S or Gpp(NH)p had only small inhibitory effects on the rate of inositol phosphate production observed over the subsequent 10 min. In contrast, addition of GDP beta S to GTP-preactivated membranes resulted in a rapid return of enzyme activity to the basal state within 60 s. Taken together, the data are consistent with the idea that P2Y receptor activation increases the rate of exchange of GTP and GTP analogs for GDP on the relevant guanine nucleotide regulatory protein. Once the active enzymic species is formed, hydrolysis of guanine nucleotide reverts the enzyme to the inactive state.     

Structural and functional studies of cross-linked Go protein subunits

The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.     

Identification of a ribosomal protein S6 kinase regulated by transformation and growth-promoting stimuli

A serine protein kinase specific for ribosomal protein S6 in 40 S subunits has been identified and purified greater than 15,000-fold (with 18% recovery) from developing chicken embryos. An analogous enzyme has also been detected in serum-stimulated chicken embryo fibroblasts. The S6 kinase was identified as a phosphoprotein of Mr approximately 65,000 based on (i) gel filtration, (ii) apparent autophosphorylation of a 65-kDa protein when several enzyme preparations were incubated with [gamma-32P]ATP in the absence of added substrate, (iii) comigration of S6 kinase activity with the autophosphorylating activity over a variety of chromatographic resins, and (iv) elution and renaturation of S6 kinase activity from the 65-kDa region of a sodium dodecyl sulfate-polyacrylamide gel. The purified protein kinase is highly specific for S6 in 40 S subunits and does not appreciably phosphorylate casein, histone H1, mixed histones, protamine, polyoma virus capsid protein, or phosphorylase a/b. These characteristics suggest that this enzyme is unrelated to other protein kinases believed to be activated in stimulated cells, including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), or Ca2+/calmodulin-dependent protein kinases. In fibroblasts, S6 kinase is activated by a variety of mitogenic agents including the tyrosine-specific protein kinase of Rous sarcoma virus, pp60v-src, phorbol esters, and growth factors. The present identification and purification of the S6 kinase should facilitate future studies aimed at elucidating the molecular mechanisms by which signals from these diverse stimuli rapidly converge upon and activate this enzyme.     

Clathrin-coated vesicles contain two protein kinase activities. Phosphorylation of clathrin beta-light chain by casein kinase II

Incubation of clathrin-coated vesicles with Mg2+-[gamma-32P]ATP results in the autophosphorylation of a 50-kDa polypeptide (pp50) (Pauloin, A., Bernier, I., and Jollès, P. (1982) Nature 298, 574-576). We describe here a second protein kinase that is associated with calf brain and liver coated vesicles. This kinase, which phosphorylates casein and phosvitin but not histone and protamine using either ATP or GTP, co-fractionates with coated vesicles as assayed by gel filtration, electrophoresis, and sedimentation. The enzyme can be extracted with 0.5 M Tris-HCl or 1 M NaCl, and can be separated from the pp50 kinase as well as the other major coat proteins. We identified this enzyme as casein kinase II based on physical and catalytic properties and by comparative studies with casein kinase II isolated from brain cytosol. It has a Stokes radius of 4.5 nm, a catalytic moiety of approximately 45 kDa, and labels a polypeptide of 26 kDa when the pure enzyme is assayed for autophosphorylation. Its activity is inhibited by heparin and not affected by cAMP, phospholipids, or calmodulin. This protein kinase preferentially phosphorylates clathrin beta-light chain. The phosphorylation is markedly stimulated by polylysine and inhibited by heparin. Isolated beta-light chain as well as beta-light chain in triskelions or in intact coated vesicles is phosphorylated. All of the phosphate (0.86 mol of Pi/mol of clathrin beta-light chain) is incorporated into phosphoserine.     

Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins

A direct interaction of alpha beta gamma trimeric GTP binding proteins (G proteins; G0 and Gs) with nucleoside diphosphate kinase (NDP kinase) was investigated with homogeneously purified proteins. There was a progressive release of 32Pi from [gamma-32P]ATP when GDP-bound G0 was incubated together with NDP kinase. The Pi release induced by the interaction of G0 with NDP kinase was not accompanied by the dissociation of GDP bound to the alpha-subunit of G0. This was a sharp contrast to G protein-catalyzed GTP hydrolysis observed with GTP as the substrate; the dissociation of bound GDP was essentially required for the following binding of the substrate, GTP, to be hydrolyzed. A kinetic analysis displayed different properties for the substrate of NDP kinase between free GDP and G protein-bound GDP. NDP kinase-dependent phosphorylation of GDP on G0 was indeed demonstrated with adenosine 5'-(3-O-thio)triphosphate as the phosphate donor; there was a formation of guanosine 5'-(3-O-thio)triphosphate-bound G0 from the ATP analogue. Moreover, purified Gs was readily ADP-ribosylated by cholera toxin in the presence of NDP kinase, ATP, and an ADP-ribosylation factor, also suggesting that the nucleotide form on Gs was certainly GTP. These results indicate that NDP kinase can transfer the gamma-phosphate of ATP directly to GDP bound to G proteins and that this phosphorylation results in the activation of the signal-coupling proteins. A possible role of the new activation mechanism of G proteins is discussed in comparison with the previously characterized GDP-GTP exchange pathway by the agonist-receptor complex.     

Identification in bovine liver plasma membranes of a Gq-activatable phosphoinositide phospholipase C.

Phosphoinositide phospholipase C (PLC) activity extracted from bovine liver plasma membranes with sodium cholate was stimulated by GTP gamma S-activated G alpha q/G alpha 11, whereas the enzyme from liver cytosol was not. The membrane-associated PLC was subjected to chromatography on heparin-Sepharose, Q Sepharose, and S300HR, enabling the isolation of the G-protein stimulated activity and its resolution from PLC-gamma and PLC-delta. Following gel filtration, two proteins of 150 and 140 kDa were found to correspond to the activatable enzyme. These proteins were identified immunologically as members of the PLC-beta family and were completely resolved by chromatography on TSK Phenyl 5PW. The 150-kDa enzyme was markedly responsive to GTP gamma S-activated alpha-subunits of G alpha q/G alpha 11 or to purified Gq/G11 in the presence of GTP gamma S. The response of this PLC was of much greater magnitude than that of the 140-kDa enzyme. The partially purified 150-kDa enzyme showed specificity for PtdIns(4,5)P2 and PtdIns4P as compared to PtdIns and had an absolute dependence upon Ca2+. These characteristics were similar to those of the brain PLC-beta 1. The immunological and biochemical properties of the 150-kDa membrane-associated enzyme are consistent with its being the PLC-beta isozyme that is involved in receptor-G-protein-mediated generation of inositol 1,4,5-triphosphate in liver.     

5-Hydroxytryptamine stimulates inositol phosphate production in a cell-free system from blowfly salivary glands. Evidence for a role of GTP in coupling receptor activation to phosphoinositide breakdown

Phosphoinositide breakdown has been linked to the receptor mechanism involved in the elevation of cytosolic Ca2+. In a cell-free system prepared from [3H] inositol-labeled blowfly salivary glands, 5-hydroxytryptamine stimulated the rapid production of inositol phosphates. Within 30 s of hormone addition, there was a 100% increase in inositol trisphosphate formation, a 70% increase in inositol bisphosphate formation, and a 90% increase in inositol monophosphate formation as compared to control homogenates incubated for the same length of time. 5-Hydroxytryptamine did not stimulate inositol or glycerol phosphoinositol formation. Half-maximal activation of inositol phosphate production was obtained with 0.33 microM 5-hydroxytryptamine. Ethylene glycol bis(beta-aminoethyl ether)-N',N',N',N'-tetraacetic acid, (EGTA) (0.3 mM) inhibited the basal formation of inositol phosphates and decreased the net accumulation of inositol bisphosphate and inositol trisphosphate due to hormone as compared to homogenates incubated in the absence of added Ca2+. EGTA, however, had little effect on the per cent stimulation of inositol phosphate production due to hormone. In homogenates, ATP, GTP or guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was required for a hormone effect. Gpp(NH)p, unlike ATP or GTP, increased the basal formation of inositol phosphates. In membranes, GTP, Gpp(NH)p, or guanosine 5'-(3-O-thio)trisphosphate (GTP gamma S) sustained a hormone effect whereas ATP was ineffective. GTP did not affect production while Gpp(NH)p and GTP gamma S increased inositol phosphate production. Half-maximal effects of Gpp(NH)p and GTP gamma S on hormone-stimulated inositol phosphate formation occurred at 10 microM and 100 nM, respectively. In the presence of 1 microM GTP gamma S, 5-methyltryptamine stimulated inositol phosphate formation within 2 s in membranes. These results indicate that in a cell-free system, GTP is involved in mediating the effects of Ca2+-mobilizing hormones on phosphoinositide breakdown.     

Regulation of protein phosphorylation in Dictyostelium discoideum

We have examined the phosphorylation of the cyclic adenosine 3':5' monophosphate (cAMP) cell surface chemotactic receptor and a 36 kDa membrane-associated protein (p36) in Dictyostelium discoideum. The activity of CAR-kinase, the enzyme responsible for the phosphorylation of the cAMP receptor, was studied in plasma membrane preparations. It was found that, as in intact cells, the receptor was rapidly phosphorylated in membranes incubated with [gamma 32P] adenosine triphosphate (ATP) but only in the presence of cAMP. This phosphorylation was not observed in membranes prepared from cells which did not display significant cAMP binding activity. cAMP could induce receptor phosphorylation at low concentrations, while cyclic guanosine 3':5' monophosphate (cGMP) could elicit receptor phosphorylation only at high concentrations. Neither ConA, Ca2+, or guanine nucleotides had an effect on CAR-kinase. It was also observed that 2-deoxy cAMP but not dibutyryl cAMP induced receptor phosphorylation. The data suggest that the ligand occupied form of the cAMP receptor is required for CAR-kinase activity. Although the receptor is rapidly dephosphorylated in vivo, we were unable to observe its dephosphorylation in vitro. In contrast, p36 was rapidly dephosphorylated. Also, unlike the cAMP receptor, the phosphorylation of p36 was found to be regulated by the addition of guanine nucleotides. Guanosine diphosphate (GDP) enhanced the phosphorylation while guanosine triphosphate (GTP) decreased the radiolabeling of p36 indicating that GTP can compete with ATP for the nucleotide triphosphate binding site of p36 kinase. Thus was verified using radiolabeled GTP as the phosphate donor. Competition experiments with GTP gamma S, ATP, GTP, CTP, and uridine triphosphate (UTP) indicated that the phosphate donor site of p36 kinase is relatively non-specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a maturation-activated myelin basic protein kinase from sea star oocytes

A meiosis-activated myelin basic protein (MBP) kinase was purified approximately 8700-fold from soluble post-germinal vesicle breakdown extracts from maturing oocytes of the sea star Pisaster ochraceus. Purification to apparent homogeneity was achieved by sequential chromatography on DEAE-cellulose, hydroxylapatite, phosphocellulose, phenyl-Sepharose, heparin-Sepharose, polylysine-Sepharose, and Mono-Q. The final product exhibited an apparent molecular mass of approximately 42 kDa by both native gradient and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this precisely correlated with the chromatographic behavior of the recovered MBP kinase activity on a Superose 6/12 column. The kinase utilized the MBP as the major substrate with little or no phosphorylation of histones (H1, H2A, or H2B), casein, phosvitin, protamine, or 40 S ribosomal proteins. The purified enzyme was relatively insensitive to high concentrations of beta-glycerol phosphate, calmodulin, EGTA, NaCl, sodium fluoride, dithiothreitol, spermine, and heparin but was quite sensitive to inhibition by metal ions such as Mn2+, Zn2+, and Ca2+. The true Km values for ATP and myelin basic protein were determined to be 58 and 25 microM, respectively, using double-reciprocal plots. The purified enzyme was unable to utilize GTP in place of ATP. The enzyme was shown to rapidly undergo autophosphorylation. The autophosphorylation was sensitive to alkali treatment implying that phosphate was incorporated on serine/threonine residues. The properties of this MBP kinase are reminiscent of a protein kinase that is also activated in a cyclic fashion at M-phase during the early cell divisions of sea star and sea urchin embryos (Pelech, S. L., Tombe, R., Meijer, L., and Krebs, E. G. (1988) Dev. Biol. 130, 26-36).     

Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity.

A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed. The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system. In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast. In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method was used to detect NDP kinase contamination in commercial enzyme preparations.     

Nucleoside diphosphate kinase activity in soluble transducin preparations biochemical properties and possible role of transducin-beta as phosphorylated enzyme intermediate.

Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17-23-kDa subunits and catalyze the reaction N1TP + N2DP --> N1DP + N2TP via formation of a histidine-phosphorylated enzyme intermediate. NDPKs are involved in the activation of heterotrimeric GTP-binding proteins (G-proteins) by catalyzing the formation of GTP from GDP, but the properties of G-protein-associated NDPKs are still incompletely known. The aim of our present study was to characterize NDPK in soluble preparations of the retinal G-protein transducin. The NDPK is operationally referred to as transducin-NDPK. Like known NDPKs, transducin-NDPK utilizes NTPs and phosphorothioate analogs of NTPs as substrates. GDP was a more effective phosphoryl group acceptor at transducin-NDPK than ADP and CDP, and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a more effective thiophosphoryl group donor than adenosine 5'-[gamma-thio]triphosphate (ATP[S]). In contrast with their action on known NDPKs, mastoparan and mastoparan 7 had no stimulatory effect on transducin-NDPK. Guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) potentiated [3H]GTP[S] formation from [3H]GDP and ATP[S] but not [3H]GTP[S] formation from [3H]GDP and GTP[S]. Depending on the thiophosphoryl group acceptor and donor, [3H]NTP[S] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. [gamma-32P]ATP and [gamma-32P]GTP [32P]phosphorylated, and [35S]ATP[S] [35S]thiophosphorylated, a 36-kDa protein comigrating with transducin-beta. p[NH]ppG potentiated [35S]thiophosphorylation of the 36-kDa protein. 32P-labeling of the 36-kDa protein showed characteristics of histidine phosphorylation. There was no evidence for (thio)phosphorylation of 17-23-kDa proteins. Our data show the following: (a) soluble transducin preparations contain a GDP-prefering and guanine nucleotide-regulated NDPK; (b) transducin-beta may serve as a (thio)phosphorylated NDPK intermediate; (c) transducin-NDPK is distinct from known NDPKs and may consist of multiple kinases or a single kinase with multiple regulatory domains.     

Putative functions of nucleoside diphosphate kinase in plants and fungi

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red–far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. In Neurospora crassa a Pro72His mutation in NDP kinase (ndk-1 ^Pro72His ) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as light-induced polarity of perithecia. In wild-type in darkness the beak was formed at random places on perithecia, and in ndk ^Pro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases.     

Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells

A protein kinase, termed microtubule-associated protein (MAP) kinase, which phosphorylates microtubule-associated protein 2 (MAP-2) in vitro and is stimulated 1.5-3-fold in extracts from insulin-treated 3T3-L1 cells has been identified (Ray, L.B., and Sturgill, T.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1502-1506). Here, we describe chromatographic properties of MAP kinase and provide biochemical characterization of the partially purified enzyme. Isolation of the enzyme is facilitated by its unusually high affinity for hydrophobic interaction chromatography matrices. The molecular weight of the partially purified enzyme was determined to be 35,000 by gel filtration chromatography and 37,000 by glycerol gradient centrifugation. MAP kinase activity of chromatographic fractions correlated precisely with the presence of a 40-kDa phosphoprotein detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MAP kinase has a Km of 7 microM for ATP and does not utilize GTP. Acetyl-CoA carboxylase, ATP citrate-lyase, casein, histones, phosvitin, protamine, and ribosomal protein S6 were all poor substrates relative to MAP-2. The enzyme is inhibited by fluoride and beta-glycerol phosphate but not by heparin. These properties of MAP kinase distinguish it from protein kinases previously described in the literature.     

Detection of G Proteins in Purified Bovine Brain Myelin

Following a previous report on detection of muscarinic receptors in myelin with the implied presence of G proteins, we now demonstrate by more direct means the presence of such proteins and their quantification. Using [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) as the binding ligand, purified myelin from bovine brain was found to contain approximately half the binding activity of whole white matter (138 +/- 9 vs. 271 +/- 18 pmol/mg of protein). Scatchard analysis of saturation binding data revealed two slopes, a result suggesting at least two binding populations. This binding was inhibited by GTP and its analog but not by 5'-adenylylimidodiphosphate [App(NH)p], GMP, or UTP. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of myelin proteins and blotting on nitrocellulose, [alpha-32P]GTP bound to three bands in the 21-27-kDa range in a manner inhibited by GTP and GTP gamma S but not App(NH)p. ADP-ribosylation of myelin with [32P]NAD+ and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholate extract of myelin subjected to chromatography on a column of phenyl-Sepharose gave at least three major peaks of [35S]GTP gamma S binding activity. SDS-PAGE and immunoblot analyses of peak I indicated the presence of Go alpha, Gi alpha, and Gs alpha. Further fractionation of peak II by diethyl-aminoethyl-Sephacel chromatography gave one [35S]GTP gamma S binding peak with the low-molecular-mass (21-27 kDa) proteins and a second showing two major protein bands of 36 and 40 kDa on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)