Bovine heart pyruvate dehydrogenase kinase stimulation by monovalent ions

The effects of monovalent ions on endogenous pyruvate dehydrogenase (PDH) kinase activity in purified bovine heart pyruvate dehydrogenase complex were investigated. Activity of PDH kinase was stimulated 1.9-, 1.95-, 1.65-, and 1.4-fold by 10 mM K+, Rb+, NH+4, and Cs+, respectively, whereas Na+ and Li+ had no effect on PDH kinase activity. The crystal radii of stimulatory ions were in the range of 1.33 to 1.69 A while the crystal radii of nonstimulatory ions were in the range of 0.6 to 0.94 A. Stimulation of PDH kinase by monovalent ions was not pH dependent. Protein dilution studies showed that monovalent ion stimulation was measurable within 10 s after protein addition to PDH kinase assays. Furthermore, stimulation occurred at all protein concentrations tested. At ATP concentrations from 12.5 to 25 microM, K+ and NH+4 stimulation was constant from 0 to 110 and 0 to 30 mM, respectively. At higher ATP concentrations, from 50 to 500 microM, K+ and NH+4 stimulation peaked at approximately 30 and 3 mM, respectively, and thereafter declined as the ion concentration increased. Maximal PDH kinase stimulation by K+ or NH+4 also declined as Na+ was increased from 0 to 120 mM, but at a fixed salt concentration of 120 mM, both K+ and NH+4 stimulated PDH kinase activity. Phosphopeptide analysis demonstrated that K+ and NH+4 stimulated phosphorylation at sites 1 and 2, but that site 3 phosphorylation was relatively constant under all conditions. Thiamin pyrophosphate and 5,5'-dithiobis-(2-nitrobenzoate) blocked monovalent ion stimulation half-maximally at 4 and 6 microM, respectively. However, neither thiamin pyrophosphate nor 5,5'-dithiobis-(2-nitrobenzoate) significantly inhibited PDH kinase activity in the absence of monovalent ions. The results indicate that heart PDH kinase stimulation by monovalent ions does not occur by changing the binding equilibrium between PDH and dihydrolipoyl transacetylase core. Instead, monovalent ions bind and exert their regulatory effects at or near the active site of PDH kinase.     

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.     

Effects of polyamines and methylglyoxal bis(guanylhydrazone) on Escherichia coli ribonucleic acid polymerase and the template activity of hepatic cell nuclei in vitro

Escherichia coli RNA polymerase was assayed with 4 mM Mg2+ and 1 mM Mn2+ using native DNA, heat-denatured DNA, histone-nucleate and isolated rat liver nuclei as the template source. With purified DNA and either or both divalent metal ions, 0.1--5 mM amine stimulated enzyme activity. Spermidine resulted in the greatest stimulation (1.7-fold at 5 mM); whereas, spermine or methylglyoxal bis(guanylhydrazone) first stimulated, then above 3 mM inhibited, the reaction. The addition of unfractionated histone to purified DNA inhibited the reaction by 90%. The subsequent addition of amines resulted in a slight stimulation in incorporation (1.5-fold) in the range of 1--3 mM amine. Alternatively, when enzyme was combined with DNA before histone, only a 20% inhibition was observed and this could be completely prevented by 3 mM spermidine. The addition of amines to isolated nuclei resulted in marked alterations in ultrastructure and Mg2+ content; however, relatively small effects on RNA polymerase activity were observed. With the E. coli enzyme, 0.1--1.0 mM amine stimulated RNA synthesis (1.5-fold) whereas, none of the amines stimulated endogeneous activity in the absence or presence of 300 mM (NH4)2SO4.     

Purification and characterization of Novikoff ascites tumor protein kinase.

A protein kinase, designed KII, has been purified 5000-fold from Novikoff ascites tumor cells. The purification procedure also allows for the purification of a second major protein kinase, designated KI, as well as RNA polymerase I and II. Purified KII has a sedimentation constant of 7.6 S and a Stokes radius of 39 A, suggesting a molecular weight of about 122000. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate suggests the enzyme is composed of subunits of molecular weights 44 000, 40 000, and 26 000 present in a molar ratio of 1:1:2. Incubation of the enzyme alone in the presence of [gamma-32P]ATP results in the phosphorylation of the 26 000-dalton subunit. Protein kinase II actively phosphorylates phosvitin, casein, and nonhistone chromosomal proteins but does not phosphorylate basic proteins such as histones or protamine to an appreciable extent. Km values of 3.6 micron for ATP and 6.5 micronM for GTP were determined in the presence of 4mM Mg2+. The enzyme is neither stimulated by cyclic adenosine 3',5'-monophosphate or cyclic guanosine 3', 5'-monophosphate nor inhibited by the regulatory subunit of rabbit muscle protein kinase. Its activity is stimulated by KCl at concentrations below 0.2 M and inhibited by higher concentrations.     

The effect of Fe2+ on the activity of human plasma protein kinase

A Fe2+ stimulatory protein kinase in human plasma is demonstrated. This enzyme was partial purified from the 100,000 X g supernatant of human plasma by using (NH4)2SO4 fractionation. The activity of this protein kinase existed in 30-50% fraction. The effect of various compounds on the activity of this partial purified protein kinase was studied. The Fe2+ stimulates 5 to 10-fold of the protein kinase activity. The maximum concentration of Fe2+ to stimulate the activity of this plasma protein kinase was 2 mM. Double reciprocal plot for the stimulation showed that Fe2+ increased the Vm but not the Km for ATP. Other divalent compounds such as MgCl2, Mg(Oac)2, MnCl2 and BaCl2 were less effective.     

Effects of cyclic adenosine 3': 5'-monophosphate on phosphoprotein kinase and phosphatase fractions prepared from rat liver nuclei.

A soluble rat liver nuclear extract containing total RNA polymerase activities also exhibits appreciable amounts of protein kinase activity. This unfractionated protein kinase catalyzes the phosphorylation of both endogenous proteins and exogenous lysine-rich histone in the presence of [γ-³²P]ATP and Mg²⁺. The optimal concentration of Mg²⁺ is 5 mm for histone phosphorylation and 25 mm for the phosphorylation of endogenous proteins. Cyclic AMP has no effect on the phosphorylation of lysine-rich histone by this unfractionated nuclear protein kinase. However, addition of cyclic AMP causes a reduction in the ³²P-labeling of an endogenous protein (CAI) which can be characterized by its mobility during SDS-acrylamide gel electrophoresis and elution in the unbound fraction of a DEAESephadex column. If CAI is first labeled with ³²P and then incubated with 10^?6m cyclic AMP under conditions where protein kinase activity is inhibited, the presence of the cyclic nucleotide causes a loss of the ³²P-labeling of this protein, implying the activation of a substrate-specific protein phosphatase. When rat liver RNA polymerases are purified by DEAE-Sephadex chromatography, protein kinase activity is found in the unbound fraction and in those column fractions containing RNA polymerase I and II. The fractionated protein kinases exhibit different responses to cyclic AMP, the unbound protein kinase being stimulated and the RNA polymerase-associated protein kinases being dramatically inhibited. A second protein (CAII) whose phosphorylated state is modified by cyclic AMP is found within the DEAE-Sephadex column fractions containing RNA polymerase II. The cyclic nucleotide in this case appears to reduce labeling of CAII by inhibition of the protein kinase activity which co-chromatographs with both CAII and RNA polymerase II. Based on molecular weight estimates, neither CAI nor CAII appears to be an RNA polymerase subunit. The identity of CAI as a protein factor whose phosphorylated state influences nuclear RNA synthesis is suggested by the fact that addition of fractions containing CAI to purified RNA polymerase II inhibits the activity of this enzyme, but only if CAI has been previously incubated in the presence of cyclic AMP.     

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.     

Purification of nuclear cAMP-independent protein kinases from rat ventral prostate

Two nuclear cAMP-independent protein kinases (designated PK-N1 and PK-N2) were purified from rat ventral-prostate and liver. The yield of enzyme units was 4-5% and 7-9% for each enzyme from the prostatic nuclei and liver nuclei, respectively. The average fold purification for prostatic nuclear protein kinase N1 and N2 was 1360 and 1833, respectively. The respective average specific activity of the two enzymes towards casein was 81,585 and 110,000 nmol 32P incorporated/hr/mg of enzyme. Protein kinase N1 comprised one polypeptide of Mr 35,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase N2 comprised two polypeptides Mr 40,000 and 30,000 of which only the Mr 30,000 polypeptide was autophosphorylated. Both enzymes were active towards casein, phosvitin, dephosphophosvitin, spermine-binding protein, and non-histone proteins in vitro. Little activity was detected towards histones. Both enzymes were stimulated by 150-200 mM NaCl. MgCl2 requirement varied with the protein substrate but was between 2-4 mM for both enzymes. With dephosphophosvitin as substrate, the apparent Km for ATP for N1 protein kinase was 0.01 mM. GTP did not replace ATP in this reaction. Protein kinase N2 was active in the presence of ATP or GTP. The apparent Km was 0.01 mM for ATP, but 0.1 mM for GTP.     

Demonstration of two protein kinases in extracts of Legionella micdadei

Protein kinases I (PK I) and II (PK II) were purified 253- and 13.5-fold, respectively, from an extract of sonically disrupted cells of Legionella micdadei by ion-exchange chromatography on QAE-Sephadex, by histone affinity chromatography, and by HPLC-gel filtration chromatography. Both enzymes catalysed the phosphorylation of calf thymus histones, with a Km of 2.7 mg ml-1 for PK I and 2.9 mg ml-1 for PK II. Histone H2b was the best protein kinase substrate for both PK I and PK II. The pH optima were 6.8 and 7.0 for PK I and PK II respectively. The Km for ATP was 0.29 mM for PK I and 0.33 mM for PK II. PK II activity was stimulated by either cAMP or cGMP, whereas PK I was inhibited by both cyclic nucleotides. The activity of PK I was unaffected by addition of calmodulin, diacylglycerol and mixtures of Ca2+ and acidic phospholipids, but these additions increased PK II activity threefold. The activity of PK II was stimulated by spermine and spermidine, but PK I was inhibited by these compounds. PK I and PK II were both strongly inhibited by heparin.     

Extracellular calcium stimulates DNA synthesis in synergism with zinc, insulin and insulin-like growth factor I in fibroblasts.

In serum-starved mouse NIH 3T3 fibroblasts cultured in 1.8 mM Ca2+-containing medium, addition of 0.75-2 mM extra Ca2+ stimulated DNA synthesis in synergism with zinc (15-60 microM), insulin and insulin-like growth factor I. Extra Ca2+ stimulated phosphorylation/activation of p42/p44 mitogen-activated protein kinases by an initially (10 min) zinc-independent mechanism; however, insulin, and particularly zinc, significantly prolonged Ca2+-induced mitogen-activated protein kinase phosphorylation. In addition, extra Ca2+ activated p70 S6 kinase by a zinc-dependent mechanism and enhanced the stimulatory effect of zinc on choline kinase activity. Insulin and insulin-like growth factor I also commonly increased both p70 S6 kinase and choline kinase activities. In support of the role of the choline kinase product phosphocholine in the mediation of mitogenic Ca2+ effects, cotreatments with the choline kinase substrate choline (250 microM) and the choline kinase inhibitor hemicholinium-3 (2 mM) enhanced and inhibited, respectively, the combined stimulatory effect of extra Ca2+ (3.8 mM total) and zinc on DNA synthesis. In various human skin fibroblast lines, 1-2 mM extra Ca2+ also stimulated DNA synthesis in synergism with zinc and insulin. The results show that in various fibroblast cultures, high concentrations of extracellular Ca2+ can collaborate with zinc and certain growth factors to stimulate DNA synthesis. Considering the high concentration of extracellular Ca2+ in the dermal layer, Ca2+ may promote fibroblast growth during wound healing in concert with zinc, insulin growth factor-I insulin, and perhaps other growth factors.     

Mouse spleen cell nuclear protein kinases and the stimulating effect of dsDNA on NHP phosphorylation by cyclic AMP-independent protein kinase in vitro

cAMP-dependent (designated as enzyme I, about 68,000 daltons) and cAMP-independent protein kinase (designated as enzyme II, about 45,000 daltons) have been partially purified from the nuclei of mouse spleen cells. Both kinases phosphorylated calf thymus histones as well as non-histone proteins (NHP) and required Mg2+ (8 mM) or Mn2+ (2 mM) for maximal activity. NEM (0.5 mM), which is an inhibitor of SH-enzymes, inhibited the histone phosphorylating activity of enzyme II by more than 90%, whereas it inhibited the activity of enzyme I by less than 10%. Moreover, the activity of enzyme II was more sensitive to high temperature than that of enzyme I. Non-histone protein (CM-III protein) served as a more effective substrate for enzyme II than histones; the Km value for CM-III protein was 34.4 micrograms/ml whereas that for histone H2a (14,300 daltons) was 155 micrograms/ml (1.08 x 10(-5) M). CM-III protein phosphorylation by enzyme II in vitro was greatly stimulated by the addition of dsDNA, but not by single-stranded DNA or bacterial ribosomal RNA. However, the phosphorylation of CM-III protein by enzyme I was less than 50% of that of histones, and there was no stimulatory effect. SDS-gel electrophoresis showed that two distinct NHPs (about 13,000 and 19,000 daltons) prepared from calf thymus chromatin were preferentially phosphorylated by enzyme II in vitro in the presence of dsDNA. This finding suggests that these two NHPs may be specific phosphate acceptors of cAMP-independent protein kinase (enzyme II) in the nuclei of mouse spleen cells.     

Isolation and study of cAMP-independent chromatin protein kinases from brain cells]

Two cAMP-independent protein kinases were purified from rat brain neuron chromatin by using extraction with ammonium sulfate with subsequent chromatography on DEAE-Sephadex A-25 and Sephadex G-150. These enzymes were identified as casein kinases NI and NII, respectively. The molecular masses of the proteins as determined by gel filtration are 4500 and 130 Da. Casein kinase NII utilizes ATP (Km = 7.5 mM) and GTP (Km = 8.5 mM) as substrates, while casein kinase NI utilizes only ATP (Km = 6 mM). The activities of the both enzymes are inhibited by Mn2+ and Ca2+, while heparin (1 microgram/ml) inhibits only casein kinase NII. The memory stimulator ethymizol (ethylnorantipheine) increases the activity of casein kinase NII only when brain proteins extracted by 0.35 M NaCl or rat liver HMG-proteins are used as reaction substrates. This substance has no effect on the phosphorylation of casein and histone HI. The role of casein kinase NII of neuronal chromatin in the realization of stimulatory effects of physiologically active substances on RNA synthesis is discussed.     

The ribonuclease activity of nucleolar protein B23.

Protein B23 is an abundant nucleolar protein and putative ribosome assembly factor. The protein was analyzed for ribonuclease activity using RNA-embedded gels and perchloric acid precipitation assays. Three purified bacterially expressed forms of the protein, B23.1, B23.2 and an N-terminal polyhistidine tagged B23.1 as well as the natural protein were found to have ribonuclease activity. However, the specific activity of recombinant B23.1 was approximately 5-fold greater than that of recombinant B23.2. The activity was insensitive to human placental ribonuclease inhibitor, but was inhibited by calf thymus DNA in a dose dependent manner. The enzyme exhibited activity over a broad range of pH with an apparent optimum at pH 7.5. The activity was stimulated by but not dependent on the presence of low concentrations of Ca2+, Mg2+ or NaCl. The Ca2+ effect was saturable and only stimulatory in nature. In contrast, Mg2+ and NaCl exhibited optimal concentrations for stimulation and both inhibited the ribonuclease at concentrations above these optima. These data suggest that protein B23 has intrinsic ribonuclease activity. The location of protein B23 in subcompartments of the nucleolus that contain preribosomal RNA suggests that its ribonuclease activity plays a role in the processing of preribosomal RNA.     

Purification of cytosolic cAMP-independent protein kinases from rat ventral prostate

Two cAMP-independent protein kinases were purified from rat ventral-prostate and liver cytosol, and were designated PK-C1 and PK-C2 to distinguish them from the nuclear protein kinases described in the preceding paper. The yield of the prostate enzymes was about 5% each, and about 10% each for the liver enzymes. The average fold purification of the prostatic enzymes was 1892 and 3176 for protein kinase C1 and C2, respectively. Their average respective specific activity towards casein was 40,111 and 67,340 nmol 32P incorporated/hr per mg of enzyme protein. protein kinase C1 comprised one polypeptide of Mr 39,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase C2 comprised three polypeptides of Mr 41,000; 38,000; 26,000. Of these only the Mr 26,000 polypeptide was autophosphorylated. The Mg2+ requirement for protein kinase C1 and C2 was between 1 and 4 mM depending on the nature of the protein substrate. Both enzymes were stimulated by 100-200 mM NaCl. Km for ATP for C1 and C2 kinases was 0.01 mM; GTP could be used only by protein kinase C2 but with a markedly lower affinity. The enzymes were active towards casein, phosvitin, dephosphophosvitin, and spermine-binding protein in vitro, but demonstrated little activity towards histones. Despite several similarities in these general properties of cytosolic protein kinases C1 and C2 with those of nuclear protein kinases N1 and N2, a number of differences are also noted.     

Exposure of HeLa DNA polymerase alpha to protein kinase C affects its catalytic properties

Protein kinase C (Ca2+/phospholipid-dependent protein kinase) purified from rat brain or endogenous to cell-free extracts from HeLa cells stimulates, by a factor of 2-3, HeLa DNA polymerase alpha but not beta or gamma. Monoclonal antibody to the kinase prevents the stimulation, and monoclonal antibody to human DNA polymerase alpha neutralizes the enhanced activity. Reduced DNA polymerase alpha activity is obtained from noncycling HeLa cells and this activity has lower fidelity when copying synthetic primer-templates than that obtained from log phase cultures. After exposure to the kinase, the fidelities and activities of the polymerase from both sources increase by 2- to 3-fold. This improved accuracy is not accompanied by the appearance of triphosphatase or DNase activities. Exposure to the protein kinase reduces the Km for activated DNA and for poly(dA-dT) but not for dNTPs. Moreover, the Vmax for activated DNA but not for poly(dA-dT) is increased approximately 2- to 3-fold. These alterations suggest a role for protein phosphorylation in modulating DNA polymerase alpha.     

Purification and partial characterization of a stimulatory factor for lamb thymus RNA polymerase II.

A heat-stable protein (HSF) that stimulates the activity of lamb thymus RNA polymerase II has been purified 2500-fold and partially characterized. This factor stimulates the activity of RNA polymerase II up to 13 times and retains complete activity when heated at 90 degrees C for 5 min. Stimulation is observed only in the presence of RNA polymerase II and requires native DNA as template. The stimulatory factor has a sedimentation coefficient of 2.7 S, a diffusion coefficient of 9.55 x 10(-7) cm2/s, and an isoelectric point of 8.0. Calculated from the sedimentation and diffusion data, the factor has a molecular weight of about 24,000. Electrophoresis of the purified factor on polyacrylamide gels in the presence of sodium dodecyl sulfate results in a single band corresponding to a molecular weight of 25,000. The number-average length of the RNA synthesized by RNA polymerase II is increased in the presence of the factor. Sedimentation velocity and exclusion chromatography experiments suggest that the stimulatory factor interacts with RNA polymerase II. These results suggest that the factor stimulates RNA synthesis through a direct interaction with RNA polymerase II. The stoichiometry of the HSF-RNA polymerase binding appears to be about 1:1. HSF is located in the nucleus, as determined by cell fractionation studies.     

Purification and kinetic properties of polynucleotide kinase from rat testes

Polynucleotide kinase (EC 2.7.1.78) has been purified from rat testes, and an approximately 2000-fold purification was obtained. The purified enzyme had an Mr of 38000 +/- 3800. The enzyme phosphorylated micrococcal nuclease-treated calf thymus DNA and (dT)10 while 5'-HO-tRNA was a very poor substrate. A certain degree of specificity towards purine-containing 5'-HO-nucleotides was observed. The polynucleotide kinase had an absolute requirement for a divalent cation. Both Mg2+ and Mn2+ could be used, but 10 mM MgCl2 gave optimal activity. The monovalent cations Na+, K+ and NH4+ all stimulated enzyme activity, and the optimal concentration was 0.1 M. The enzyme was inhibited by inorganic phosphate, pyrophosphate and sulphate. A 50% inhibition was obtained with 20, 0.3 and 2 mM, respectively. At 2 mM MgCl2, 1 mM spermine enhanced the enzyme activity 3-times. The apparent KATP was estimated to be 36 microM and KHO-DNA was found to be 2 microM.     

Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.     

A plasmodium protein kinase that is developmentally regulated, stimulated by spermine, and inhibited by quercetin

Plasmodium berghei-infected murine red cells possess protein kinase activity that is associated with the isolated parasites. Schizonts contain significantly higher levels of this protein kinase than the more immature forms, suggesting a relationship between this enzyme activity and parasite development. Partially purified protein kinase has a Km for ATP of approximately 30 microMs, whereas the Km for GTP is approximately 300 microMs and the substrate preference is phosvitin greater than casein much greater than histone greater than protamine. The Mg2+ optimum is 10-20 mM, and the protein kinase activity is stimulated by the polyamines spermine and spermidine. The flavone, quercetin, inhibits the protein kinase activity in a competitive manner with respect to ATP (Ki approximately 3 microMs), and P chabaudi also has a very similarly regulated protein kinase. Protein kinases from both species are very similar to the type I casein kinase.