Phosphotransferase Reaction Research Articles

Inhibition of the herpes simplex virus-coded thymidine kinase-complex by 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) and 9-(2-hydroxyethoxymethyl)guanine-monophosphate (acyclo-GMP).

The thymidine kinase-complex isolated from herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) is associated with the following enzyme activities:ATP:dThd (dCyd) deoxypyrimidine kinase, ATP:dTMP thymidylate kinase, ADP:dThd- and AMP:dThd 5'-phosphotransferase. In kinetic experiments it is shown that ara-AMP inhibits AMP:dThd- and ADP:dThd phosphotransferase activity, while acyclo-GMP impairs ADP:dThd phosphotransferase reaction only; the inhibition was found to be non-competitive. The functional subunit ATP:dThd kinase was not affected by either compound.

Some evidence for the reversibility of the cholinephosphotransferasecatalysed reaction in developing linseed cotyledons in vivo

The relative proportions of3H and32P incorporated into the glycerol and phosphorus moieties of individual phospholipids of linseed cotyledons doubly-labelled with [2-3H]glycerol and32PO4 differed considerably-Whereas [3H]glycerol was incorporated predominantly into PC,32PO4 was not and the ratio of3H/32P in this phospholipid was appreciably greater than in other phospholipids. Neither [methyl-14C]choline nor32PO4was incorporated into the different molecular species of PC in the same relative proportions as [pH]glycerol, and there was more radioactivity in the phosphorylcholine moiety relative to the glycerol moiety in the highly unsaturated molecular species. The specific radioactivities of the phosphorus moieties of most PC species were similar at a particular harvest, whereas the glycerol specific radioactivities of highly unsaturated species were lower than those of the more saturated species. Little [14Ccholine accumulated in CDPcholine in cotyledons supplied with [methyl-14Ccholine and the amount of radioactivity in this compound was less than 1% of that in phosphorylcholine plus PC. The specific radioactivity of phosphorylcholine was appreciably greater than those of the other choline derivatives. Comparisons of the changes with time in the specific radioactivities of the C18 unsaturated fatty acids in PC and 1,2-di acylglycerol in cotyledons labelled with [14Cacetate and of the glycerol moieties of the individual species these two lipids in cotyledons labelled with [3H]glycerol indicated that an exchange of diacylglycerol must occur between PC and diacylglycerol pools in the tissue. It is argued that each of the independent observations described can best be explained as resulting from the reversibility of the choline phosphotransferase reaction in the cotyledons.

Retinal inhibits TPA activated, calcium-dependent, phospholipid-dependent protein kinase (“C” kinase)

RAW264 macrophage-like cells contain a kinase which is dependent on Ca ++ and phosphatidylserine for activity (C kinase) and is stimulable by the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate. Retinal inhibits the activity of the tumor promoter-activated kinase in a concentration-dependent manner. The apparent Ki for inhibition is 1 × 10 −5M. Retinal is not a general inhibitor of phosphotrans-ferase reactions as it did not inhibit the activity of purified cyclic AMP-dependent protein kinase. It is possible, therefore, that the action of retinoids to antagonize tumor promoter effects on cell function may be mediated at the level of regulation of C-kinase.

6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase from rat liver.

Rat liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated by [gamma-32P]ATP plus fructose-6-P or [2-32P]fructose-2,6-P2. The radioactivity co-migrated with homogeneous 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase during sodium dodecyl sulfate-disc gel electrophoresis and the phosphoenzyme was acid-labile and base-stable. Hydrolysis of the phosphate group from phosphoenzyme prepared with either donor and the hydrolysis of their tryptic phosphopeptides depended on pH similarly. The pH dependence suggested that phosphate was linked to the N3 of a histidine residue. Co-electrophoresis and co-chromatography of alkaline hydrolysates of the labeled phosphoenzyme prepared from either substrate allowed the definitive identification of 3-phosphohistidine in the degradation products. Modification of the enzyme with diethylpyrocarbonate inactivated both the phosphotransferase and phosphohydrolase activities and suppressed the phosphorylation of the enzyme by ATP and fructose-6-P or by fructose-2,6-P2. Trypsin digestion of the phosphoenzyme formed upon incubation with ATP or fructose-2,6-P2 yielded an identical phosphopeptide after high pressure liquid chromatography. All the above data are consistent with this enzyme catalyzing both phosphohydrolase and phosphotransferase reactions with the mediation of phosphohistidine in the reaction scheme(s).

Activation of a cyclic AMP-independent protein kinase by an endogenous ATP-requiring protease from lymphosarcoma cells.

The activation of a cyclic AMP-independent protein kinase by an endogenous protease is described. The H4 phosphotransferase (Masaracchia, R. A., Kemp, B., and Walsh, D. A. (1977) J. Biol. Chem. 252, 7109-7117) from lymphosarcoma cells was isolated in a nonactive form. Activation required ATP and Mg2+ and was shown to be time-dependent. Although Mn2+ was capable of substituting for Mg2+ in the protein kinase reaction, no activation was observed when Mn2+ replaced Mg2+. The protein substrate histone H4 inhibited phosphotransferase activation at concentrations greater than 60 microM. The inhibition was complete in the presence of 100 microM H4. Comparable concentrations of bovine serum albumin did not inhibit the activation. The selective dependence on Mg2+ suggested separate activating and phosphotransferase activities. This was confirmed by heat denaturation in which the activation reaction was shown to be more sensitive to heat inactivation than was the phosphotransferase reaction. The activating enzyme was separated from the protein kinase by column chromatofocusing in the pH range 7-4. The pI of the activating enzyme was greater than 7.0. The pI values of the activated and nonactivated phosphotransferase were 4.8 and 5.3, respectively. The apparent molecular weight of the nonactivated phosphotransferase was 68,000; the activated enzyme was eluted from an S-200 Sephadex column with an apparent Mr = 52,000. Despite many similarities to a protease-activated Ca2+/phospholipid-dependent enzyme isolated from lymphocytes (Ogawa, Y., Takai, Y., Kawahara, Y., Kimura, S., and Nishizuka, Y. (1981) J. Immunol. 127, 1369-1374), the H4 phosphotransferase was not activated by Ca2+, phospholipids, or any combination thereof.

Characterization of phosphorylated and native cGMP-dependent protein kinase.

Pure preparations of cGMP‐dependent protein kinase are autophosphorylated in the presence of cAMP and cGMP. The stoichiometry and functional significance of this reaction has been studied. Pure preparations of cGMP‐dependent protein kinase contained 1.4 ± 0.1 (7) mol phosphate/mol subunit as determined by chemical phosphate analysis. The protein‐bound phosphate was hydrolyzed by 1 M NaOH, but was stable in 0.1 M HCl or 0.8 M NH4OH or hot 16% trichloracetic acid. In the presence of cAMP and [γ‐32P]ATP. Mg another 2.5 ± 0.2 (5) mol phosphate/mol subunit were incorporated into the enzyme yielding a total of 4.2 ± 0.06 (5) mol phosphate/mol subunit. cGMP also stimulated the phosphorylation of the enzyme although to a lower extent than cAMP. The autophosphorylation reaction was half‐maximally stimulated at 0.12 μM and 7.0 μM cGMP and cAMP, respectively. cAMP‐dependent autophosphorylation of the native enzyme did not change the apparent KA for cGMP in a phosphotransferase assay (KA= 0.15 μM) but decreased the KA for cAMP from 19.6 μM to 1.75 μM. Phosphorylation did not affect the apparent Km for the substrate peptide but doubled the apparent V of the phosphotransferase reaction determined in the presence of saturating concentrations of cGMP or cAMP. Phosphorylation did not change the affinity of the enzyme for cGMP, when cGMP binding was determined in the absence of ATP (apparent Kd= 18 nM). ATP · Mg decreased the affinity of the native enzyme for cGMP about threefold to an apparent Kd of 54 nM; in contrast, ATP · Mg had only a minimal effect on the binding kinetics of the phosphorylated enzyme. These results support the notion that autophosphorylation of cGMP‐dependent protein kinase considerably influences the kinetic parameter of the enzyme.

34] Casein kinase II

This chapter describes casein kinase II. A multipotential casein kinase, casein kinase II, has been identified in both the nucleus and cytoplasm of a wide variety of eukaryotes. Casein kinase II is structurally and chromatographically distinct from casein kinase I, but like the latter enzyme, phosphorylates acidic substrates. Casein kinase II phosphorylates both seryl and threonyl residues; the recognition sequence for the enzyme two acidic residues following the phosphorylatable residue and is identified as Ser(Thr)- Glu(Ser-P)-Asp(Glu). Casein kinase II utilizes both adenosine triphosphate (ATP) and guanosine triphosphate (GTP) as phosphoryl donors in the phosphotransferase reaction. Regulation of the type II casein kinase appears to be quite complex. Monovalent cations stimulate casein kinase activity with most of the substrates and polyamines are shown to increase activity. Casein kinase II is shown to phosphorylate translational initiation factors, mRNP particles, spectrin, glycophorin, RNA polymerase, high-mobility-group protein 17, glycogen synthase, troponin T, and the regulatory subunit of the type II, cAMP-dependent protein kinase. In addition, it is postulated that casein kinase II may function to integrate total cell metabolism by regulating phosphorylation of a number of proteins in different metabolic pathways.

Rapid, isocratic separation of purine nucleotides using strong anion-exchange high-performance liquid chromatography

A method is presented for the rapid, isocratic separation of purine nucleotides using strong anion-exchange high-performance liquid chromatography at ambient temperature. The last peak of interest guanosine 5′-triphosphate (GTP) is eluted within 30 min and immediate reinjection is possible. All adenine and guanine nucleotides can be assayed with a single injection without the use of a gradient for elution. The procedure is particularly useful for the assay of NTP:AMP phosphotransferase reactions and/or the determination of changes in size of cellular purine nucleotide pools and computation of energy charges. An Altex Ultrasil AX prepacked column was used, and virtually identical results were obtained under similar conditions with the Whatman Partisil-10 SAX column. The eluting solution was 200 m M potassium phosphate, pH 6.85.

Tyrosine phosphorylation sites common to transforming proteins encoded by Gardner and Snyder-Theilen FeSV

The Gardner and Snyder-Theilen strains of feline sarcoma virus (FeSV) encode 110,000 molecular weight ( M r ) polyproteins as their primary translational products. Cells transformed by a variant of Snyder-Theilen FeSV express an 85,000 M r polyprotein. Single tyrosine phosphorylation sites identified within each of these viral gene products are shown to represent major in vitro acceptors phosphorylated by the polyprotein associated protein kinases. By two-dimensional tryptic peptide analysis, these acceptor sites are highly related although exhibit minor differences in map position, while by analytical high-performance liquid chromatography, all three elute at approximately the same acetonitrile concentration (21–23%). The tyrosine acceptor sites in these FeSV-encoded polyproteins have been localized by sequential Edman degradation at a position seven amino acid residues distal to their trypsin cleavage site. The major tyrosine acceptors, two serine phosphorylation acceptors common to the p12 structural components of all three polyproteins, a single major phosphothreonine site, and several minor less well-resolved acceptor sites are shown to be phosphorylated in vivo. In addition to the major tyrosine acceptor site initially phosphorylated in vitro, phosphorylation of secondary tyrosine acceptors is shown to occur to proportionately greater extents when in vitro phosphotransferase reactions are carried out for longer times or at higher temperatures.

Cyclic AMP independent protein kinases from soybean cotyledons ( Glycine max, L.)

Two cAMP-independent protein kinases which phosphorylate casein were isolated from post-ribosomal supernatant of soybean cotyledons. They were referred to as casein kinase I (CK I) and casein kinase II (CK II) based on their elution from DEAE cellulose column and purified to 350- and 285- fold, respectively. SDS-polyacrylamide gel electrophoresis data for CK I indicated that it is a single subunit protein of 39 000 daltons, while CK II was observed to be made up of 3 subunits of 52 000, 37 000 and 35 000 daltons. Both CK I and CK II use casein as substrate preferentially over all other substrates tested and CK II phosphorylates 40 S ribosomal subunits and 40 S ribosomal subunit proteins. Both the enzymes were found to undergo autophosphorylation in the presence of [γ- 32P] ATP, phosphorylating CK I and 35 000-dalton protein band of CK II. The enzyme utilized ATP in preference to GTP in phosphotransferase reactions.

The role of the membrane in the regulation of activity of microsomal glucose-6-phosphatase.

The factors regulating glucose-6-phosphatase (EC 3.1.3.9) activity and substrate specificity in hepatic microsomes were studied by determining the rate-limiting reaction for the hydrolysis of glucose-6-P, and by examining the effect of detergent activation on phosphotransferase activity. Examination of the pre-steady state kinetics of glucose-6-phosphatase revealed that the steady state rate is determined by the rate of hydrolysis of the enzyme-P intermediate. Treatment of the enzyme with detergent does not alter the extent of the rapid release of glucose per mg of protein, but activates the steady state rate of catalytic turnover. Specificity of the enzyme was evaluated by comparing the effects of mannose and glucose as phosphate acceptors in the phosphotransferase reaction catalyzed by glucose-6-phosphatase. Untreated glucose-6-phosphatase discriminates against mannose as compared with glucose in that mannose and glucose bind to the enzyme-P intermediate of untreated enzyme, but mannose is not an acceptor of Pi. Mannose is an acceptor, however, after treatment of microsomes with detergent. These data cannot be explained in terms of the currently accepted "compartmentation" model for the regulation of glucose-6-phosphatase. The detergent-induced changes in kinetic properties appear to reflect alterations in the intrinsic characteristics of glucose-6-phosphatase, which could result from interaction with its membrane environment.

Characterization of endogenous protein phosphorylation in isolated rat liver lysosomes.

Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphorylation activities. The transfer of phosphate to membrane fractions from [gamma-32P]ATP was analyzed by gel electrophoresis under acidic denaturing conditions. Two phosphopeptides were detected, with molecular weights of 3,000 and 14,000. Phosphorylation of these proteins was unaffected by the addition of cAMP, cGMP, or the heat-stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP-dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14,000-dalton 32P-labeled product was formed rapidly in the presence of low concentrations (250 microM) of either Ca2+ or Mg2+. This product was labile under both acidic and alkaline conditions, suggesting that this protein contains an acyl phosphate, present presumably as a catalytic intermediate in a phosphotransferase reaction. The lower molecular weight species required a high concentration (5 mM) of Mg2+ for phosphorylation, and micromolar concentrations of Ca2+ stimulated the Mg2+-dependent activity. The addition of Ca2+ and calmodulin stimulated the phosphorylation reaction to a greater extent than with Ca2+ alone. This activity was strongly inhibited by 0.2 mM LaCl3 and to a lesser extent by 50 microM chlorpromazine or trifluoperazine. These results suggest that the 3000-dalton peptide may be phosphorylated by a Ca2+, calmodulin-dependent kinase associated with the lysosomal membrane.

Effect of cyclic AMP-dependent protein kinases on gene expression

Mechanisms for regulating activation of cyclic AMP-dependent protein kinase were investigated and a possible biological role of this enzyme was elucidated. Autophosphorylation of cyclic AMP-dependent protein kinase was shown to occur via an intramolecular mechanism: the regulatory subunit undergoes phosphorylation only within the holoenzyme. The phosphoform of the catalytic subunit has the capacity to phosphorylate the regulatory subunit. The phosphotransferase reaction and the reaction of autophosphorylation were found to proceed with the involvement of the same active site. Autophosphorylation was demonstrated to lead to almost complete dissociation of the holoenzyme under the influence of cyclic AMP. Circular dichroism spectra of the phosphorylated and nonphosphorylated forms of protein kinase were studied. The relative content of the secondary structure elements in proteins was estimated and conformational changes were detected in the enzyme upon its interaction with cyclic AMP. By the method of circular dichroism it was also shown that cyclic AMP and its 8-Br-substituted analog were bound in the cyclic AMP-binding site of the protein kinase in anti-conformation. The data obtained suggest the change of conformation of the 8-Br-cyclic AMP molecule during activation of protein kinase: while having a preferential syn-conformation in the free state in solution, the molecule of 8-Br-cyclic AMP acquires anti-conformation in the fixed complex with the regulatory subunit. A study was made of nuclear translocation of cyclic AMP-dependent protein kinase and its subunits, as well as of the binding of these proteins to metaphase chromosomes. The CHO cell cultures were treated with 3H-labeled protein kinase and its subunits. The results indicate that cyclic AMP-dependent protein kinase becomes translocated into the nucleus in a dissociated state and that the subunits have specific binding sites on chromatin. Transformation of normal mouse fibroblasts by virus SV 40 interferes with the nuclear translocation of the regulatory subunit. The process is restored when the level of cyclic AMP in the system is increased. Binding of the regulatory subunit to metaphase chromosomes of cells transformed by virus SV 40 does not change. In the case of spontaneous cancer (KB cells) translocation of the regulatory subunit remains unaffected, whereas acceptance of the protein by the metaphase chromosomes is impeded. The results of this work suggest that compartmentalization of cyclic AMP-dependent protein kinase, and particularly of its regulatory subunit, in the cell is highly significant for cellular processes. Disorders arising as a result of neoplastic transformation involve changes in nuclear translocation of the regulatory subunit and in its binding to the structural elements of the genome. The effect of the regulatory subunit of cyclic AMP-dependent protein kinase on the process of transcription in vitro was shown. The regulatory subunit was found to increase the number of initiation sites on chromatin for RNA polymerase. In this connection, an attempt was made to detect more directly expression of the genome by the regulatory subunit and to identify the final protein product formed. The regulatory subunit induced a specific synthesis of new proteins, the direct and first response of 3T3 cells to the introduction of the regulatory subunit being the synthesis of protein P-15. The molecular weight of the protein was 15,000, the isoelectric point 6.3. The electrophoretic analysis of the cytosol of SV 40-3T3 cells revealed a general derepression of the genome of the virus-transformed cells. A protein identical with P-15 was detected to be present in SV 40-3T3 cells. The treatment of these cells with the regulatory subunit as well as with cyclic AMP separately did not affect the synthesis of P-15, whereas introduction of the cyclic AMP-regulatory subunit complex caused a significant expression of protein P-15. The data obtained indicate that the protein synthesis is dependent on nuclear translocation of the regulatory subunit.

18] Kinetic examination of enzyme mechanisms involving branched reaction pathways—A detailed consideration of multifunctional glucose-6-phosphatase

Publisher Summary This chapter discusses the kinetic examination of enzyme mechanisms involving branched reaction pathways–that is multifunctional glucose-6-phosphatase. The kinetic mechanism applicable generally to both hydrolytic and synthetic activities of glucose-6-phosphatase phosphotransferase is a specific adaptation of the generally applicable kinetic mechanism for enzymes with modified Ping Pong Bi Bi kinetics. The mechanism is based on kinetic analysis, both experimental and theoretical, of various aspects of this complex system. This differs from the usual Ping Pong Bi Bi mechanism applicable to certain phosphotransferase reactions involving the hydrolysis of phosphoryl-enzyme as an alternative to phosphoryl transfer. Mechanism is based on the kinetic observations; data in all cases are plotted in double-reciprocal fashion.

Identification of the catalytic subunit of casein kinase II by affinity labeling with 5'-p-fluorosulfonylbenzoyl adenosine.

Casein kinase II, purified from reticulocytes, was covalently labeled with the ATP affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine. The reaction was monitored by the decrease in enzyme activity and showed saturation kinetics with respect to the sulfonyl compound. This suggested a rapid equilibrium was established between the enzyme and affinity reagent prior to a slower, rate-determining step in the overall inactivation process. The enzyme was protected from the covalent modification by ATP and a series of ATP analogs. Their effectiveness in preventing inactivation by the affinity labeling reagent paralleled their ability to function as inhibitors of the phosphotransferase reaction. When radioactive p-fluorosulfonyl [14C]benzoyl adenosine was used to inactivate the enzyme, the alpha subunit was labeled and incorporation of radioactivity into the alpha subunit was blocked when ADP was included in the reaction mixture. Thus the ATP binding site of casein kinase II was shown to be contained within the domain of the alpha subunit of the alpha 2 beta 2 complex.

Effect of cytidine diphosphate choline (CDP-choline) on ischemia-induced alterations of brain lipid in the gerbil.

Brain ischemia was produced in gerbils (Meriones unguiculatus) by the bilateral ligation of the carotid arteries. Definite changes in the energy status of brain demonstrated that carotid occlusion was effective. Five minutes before ligation, an intraventricular injection of either saline or cytidine disphosphate choline (CDP-choline, 0.6 micromol/brain, 3 microliter) was given to groups of animals. Control animals, with and without CDP-choline, together with the ischemic groups, were decapitated directly into liquid nitrogen; 10 min after arterial ligation. Brain free fatty acids, neutral lipids and phospholipids, which were labeled in vivo by the intraventricular injection of [1-14C]arachidonic acid (0.4-0.6 micro Ci, 6-9 nmol) 2 hr prior to ligation, were extracted, purified, and separated by thin-layer chromatographic procedures. The CDP-choline treatment noticeably corrected the increase of total and individual fatty acids due to ischemia and the increase of their radioactivity content. The changes in neutral lipids, particularly in the diacyl glycerol fraction, were also corrected by the injection of the nucleotide. CDP-choline partially reversed the decreased of brain phosphatidylcholine and of its labeling, which was due to ischemia. All the data indicate that the prior injection of CDP-choline stimulates the choline phosphotransferase reaction of brain towards synthesis of phosphatidylcholine and prevents the release of free fatty acids, particularly of arachidonic acid, associated with ischemia.

The specific interaction of the Rous sarcoma virus transforming protein, pp60 src, with two cellular proteins

Sera from rabbits bearing tumors induced by Rous sarcoma virus (RSV) were previously found to contain antibody to the RSV transforming protein, pp60 src. Two additional transformation-specific phosphoproteins from RSV-transformed avian cells are immunoprecipitated with these sera. These proteins, having molecular weights of 90,000 (pp90) and 50,000 (pp50), are not precipitated from uninfected or transformation-defective virus-infected cells and are not related to any RSV structural proteins. Neither pp50 nor pp90 shares any partial or complete proteolytic cleavage peptides with pp60 src, suggesting that pp90 and pp50 do not represent either a precursor or a cleavage product of pp60 src. Sedimentation analysis of RSV-transformed cell lysates on glycerol gradients revealed that the RSV pp60 src protein is present as two forms, one of which represents the majority (95%) of pp60 src and sediments as a monomer, 60,000 molecular weight protein and the other of which sediments with pp90 and pp50 as an apparent 200,000 molecular weight complex. Lysates from cells transformed by viruses containing a temperature-sensitive defect in the src gene contain a greater percentage of pp60 src associated with pp90 and pp50 under both permissive (35°C) and nonpermissive (41°C) conditions compared to wild-type virus-infected cell lysates. Phosphoserine and phosphotyrosine were found associated with pp60 src molecules that sedimented as a monomer, whereas pp60 src molecules that are complexed with pp90 and pp50 contain phosphoserine and greatly reduced amounts of phosphotyrosine. Only the monomer form of pp60 src is capable of phosphorylating IgG in the immune complex phosphotransferase reaction. Normal uninfected chicken cells contain a protein that shares identical partial proteolytic cleavage peptides with the pp90 protein immunoprecipitated from RSV-transformed cells. This pp90 protein is one of the major cytoplasmic proteins in uninfected cells. Antibody directed against pp90 also immunoprecipitates pp60 src and pp50 from lysates of RSV-transformed chicken cells.

The autophosphorylation reaction in the mechanism of activation of pig brain cyclic AMP-dependent protein kinase

Autophosphorylation of cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was shown to occur via an intramolecular mechanism: the regulatory subunit undergoes phosphorylation only within the holoenzyme. The phospho form of the catalytic subunit has the capacity to phosphorylate the regulatory subunit. The phosphotransferase reaction and the reaction of autophosphorylation were found to proceed with the involvement of the same active site. The activation constant of phospho- and dephosphoprotein kinase under the influence of cyclic AMP and the dissociation constant of the cyclic AMP complex with phospho- and dephospho forms of the holoenzyme were estimated. Autophosphorylation was demonstrated to lead to almost complete dissociation of the holoenzyme under the influence of cyclic AMP. Circular dichroism spectra of the phosphorylated and non-phosphorylated forms of protein kinase were studied. The relative content of the secondary structure elements in proteins was estimated and conformational changes were detected in the enzyme upon its interaction with cyclic AMP. The anti-conformation of the cyclic nucleotide fixed in the complex with the phospho form of the regulatory subunit is suggested.

Studies on the mechanism of action of the histone kinase dependent on adenosine 3',5'-monophosphate. Interaction of ATP with the catalytic subunit of the pig-brain enzyme: application of the quenched-flow technique.

A significant release of inorganic phosphate from ATP in the presence of the cAMP-dependent pig-brain histone kinase was detected. The high degree of homogeneity of the enzyme preparations used, identity of Michaelis constants (Km for ATP = 12 microM), the close values of cAMP activation constants (48 nM and 51 nM) for the phosphotransferase and ATPase activities, respectively, are all evidence that ATP decomposition is catalysed by the histone kinase under study. The ATPase activity observed supports the ping-pong bi-bi mechanism established earlier for the phosphotransferase reaction and can be regarded as due to decomposition of the phosphoryl enzyme. The transient and steady-state phases of the ATP hydrolysis were studied. The simplest reaction pathway can be described in terms of a three-step mechanism. The close values of the rate constant for the elementary stages of the ATPase reaction obtained in the nucleophile competition study and by computer simulation of the quenched-flow kinetics give further support for the mechanism proposed. The phosphoryl enzyme decomposition was shown to be a rate-limiting step under the experimental conditions used (pH 7.8-8.0).

Effect of denervation on the endogenous phosphorylating activity in the cytosol of rat skeletal muscle

An endogenous phosphorylating activity is demonstrated in the cytosol from soleus muscle of the rat which is markedly stimulated after severing the motor nerve fibers to this muscle. The [γ- 32P]ATP phosphotransferase reaction is heat-labile, dependent upon Mg 2+ but not Ca 2+ or cyclic GMP, inhibited by a cyclic AMP dependent protein kinase inhibitor, and directly related to the amount of cytosolic protein which provides the endogenous source of both the protein kinase enzyme, ATP, cyclic AMP and phosphorylatable protein substrate. The time-course of the delayed transitory stimulation of the cytosolic phosphorylating activity of the denervated soleus may involve neurotropic factors.