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PubMed1979

Digoxin induced release of creatine kinase from isolated guinea-pig hearts.

In isolated perfused guinea-pig hearts, digoxin produced a concentration dependent release of creatine kinase (ATP-creatine-transphosphorylase; CK). A corresponding decrease of the CK activity in the myocardium was obtained. The enzyme release seems to be a sign of glycoside intoxication, as its extent paralleled the severity of digoxin induced arrhythmias. Especially high CK activities were liberated when ventricular fibrillation occurred. Likewise, electrically induced fibrillation, in control hearts, led to enzyme release. However, the digoxin effect was not matched. Reserpine pretreatment antagonized the CK release by electrical fibrillation, whereas it increased excessively the enzyme liberating effect of higher digoxin concentrations. Also, propranolol decreased the enzyme release due to electrical fibrillation. The glycoside induced CK liberation, however, was not diminished, although the ventricular fibrillation was prevented. Increase of the potassium concentration of the perfusion fluid prevented the glycoside induced fibrillation, and reduced the enzyme release. The significance of the enzyme loss from the myocardium, and the mechanisms of enzyme release are discussed.

Adrenergic beta-Antagonists↗

PubMed1979

Enzymatic properties of the Ca2+-binding glycoprotein isolated from preosseous cartilage.

The Ca2+-binding glycoprotein isolated from preosseous cartilage shows also alkaline phosphatase activity. The purification procedure indicates that the enzyme is inhibited in crude extract and conceivably in the intact tissue; the activity may be controlled by the proteoglycans present in the matrix. Other substrates are hydrolyzed by the purified enzyme in addition to p-nitrophenylphosphate; the highest specific activity was measured with ATP and pyrophosphate (PPi) at pH 7.5 and 9.0 Mg2+ induces an activation of ATP and PPi hydrolysis; Ca2+ activates hydrolysis of ATP but inhibits that of PPi. The glycoprotein shows also transphosphorylase activity, L-serine being the best phosphate acceptor. The release or transfer of Pi catalyzed by the glycoprotein can be an important step in calcium phosphate precipitation.

Alkaline Phosphatase↗

PubMed1961

Immunoenzymological evidence suggesting a change in conformation of adenylic acid deaminase and creatine kinase during substrate combination.

The kinetics of inhibition of 5'-adenylic acid deaminase and creatine-ATP transphosphorylase by their respective antibodies are studied and rate constants of combination are ascertained. It is shown that the single substrate 5'-adenylic acid (AMP) of deaminase "protects" the enzyme against antibody inhibition. However, phosphate, a competitive inhibitor of the highly specific deaminase, enhances combination with antibody. With creatine kinase, however, addition of either of the substrates, alone or in combination with the required magnesium, each of which separately bind to the enzyme, does not prevent inhibition of the enzyme by its antibody. However, the "working" enzyme combined with all substrates is "protected" against antibody inhibition.

Adenosine Monophosphate↗

PubMed1978

Phosphorylation of adenosine monophosphate in the mitochondrial matrix.

The origin of the GTP needed for th phosphorylation of AMP in the mitochondrial matrix was investigated. When short-chain fatty acids are metabolized by hepatocytes, AMP is readily formed within the matrix by the butyryl-CoA ligase (AMP-forming) reaction (EC 6.2.1.2). The rate of matrix AMP formation in rat hepatocytes was calculated from the rate of ketone-body formation. The rate of the reconversion of matrix AMP into ADP by GTP-AMP transphosphorylase is limited by the rate of supply of GTP. GTP can be formed either by succinic thiokinase (EC 6.2.1.4) or by nucleoside diphosphokinase (EC 2.7.4.6). The rate of the succinic thiokinase reaction was calculated from turnover of the tricarboxylic acid cycle and this was calculated from the rate of O2 consumption and ketone-body formation. The results show that nucleoside diphosphokinase can make a major contribution (up to 80%) to the supply of GTP under the test conditions.

Adenosine Monophosphate↗

PubMed1978

Role of nucleotides in tubulin polymerization: effect of guanosine 5'-methylene diphosphonate.

Incubation of purified rat brain tubulin with guanosine 5'-methylene diphosphonate [GMP(CH2)P] (1 mM), a GDP analog resistant to hydrolysis, results in the polymerization of 20-30% of the total tubulin present. Analogous incubations with GDP (1 mM) do not result in tubulin polymerization. Polymerization with GMP(CH2)P occurs in the presence of alkaline phosphatase (EC 3.1.3.1) under conditions that completely hydrolyze the likely phosphate donors (GTP, GDP, and GMP) as well as the potential product [GMP(CH2)PP] of the transphosphorylase activity present in purified tubulin preparations. Tubulin polymerization in vitro thus can occur in the absence of gamma-phosphate and phosphate bond hydrolysis at the exchangeable nucleotide-binding site of tubulin. Polymerization of tubulin by GMP(CH2)P is neither prevented nor reversed by concentrations of calcium (2 mM) that prevent microtubule assembly and disrupt already formed microtubules induced by GTP. However, tubulin polymerized with GMP(CH2)P is readily depolymerized by cold (4 degrees, 30 min). The possible involvement of GTP alpha-beta bond hydrolysis must be considered seriously as playing a role in the process of microtubule depolymerization.

Alkaline Phosphatase↗

PubMed2002

Thermodynamics of the pyruvate kinase reaction and the reversal of glycolysis in heart and skeletal muscle.

The effect of temperature, pH, and free [Mg(2+)] on the apparent equilibrium constant of pyruvate kinase (phosphoenol transphosphorylase) (EC ) was investigated. The apparent equilibrium constant, K', for the biochemical reaction P-enolpyruvate + ADP = ATP + Pyr was defined as K' = [ATP][Pyr]/[ADP][P-enolpyruvate], where each reactant represents the sum of all the ionic and metal complexed species in M. The K' at pH 7.0, 1.0 mm free Mg(2+) and I of 0.25 m was 3.89 x 10(4) (n = 8) at 25 degrees C. The standard apparent enthalpy (DeltaH' degrees ) for the biochemical reaction was -4.31 kJmol(-1) in the direction of ATP formation. The corresponding standard apparent entropy (DeltaS' degrees ) was +73.4 J K(-1) mol(-1). The DeltaH degrees and DeltaS degrees values for the reference reaction, P-enolpyruvate(3-) + ADP(3-) + H(+) = ATP(4-) + Pyr(1-), were -6.43 kJmol(-1) and +180 J K(-1) mol(-1), respectively (5 to 38 degrees C). We examined further the mass action ratio in rat heart and skeletal muscle at rest and found that the pyruvate kinase reaction in vivo was close to equilibrium i.e. within a factor of about 3 to 6 of K' in the direction of ATP at the same pH, free [Mg(2+)], and T. We conclude that the pyruvate kinase reaction may be reversed under some conditions in vivo, a finding that challenges the long held dogma that the reaction is displaced far from equilibrium.

Animals↗

PubMed1993

Metabolism of thiamine triphosphate in rat brain: correlation with chloride permeability.

Our results show that a net synthesis of thiamine triphosphate (TTP) can be demonstrated in vitro using rat brain extracts. The total homogenate was preincubated with thiamine or its diphosphate derivative (TDP), centrifuged, and washed twice. With TDP (1 mM) as substrate, a 10-fold increase in TTP content was observed in this fraction (nuclear fraction, membrane vesicles). A smaller, but significant, increase was observed in the P2 fraction (mitochondrial/synaptosomal fraction). In view of the low TTP content of our fractions, it was carefully assessed that authentic TTP was being formed. Incorporation of radioactivity from [beta-32P]TDP and [gamma-32P]ATP in TTP suggests that these two compounds are its precursors. Furthermore, TTP synthesis was inhibited by ADP and relatively low concentrations of Zn2+. These results suggest that TTP synthesis is catalyzed by an ATP:TDP transphosphorylase rather than by the cytoplasmic adenylate kinase that may be present in the vesicles. After osmotic lysis of the vesicles at alkaline pH, TTP was recovered in protein-bound form. Concomitantly, a soluble thiamine triphosphatase, with alkaline pH optimum, was also released from the vesicles. No net synthesis could be obtained in the cytosolic fraction or in detergent-solubilized systems. Like TTP synthesis, chloride permeability of the vesicles was increased when the homogenate had been incubated with thiamine and particularly with TDP. Our results suggest a regulatory role of TTP on chloride permeability, but the target remains to be characterized.

Animals↗

PubMed1999

Biotechnological implications of the interactions between magnesium and calcium.

At the biochemical level, magnesium and calcium are known to act antagonistically towards each other. For example, many enzymes whose activities critically depend on sufficiency of intracellular magnesium, especially transphosphorylases, will be detrimentally affected by small increments in levels of cellular calcium ions. Growth of cells, cell division cycle progress and intermediary metabolism are also absolutely dependent on the bioavailability of magnesium, which can be compromised if excess calcium is present. Many biotechnological processes, therefore, which fundamentally exploit cellular growth and metabolism, will be influenced strongly by relative concentrations of the two cations and the ratio of bioavailable magnesium to calcium in external growth media. This paper surveys the interactive effects of magnesium and calcium at the bioinorganic level and discusses some examples of how these interactions may play important roles in governing the physiology of cells which are widely exploited in modern industrial bioprocesses.

Biological Transport↗

PubMed1975

Interaction of guanosine nucleotides with elongation factor 2. I. Equilibrium dialysis studies.

Binding of the guanosine nucleotides, GDP and GTP, to elongation factor 2 (EF-2) from rat liver was studied by equilibrium dialysis. It was found that the enzyme has one binding site for GDP with a dissociation constant of 4 times 10--7 M. The examination of GTP binding was difficult due to the simultaneous presence of GDP and GTP even in purified GTP preparations. This problem was further magnified by traces of GTPase in the enzyme preparation. However, by analyzing the incubation mixtures by thin layer chromatography the fraction of the total nucleotide binding to EF-2 which was due to GDP could be determined and corrected for. A GTP binding curve, corrected for GDP binding, and GTP hydrolysis extrapolated to one binding site with a dissociation constant of approximately 2 times 10--6 M. The nonhydrolyzable GTP analogue, theta, gamma-methylene-guanosine-5-triphosphate, also bound to EF-2 in a 1:1 ratio. During the studies of GTP binding to EF-2 it was observed that the enzyme preparation contained a GTP-GDP transphosphorylase activity. It was initially thought that this was a novel property of EF-2, but when the activity was followed during purification of EF-2 it was whown that it was an impurity in the EF-2 preparation. ATP as well as GTP can serve as a phosphate donor in the transphosphorylation reaction; this might suggest that regeneration of GTP from GDP can take place via this pathway.

Animals↗

PubMed1981

Special capabilities of the sexes: differences in the molecular repertoire of mammal brain, heart and skeletal muscle.

World records for men's and women's (anaerobic) 200 meter dash are 19.67 and 21.9 seconds respectively. Here the transphosphorylation mechanisms are operative and not the glycolytic anaerobic and aerobic ones. The purpose of this paper is to try to find molecular differences during the neuro-muscular sexual differentiation in the Wistar rat. Creatine kinase (CPK: EC 2.7.3.2) as model transphosphorylase and the protein synthesis pattern, determined from the post 125,000 x g cytosol fraction of the brain, heart and masseter muscle, were chosen as molecular monitors. The CPK specific activity profiles showed differences among tissues and sexes. In rats with similar weights between 200 and 320 g, the epigenetic control of protein synthesis in the studied tissues, reflected genome differential activation depending on sex and age. The electrophoretograms differed in regard to the number and position of the protein bands of homologous tissues of different sexes. As an original finding, against the generally accepted criterion (the brain fundamentally synthesizes BB-CPK isoenzyme), MB-CPK and MM-CPK isoenzymes appeared in the brain of both sexes. The molecular sexual differences in the studied tissues determine functional and anatomical differences.

Animals↗

PubMed1984

A kinetic analysis of the interaction of elongation factor Tu with guanosine nucleotides and elongation factor Ts.

The interaction of elongation factor Tu (EF-Tu) and elongation factor Ts (EF-Ts) from Escherichia coli has been investigated by kinetic methods. It was found that EF-Ts purified on an EF-Tu affinity column contained a transphosphorylase activity which could transfer the gamma-phosphate of GTP to [3H]GDP. However, this activity showed different sensitivities to heat and N-ethylmaleimide compared to the EF-Ts activity. Using the chromophoric GDP analogue, 2-amino-6-mercaptopurine riboside 5'-diphosphate (thioGDP), spectrophotometric titrations and stopped-flow experiments enabled the interaction of EF-Tu X thioGDP with EF-Ts and of EF-Tu X EF-Ts with thioGDP to be investigated. The results were analyzed according to the scheme of Chau et al. (Chau, V., Romero, G., and Biltonen, R.L. (1981) J. Biol. Chem. 256, 5591-5596). (Formula: see text) Values for the rate constants obtained were k1 greater than or equal to 2 X 10(8) M-1 s-1, k-1 greater than or equal to 2600 s-1, k2 = 500 s-1, and k-2 = 4 X 10(5) M-1 s-1. The most notable feature of these results is that EF-Ts binds to EF-Tu X thioGDP at a rate approaching that expected for a diffusion-controlled reaction whereas thioGDP binds to EF-Tu X EF-Ts several orders of magnitude more slowly than this. The relevance of these results to the interactions involving GDP is discussed.

Escherichia coli↗

PubMed1980

ATP-induced aggregates of tubulin rings.

Aggregates of double-walled rings (46 nm diameter) were formed upon warming (37 degrees C) ion exchange purified bovine brain tubulin (1.5 to 2.0 mg/ml) in the presence of 1.0 mM ATP and 5.0 mM Mg2+. The formation of aggregated rings was blocked by GDP (1.0 mM), but when GTP (1.0 mM) was added subsequently, the block was overcome. Warming in the presence of ATP and GTP simultaneously produced a mixture of microtubules and aggregated rings. The tubulin preparations used were demonstrated to be devoid of transphosphorylase, and it is therefore clear that the action of ATP in the induction of aggregated rings was not mediated by transphosphorylation. These results are consistent with an interaction of nucleotide with tubulin at a site distinct from the previously described N- and E-sites.

Adenosine Triphosphate↗

PubMed1959