STUDIES ON THE MECHANISM OF ACTION OF ADENOSINE 5′-TRIPHOSPHATE-CREATINE PHOSPHOTRANSFERASE. INHIBITION BY MANGANESE IONS AND BY <i>P</i>-NITROPHENYL ACETATE

Dc, Watts

doi:10.1042/bj0890220

Cited by 29 publications

(3 citation statements)

References 19 publications

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“…Other known inhibitors of the phosphotransferase enzyme (Watts, 1963 and personal communication) were tested on muscle. Manganese chloride (2 mM) (Expt.…”

Section: Active Ion Movements and Phosphate Esters 99mentioning

confidence: 99%

Consumption of high‐energy phosphates during active sodium and potassium interchange in frog muscle

Dydyńska¹,

Harris²

1966

The Journal of Physiology

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SUMMARY1. Potassium-depleted muscles have been analysed for cations, phosphocreatine, adenosine triphosphate and lactate before or after an exposure to a medium with 10 mm potassium salt.2. The net movements of sodium out and potassium in when the system is anaerobic but not otherwise poisoned are accompanied by break-down of phosphocreatine and formation of lactate.3. In bicarbonate media oligomycin has little perceptible effect upon these observed changes, which is taken to indicate that mitochondrial phosphorylation is not essential. An inhibition by oligomycin was noted in media buffered with Tris.4. Dinitrofluorobenzene, which poisons creatine phosphotransferase, leads to the cation changes being accompanied by break-down of ATP and formation of lactate. This indicates that ATP is more directly concerned with energizing the ion movements than is phosphocreatine.5. lodoacetate inhibits the glycolytic process and the ion movement is then accompanied by more phosphocreatine break-down than in the other conditions; the level of ATP also falls.6. The mean number of sodium ions moved out is closely equal to the number of potassium ions moved in. Conditions mentioned in (2) and (3) above lead to about 2-5 sodium ions being moved out per high-energy phosphate bond hydrolysed provided allowance is made for the glycolytic resynthesis of ATP.7. Some measurements of membrane potential under comparable conditions of ion movement are reported and these are used to calculate the energy requirement of the process of sodium-potassium interchange.

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“…Other known inhibitors of the phosphotransferase enzyme (Watts, 1963 and personal communication) were tested on muscle. Manganese chloride (2 mM) (Expt.…”

Section: Active Ion Movements and Phosphate Esters 99mentioning

confidence: 99%

Consumption of high‐energy phosphates during active sodium and potassium interchange in frog muscle

Dydyńska¹,

Harris²

1966

The Journal of Physiology

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“…Manganese chloride in the assay led to a complete loss of activity. A n animal creatine kinase is also inhibited by Mn2+ ions (WATTS 1963). This inhibition was interpreted as a Mn2+ catalyzed oxidation of sulfhydryl groups in the substrate binding sites of the enzyme.…”

Section: 3mentioning

confidence: 99%

Dihydroxyacetone kinase of methanol‐assimilating yeasts II. Partial purification and some properties of dihydroxyacetone kinase from Candida methylica

Babel¹

1981

J of Basic Microbiology

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Dihydroxyacetone kinase (DHAII) from the cell-free extract of methanol-grown Candida rnethylica was partially purified about 100-fold by a procedure employing streptomycine sulfate fractionation, ammonium sulfate fractionation, negative absorption on Cibacron blue F3G-A sephadex G 200 and DEAE-cellulose column chromatography. The enzyme was stable in 50 mnf Tris-HC1 buffer p H 7.5 containing 60% glycerol at -18 "C.The p H optimum for the activity of DHAK from C. methylica was 7.5. The purified enzyme phosphorylated dihydroxyacetone four times faster than D,L-glyceraldehyde. The apparent MI-CHAELIS-MENTEN constants for dihydroxyacetone and D,L-glyceraldehyde were 0.011 mM and 0.024 mM. Other C, compounds including glycerol were not phosphorylated. I T P and U T P were used as phosphate donors with a reaction rate of 11% and 3.1%, respectively, in relation t o ATP, whereas the reaction rates of DHAK from C. methylica with CTP or GTP were much lower than 1%. The reaction of DHAK depends upon the presence of divalent cations in the assay. The highest activity was found with MgZt ions. The reaction rates with Co2+ or Caz+ ions were only 57.3% and 30.3%, respectively, in relation t o the assay with magnesium ions. Manganese chloride in the assay led t o a complete loss of activity.The phosphorylation of glyceraldehyde and dihydroxyacetone by means of triokinases plays an important role in the metabolism of glycerol via NAD+-and NADP+-linked glycerol dehydrogenases and glycerol oxidase in niicroorganisms (LIN 1976, TOM et al. 1978. Recently it was found that dihydroxyacetone is an intermediate in the assimilation of methanol by yeasts, and high activities of dihydroxyacetone kinase were measured in crude extracts from methanol-grown yeasts ( VAN DIJXEN et al. 1978, BABEL and LOFFIIAGEN 1979). However, the knowledge about the kinetic and regulatory properties of triokinases from microorganisms is very limited. Previously we have investigated the regulation of the dihydroxyacetone kinase from the methanol-grown yeast Candida methylica in situ (HOFMANN and BABEL 1980). The present paper will describe the procedure for purification, the substrate specificity and the kinetic properties of the enzyme from Candida methylica. Materials and methodsCarLdida methylica was obtained from Dr. Y. A. TROTSENKO, Pushchino, USSR, and was grown as reported previously (HOFMANN and BABEL 1980). For the preparation of cell-free extracts cells were harvested by centrifugation, washed twice with 20 miw Tris-HC1 buffer (pH 7.5) and resuspended in the same buffer. The cell suspension was passed three times through a FRENCH pressure cell (9.9 kN m-2) followed by centrifugation of the homogenate at 20,000 g for 20 min. Protein concentration was determined by the method of protein-dye binding (BRADFORD 1976) in which human serum albumin as a standard was used. The enzyme activity of dihydroxyacetone kinase was measured at 30 "C using a method based on that of HEINZ a.nd LAMPRECHT (1961). The assay mixtures consisted of 100 miv imidazole-HC1 buffe...

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“…The involvement of a lysine residue (Kassab, Ronstan & Pradel, 1968;Mahowald, 1969) and of a histidine residue in catalysis have also been reported. Changes in the conformation of the protein upon binding of substrate have been inferred from the alteration of the chemical reactivities of sulf hydryl (0 'Sullivan, Diefenbach & Cohn, 1966) and other groups (Watts, 1963;Lui & Cunningham, 1966) and modification of other physicochemical parameters (Lui & Cunningham, 1966;Jacobs & Cunningham, 1968). Recent kinetic experiments using the temperature jump method (Hammes & Hurst, 1969) also indicate that the binding of nucleotides and of metal nucleotides is accompanied by a conformational change in the enzyme-substrate complex.…”

Section: Properties Of the Enzymementioning

confidence: 99%

Magnetic resonance studies of enzymesubstrate complexes with paramagnetic probes as illustrated by creatine kinase

Cohn

1970

Quart. Rev. Biophys.

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Only two spectroscopic methods are capable of detecting individual atoms in macromolecular systems, X-ray diffraction in the crystalline state and nuclear magnetic resonance (NMR) in the liquid state. For an enzyme-substrate complex, X-ray diffraction can yield information on the geometric structure at the active site and nuclear magnetic resonance absorption can, in principle, yield information on the electronic structure at the active site and on the conformation of enzyme-substrate complexes. Both types of information are needed for unraveling the mechanism of enzyme catalysis on the molecular level. The exciting successes of X-ray diffraction in delineating active sites are already established; NMR, a comparative latecomer among spectroscopic techniques is just beginning to demonstrate its potentialities. McDonald & Phillips have summarized in their excellent review (1969) the work on direct observation of hydrogen atoms (protons) by NMR spectroscopy which has advanced our knowledge of protein structure. The extensive studies of Jardetzky and his co-workers on the NMR of ribonuclease and its inhibitor complexes has culminated in a suggested mechanism of catalytic action (Roberts et al. 1969).

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STUDIES ON THE MECHANISM OF ACTION OF ADENOSINE 5′-TRIPHOSPHATE-CREATINE PHOSPHOTRANSFERASE. INHIBITION BY MANGANESE IONS AND BY P-NITROPHENYL ACETATE

Cited by 29 publications

References 19 publications

Consumption of high‐energy phosphates during active sodium and potassium interchange in frog muscle

Consumption of high‐energy phosphates during active sodium and potassium interchange in frog muscle

Dihydroxyacetone kinase of methanol‐assimilating yeasts II. Partial purification and some properties of dihydroxyacetone kinase from Candida methylica

Magnetic resonance studies of enzymesubstrate complexes with paramagnetic probes as illustrated by creatine kinase

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