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D C Watts

Publications and source records attributed to D C Watts.

At least 181 records · Page 10Linked to original sources
PubMed1973

Properties and mechanism of action of creatine kinase from ox smooth muscle. Anion effects compared with pyruvate kinase.

1. An improved purification procedure for the brain-type creatine kinase from ox smooth muscle is described. 2. Michaelis constants show the characteristic dependence on the concentration of the second substrate: the derived constants are compared with those for the enzyme from ox brain. 3. Inhibition by iodoacetamide gives a biphasic curve and the total extent of the reaction depends on the enzyme concentration. The rate of inhibition at pH8.6 is not affected by creatine plus MgADP or by a range of simple anions. Addition of creatine plus MgADP plus either NO(3) (-) or Cl(-) ions affords 71.5 and 44% protection respectively. ADP could be replaced by 2-deoxy-ADP but not by alphabeta-methylene ADP, XDP, IDP, GDP or CDP. Nucleotides that did not protect would not act as substrates. 4. Difference-spectra measurements support the interpretation that addition of NO(3) (-) ions to the enzyme-creatine-MgADP complex causes further conformational changes in the enzyme accompanying the formation of a stable quaternary enzyme-creatine-NO(3) (-)-MgADP complex that simulates an intermediate stage in the transphosphorylation reaction. However, the enzyme structure is partially destabilized by quaternary-complex formation. IDP apparently fails to act as a substrate because it cannot induce the necessary conformational change. This behaviour is compared with that of rabbit skeletal muscle creatine kinase. 5. With pyruvate kinase from rabbit muscle, anions activate in the absence of an activating cation and either inhibit or have no effect in its presence. 6. Both activation and inhibition were competitive with respect to the substrate, phosphoenolpyruvate, and curved double-reciprocal plots were obtained. The results may be interpreted in terms of co-operatively induced conformational changes, and this is supported by difference-spectra measurements. However, the Hill coefficient of 1 was not significantly altered. 7. Inhibition by lactate plus pyruvate is less than additive, indicating that both bind to the same site on the enzyme, whereas that by lactate plus NO(3) (-) is additive, indicating binding at separate sites. It is inferred that a quaternary enzyme-pyruvate-NO(3) (-)-MgADP complex could form, but no evidence was obtained to suggest that it possessed special properties comparable with those found with creatine kinase. The implications of these findings for the unidirectional nature of the mechanism of pyruvate kinase is discussed. 8. Lactate or alpha-hydroxybutyrate could not act instead of pyruvate to form a stable quaternary complex, although both activate the K(+)-free enzyme. Only the former inhibits the K(+)-activated enzyme. The activating cation both lowers the Michaelis constant for phosphoenolpyruvate and tightens up the specificity of its binding site.

Adenosine Diphosphate↗
PubMed1973

Comparison of the turnover patterns of total and individual muscle proteins in normal mice and those with hereditary muscular dystrophy.

1. The incorporation of amino acids into hindleg muscle proteins of normal and dystrophic mice was measured (1/2)h to 16 days after administration of the radioactive pulse. 2. Dystrophic animals showed a faster initial rate of incorporation into total and soluble proteins in the first few hours after injection, but the extent of incorporation relative to the size of the amino acid pool was similar in both. There was little difference between the overall degradation rates although this started later in the dystrophic proteins. An initial fast phase of degradation reached a plateau after 3 days whereupon the residual label in the protein remained constant up to 16 days after injection. 3. Analyses of individual radioactive proteins fractionated by polyacrylamide-gel electrophoresis showed that the distribution of label was similar in all the soluble proteins from normal and dystrophic muscle. Time-course experiments revealed that in dystrophic mice the two major soluble proteins of the muscle, creatine kinase and adenylate kinase, initially incorporated 2-3 times more label relative to the initial size of the precursor pool. This label was then lost equally rapidly and the final plateau value was much less than that in normal mice. This initial peak of activity was not observed in normal mice. 4. A group of dehydrogenases showed similar initial turnover patterns in both dystrophic and normal mice but the final plateau value was much higher in the former. 5. The results provide support for the hypothesis that there is no obvious defect in the protein synthetic machinery of dystrophic muscle. However, certain proteins do show anomalous turnover patterns relative to those in normal animals. A single structural gene mutation giving rise to one particularly unstable and readily degradable muscle protein is excluded as the cause of the dystrophy.

Adenosine Monophosphate↗
PubMed1972

Purification and properties of adenosine triphosphate-creatine phosphotransferase from muscle of the dogfish Scylliorhinus canicula.

1. Creatine kinase occurs in high concentration in the soluble proteins of dogfish muscle. A fourfold purification gives essentially pure enzyme but with a low specific activity. This appears to be a property of the native enzyme and not a result of the isolation procedures used. 2. The amino acid composition is similar to that of other phosphagen kinases, but the enzyme differs from mammalian creatine kinases in having four thiol groups readily reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid). Titration of two thiol groups is accompanied by almost complete loss of activity. The remaining two thiol groups react at different rates, suggesting that modifying the third thiol group affects the reactivity of the fourth thiol group. 3. The enzyme is markedly protected against inactivation by iodoacetamide by MgATP or MgADP. Addition of creatine to MgADP decreases protection, but the further addition of Cl(-) restores protection to the original value. The quaternary MgADP-creatine-enzyme-nitrate complex protects very strongly as is found for the rabbit enzyme. The involvement of the conformational state of the enzyme in such effects is discussed. 4. Creatine kinase from both dogfish and rabbit is equally sensitive to urea denaturation. Urea protects the dogfish enzyme by about 9% against inhibition by iodoacetamide. 5. The formation of a hybrid between the dogfish and rabbit enzymes in vitro has been demonstrated. 6. At high substrate concentrations the dogfish enzyme shows apparent ordered kinetics. The effect of temperature on V(max.) and the Michaelis constants for MgATP and creatine were determined. These and changes in the apparent activation energy suggest that limited adaptation has occurred commensurate with physiological need.

Adenosine Diphosphate↗
PubMed1971

Inhibition of adenosine 5'-triphosphate-creatine phosphotransferase by substrate-anion complexes. Evidence for the transition-state organization of the catalytic site.

1. The substrate combination creatine-MgADP does not significantly protect creatine kinase against inhibition by iodoacetamide in the absence of small anions. 2. Small anions can be divided into three groups according to the way in which they affect creatine kinase: I, acetate reversibly increases enzyme activity in the forward reaction but does not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP; II, planar anions and some halides (HCO(3) (-), HCO(2) (-), NO(3) (-), NO(2) (-), Cl(-), Br(-), F(-)) in the presence of creatine plus MgADP protect the enzyme from inhibition by iodoacetamide; III, tetrahedral anions (SO(4) (2-), HPO(4) (2-), ClO(4) (-), BF(4) (-)) and iodide do not affect the rate of inhibition by iodoacetamide in the presence of creatine plus MgADP but may decrease the protection by class II anions under these conditions. Anions of class II and class III also reversibly inhibit enzyme activity. 3. It is concluded that class II anions form a stable and inactive quaternary enzyme-creatine-MgADP-anion complex and this is responsible for the effect attributed by previous workers to the ternary complex lacking anion. Formation of this complex, particularly in the forward reaction, can lead to markedly non-linear enzyme progress curves. Some previous observations are re-appraised in the light of these findings. 4. From the behaviour of chloride and nitrate ions, and the marked lowering of the K(i) values for creatine and MgADP they produce, it is inferred that planar or monoatomic anions act in the quaternary complex by simulating the transferable phosphoryl group in the transition state (or another intermediate state) of the reaction. 5. It is suggested that, in the course of the reaction, the tetrahedral phosphate-binding site for the transferable phosphoryl group of the substrate (that also binds class II and class III anions) changes into a trigonal bipyramid site (also occupied by class II anions). This strains the phosphoryl group to adopt the transitional sp(3)d hybridized state and must contribute significantly to the low activation energy of the reaction. 6. Catalysis is deduced to proceed by an ;in line' transfer reaction and from the effects of class II anions it is possible to estimate the approximate dimensions of the anionic site in the transition-state complex. 7. The specific protecting effect of an equilibrium mixture of substrates against inhibition by iodoacetamide provides further evidence for the conformational change suggested above as a step in the catalytic process.

Adenosine Diphosphate↗
PubMed1971

Influence of pH on aggregation and protein binding of barbituric acid and amylobarbitone.

1. Cryoscopic methods indicated that barbituric acid exists in aqueous solution as a monomer. Amylobarbitone is a monomer at pH 8, but appears to be polymerized at pH 2. The size of the oligomers increases with drug concentration.2. Using a non-equilibrium dialysis technique supportive evidence for the monomeric form of barbituric acid and the polymerization of amylobarbitone was obtained. The degree of polymerization appeared to increase with fall in pH. Binding constants for these barbiturates with bovine serum albumin were derived, but in acid media no binding was observed.

Amobarbital↗
PubMed1970

Properties and reaction with iodoacetamide of adenosine 5'-triphosphate-creatine phosphotransferase from human skeletal muscle. Further evidence about the role of the essential thiol group in relation to the mechanism of action.

1. The purification of creatine kinase from human and monkey skeletal muscle by horizontal electrophoresis on Sephadex blocks is described. 2. The purified enzymes are shown to have similar chemical and kinetic properties to the rabbit muscle enzyme and a common mechanism is inferred. 3. Iodoacetamide has a similar apparent second-order inhibition constant with the human and rabbit enzymes, but the inhibition does not go to completion with the former. This is even more marked with the monkey enzyme, which has more reactive thiol groups, but inhibition is only about 50%. 4. Single substrates have little effect on the inhibition by iodoacetamide, but with the primate enzymes, in contrast with the rabbit enzyme, high concentrations of ADP-Mg(2+) plus creatine convert the essential thiol group from being pH-independent into one with a normal ionization. Low concentrations of ADP-Mg(2+) plus creatine first enhance the rate of inactivation, but cause protection as the reaction proceeds. These results are interpreted to indicate an activation of the thiol group on the subunit to which the substrates bind and a co-operatively induced decrease in the activity of the thiol group on the other subunit which lacks substrates. 5. The effects of a substrate equilibrium mixture on the rate of inhibition are essentially those of ADP-Mg(2+) plus creatine. 6. Since no substrate combination affords significant protection to the thiol group associated with the catalytic site to which the substrates are bound, it is concluded that any mechanism involving the thiol group in a direct participation in the transition-state complex of the catalytic reaction must be abandoned unless the transition state is only a small part of the time taken for one catalytic cycle.

Adenosine Triphosphate↗
PubMed1969

Comparison of the protein-synthesizing machinery in the skeletal muscle of normal and dystrophic Bar Harbor mice.

1. Although the total weight of leg muscle increased with the age of a normal mouse the DNA and RNA content per leg did not change significantly. 2. The weight of leg muscle from a dystrophic mouse was only about 45% of that from a normal mouse but the DNA and RNA contents were the same and hence similar DNA/RNA ratios were obtained. 3. The total ribosome contents of normal and dystrophic mice were the same on a whole-leg basis, and for both the free ribosomes were about 60% of the total. However, comparison with similar data from liver suggested that some loss of ribosomes occurred during the isolation procedure. 4. The polyribosome patterns obtained by density-gradient centrifugation were the same for normal and dystrophic muscle, and comparable polyribosome fractions of different sizes obtained from such gradients had similar capacities for the incorporation of radioactive amino acids in a standard protein-synthesizing system. 5. By using a standard protein-synthesizing system with normal polyribosomes similar extents of incorporation were found with normal- or dystrophic-muscle pH5 fraction or partially purified transfer RNA preparation. 6. It is concluded that there is no absolute difference between the protein-synthesizing systems of normal and dystrophic mouse muscle and that the observed apparent differences result from concentration differences caused by changes in muscle volume. 7. A possible cause of the failure of dystrophic muscle to resynthesize myofibrils is also suggested.

Amino Acids↗
PubMed1969

The influence of psychotropic drugs on aldole, mitochondrial malic dehydrogenase and Mg++na+K+ adenosine triphosphatase.

1. The experiment investigated the effects of psychotropic drugs on enzymes in vitro.2. Chlorpromazine inhibits mitochondrial malate dehydrogenase in concentrations of 1.4x10(-4)M and above. No effect was observed below this concentration.3. A sharp inflection in the [unk]/drug plot was also seen with chlorpromazine (1.4 x 10(-4)M), trifluoperazine (1 x 10(-5)M), prochlorperazine (1 x 10(-4)M), and amylobarbitone (8 x 10(-3)M) acting on rat brain (Na(+)-K(+)-) Mg(++)-ATPase and with chlorpromazine (10(-4)M) and imipramine (3 x 10(-4)M) acting on aldolase.

Adenosine Triphosphatases↗