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

1970 ◽  
Vol 117 (3) ◽  
pp. 513-523 ◽  
Author(s):  
I. Kumudavalli ◽  
B. H. Moreland ◽  
D. C. Watts
Keyword(s):  
Skeletal Muscle ◽  
Transition State ◽  
Thiol Group ◽  
Equilibrium Mixture ◽  
Common Mechanism ◽  
Kinetic Properties ◽  
Rabbit Muscle ◽  
Muscle Enzyme ◽  
Similar Chemical ◽  
High Concentrations

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–Mg2+ plus creatine convert the essential thiol group from being pH-independent into one with a normal ionization. Low concentrations of ADP–Mg2+ 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–Mg2+ 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.

scholarly journals Kinetic properties of cerebral pyruvate kinase

1974 ◽  
Vol 141 (1) ◽  
pp. 165-171 ◽  
Author(s):  
Peter C. Nicholas ◽  
Herman S. Bachelard
Keyword(s):  
Pyruvate Kinase ◽  
Enzymic Activity ◽  
Kinetic Properties ◽  
Muscle Enzyme ◽  
Mammalian Liver ◽  
Substrate Saturation ◽  
Enzyme Stimulation ◽  
High Concentrations ◽  
The Brain ◽  
Guinea Pig Brain

Partly purified guinea-pig brain pyruvate kinase is not activated by fructose 1,6-diphosphate and gives hyperbolic substrate-saturation curves with phosphoenolpyruvate. It is therefore different from the L-type pyruvate kinase of mammalian liver. Inhibition by MgATP2- was competitive for MgADP- but not for phosphoenolpyruvate, and the enzyme is therefore different from the M-type pyruvate kinase, which is said to be competitively inhibited by MgATP2- with respect to both substrates. The Ki(MgATP2-) value of approx. 8mm for the brain enzyme is higher than the values (about 2mm) reported for the muscle enzyme. Stimulation of enzymic activity was observed at low (1–2mm) concentrations of MgATP2-. Substrate kinetic constants were Km (MgADP-)=0.47mm, Km (phosphoenolpyruvate)=0.08mm. Free Mg2+ at very high concentrations (over 10mm) was inhibitory (Ki=20–32mm). Neither ADP3- nor 5′-AMP2- inhibited the activity. The brain enzyme was concluded to be different from both the M-type and the L-type of other mammalian organs such as muscle and liver.

Modifizierung von Glycerinaldehyd-3-phosphat-Dehydrogenase aus Kaninchen-Skelettmuskel mit dem Coenzymanalogen [3-(3-Bromacetylpyridinio)-propyl]-adenosin-pyrophosphat / Modification of Glyceraldehyde-3-phosphate Dehydrogenase from Rabbit Skeletal Muscle by [3-(3-Bromoacetylpyridinio)-propyl]-Adenosine Pyrophosphate

1977 ◽  
Vol 32 (1-2) ◽  
pp. 85-92b ◽  
Author(s):  
Gerhard Dietz ◽  
Christoph Woenckhaus ◽  
Rainer Jaenicke ◽  
Inge Schuster
Keyword(s):  
Skeletal Muscle ◽  
Quaternary Structure ◽  
Enzymic Activity ◽  
Rabbit Skeletal Muscle ◽  
Rabbit Muscle ◽  
Irreversible Loss ◽  
Muscle Enzyme ◽  
Active Site Cysteine ◽  
Complete Inactivation ◽  
Inhibited Enzyme

The NAD analogue [3-(3-acetylpyridinio)-propyl] adenosine pyrophosphate forms enzymically inactive complexes with glyceraldehyde-3-phosphate dehydrogenase from yeast and rabbit skeletal muscle. In the latter enzyme four mol of the analogue are bound with equal affinity inhibiting the enzyme in a competitive way: KI = 0.3 mM as compared to the dissociation constant KD=O.6 mм. The brominated derivative [3- (3-bromoacetylpyridinio) -propyl] adenosine pyrophosphate is co­valently bound to both enzymes causing irreversible loss of enzymic activity. Complete inactivation of the enzyme from muscle requires two moles of the analogue per mol of tetramer. The remaining two sites are still able to bind two mol of NAD+ without regain of enzymic activity. In the case of the yeast enzyme four mol of the analogue are bound. Inactivation of the rabbit muscle enzyme is accompanied by the disappearance of two out of four highly reactive sulfhydryl groups; in the yeast enzyme the four active site cysteine residues are still able to react with DTNB1 the reactivity being diminished significantly. Hybrid formation between the native enzymes from yeast and skeletal muscle is not affected by the modification of the enzyme. Similarly the sedimentation properties of the covalently modi­fied enzyme are indistinguishable from those of the native molecule. This indicates that both the native and the irreversibly inhibited enzyme are identical regarding their quaternary structure.

scholarly journals The mechanism of phosphoglucomutase from Micrococcus lysodeikticus

1974 ◽  
Vol 137 (3) ◽  
pp. 453-461 ◽  
Author(s):  
John B. Clarke ◽  
Hubert G. Britton
Keyword(s):  
Quantitative Agreement ◽  
Rabbit Muscle ◽  
Muscle Enzyme ◽  
Previous Evidence ◽  
Micrococcus Lysodeikticus ◽  
Sequential Mechanism ◽  
Diphosphate Glucose ◽  
High Concentrations ◽  
Intramolecular Transfer ◽  
Substrate Concentrations

The mechanism of the phosphoglucomutase from Micrococcus lysodeikticus was investigated. Induced-transport tests at low substrate concentrations (0.15mm) showed co-transport of the 32P label but no induced transport of the 14C label, which is in quantitative agreement with a phosphoenzyme mechanism with a rapid isomerization of the phosphoenzyme. The results excluded an intramolecular transfer of phosphate and could only have been compatible with a sequential mechanism if the Km for glucose 1-phosphate had been over 20 times smaller than the measured value. The results of induced-transport tests at intermediate concentrations (1mm) with both labels agreed quantitatively with a phosphoenzyme mechanism, and induced-transport tests with 14C-labelled substrates at high concentrations (26mm) indicated that the rate constants for isomerization of the phosphoenzyme must be greater than about 3×106s-1. Consistent with these findings is the fact that 14C label exchanged between the substrates twice as rapidly as the 32P label at chemical equilibrium. Further, since the 14C label exchanged between the substrates about ten times more rapidly than between the substrates and glucose 1,6-diphosphate, glucose 1,6-diphosphate is not an obligatory intermediate in the interconversion of the substrates. It is concluded that, contrary to previous evidence, the mechanism of the enzyme from M. lysodeikticus is essentially that of the rabbit muscle enzyme. To account for the rapid isomerization of the phosphoenzyme in both cases a mechanism is proposed in which there is no formal isomerization of the phosphoenzyme.

scholarly journals Adenylate metabolism in the heart. Regulatory properties of rabbit cardiac adenylate deaminase

1979 ◽  
Vol 182 (2) ◽  
pp. 361-366 ◽  
Author(s):  
R Barsacchi ◽  
M Ranieri-Raggi ◽  
C Bergamini ◽  
A Raggi
Keyword(s):  
Skeletal Muscle ◽  
Kinetic Properties ◽  
Amp Deaminase ◽  
Alkaline Ph ◽  
Muscle Enzyme ◽  
Adenylate Deaminase ◽  
Activated State ◽  
Maximal Activation ◽  
Regulatory Properties

The kinetic properties of a 300-fold purified cardiac AMP deaminase were studied and compared with those of the corresponding enzyme from skeletal muscle. The heart enzyme is activated by ATP and less efficiently by ADP, and is inhibited by Pi, phosphocreatine and GTP. ATP, even at micromolar concentrations, is able to abolish the effects of the inhibitors. The affinity of the enzyme for AMP is low in the absence of activators (Km 3.1 mM), but, in the presence of ATP, becomes as high as that of skeletal-muscle AMP deaminase (Km 0.4 mM). The maximal activation by ATP is observed at alkaline pH (pH 7.5-8.0). Under the same conditions ATP is maximally inhibitory for skeletal-muscle enzyme. These results suggest that AMP deaminase in the heart is always in the activated state, whereas in skeletal muscle the enzyme is active only during exhaustive contractions.

Effect of training on insulin binding to rat skeletal muscle sarcolemmal vesicles

1986 ◽  
Vol 250 (5) ◽  
pp. E570-E575
Author(s):  
G. K. Grimditch ◽  
R. J. Barnard ◽  
S. A. Kaplan ◽  
E. Sternlicht
Keyword(s):  
Skeletal Muscle ◽  
Insulin Sensitivity ◽  
Exercise Training ◽  
Insulin Binding ◽  
Whole Body ◽  
Rat Skeletal Muscle ◽  
Binding Studies ◽  
Muscle Enzyme ◽  
Intravenous Glucose ◽  
High Affinity

We examined the hypothesis that the exercise training-induced increase in skeletal muscle insulin sensitivity is mediated by adaptations in insulin binding to sarcolemmal (SL) insulin receptors. Insulin binding studies were performed on rat skeletal muscle SL isolated from control and trained rats. No significant differences were noted between groups in body weight or fat. An intravenous glucose tolerance test showed an increase in whole-body insulin sensitivity with training, and specific D-glucose transport studies on isolated SL vesicles indicated that this was due in part to adaptations in skeletal muscle. Enzyme marker analyses revealed no differences in yield, purity, or contamination of SL membranes between the two groups. Scatchard analyses indicated no significant differences in the number of insulin binding sites per milligram SL protein on the high-affinity (15.0 +/- 4.1 vs. 18.1 +/- 6.4 X 10(9)) or on the low-affinity portions (925 +/- 80 vs. 884 +/- 106 X 10(9)) of the curves. The association constants of the high-affinity (0.764 +/- 0.154 vs. 0.685 +/- 0.264 X 10(9) M-1) and of the low affinity sites (0.0096 +/- 0.0012 vs. 0.0102 +/- 0.0012 X 10(9) M-1) also were similar. These results do not support the hypothesis that the increased sensitivity to insulin after exercise training is due to changes in SL insulin receptor binding.

scholarly journals Regulation of glycogen synthase. Dephosphorylation of the skeletal muscle enzyme.

1978 ◽  
Vol 253 (8) ◽  
pp. 2540-2545 ◽  
Author(s):  
N.J. Hutson ◽  
B.S. Khatra ◽  
T.R. Soderling
Keyword(s):  
Skeletal Muscle ◽  
Glycogen Synthase ◽  
Muscle Enzyme
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