Turnover of Several Glycolytic Enzymes in Rabbit Heart, Soleus Muscle and Liver (original) (raw)
Related papers
Journal of Biological Chemistry
Methods are described for the purification, close to homogeneity, of rabbit liver glycogen synthase in forms dependent on (D-form) or independent (I-form) of glucose-6-P for activity. In previous studies (Camici, M., DePaoli-Roach, A. A., and Roach, P. J. (1982) J. Biol. Chern. 257,9898-9901), the D-form enzyme was shown to have apparent subunit molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Mapp) of 90,000 and to be susceptible to partial proteolytic degradation. We report here that the purified I-form consisted of a single polypeptide of Map = 86,000, even when protease inhibitors were present during the purification. However, appropriate phosphorylation of the I-form enzyme led to a decrease in the electrophoretic mobility of the subunit to generate a species of Mapp = 90,000, identical to that of the D-form. Exposure of the I-form enzyme (subunit Mapp = 85,000) to trypsin caused degradation in the sequence 85,000 + 82,000 + 79,000 + 72,000;
Archives of Biochemistry and Biophysics, 1988
Preliminary characterization of the "glycolytic complex," formed in trout white muscle, revealed that phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are bound to particulate matter largely by ionic interactions; increasing neutral salt or charged metabolite concentrations released bound PFK and GAPDH. GAPDH was consistently solubilized at lower salt concentrations, indicating that it is not bound as tightly as PFK, but both enzymes were readily solubilized at physiological concentrations of salts and metabolites. pH titrations indicated that PFK binding is dependent on group(s) with a pK, of 7.3 in 30 IIIM imidazole. PFK binding increased at lower pH values; at 150 mM KC1 the apparent p& value is 6.5. Experiments with polyethylene glycol8000 (PEG), which is used to mimic the high in vivo protein concentrations under in vitro conditions, showed that the binding of PFK and GAPDH increased with increasing PEG concentrations. Interestingly, at 5% PEG, only the PFK binding response depended on the ionic composition of the medium-with increased binding occurring at the pH of the exhausted muscle and decreased binding at control pH values. These results suggested that only PFK reversibly bound to cellular structures in response to changing conditions and disagrees with previous studies showing binding of several glycolytic enzymes as measured using the dilution method (F. M. Clarke, F. D. Shaw, and D. J. Morton (1980) Biochem. J. 186,105-109). In order to determine whether artifactual binding was measured by the dilution method, two new methodologies were employed to measure enzyme binding in viva: (a) whole muscle slices were pressed to quickly extrude cellular juice, and (b) muscle strips were finely minced and centrifuged to liberate cytoplasmic contents. Both methods indicated that, under physiological conditions, up to 70% of the total cellular phosphofructokinase may be bound, but other glycolytic enzymes are bound to a lesser extent (lo-30%). This result contrasts those obtained with the dilution method, and suggests that dilution of cellular contents may result in an overestimation of the percentage of enzyme associated with cellular structures; this is dramatically shown for glyceraldehyde-3-phosphate dehydrogenase. The viability of the glycolytic complex in trout white muscle is discussed in light of the decreased binding measured using these new methodologies. Q 13% Academic press, I~~.
Rabbit liver glycogen synthase. Susceptibility of the enzyme subunit to proteolysis
Journal of Biological Chemistry
Rabbit liver glycogen synthase has been purified close to apparent homogeneity in a form dependent on glucose-6-P for full activity. From analyses of the purified enzyme by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, some five polypeptides, apparent molecular weights in the range 79,000 to 90,000, correlated with enzyme activity. The relative abundance of the species varied in different preparations but enzyme could be prepared that was composed almost entirely of a 90,000-dalton polypeptide. Treatment of such enzyme with trypsin generated smaller polypeptides in the sequence 90,000 -+ 85,000 + 82,000 -+ 80,000; concomitantly, the enzyme was activated when assayed either in the presence or absence of glucose-6-P. Tryptic proteolysis caused as much as a 16-fold increase in Vmax and a 20-fold increase in the concentration of its substrate, UDP-glucose, necessary for half-maximal activity. The concentration of the activator, glucose-6-P, needed for half-maximal stimulation was decreased 3.S-fold. We propose that rabbit liver glycogen synthase in a glucose-6-P-dependent form has a subunit of apparent molecular weight approximately 90,000, larger than previously reported, and that the enzyme is sensitive to proteolytic degradation.
A titrimetric assay for glycogen phosphorylase
Analytical Biochemistry, 1973
A titrimetric method for the assay of glycogen phosphorylase is presented in which a direct and continuous course of reaction is obtained over a wide range of enzyme concentrations (7.2-378.3 pg/ml). The method resulted in rates which were in agreement with those obtained using the inorganic phosphate method, and the expected value of the equilibrium concentration ratio of inorganic phosphate to glucose-l-phosphate was obtained. The method can be extended to higher concentrations, and it can be used to measure the rate in either direction. The K, and V,., values of each substrate, glucose-l-phosphate and inorganic phosphate, were determined.
European journal of biochemistry / FEBS, 1973
Cytoplasmic NAD-linked glycerol-3-phosphate dehydrogenase was isolated from rabbit renal adipose tissue in a preparation that was apparently homogeneous by preparative isoelectric focusing and polyacrylamide-disc-gel electrophoresis. The adipose tissue and skeletal muscle enzymes were indistinguishable by disc-gel electrophoresis, immunological comparisons, apparent K , values for substrates and coenzymes, isoelectric point in preparative isoelectricfocusing runs, and apparent molecular weight and Stokes radius on polyacrylamide-gel-filtration columns. The ultraviolet spectrum of the adipose tissue enzyme was identical to that of the muscle enzyme including the broad maximum at 275nm indicative of the non-protein component characteristic of the muscle enzyme. Crude homogenates of rabbit adipose tissue and muscle electro-focused in polyacrylamide gels and stained for glycerol-3-P dehydrogenase activity showed four major zones present in slightly different proportions in the two homogenates, but fully superimposable in runs on mixed tissue homogenates.
The interaction of muscle glycogen phosphorylase b with glycogen
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1982
Interaction of muscle glycogen phosphorylase b (EC 2.4.1.1) with glycogen wa~ , studied by sedimentation, stopped-flow and temperature-jump methods. The equilibrium enzyme concentration was determined by sedimentation in an analytical ultracentrifuge equipped with absorption optics and a photoelectric scanning system. The maximum adsorption capacity of pig liver glycogen is 3.64/tmol dimeric glycogen phosphorylase b per g glycogen, which corresponds to 20 dimeric enzyme molecules per average glycogen molecule of M r 5.5.106 . Microscopic dissociation constants were determined for the enzyme-glycogen complex within the temperature range from 12.7 to 30.0°C. Enzyme-glycogen complexing is accompanied by increasing light scattering and its increment depends linearly on the concentration of the binding sites on a glycogen particle that are occupied by the enzyme. Complex formation and relaxation kinetics are in accordance with the proposed bimolecular reaction scheme. The monomolecular dissociation rate constant of the complex increases as the temperature increases from 12.7 to 30.0°C, whereas the bimolecular rate constant changes sligtly and is about 10 s M -l's-t. These data point to the possibility of diffusional control of the complex formation.