[Molecular hybridization of native and modified subunits of lactate dehydrogenase isoenzymes and aldolase A in rats].
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Experimental conditions for the molecular hybridization in vitro between iodine and native subunits of isoenzymes 1 and 5 of lactate dehydrogenase (LDH) are described. It is also shown that the covalently fixed on the polyacrylamide beads rat J125 labelled LDH-5 and J125 labelled aldolase A, under conditions of complete dissociation of the quaternary structure of these enzymes, only one of the four subunits remain bound with the beads. Subunit of LDH-5, which is covalently bound with the polyacrylamide beads, is capable to hybridize (reassociated) with 3 native subunits. In addition, the immobilized LDH-5 subunits and aldolase A are capable to hybridize with J125 labelled subunits of these enzymes. Thus, when thyrosine, lysine and N-terminal amino acids are modified, subunits of LDH-5 and aldolase A retain their capacity to restore their quaternary structures.Keywords:
Protein quaternary structure
Polyacrylamide
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Experimental conditions for the molecular hybridization in vitro between iodine and native subunits of isoenzymes 1 and 5 of lactate dehydrogenase (LDH) are described. It is also shown that the covalently fixed on the polyacrylamide beads rat J125 labelled LDH-5 and J125 labelled aldolase A, under conditions of complete dissociation of the quaternary structure of these enzymes, only one of the four subunits remain bound with the beads. Subunit of LDH-5, which is covalently bound with the polyacrylamide beads, is capable to hybridize (reassociated) with 3 native subunits. In addition, the immobilized LDH-5 subunits and aldolase A are capable to hybridize with J125 labelled subunits of these enzymes. Thus, when thyrosine, lysine and N-terminal amino acids are modified, subunits of LDH-5 and aldolase A retain their capacity to restore their quaternary structures.
Protein quaternary structure
Polyacrylamide
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A study has been made of the effects of phytohaemagglutinin on the gene expression of the glycolytic enzymes in cultured human lymphocytes. All the enzymes were found to show an average increase in activity of between 160% and 360% in stimulated cells, but the increases were greater for the enzymes comprising the second half of the pathway. The enzyme activities in stimulated cells, cultured for 72 h, were similar to the activities measured in long-term lymphoid lines. Starch-gel electrophoresis was used to examine the isozyme patterns of the enzymes before and after exposure of the lymphocytes to PHA. Six of the enzymes showed isozyme patterns unchanged by stimulation. Four of the enzymes, aldolase, triosephosphate isomerase, enolase and lactate dehydrogenase, showed different isozyme patterns in stimulated cells from those seen in uncultured or unstimulated cells. The electrophoretic results showed a good correlation in isozyme pattern between uncultured lymphocytes and cultured unstimulated lymphocytes, and between PHA-stimulated lymphocytes and long-term lymphoid lines.
Galton's problem
Phytohaemagglutinin
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The rat glucose 6-phosphate dehydrogenase isozymes of cell sap origin designated D, F 1 , F 2 and F 3 were found to be interconvertible with each other in the presence or absence of mercaptans. The D and F 3 enzymes were the oxidized, the F 1 was the reduced and the F 2 was the intermediate form. Molecular weight estimation by the polyacrylamide gel electrophoresis and Sephadex G-200 methods demonstrated that the D enzyme had twice the molecular weight of the F enzymes. The mitochondrial enzyme which moved as fast as the F 3 enzyme of cell sap origin was not modified with mercaptans. Together with previous data, the present results caution in the interpretation of zymograms.
Sephadex
Polyacrylamide
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The acetohydroxy acid synthase (AHAS) isozymes from enterobacteria are each composed of a large and small subunit in an alpha 2 beta 2 structure. It has been generally accepted that the large (ca. 60-kDa) subunits are catalytic, while the small ones are regulatory. In order to further characterize the roles of the subunits as well as the nature and the specificities of their interactions, we have constructed plasmids encoding the large or small subunits of isozymes AHAS I and AHAS III, each with limited remnants of the other peptide. The catalytic properties of the large subunits have been characterized and compared with those of extracts containing the intact enzyme or of purified enzymes. Antisera to the isolated subunits have been used in Western blot (immunoblot) analyses for qualitative and semiquantitative determinations of the presence of the polypeptides in extracts. The large subunits of AHAS isozymes I and III have lower activities than the intact enzymes: Vmax/Km is 20 to 50 times lower in both cases. However, for AHAS I, most of this difference is due to the raised Km of the large subunit alone, while for AHAS III, it is due to a lowered Vmax. The substrate specificities, R, of large subunits are close to those of the intact enzymes. The catalytic activity of the large subunits of AHAS I is dependent on flavin adenine dinucleotide (FAD), as is that of the intact enzyme, although the apparent affinities of the large subunits alone for FAD are 10-fold lower. Isolated subunits are insensitive to valine inhibition. Nearly all of the properties of the intact AHAS isozyme I or III can be reconstituted by mixing extracts containing the respective large and small subunits. The mixing of subunits from different enzymes does not lead to activation of the large subunits. It is concluded that the catalytic machinery of these AHAS isozymes is entirely contained within the large subunits. The small subunits are required, however, for specific stabilization of an active conformation of the large subunits as well as for value sensitivity.
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85 yaks from Jiali and Yadong in Tibet were used to observ the serum isozymes of lactate dehydrogenase (LDH)by polyacrylamide gel electrophoresis, 5 bands of LDH isozymes (LDH_(1)-LDH_(5))were observed on the gels, and it was discovered that there exist three genetic variants (A、B and C)in H subunit of LDH, the C variant took 82.35 percent of the total. Also, the relative activity of LDH_(2)in the serum were significantly lower with A and B type of H subunit than that with C type in Jiali region, Its mechanism and significance remains further investigation.
YAK
L-Lactate dehydrogenase
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Triosephosphate isomerase
Phosphoglycerate kinase
Phosphoglycerate mutase
Glucose-6-phosphate isomerase
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The intraccellular proteins of animal cells are continuously turning over; therefore, the concentration of a given protein is regulated both at the level of protein synthesis and at the level of protein degradation. Studies on the relative rates of turnover of isoenzymes, such as those of aldolase and lactate dehydrogenase, may help to clarify the mechanisms involved in protein turnover. The isoenzymes and subunit types are very similar proteins, and are located within the same intracellular compartments; yet, the concentrations of these proteins are independently regulated. The present paper describes the roles of synthesis and degradation in regulating aldolase isoenzyme concentrations in avian brain...
Protein Degradation
Mammalian brain
Protein turnover
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The copolymerization method of immobilization was used to obtain preparations of enzymes covalently incorporated in polyacrylamide gel. They possess properties making them suitable for practical use. First, the preparations are hundreds of times more stable against irreversible thermoinactivation than native enzymes. Second, on immobilization, the reversible conformational changes which also lower enzyme activity at elevated temperatures are completely suppressed. As a result, the temperatures of maximum activity for trypsin and alpha-chymotrypsin covalently entrapped in polyacrylamide gel are 75 and 70 degrees C, respectively-25 and 30 degrees C higher than the corresponding values for the native enzymes. Therefore, the copolymerized enzyme preparations have a high operational stability at elevated temperatures.
Polyacrylamide
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Hexokinase
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