Effects of fixation and substrate protection on the isoenzymes of aspartate aminotransferase studied in a quantitative cytochemical model system.
The cytochemical technique of Lee and Torack for the demonstration of aspartate aminotransferase activity was tested on a model system consisting of either total liver homogenate or the mitochondrial or soluble cytoplasmic fraction, incorporated in polyacrylamide film. After incubation of portions of film in a medium of alpha-ketoglutarate, L-aspartate, and lead nitrate, the lead oxaloacetate formed was converted to lead sulfide. The absorbance determined at 520 nm in a film spectrophotometer and expressed in terms of unit weight of film provided a measure of the contained enzymatic activity, and was directly proportional to the concentration of chemically determined oxaloacetate in the film. Both mitochondrial and "soluble" isozymes of aspartate aminotransferase reacted with the cytochemical media to a quantitatively similar degree, but were considerably inactivated after 15 min of treatment with 1% glutaraldehyde or 3.7% formaldehyde in imidazole buffer, the rate of inactivation being greater for the soluble isozyme. Application of the principle of substrate protection delayed inactivation. Thus, for both isozymes the rate of inactivation decreased if ketoglutarate was added to the fixative. Similarly, it was shown that the optimal incubation medium for the demonstration of the soluble isozyme must contain 4 mM of alpha-ketoglutarate and 20 mM of L-aspartate. Under these conditions the turnover-number for the cytochemical system is 70% of the value obtained from biochemical estimations. Cytochemical K(m) values differed for each isozyme and were in accord with values determined by biochemical techniques, indicating that the model system can be used as a link between biochemical and cytochemical data in enzymatic studies.
D000447:Aldehydes / Q000494:pharmacology; D000818:Animals; D001219:Aspartate Aminotransferases / Q000032:analysis*; D001224:Aspartic Acid / Q000494:pharmacology; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003470:Culture Media; D003593:Cytoplasm / Q000201:enzymology; D005557:Formaldehyde / Q000494:pharmacology; D005782:Gels; D006651:Histocytochemistry; D007093:Imidazoles / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis* / Q000037:antagonists & inhibitors; D007656:Ketoglutaric Acids / Q000494:pharmacology; D007854:Lead; D008099:Liver / Q000166:cytology / Q000201:enzymology; D008722:Methods; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology; D008954:Models, Biological; D009566:Nitrates; D010071:Oxaloacetates / Q000032:analysis; D051381:Rats; D013053:Spectrophotometry; D013440:Sulfides; D013997:Time Factors; D014020:Tissue Extracts
D000447:Aldehydes; D003470:Culture Media; D005782:Gels; D007093:Imidazoles; D007527:Isoenzymes; D007656:Ketoglutaric Acids; D009566:Nitrates; D010071:Oxaloacetates; D013440:Sulfides; D014020:Tissue Extracts; D005557:Formaldehyde; D007854:Lead; D001224:Aspartic Acid; D001219:Aspartate Aminotransferases