J. S. Nisselbaum

IMMUNOCHEMICAL STUDIES OF FUNCTIONALLY SIMILAR ENZYMES

For many years attempts have been made to demonstrate differences among functionally similar enzymes from different tissues of the same species. Chemical, kinetic, physical, and immunochemical methods have been employed. For example, 0.8 per cent formaldehyde inhibits red-cell acid phosphatase completely, but has no effect on prostatic acid ph0sphatase.l Kaplan and his associates have characterized the lactic dehydrogenases from different tissues by the relative rates of reduction of diphosphopyridine nucleotide (DPN) and DPN analogues? Liver phosphoprotein phosphatase of lysosomes has been shown to differ from that in the soluble phase with respect to the K, values obtained with a-casein and &casein, as well as on the basis of the ratios of V,,,. a-casein: V,,,. p-casein? Several zones of lactic dehydrogenase activity in human serum were demonstrated electrophoretically in 1957 by Vesell and Bearn; the relative amounts of lactic dehydrogenase in the zones were changed in myocardial infarction and in le~kemia .~ In 1943, Kubowitz and Ott5 attempted to differentiate enzymes from normal and tumor tissues in a single species by immunochemical techniques. They found that the lactic dehydrogenase isolated from either rat muscle or Jensen sarcoma of the rat was inhibited equally by rooster antiserum to the rat-muscle enzyme, and that the inhibition by the antiserum was competitive with the substrate, pyruvate. Henion and Sutherland6 showed that rooster antisera to dog-liver and dog-heart phosphorylases most powerfully inhibited the homologous enzyme in each case, and had less effect upon the phosphorylases from other dog tissues, as well as from heart and liver of other species. McGeachin and Reynolds’ ,* have recently reported that antisera produced in rabbits, roosters, or rats against hog pancreatic a-amylase inhibited hog sali-

Isozymes of aspartate aminotransferase in tissues and blood of man

Available literature on the characteristics of alanine and aspartate aminotransferases has been briefly reviewed. Our own studies have concerned themselves with the isozymes of aspartate aminotransferase in the tissues and blood of man. The anionic (supernatant) and the cationic (mitochondrial) isozymes of liver and heart have been prepared, and antisera to each of the isozymes from heart have been produced. The values for Km(l-aspartate) were the same for the anionic isozyme of heart or liver and were distinctly higher than the values that were obtained for the cationic components. Conversely, the values for Km(α-ketoglutarate) were the same for the anionic isozyme of heart or liver and were distinctly lower than the values that were obtained for the cationic components. Antiserum to each of the two isozymes from heart inhibited the homologous isozyme from heart or liver specifically and practically completely. Hemolysates of washed human mature erythrocytes yielded, in general, one band, anionic in migration. This component had values for Km(l-aspartate) and Km(α-ketoglutarate) and immunochemical characteristics that were essentially the same as those obtained for the anionic component of liver or heart. The observation that a cationic band was present in the blood of an individual after blood donation led to the study of experimentally produced reticulocytosis in rabbits. The total erythrocyte aspartate aminotransferase activity increased five- to sixfold, and a cationic mitochondrial isozyme appeared. The relationship of the electrophoretic pattern of aspartate transaminase in human tissues to the electrophoretic pattern in the serum was also studied. Starch paste electrophoresis was performed on homogenates of forty specimens from ten different tissues and two tumors. The aminotransferase was distributed into two main areas, one in the region of the gamma globulin and the other in the alpha-beta globulin region. The serum aspartate aminotransferase in four normal persons and in twenty patients with neoplastic disease with elevated serum enzyme activity was completely confined to one area—the alpha-beta globulin region. Fourteen patients also exhibited a cationic component in the serum. The appearance of this component appeared to be associated with an acute phase of the patients disease and its disappearance with the subsidence of this phase. The general role of the cationic, mitochondrial and the anionic, supernatant isozymes in regulation of cell metabolism is discussed briefly.

Regulation of aspartate amino-transferase isozymes by glyceraldehyde-3-phosphate

Abstract This review concerns our work on glyceraldehyde-3-P as a time-dependent inhibitor of rat liver aspartate aminotransferase isozymes, and the possibility that glyceraldehyde-3-P may function as a rapidly acting regulator of aspartate aminotransferase. The d -isomer and the dl -racemate of glyceraldehyde-3-P were equally effective for each isozyme. Study of several glycolytic intermediates indicated that the conjoint presence of the free aldehyde and the phosphoryl residue was necessary for inhibition. Maximum inhibition of the anionic isozyme occurred at pH values from 8.4 to 10.3; the cationic isozyme was optimally inhibited at pH 7.4. Keto acid substrates decreased the inhibition, whereas amino acid substrates accentuated it. Inhibition of the cationic isozyme was completely competitive with respect to α-ketoglutarate ( K i = 0.98 m m ) and oxaloacetate (Ki not measurable), and completely noncompetitive with respect to l -aspartate ( K i = 0.084 m m ) and l -glutamate ( K i = 0.11 m m ). Inhibition of the anionic isozyme was mixed partially competitive-partially noncompetitive with respect to α-ketoglutarate ( K i = 1.9 m m ) and oxaloacetate ( K i = 1.5 m m ) and partially noncompetitive with respect to l -aspartate ( K i = 0.39 m m ) and l -glutamate ( K i = 0.57 m m ). These data suggest that both keto acids bind to the isozymes at a single site and compete with glyceraldehyde-3-P for that site, whereas the amino acids bind to a site other than the one for which keto acids and glyceraldehyde-3-P compete. Homologues of glyceraldehyde-3-P were also investigated. Ribose-5-P, fructose-6-P and glucose-6-P did not inhibit either isozyme since they exist as the internal hemiacetals. d -erythrose-4-P was a time-dependent inhibitor of both isozymes. Inhibition was completely competitive with respect to α-ketoglutarate, K i = 3.08 m m and 1.4 m m for the anionic and cationic isozymes, respectively, and was completely noncompetitive with respect to l -aspartate, K i = 0.334 m m and 0.135 m m for the anionic and cationic isozymes, respectively. Inhibition by glycolaldehyde-P was not time-dependent and was completely competitive with respect to α-ketoglutarate and uncompetitive with respect to l -aspartate for both isozymes. The Ki values were 0.77 m m and 1.01 m m for the anionic and cationic isozymes, respectively. We propose that glyceraldehyde-3-P and its homologues inhibit aspartate aminotransferase isozymes by forming a Schiff base with one of the ϵ-amino groups of lysine at the enzymically active site. Aspartate would potentiate inhibition by converting the enzyme to the pyridoxamine form, thereby exposing a second ϵ-amino lysyl group which would react with the inhibitor. Evidence for a Schiff base was obtained by NaBH4 reduction of apoisozymes in the presence of dl -glyceraldehyde-3-P. This prevented restoration of activity upon addition of pyridoxal-5′-P. It seems likely that the divalent phosphoryl group on the inhibitor molecule is involved in the competition at the keto acid binding site. Our results suggest the possibility that glyceraldehyde-3-P may be implicated in the regulation, in vivo, of gluconeogenesis as well as other metabolic pathways by affecting the activity of the isozymes of aspartate aminotransferase.