Evidence of a Critical Histidine Residue in Soluble Aspartic Aminotransferase

Photooxidation of extramitochondrial α-aspartate aminotransferase in the presence of methylene blue or Rose bengal leads to a loss of enzymatic activity which follows first order kinetics. Amino acid analysis shows that histidine is the only amino acid residue significantly affected by photooxidation. Of the 8 histidine residues present in the enzyme monomer, 2 are oxidized rapidly at a rate identical with that of the activity loss, while the other 6 are destroyed much more slowly. The pH dependence of the rate of the photo-induced inactivation of the enzyme corresponds to that expected for the photooxidation of imidazole groups. The behavior of the enzyme in Sephadex G-200 is identical before and after extensive photooxidation, while the starch gel electrophoretic pattern changes after photooxidation. It is concluded that the loss of enzyme activity caused by photooxidation is related to the destruction of 1 histidine residue

Isolation and Characterization of Multiple Forms of Glutamate-Aspartate Aminotransferase from Pig Heart

Abstract At least four different protein fractions provided with aspartate aminotransferase activity can be isolated from pig heart cytoplasm. No difference is detected in the sedimentation coefficient, immunodiffusion properties, and primary structure (as studied by quantitative amino acid analysis and peptide mapping of tryptic digests) of the three main fractions, called, respectively, α, β, and γ in their order of increasing anodic mobility on starch gel electrophoresis. The three main fractions differ significantly in specific activity, kinetics of recombination of the apoenzyme with the coenzyme, and behavior in 8 m urea. The most visible difference among them is in the way in which the coenzyme is bound to the enzyme protein. In the α and β fractions, pyridoxal phosphate is bound mostly in an "active" mode, characterized by absorption peaks at 362 or 430 mµ (depending upon the pH) and by the capacity to transaminate reversibly with the amino acid substrates. In the γ form, most of the coenzyme is bound in a nonactive mode, characterized by an absorption peak at 340 mµ and by the incapacity to react with amino acid substrates. Upon aging, the α and β fractions lose some activity, while a portion of their coenzyme becomes bound in the inactive mode. Even after these changes have occurred, the various fractions maintain their electrophoretic individuality, which is also preserved after resolution and after treatment with concentrated urea. Electrophoretic analyses carried out on a water extract of a single heart taken immediately after the death of the animal reveal the presence of the various subforms; this indicates either that the various subforms exist as such in the living cell or, if they are artifacts, that they must be formed very early in the preparation and under extremely mild conditions