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Ultracentrifugation studies show that, at concentrations of about 0.1 mM the aspartate aminotransferase of pig heart muscle does not dissociate into subunits either in 6 M guanidine hydrochloride, pH 7.5, or 8 M urea solution, pH 7.5. On prolonged treatment (8 weeks, 0°) with urea, however, complete dissociation occurs and the measured value of the molecular weight drops to approx. 40,000. The protein so obtained is enzymically inactive and migrates on electrophoresis in starch gel at pH 8.0 as a single, fast-moving band. Gel filtration experiments show that the holoenzyme (pyridoxal form) partly dissociates into two subunits at a concentration of about 3 nM. The specific activity of the enzyme does not change in this region; the monomer and dimer have, therefore, the same catalytic activity per mole of co factor. The apoenzyme ks unstable at high dilution and its dissociation has not been directly demonstrated by gel filtration. However, indirect evidence obtained from a study of the kinetics of its recombination with cofactor suggests that dissociation occurs at a concentration of about 50 nM. [ABSTRACT FROM AUTHOR]

Convenient methods for the purification of the soluble aspartate aminotransferase from pig heart muscle are described. The enzyme obtained is homogeneous in the ultracentrifuge and to starch gel electrophoresis at about pH 9.0. At lower pH, as first described by Fasella et. al. [1], a number of electrophoretically distinct species can be detected. Some of the properties of these are reported. The currently accepted value of the molecular weight of the enzyme is incorrect: ultracentrifuge studies gave a value of 78,600 ± 2,400 (23 determinations). Gel filtration experiments led to a similar value. Titration of apoenzyme with cofactor gives values of the equivalent weight of the protein which vary with the enzumic specific activity. Extrapolation to the maximum specific activity leads to a value for the equivalent weight of about 40,000. The enzyme has, therefore, two cofactor binding sites. [ABSTRACT FROM AUTHOR]

A method has been developed for specific enzymic cleavage of polypeptides at the N-terminal side of modified cysteine residues. Lysine residues are blocked by trifluoroacetylation and cysteine residues subsequently converted to the 2-aminoethyl derivatives. Digestion of the modified polypeptide with the lysine-specific protease from Armillaria mellea (patented by Walton et al., 1972) occurs only at 2-aminoethylcysteine residues. With the beta chain of human haemoglobin, which contains 2 cysteine and 11 lysine residues, cleavage was observed at both modified cysteines but at none of the lysines. In the case of a polypeptide from bee venom which contains 4 half-cystine and 5 lysine residues, cleavage occurred at only 2 of the modified cysteines and also at 2 lysine residues. The pattern of cleavage in the latter case can be interpreted in terms of the amino acid sequence of the polypeptide.

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.