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Treatment of mitochondrial aspartate aminotransferase from rat liver with trypsin leads to specific cleavage of the bonds between residues 26 and 27, and residues 31 and 32. The proteolysed enzyme has only a small residual catalytic activity, but retains a conformation similar to that of the native form as judged by accessibility and reactivity of cysteine residues. Proteolysis abolishes the ability of the enzyme either to bind to mitochondria or to be imported into the organelles. This suggests that the N-terminal segment of the native enzyme is essential for both of these functions, at least in the model system used to study the import process.

To gain further insight into the mitochondrial receptor area which allows selective uptake of both purified aspartate aminotransferase and malate dehydrogenase into mitochondria, the inhibition of metal complexing agents such as bathophenanthroline and tiron on the uptake of both enzymes has been investigated. In view of the nature of the inhibition found, we propose the existence of metal ion(s) at or near the aspartate aminotransferase, but far from the malate dehydrogenase binding site.

Carboxymethylated aspartate aminotransferase was digested with a proteinase claimed to be specific for lysine residues. Complete cleavage occurred at 12 of the 19 lysine residues in the protein, but at the remaining seven residues cleavage was either restricted or absent. In addition, cleavage was observed at three of the 26 arginine residues. These results are discussed with reference to the amino acid residues adjacent to points of complete or restricted cleavage. The complete primary structure of aspartate aminotransferase, based on these and other studies, is given. Evidence for the assignment of some acid and amide side chains has been deposited as Supplementary Publication SUP 50050 (11 pp.) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5. The evidence for the assignment of residue 366 was less conclusive than for the other acid and amide side chains and is, therefore, given in the main paper.

Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system.

1. It was previously shown [Passarella, Marra, Doonan & Quagliariello (1980) Biochem. J. 192, 649-658] that, when mitochondrial malate dehydrogenase from rat liver is incubated with sulphite-loaded mitochondria from the same source, uptake of the enzyme occurs, as judged by a fluorimetric assay of intramitochondrial enzyme activity. Confirmation of sequestration of the enzyme inside the organelles is provided by its proteinase-resistance after uptake. 2. Enzyme uptake into mitochondria is inhibited by enzyme treatment with mersalyl at concentrations that do not affect its catalytic activity. 3. Enzyme uptake is energy-dependent, as shown by inhibition of the process by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and by antimycin. ATP and oligomycin, on the other hand, both stimulate the process, but stimulation by ATP is inhibited by oligomycin. These results suggest that uptake depends on maintenance of transmembrane ion gradient rather than direct ATP involvement. 4. Measurements of delta psi by means of the ‘redistribution signal’ probe safranine suggest no dependence of malate dehydrogenase uptake on membrane potential. 5. Comparison of the effects of the ionophores valinomycin, nonactin, gramicidin and nigericin shows that uptake depends on maintenance of a transmembrane pH gradient.

1. The effects of various inhibitors of electron transport and of oxidative phosphorylation and the effects of ionophores on the uptake of native aspartate aminotransferase into mitochondria were investigated. 2. Both antimycin and cyanide completely inhibited the uptake of the enzyme. On the other hand, uptake was stimulated to ATP and by oligomycin; however, the stimulation by ATP is inhibited by oligomycin. 3. The effects of ionophores of the valinomycin type in media containing K+ ions depended on the conditions used. Valinomycin alone stimulated the uptake of the enzyme, but in the presence of phosphate ions uptake was abolished. Nonactin was without effect at a low K+ concentration, but was stimulatory at 100 mM-KCl. Gramicidin also stimulated the uptake process. 4. Nigericin completely abolished uptake of aspartate aminotransferase into mitochondria. 5. The uptake of te enzyme was decreased by 18% in the absence of inhibitors or ionophores when the external pH was increased from 6.9 to 7.6. 6. These results indicate that ATP is not directly involved in the uptake of aspartate aminotransferase into mitochondria, neither is there a requirement for a cation gradient. Rather the uptake depends on the maintenance of a pH gradient across the mitochondrial inner membrane.

Kinetic studies were made of the hydrolysis by trypsin of alpha-N-acetylglycyl-L-lysine methyl ester and of its neutral analogue alpha-N-acetylglycyl-epsilon-N-formyl-L-lysine methyl ester. The latter substance is a moderately good substrate for trypsin, and this observation is discussed in terms of the substrate specifically of the enzyme. The actions of trypsin and of the lysine-specific proteinase from Armillaria mellea on both a native and a formylated polypeptide substrate were compared. Both enzymes were found to hydrolyse specifically bonds to epsilon-N-formyl-lysine in the formylated substrate.

1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied.

1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.

The threonine dehydratase extracted from sheep liver is in an essentially inactive form, referred to here as the precursor, but can be activated by incubation at high temperature in alkaline solution. A method for purification of the activated enzyme to a state approaching homogeneity has been devised. The activated enzyme catalyses the deamination of serine but rapidly loses activity during the process. The enzyme inactivated by incubation with serine can be reactivated under the same conditions as those described for the precursor, the kinetic parameters being the same in both cases. Individual sheep livers differ widely in their content of threonine dehydratase, and the possible role of dietary factors in this variability has been examined. The average amount of the enzyme in the livers of sheep fed on high-plant-protein diets did not differ significantly from that in livers obtained from slaughterhouses, but was higher than that in livers from animals fed only on grass or normal concentrates. Large increases in enzyme concentration were, however, obtained when sheep were injected intraperitoneally with a protein hydrolysate. The enzyme from the livers of these sheep could be purified by the same technique and to the same specific activity as the enzyme from the livers of control sheep.

External suppressors, sufS, of a -1 frameshift mutant cause ribosomes to shift into the -1 frame when reading the sequence CAG GGA GUG. The resulting product is not Gln-Gly-Val but Gln-Gly-Ser with Ser being encoded by the underlined AGU. The alleles investigated are approximately 2% efficient in causing frameshifting. Two other suppressors, hopR and hopE of the same -1 frameshift mutant, cause some ribosomes reading the sequence GUG UG to decode a single amino acid, Val, from the five nucleotides. The possibility is considered that peptidyl-tRNA(Val) dissociates from the mRNA, but re-pairs in a triplet manner after the mRNA slips forward by two bases.

Peptides obtained by tryptic digestion of carboxymethylated and maleylated aspartate aminotransferase and of the aminoethylated enzyme were isolated and the complete amino acid sequences of most of them were determined. Digestion of the carboxymethylated protein with pepsin produced a complex mixture of peptides that allowed some overlapping of the tryptic peptides (Fig. 4); in addition, peptides were obtained that had not been found in either of the tryptic digests. From these studies about 400 amino acid residues were identified. Experimental details and confirmatory data for the results presented here are given in a supplementary paper that has been deposited as Supplementary Publication 50011 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. (1972) 126, 5.