Induction of cytosolic aspartate aminotransferase by a high-protein diet

Induction of cytosolic aspartate aminotransferase (glutamic oxaloacetic transaminase) was observed in rat liver on administration of a high-protein diet. The enzyme activity in the liver of rats given 60% and 80% protein diet increased to 1.8- and 1.9-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of functional sGOT mRNA, measured by in vitro translation. Whereas the activity of mitochondrial aspartate aminotransferase did not increase. Induction of cytosolic aspartate aminotransferase was also detected immunocytochemically.     

Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.     

Experimental diabetes causes mitochondrial loss and cytoplasmic enrichment of pyridoxal phosphate and aspartate aminotransferase activity

The streptozotocin diabetic rat was selected as a model to study how insulin deficiency alters vitamin B6 utilization by focusing on pyridoxal phosphate levels and aspartate aminotransferase activities in liver tissues. Diabetes of 15 weeks' duration lowered plasma pyridoxal phosphate levels by 84%. Normal plasma pyridoxal phosphate was 480 pmole/ml. Fractionation of liver into mitochondrial and extramitochondrial compartments demonstrated that diabetes caused a 43% diminution in mitochondrial pyridoxal phosphate per gram of liver. There was no cytoplasmic change in these diabetic rats. Mitochondrial aspartate aminotransferase activity was decreased 53% per gram of diabetic liver and cytoplasmic aspartate aminotransferase activity was elevated 3.4-fold. Damage to diabetic mitochondria during preparation procedures could not account for the rise in cytoplasmic aspartate aminotransferase activity. Electrophoresis showed that in the diabetic cytoplasm both cathodal and anodal forms of the enzyme were elevated. Speculations concerning mitochondrial loss and cytoplasmic gain of enzyme activity as well as those on the reduction of plasma pyridoxal phosphate in the diabetic rat are presented.     

Rat cytosolic aspartate aminotransferase: regulation of its mRNA and contribution to gluconeogenesis

Induction of cytosolic aspartate aminotransferase (cAspAT) was observed in rat liver on administration of a high-protein diet, or glucagon and during fasting. The enzyme activity in the liver of rats given 80% protein diet or glucagon injection during starvation increased to 2- to 2.4-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of hybridizable cAspAT mRNA, measured by blot analysis using the cloned rat cAspAT cDNA as a probe. No increase in the enzyme was detected in kidney, heart, brain, or skeletal muscle. The activity of mitochondrial aspartate aminotransferase (mAspAT) did not increase. Induction of cAspAT was observed when glucose metabolism tended toward gluconeogenesis. The physiological function of the induction of cAspAT is considered to be to increase the supply of oxaloacetate as a substrate for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) [EC 4.1.1.32] for gluconeogenesis.     

Purification of rat liver mitochondrial aspartate aminotransferase and separation of its isoforms utilizing high-performance liquid chromatography

A rapid method for purification of mitochondrial aspartate aminotransferase from rat liver employing high-performance liquid chromatography is reported. The product is purified 80-fold with a recovery greater than or equal to 50% in a single day. The amino acid composition, N-terminal amino acid sequence, specific activity, and spectral characteristics of the isolated enzyme are similar to those previously reported for this protein. The protein is homogeneous by standard electrophoretic and chromatographic criteria, but can be resolved into at least five isoforms by a carboxymethylated resin column using high-performance liquid chromatography. The principal isoform initially isolated is converted into two additional isoforms with lower specific activity upon storage at 4 degrees C.     

Distribution of aspartate aminotransferase activity in yeasts, and purification and characterization of mitochondrial and cytosolic isoenzymes from Rhodotorula minuta [corrected

The distribution of aspartate aminotransferase activity in yeasts was determined. The number of species of the enzyme in each yeast was determined by zymogram analysis. All the yeasts, except for the genus Saccharomyces, showed two or three activity bands on a zymogram. From among the strains, Rhodotorula minuta [corrected] and Torulopsis candida were selected for examination of the existence of yeast mitochondrial isoenzymes, because these strains showed two clear activity bands on the zymogram and contained a high amount of the enzyme. Only one aspartate aminotransferase was purified from T. candida: the component in the minor band on the zymogram was not an isoenzyme of aspartate aminotransferase. On the other hand, two aspartate aminotransferases were purified to homogeneity from R. minuta [corrected]. The components in the main and minor activity bands on the zymogram were identified as the mitochondrial and cytosolic isoenzymes, respectively, in a cell-fractionation experiment. The enzymatic properties of these isoenzymes were determined. The yeast mitochondrial isoenzyme resembled the animal mitochondrial isoenzymes in molecular weight (subunits and native form), absorption spectrum, and substrate specificity. The amino acid composition was closely similar to that of pig mitochondrial isoenzyme. Rabbit antibody against the yeast mitochondrial isoenzyme, however, did not form a precipitin band with the pig mitochondrial isoenzyme.     

Intramitochondrial location and some characteristics of chicken liver aspartate aminotransferase

Chicken liver mitochondrial aspartate aminotransferase was found to be located in the intermembrane space and bound to the inner mitochondrial membrane. Purification of two mitochondrial fractions containing aspartate aminotransferase activity was performed. Both fractions showed similar chromatographic behaviour and identical isoelectric point and molecular weight values. There were no significant differences in the general kinetic mechanism, Km values, substrates inhibition and effect of various anions on the activity of mitochondrial aspartate aminotransferase purified from both fractions.     

Cell-free synthesis of a putative precursor of mitochondrial aspartate aminotransferase with higher molecular weight

Mitochondrial aspartate aminotransferase was synthesized in a cell-free system using polysomal mRNA from chicken heart. Its primary translation product has a higher molecular weight than the subunit of the mature α₂-dimer (ΔMW ～3000). The synthesis of a precursor most likely relates to the translocation of the protein from its site of synthesis, i.e., cytosolic ribosomes, into the mitochondrial matrix.     

Functional inhibition of cytosolic and mitochondrial aspartate aminotransferase by l-2-amino-4-methoxy-trans-3-butenoic acid in isolated rat hepatocytes and mitochondria

The transaminase inhibitor l-2-amino-4-methoxy-trans-3-butenoic acid (AMB) decreased aspartate aminotransferase activity by approximately two-thirds in isolated rat liver mitohondria incubated with succinate, ammonia, and ornithine. Aspartate production by the mitochondria was unaffected over the 30-min incubation period, indicating that mitochondrial aspartate aminotransferase activity is normally far in excess of that required for maximal rates of aspartate production. In rat hepatocytes incubated with lactate, ammonia, and ornithine the inhibition of both the cytosolic and mitochondrial isozymes of aspartate aminotransferase by AMB was partially blocked by the presence of ammonia and ornithine. When pyruvate was substituted for lactate as a carbon source with isolated hepatocytes, the presence of ammonia and ornithine blocked the inhibition by AMB of the mitochondrial but not the cytosolic isozyme of aspartate aminotransferase. Urea formation by cells incubated with lactate, ammonia, and ornithine was unaffected by AMB unless the cells were preincubated with the inhibitor prior to the addition of substrates. However, urea formation by cells incubated in the presence of pyruvate, ammonia, and ornithine was inhibited strongly by AMB even without preincubation. The results suggest that the stimulation of ureogenesis from ammonia and ornithine by pyruvate involves the cytosolic isozyme of aspartate aminotransferase. In contrast, the stimulation of ureogenesis elicited by lactate primarily involved mitochondrial aspartate aminotransferase.     

Two forms of aspartate aminotransferase in rat liver and kidney mitochondria

1. Butan-1-ol solubilizes that portion of rat liver mitochondrial aspartate aminotransferase (EC 2.6.1.1) that cannot be solubilized by ultrasonics and other treatments. 2. A difference in electrophoretic mobilities, chromatographic behaviour and solubility characteristics between the enzymes solubilized by ultrasonic treatment and by butan-1-ol was observed, suggesting the occurrence of two forms of this enzyme in rat liver mitochondria. 3. Half the aspartate aminotransferase activity of rat kidney homogenate was present in a high-speed supernatant fraction, the remainder being in the mitochondria. 4. A considerable increase in aspartate aminotransferase activity was observed when kidney mitochondrial suspensions were treated with ultrasonics or detergents. 5. All the activity after maximum activation was recoverable in the supernatant after centrifugation at 105000g for 1hr. 6. The electrophoretic mobility of the kidney mitochondrial enzyme was cathodic and that of the supernatant enzyme anodic. 7. Cortisone administration increased the activities of both mitochondrial and supernatant aspartate aminotransferases of liver, but only that of the supernatant enzyme of kidney.     

Effect of vitamin B6 on the synthesis and degradation of aspartate aminotransferase in chicken embryo fibroblasts

The effect of pyridoxal depletion and supplementation on the intracellular level of mitochondrial and cytosolic aspartate aminotransferase in cultured chicken embryo fibroblasts was examined. No apoenzyme was detected in cells grown in the presence of pyridoxal, and the specific activity of total enzyme did not vary profoundly from primary to quaternary cultures. Under pyridoxal depletion, up to 40% apoenzyme was found in tertiary cultures which was entirely due to the mitochondrial isoenzyme. Cytosolic apoenzyme was never detected. Total aspartate aminotransferase relative to total protein was increased 2-fold in secondary cultures; only the mitochondrial isoenzyme contributed to the increased specific activity. The cytosolic isoenzyme decreased steadily and was below the limit of detection in quaternary cultures. The changes are attributed to an increased and decreased synthesis of mitochondrial and cytosolic isoenzyme, respectively. No induction of either isoenzyme was observed after incubating the cells with different hormones and substrates. In secondary cultures, no degradation of mitochondrial isoenzyme could be detected under pyridoxal deficiency or supplementation during 4.4 days, an interpassage duration. The cytosolic aspartate aminotransferase was degraded initially with an apparent half-life of approximately 0.9 day under both sets of conditions. The pronounced stability of mitochondrial aspartate aminotransferase, even though one-third of it was present as apoenzyme, excludes the formation of the apoform to be the rate-limiting step in its degradation. The present results show that pyridoxal affects the synthesis of mitochondrial and cytosolic aspartate aminotransferase, but differently.     

Cycloheximide causes increased accumulation of translatable mRNA for tyrosine aminotransferase and tryptophan oxygenase in livers of cortisol-treated rats.

Messenger RNA activities for two cortisol-inducible enzymes, tyrosine aminotransferase and tryptophan oxygenase, have been determined by translation in a wheat germ system. The effects of cycloheximide on the two mRNA activities have been evaluated. Cortisol leads to an increase of the translatable mRNAs for tyrosine aminotransferase and tryptophan oxygenase with a maximum at approximately 6 h. Cycloheximide was administered 4 h after treatment with cortisol; 2 h later, the activities of tyrosine aminotransferase and tryptophan oxygenase mRNA had increased five-fold and two-fold, respectively, compared to the activities reached with cortisol alone. Thereafter the amount of the two translatable mRNAs declined, though 14 h after cortisol administration the mRNA activities were still several fold higher than in control animals. Application of alpha-amanitin together with cycloheximide did not prevent an increased accumulation of specific translatable mRNAs. The increase in tyrosine aminotransferase and tryptophan oxygenase activity by cortisol was immediately blocked by cycloheximide. Whereas tryptophan oxygenase activity rapidly declined after cycloheximide application, tyrosine aminotransferase activity remained at the same level. Approximately 4 h thereafter, both enzyme activities increased again.     

Expression of cDNAs encoding the precursor and the mature form of chicken mitochondrial aspartate aminotransferase in Escherichia coli

Both the precursor and the mature form of chicken mitochondrial aspartate aminotransferase were synthesized in Escherichia coli. The precursor was found to sediment quantitatively together with insoluble cell material. In contrast, mature mitochondrial aspartate aminotransferase could be readily extracted from the cells and was indistinguishable from the enzyme isolated from chicken heart in all respects tested: specific activity 230 units mg-1; Mr 2 X 45,000; pI greater than 9; NH2-terminal sequence SSWWSHVEMG, the initiator methionine having been removed by the bacteria. Thus, the polypeptide chain representing mature mitochondrial aspartate aminotransferase is an autonomous folding unit which attains its functional spatial structure independently of the presence of the prepiece, trans-membrane passage, and proteolytic processing.     

Translational and pretranslational control of ornithine aminotransferase synthesis in rat liver

The mitochondrial matrix enzyme, ornithine aminotransferase, is induced in rat liver by the administration of a diet high in protein and by glucagon. The rate of synthesis of the enzyme is increased 100-fold in the livers of rats maintained on a 60% relative to a 0% protein diet, whereas the levels of functional and hybridizable mRNA measured by in vitro translation and through the use of a cloned cDNA probe increased by only 2- to 6-fold and 2- to 3-fold, respectively. Under conditions of glucagon induction that resulted in a 10- to 12-fold increase in the rate of enzyme synthesis, the relative level of functional ornithine aminotransferase mRNA increased by only 2-fold, and the level of hybridizable mRNA actually decreased. The rate of polypeptide chain elongation and the relative number of ornithine aminotransferase nascent chains on polysomes were 2-fold and 23-fold greater, respectively, in hepatocytes derived from 60% relative to 0% protein-fed rats. Using these data, a 23-fold increase in the translational efficiency of the mRNA was calculated. This increase, along with a 2-fold increase in the mRNA level, completely account for the 40-fold increase in the rate of ornithine aminotransferase synthesis observed in hepatocytes derived from 60% protein-fed rats. We conclude that ornithine aminotransferase synthesis is regulated at both a translational and a pretranslational level in rat liver.     

Nonidentity of the Aspartate and the Aromatic Aminotransferase Components of Transaminase A in Escherichia coli

Tyrosine, added to the growth medium of a strain of Escherichia coli K-12 lacking transaminase B, repressed the tyrosine, phenylalanine, and tryptophan aminotransferase activities while leaving the aspartate aminotransferase activity unchanged. This suggested that the aspartate and the aromatic aminotransferase activities, previously believed to reside in the same protein, viz. transaminase A, are actually nonidentical. Further experiments showed that, upon incubation at 55 C, the aspartate aminotransferase of crude extracts was almost completely stable, whereas the tyrosine and phenylalanine activities were rapidly inactivated. Apoenzyme formation was faster, and apoenzyme degradation proceeded more slowly with aspartate aminotransferase than with tyrosine aminotransferase. Electrophoresis in polyacrylamide gels separated the aminotransferases. A more rapidly moving band contained tyrosine, phenylalanine, and tryptophan aminotransferases, and a slower band contained aspartate aminotransferase. A mutant of E. coli K-12 with low levels of aspartate aminotransferase exhibited unchanged levels of tyrosine aminotransferase. Thus, transaminase A appears to be made up of at least two proteins: one of broad specificity whose synthesis is repressed by tyrosine and another, specific for aspartate, which is not subject to repression by amino acids. The apparent molecular weights of both the aspartate and the aromatic aminotransferases, determined by gel filtration, were about 100,000.     

The effect of testosterone on citrate synthesis and citrate oxidation and a proposed mechanism for regulation of net citrate production in prostate

Citrate oxidation by rat ventral prostate was reduced by castration and increased by testosterone administration. Similarly, the mitochondrial aconitase activity was decreased by castration; whereas cytosol aconitase was unaffected. The rate of citrate oxidation is extremely low in prostate. Castration also decreased mitochondrial aspartate aminotransferase activity while having no effect on the cytosol isoenzyme. Testosterone markedly stimulated the net production of citrate from aspartate plus glutamate by prostate mitochondria. These studies support the proposal that aspartate is a major source of oxalacetate for citrate production, and that a "glutamate-aspartate-citrate" pathway may be functional in prostate mitochondria. In addition, testosterone can regulate citrate production by a specific effect on mitochondrial aspartate aminotransferase activity. Testosterone also regulates the flux of citrate through the Krebs cycle, but this represents only a small proportion of the citrate accumulated. These conditions would be consistent with the function of prostate epithelium in accumulating and secreting citrate.     

Kinetic studies of the uptake of aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro.

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.     

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver.

A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.