Purification and properties of alpha-aminoadipate aminotransferase from rat liver and kidney mitochondria.

Recently we reported an affinity chromatography method to purify alpha-aminoadipate aminotransferase (AadAT) activity from rat kidney supernatant fraction. Using the same affinity column, we purified AadAT activities from rat kidney and liver mitochondria. The physical and kinetic properties such as pH optima, Km for substrates, molecular weight, subunit structure, isoelectric pH, electrophoretic mobility and inhibition by dicarboxylic acids of mitochondrial AadAT were similar to those of the AadAT from rat kidney supernatant fraction. These results indicate that AadAT from different subcellular fractions is structurally and immunologically identical.     

Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase

Summary The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane.     

Effect of pyridoxine-deficiency on the turnover of aspartate aminotransferase isozymes in rat liver

DL-[14C]Leucine or L-[3H]leucine was injected intraperitoneally into pyridoxine-deficient and control rats, and the subsequent incorporation of the radioactivities into aspartate aminotransferase (AspAT) isozymes and the total soluble protein in the liver was measured. AspAT in the cytosol (AspATc) was separated into 3 subfractions with different characteristics on chromatofocusing. The results showed that in the liver of pyridoxine-deficient rats, the syntheses of all 3 subfractions of AspATc and degradation of AspATc (total) were increased, but that the syntheses and degradation of the total soluble protein and mitochondrial AspAT (AspATm) were not much different from those in control rats. The half-lives of soluble protein and AspATm were calculated to be 3.26-3.72 and 5.02-6.67 days, respectively, in both groups, and that of AspATc in control liver was found to be 4.78 days. The rate of degradation of AspATc in pyridoxine-deficient rat liver could not be calculated, because its kinetics were very complicated; there were apparently at least 2 components with different rates of degradation. Thus pyridoxal 5'-phosphate (PLP) apparently affects both the synthesis and degradation of AspATc, but does not affect the turnover of AspATm in rat liver.     

Regulation of 2-oxoglutarate metabolism in rat liver by NADP-isocitrate dehydrogenase and aspartate aminotransferase

Kinetic and regulatory properties of NADP-isocitrate dehydrogenase (NADP-IDH) and aspartate aminotransferase (AsAT) responsible for 2-oxoglutarate metabolism in the cytoplasm and mitochondria of rat liver were studied. Based on the subcellular location of these enzymes and their kinetic parameters (Km, Ksi) obtained with highly purified enzyme preparations, it is suggested that synthesis of 2-oxoglutarate should be mainly determined by cytoplasmic NADP-IDH (86% of the total activity in the cell), whereas its utilization should depend on cytoplasmic AsAT (78% of the total activity). AsAT from the rat liver was specified by substrate inhibition and also by changes in the enzyme affinity for the substrates under the influence of some intermediates of the tricarboxylic acid cycle: isocitrate, succinate, fumarate, and citrate. Key intermediates of nitrogen metabolism (glutamate, glutamine, and aspartate) are involved in the regulation of NADP-IDH and AsAT. These enzymes are regulated oppositely, and the catalytic activity of one enzyme can be stimulated concurrently with a decrease in the activity of the other. Obviously, carbon and nitrogen metabolism in the rat liver can be controlled through redistribution of 2-oxoglutarate between different metabolic processes via regulatory mechanisms influencing differently located forms of NADP-IDH and AsAT.     

Immunogold localization of mitochondrial aspartate aminotransferase in mitochondria and on the cell surface in normal rat tissues

Mitochondrial aspartate aminotransferase (mAspAT) (E.C. 2.6.1.1), an important enzyme in amino acid metabolism, is identical to a fatty acid-binding protein (FABPpm) isolated from plasma membranes of several cell types. Employing a monospecific polyclonal antibody to rat mAspAT, we have used immunogold electron microscopy to study the subcellular distribution of mAspAT in various mammalian tissues. Immunogold labeling of rat tissue sections embedded in LR Gold resin showed strong labeling of mitochondria in all tissues examined (viz. liver, pancreas, pituitary, spleen, heart, kidney, submandibular gland). In addition, strong and specific labeling was also observed at a number of non-mitochondrial sites including various locations in kidney, such as on cell surface in distal tubules and cortical collecting ducts, in condensing vacuoles, along cell boundaries between adjoining cells, and in endothelial cells lining capillaries in the glomerulus. Surface labeling due to mAspAT was also seen in arteriolar endothelial cells and in lymphocytes. These findings support the previous identification of mAspAT as both a mitochondrial enzyme and a plasma membrane protein. It is suggested that in accordance with its established role in other cells and tissues, the surface-located mAspAT in kidney and endothelial cells is involved in the fatty acid transport process. The dual-localization of mAspAT, as well as a large number of other mitochondrial proteins (viz. Hsp60, Hsp10, Cytochrome c, TRAP-1 and P32 (gC1q-R)) in recent studies, within both mitochondria and at various specific extramitochondrial sites raises fundamental questions about the role of mitochondria in cell structure and function, and about the mechanisms that exist in normal cells for protein translocation from mitochondria to other compartments. These results have implications for the role of mitochondria in apoptosis and different diseases.     

Generation of aspartate aminotransferase multiple forms by deamidation.

The development of aspartate aminotransferase subforms in vitro was followed by densitometry after thin-film isoelectric focusing. At the same time ammonia production was measured. Each reaction can be expressed in terms of a first-order process in which 2 mol of glutamine or asparagine/mol of dimer are deamidated with a half time of 22 days. The more negatively charged subforms developed in vitro were almost fully active. Another process occurred leading to inactivation by coenzyme modification, and this was independent of deamidation. Although the enzyme formed absorbed maximally at 340nm, it was different from the naturally occurring inactive enzyme that absorbs at this wavelength.     

Transport of glutamate and aspartate across the membranes of rat liver mitochondria

Chromatographic analyses have indicated that aspartate and glutamate constitute from 50–70% of the total free amino acids in freshly isolated mitochondria. Radioactive tracer studies indicate that while the l-isomers of glutamate and aspartate are rapidly accumulated by mitochondria, the d-isomers of these amino acids do not penetrate the mitochondrial membrane. The action of two inhibitory compounds, 1-fluoro-2,4-dinitrobenzene (Sanger's reagent) and tannic acid, on the transport of l-glutamate and l-aspartate has been examined. A marked inhibition of l-glutamate transfer by 1-fluoro-2,4-dinitrobenzene is observed. A corresponding effect on the transport of either l-aspartate or the anionic substrate, succinate has not been found. Tannic acid, an agent previously known to inhibit certain carrier-mediated solute fluxes in mitochondria, is shown also to inhibit the uptake of both l-glutamate and l-aspartate. These findings are consistent with the view that the mitochondrial membranes of rat liver cells contain distinct, stereospecific transport mechanisms for aspartate and glutamate.     

Immunocytochemical demonstration of serine:pyruvate aminotransferase in peroxisomes and mitochondria of rat kidney

Summary The light- and electron-microscopic localization of serine:pyruvate aminotransferase (SPT) in rat kidney was studied using immunoenzyme and protein A-gold techniques. Rat kidneys were fixed by perfusion through the abdominal aorta and small tissue slices were embedded in Epon, Lowicryl K4M, or LR Gold. The Epon was removed from the semithin sections, which were then stained using the immunoenzyme technique. Ultrathin sections of Lowicryl K4M- or LR gold-embedded materials were labeled using the protein A-gold technique. At light microscopy, discrete granular reaction deposits were exclusively present in the proximal tubule, all of whose segments were positive for SPT. A weakly positive reaction was observed in the distal tubules. At electron microscopy, gold particles indicating the antigenic sites for SPT were confined to the peroxisomes and mitochondria. The labeling intensity of both organelles was dependent on the embedding resins used. The labeling of Lowicryl K4M-embedded material was weaker than that of LR gold-embedded material; Quantitative analysis confirmed this result. Our results indicate that, in rat kidney, the main intracellular sites for SPT are peroxisomes and mitochondria of the proximal tubule.     

A quantitative study of the subcellular distribution of aspartate aminotransferase isozymes in rat liver

• 1.1. Between 15 and 19% of the aspartate aminotransferase activity of rat liver is of cytosolic origin; the remainder is localised entirely in the mitochondria.
• 2.2. Mitochondria do not contain detectable levels of the cytosolic isozyme or vice versa.
• 3.3. In solutions of low ionic strength, damaged mitochondria release only small amounts of aspartate aminotransferase. but the enzyme is released quantitatively by exposure to high concentrations of salt.

     

Hormonal regulation of ornithine aminotransferase biosynthesis in rat liver and kidney.

The relative rates of ornithine aminotransferase (OAT) synthesis in vivo were studied by pulse-labeling rats with [4,5-³H]leucine, isolating the mitochondrial enzyme protein by immunoprecipitation with a monospecific antibody, dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels, and determining the radioactivity in OAT. After 4 days of treatment with triiodothyronine (T₃), both the enzyme activity level and the relative synthetic rate of OAT in rat kidney were elevated over twofold. The level of hepatic OAT activity was unaffected by this treatment. Thyroidectomy caused a 50% drop in the basal level of OAT activity and synthesis in kidney but not in liver. Although the basal levels of activity and synthesis of both renal and hepatic OAT were unaffected by adrenalectomy, the glucagon induction of the enzyme in liver was enhanced by about one-third and the T₃ induction in kidney was suppressed 50% by this operation. After 4 days of treatment with estrogen, both the enzyme activity level and the relative synthetic rate of OAT in male rat kidney were elevated nearly 10-fold. Hepatic OAT activity and synthesis were unaffected by this regimen. Thyroidectomy almost completely abolished the estrogen induction of OAT in kidney. OAT induction by estrogen could be restored by treating thyroidectomized rats with T₃. Simultaneous administration of T₃ plus estrogen to intact rats produced a multiple effect, resulting in a striking 20-fold induction of renal OAT. Although administration of either T₃ or estrogen causes an increase in the synthesis of immunoprecipitable OAT protein in rat kidney, each of these hormones may induce OAT by a different mechanism.