Mitochondrial Isoenzyme Of Aspartate Aminotransferase Research Articles

Regulation of glutamate‐synthesizing enzymes by NMDA and potassium in cerebellar granule cells

The presence of 25 mm potassium (KCl) or N-methyl-d-aspartate (NMDA) in cultured cerebellar granule neurons (CGN) induces a trophic effect, including a specific regulation of the enzymes involved in the glutamate neurotransmitter synthesis. In this study we explored the effect of these conditions on the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase (AAT), and phosphate-activated glutaminase (PAG) in CGN. We found that NMDA and KCl increased the AAT total activity by 40% and 70%, respectively. This effect was mediated by an augmentation in the protein levels (68% by NMDA, 58% by KCl). NMDA raised the Vmax and KCl raised both the maximol velocity (Vmax) and Michaelis constant (Km) of AAT. NMDA increased cytosolic AAT activity by 30% and mitochondrial activity by 70%; KCl increased cytosolic and mitochondrial AAT activity by 60% and 100%, respectively. This activation was also related to an increase in the protein levels. The effect of both conditions on the activity and protein levels were more pronounced in mitochondrial than cytosolic AAT and the increment elicited by KCl was higher in both isoforms than that produced by NMDA. The PAG and AAT mRNA levels were also regulated by incubation with NMDA and KCl similarly to the observed changes in the protein levels. These results suggest that NMDA receptor stimulation during CGN development differentially regulates the two AAT isoenzymes involved in the maturation of CGN and that the regulation of both AAT and PAG occurs also at the mRNA expression level, suggesting the involvement of a mechanism of gene expression regulation.

Enzymes and circulating proteins as markers of alcohol abuse

The identification of alcohol abuse is an important social and clinical objective for which various biochemical procedures have been utilized, serum enzymes and circulating proteins being predominant. Tests are required to detect alcohol abuse as screening procedures in the general population as well as for the specific diagnosis of those presenting as hospital inpatients or outpatients, especially when liver disease is present or suspected. The amino-transferases are of limited value, although the mitochondrial isoenzyme of aspartate amino-transferase has been strongly advocated and is quite useful in detecting alcoholics among patients with liver disease. Gammaglutamyl transferase, by contrast, is raised in all forms of liver disease but can identify 30–50% of those consuming excessive amounts of alcohol before organic damage becomes manifest. Serum carbohydrate-deficient transferrin (CDT) is raised in many alcohol abusers without and most with liver damage, but is rarely elevated in other forms of liver disease. Haemoglobin-associated acetaldehyde, the newest biochemical index to be evaluated in alcoholics, is one of several adducts formed by the reaction of acetaldehyde with various proteins, and antibodies to these adducts may contribte, at least in part, to immunological tissue damage provoked by chronic excessive consumption of alcohol. Its assay is technically complex and it appears to be present in higher concentrations in heavy drinkers than in those who fulfill the criteria of addictive alcohol abuse. Many other markers have been introduced in the last decade but the search for a reliable index continues. CDT comes closest at the present time to matching the desired specificity, although it is of limited value in screening unselected non-hospitalized subjects.

Enzymatic determinations in acute rejection after liver transplantation: preliminary report on necrosis index

The catalytic activities of some mitochondrial and cytoplasmic enzymes were measured in plasma from 19 patients after orthotopic liver transplantation, in order to detect and monitor the evolution of hepatocellular damage and to predict liver rejection. The enzymatic activities determined were: mitochondrial isoenzyme of aspartate aminotransferase, glutamate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, γ-glutamyltranspeptidase and alkaline phosphatase. The results of all enzymatic activities were normalized by expressing them as multiples of the upper limit of the relevant reference range and then the necrosis index (NI) has been calculated. The proposed NI consists of percent ratio of the normalized mitochondrial enzymatic activities over the sum of cytoplasmic and mitochondrial normalized activities. We observed that NI values higher than 30% correctly identified all but two acute rejection events which were documented by liver biopsies showing a diagnostic sensitivity of 90%, specificity of 78% and a predictive value of 90%.

Activities of mitochondrial aspartate Aminotransferase and creatine kinase isoenzyme MB in serum following coronary bypass surgery

The mitochondrial isoenzyme of aspartate aminotransferase showed only slight increases in serum of twentyseven patients after uncomplicated coronary bypass surgery, which contrasted the rapid and substantial increases in creatine kinase MB. In seven patients suffering perioperative infarction or serious complications, substantial increases in mitochondrial aspartate aminotransferase were detected and the elevations in creatine kinase MB were prolonged. Mitochondrial aspartate aminotransferase may appear as a specific marker of myocardial necrosis following coronary bypass surgery. The elevations of creatine kinase and creatine kinase MB were detected as early as 5 minutes after onset of coronary reperfusion and slightly higher activities were measured in coronary sinus blood than in systemic blood sampled simultaneously. Increases in mitochondrial aspartate aminotransferase, however, could first be measured 8 hours after reperfusion.

Desialylated transferrin and mitochondrial aspartate aminotransferase compared as laboratory markers of excessive alcohol consumption

Concentrations of both desialylated transferrin (dTf) and the mitochondrial isoenzyme of aspartate aminotransferase (EC 2.6.1.1, mAST) have been claimed to be increased in sera of alcoholic subjects. To investigate the diagnostic usefulness of these new biochemical markers of alcoholism and to compare them with more conventional markers, we measured dTf and mAST in the sera of controls, alcoholic subjects, and patients with nonalcoholic liver diseases (NALD). Alcoholic subjects had significantly (P less than 0.001) higher ratios of dTf to total transferrin than did either healthy controls or patients with NALD (sensitivity 81%, specificity 97%). The mAST was increased in 92% of alcoholic subjects but also in 48% of patients with NALD. The mAST/total AST ratio differentiated the alcoholic subjects from those with NALD (P less than 0.001) with a sensitivity of 92%, but the specificity was only 70%. In contrast, the conventional markers were less sensitive and less specific. We conclude that the best available single laboratory marker for current heavy alcohol consumption is the ratio dTf/total transferrin.

The unfolding and attempted refolding of mitochondrial aspartate aminotransferase from pig heart

The unfolding of the mitochondrial isoenzyme of aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) has been studied. By a number of criteria (enzyme activity, protein fluorescence, c.d., thiol-group reactivity), the enzyme was judged to be almost completely unfolded in 2 M-GdnHCl. On dilution of the GdnHCl, no re-activation of the enzyme could be observed, whether or not pyridoxal 5'-phosphate and dithiothreitol were present. The behaviour of the mitochondrial isoenzyme is in marked contrast with that of the cytoplasmic isoenzyme [West & Price (1989) Biochem. J. 261, 189-196], despite the similarities in the amino acid sequences and tertiary structures of the two isoenzymes. The implications of these findings for the process of folding and assembly of the mitochondrial isoenzyme in vivo are discussed.

Simultaneous purification and characterization of aspartate aminotransferase isoenzymes from chicken liver.

Cytosolic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) were purified to homogeneity from chicken liver, without previous fractionation of the subcellular components. The procedure includes initial heat treatment and ammonium sulfate fractionation. The two isoenzymes can then be separated by a DEAE-Sepharose chromatography using a linear gradient of L-aspartate (reaction substrate). The separated fractions can be further purified by a parallel step with HA-Ultrogel prior to octyl-Sepharose (c-AAT) and CM-Sepharose (m-AAT) chromatographies. Michaelis constants, pI values, inhibition by adipate and subforms generation with time were studied for both isoenzymes.

Separate enzymatic microassays for aspartate aminotransferase isoenzymes

The properties of the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase were studied using a commercial preparation of the cytosolic isoenzyme, a mitochondrial preparation, and whole brain homogenate. Based on these properties, microassays were developed and shown to be highly specific and quantitatively accurate for measuring the activity of either the cytosolic or mitochondrial isoenzyme in microgram quantities of tissue. The assays have been successfully applied to homogenates of a wide variety of tissues. They can be used to measure the activities of aspartate aminotransferase isoenzymes in sub-microgram samples of freeze-dried tissue.

The primary structure of the precursor of chicken mitochondrial aspartate aminotransferase. Cloning and sequence analysis of cDNA.

The mitochondrial isoenzyme of aspartate amino-transferase (mAspAT; subunit Mr 45,000) is synthesized on free polysomes in the cytosol as a precursor of higher Mr (pre-mAspAT; Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345). We have isolated three overlapping cDNA clones that correspond almost to the full length of pre-mAspAT mRNA with 100 nucleotides at the 5' end missing. The mRNA is 2.1 kilobase pairs long and has a 3' noncoding region of 0.7 kilobase pairs. The cDNAs code for the 401 amino acid residues of mAspAT plus an NH2-terminal pre-piece. Deviations from the reported amino acid sequence were found at positions 154 and 202 where the cDNA specifies Gln instead of Glu. The pre-piece consists of 22 amino acid residues, among them 4 arginine and no acidic residues.

A comparative study on enzymatic data from myocardial biopsies and cardiac function in patients who underwent cardiac surgery.

Twenty-three patients underwent cardiac surgery for valve replacement, valve reconstruction, aorto-coronary bypass grafting, aneurysmectomy or combinations of these. Excised cardiac tissue was obtained from left ventricular (LV) papillary muscle (17 patients), LV outflow tract (3 patients), or LV aneurysms (3 patients). A total of 34 myocardial samples, collected from excised cardiac tissue, were analysed for creatine kinase (CK), CK-isoenzymes, cytoplasmic and mitochondrial isoenzymes of aspartate aminotransferase (cAST and mAST, respectively), and lactate dehydrogenase (LDH) isoenzymes. Myocardial CK activity correlated positively with preoperative LV ejection fraction (p less than 0.001), negatively with the preoperatively measured extent of LV wall motion abnormalities (p less than 0.001), and negatively with preoperative LVEDP (p less than 0.02). Myocardial CK activity was negatively correlated with preoperative validity class (p less than 0.005). However, no correlation existed between myocardial CK activity and postoperative validity. Excluding the biopsies from LV aneurysms, myocardial CK activity was positively correlated with the fraction H-subunits in LDH (p = 0.02), and was negatively correlated with the fraction mAST in total AST (p less than 0.005). While cAST activity was proportional to CK activity in the biopsies from VL papillary muscle and LV outflow tract, mAST activity declined only with 2.4 +/- 1.2% per 10% fall of CK. The increase of mAST/cAST ratio with decreasing CK, together with the decrease of LDH-H/LDH and CK-M/CK ratios with decreasing CK, indicated the presence of an adaptation process in a myocardium with low CK activity rather than a process of necrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum activity of mitochondrial aspartate aminotransferase: a sensitive marker of alcoholism with or without alcoholic hepatitis.

Serum activity of the mitochondrial isoenzyme of aspartate aminotransferase (mAST) was measured with an immunological method in 74 subjects. Fourty-six were chronic alcoholics with (30) or without (16) obvious alcoholic liver disease; 28 were nonalcoholic controls among whom 14 had acute or chronic viral hepatitis, the remaining 14 being healthy individuals. Mean mAST activity was much higher in all the alcoholic subjects, with or without liver disease, 10.4 and 1.95 units per liter, respectively, than in the healthy controls (0.43, p less than 0.001). The mean mAST to total AST ratio was similar in the healthy controls and in the patients with viral hepatitis (2.98 and 3.19%, NS), whereas it was about 4 times higher in the alcoholics with a sensitivity which reached 93% in the patients with alcoholic liver disease and 100% in those without. Both gamma-glutamyl transpeptidase and glutamate dehydrogenase serum activities were far less sensitive and specific. As almost all chronic alcoholics had similar abnormal values of mAST/total AST ratio, this leads to question whether "normal" liver may really exist in any of such subjects.

10] Pyridoxal phosphate-binding site in enzymes: Reduction and comparison of sequences

Publisher Summary This chapter illustrates pyridoxal phosphate-binding site in enzymes. In pyridoxal phosphate-dependent enzymes, the formyl group at position 4 of pyridoxal-P forms an aldimine bond with the ɛ -amino group of a specific lysyl residue. Involvement of a single particular lysyl residue indicates that there is a specific domain structure favorable for stabilizing the aldimine bond which, otherwise, is readily hydrolyzed. X-Ray analyses of both cytosolic and mitochondrial isoenzymes of aspartate aminotransferase, representative pyridoxal-P-dependent enzymes, provide details of interaction between pyridoxal-P and the enzyme side chains in the coenzyme-accommodating active site. Only the Re side of the aldimine bond is open to solvent, the Si face being shielded from solvent. This stereospecific status of the bound coenzyme has been remarkably identical among a variety of pyridoxal-P-dependent enzymes. At neutral pH, pyridoxal-P absorbs at 390 nm, which shifts more or less to a longer wavelength (410-430 nm) upon the formation of an aldimine linkage with amino compounds or apoenzymes. Pyridoxal-P-aldimine bond is reduced by borohydride or borocyanohydride under mild conditions. The reduction renders the aldimine bond highly stable to hydrolysis. The resulting reduced enzyme is fragmented by either proteolytic or chemical cleavage, followed by isolation of a phosphopyridoxyl peptide and its structural determination.

The proportion of mitochondrial isoenzyme of aspartate aminotransferase is not elevated in Reye's syndrome.

We reexamined a previously reported, highly specific increase in the relative proportion of the mitochondrial isoenzyme of aspartate aminotransferase (AST) in the serum of patients with Reye's Syndrome. Using ion exchange chromatography, we measured mitochondrial, cytosolic, and total AST in serum samples from (1) 10 patients early in the course of Reye's Syndrome; (2) nine controls with normal serum AST; and (3) seven controls with other diseases causing an increase in serum AST. The mitochondrial percentage (2.8 +/- 2.0%) in Reye's Syndrome was significantly lower (P less than 0.05) than that of both the normal control group (6.1 +/- 7.1%) and the group with increased AST (5.6 +/- 4.0%). We thus failed to confirm the previous report of a specific increase in the % of mitochondrial isoenzyme in Reye's Syndrome, and conclude that the % of mitochondrial isoenzyme is not likely to be a useful marker of (or predictor for progression to) Reye's Syndrome.

Independent quantitation of the mitochondrial and the cytosolic isoenzyme of aspartate aminotransferase in chicken tissues by radioimmunoassays.

Two specific and sensitive radioimmunoassays for the independent determination of the cytosolic and mitochondrial isoenzyme of aspartate aminotransferase from chicken are described. The assays use 125I-labeled aspartate aminotransferases as tracer antigens, rabbit antisera, and goat anti-rabbit IgG antisera for indirect immunoprecipitation. The detection limit for the cytosolic isoenzyme was 177 pg and for the mitochondrial isoenzyme 83 pg. The assays were used for measuring the concentrations of the aspartate aminotransferase isoenzymes in serum, cultured fibroblasts, and various tissues of chicken. Both isoenzymes in the different tissue extracts proved immunologically identical with the corresponding isoenzymes isolated from heart.

Isolation and purification of aspartate aminotransferase isoenzymes from human liver by chromatography and isoelectric focusing.

To purify cytoplasmic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) from human liver. We used heat treatment, ammonium sulfate precipitation, anion- and cation-exchange chromatography, affinity chromatography, and isoelectric focusing. Final preparations of the isoenzymes were homogeneous, with specific activities of 198 and 208 kU/g for the cytoplasmic and the mitochondrial enzymes, respectively. The mitochondrial isoenzyme focused as a single band with a pl value of 9.60, whereas the cytoplasmic isoenzyme had subforms with pl values of 5.22, 5.42, and 5.62 at 4 degrees C. In Tris . HCl buffer, both isoenzymes have an activity maximum at pH 7.8. In [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bistris) buffer, however, the mitochondrial isoenzyme also showed an optimum pH of 6.7.

Studies on the Heterotropic Interaction of Hemoglobin1

A procedure is described for the large-scale preparation of the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase from pig heart. The procedure consists of: 1. extraction of both isoenzymes by heat treatment of homogenates prepared from minced and frozen heat muscle; 2. separation of each isoenzyme on a hydroxyapatite column; 3. purification of each isoenzyme by combinations of heat treatment, ammonium sulfate fractionation and chromatography on ion-exchange cellulose columns. Purified preparations of each isoenzyme thus obtained were homogeneous proteins as judged from their spectral properties and behavior on polyacrylamide gel electrophoresis. Using the present procedure, 1.2 g of the cytosolic isoenzyme and 1.7 g of the mitochondrial isoenzyme were obtained from 20 kg of minced heart muscle.

Isolation, crystallization and preliminary crystallographic data of aspartate aminotransferase from chicken heart mitochondria.

Abstract The mitochondrial isoenzyme of aspartate aminotransferase (E.C. 2.6.1.1) has been isolated from chicken heart in an electrophoretically and immunologically homogeneous form. Large, well-diffracting single crystals of this enzyme, a dimeric molecule with a molecular weight of 90,000, have been grown by vapour phase diffusion against polyethylene glycol solutions. The crystals belong to space group P1. The unit cell, with the dimensions a = 55.6 A , 6 = 58.7 A , c = 76.0 A , α = 85.3 °, β = 109.2 °, γ = 115.6 °, contains a single dimer. The diffraction pattern extends to at least 2.1 A resolution.

Interspecies comparison of cytosolic and mitochondrial aspartate aminotransferases. Evidence for a more conservative evolution of the mitochondrial isoenzyme.

The degree of structural similarity between the mitochondrial isoenzymes of aspartate aminotransferase from pig heart and chicken heart was determined by means of their immunological cross-reactivity and compared with the degree of similarity between the cytosolic isoenzymes from the same two species. Quantitative microcomplement fixation revealed a remarkable similarity of the two mitochondrial isoenzymes corresponding to an immunological distance of 104. The structures of the two cytosolic isoenzymes, on the other hand, diverge with an immunological distance of 203. The apparent conservatism of mitochondrial aspartate aminotransferase indicates additional evolutionary constraints on the structure of this organelle-confined isoenzyme.

Inactivation of aspartate aminotransferase during transamination with chloropyruvate. Evidence against modification of Cys 390 in cytosolic isoenzyme

Both the cytosolic and mitochondrial isoenzyme of aspartate aminotransferase from pig heart were inactivated during transamination with chloropyruvate. Inactivation occurred with L-alanine as the amino group donor in the presence of potassium formate. When L-glutamate or L-aspartate was employed as the amino group donor in the transamination reaction with chloropyruvate, no inactivation occurred. This is in contrast to the case of inactivation by bromopyruvate (Okamoto, M. & Morino, Y. (1973) J. Biol. Chem. 248 , 82–90) where these natural dicarboxylic amino acid substrates were effective in the transamination reaction leading to syncatalytic inactivation (Birchmeier, W. & Christen, P. (1974) J. Biol. Chem. 249 , 6311–6315). The Cys 390 in the cytosolic isoenzyme which was modified in the syncatalytic inactivation was not modified under the present condition for inactivation with either chloropyruvate or bromopyruvate.

Syncatalytic sulfhydryl group modification in mitochondrial aspartate aminotransferase from chicken and pig heart

One sulfhydryl group of the mitochondrial isoenzyme of aspartate aminotransferase from both chicken and pig heart exhibits syncatalytic reactivity changes similar to those found previously in the cytosolic isoenzyme from pig heart (Birchmeier, W., Wilson, K.J., and Christen, P. (1973) J. Biol. Chem. 248 , 1751–1759). The reactivity of the only titratable sulfhydryl group toward 5,5′-dithiobis-(2-nitrobenzoate) is at a minimum in the free pyridoxal and pyridoxamine form of the enzyme and is increased by approximately one order of magnitude when covalent enzyme-substrate intermediates are formed. The modification of the sulfhydryl group does not affect enzymatic activity. This finding supports the earlier conclusion that the syncatalytic reactivity changes are not due to a direct participation of this group in the active site but rather to conformational adaptations of the enzyme-coenzyme-substrate compound occurring in the catalytic mechanism of aspartate aminotransferases.