Aspartate Aminotransferase Isoenzymes Research Articles

Evaluation and comparison of a column-chromatographic and an electrophoretic technique for separation of the two isoenzymes of aspartate aminotransferase (AST)

Evaluation and comparison of a column-chromatographic and an electrophoretic technique for separation of the two isoenzymes of aspartate aminotransferase (AST)

Quantitation of aspartate aminotransferase isoenzymes by immunologic methods: Use of antibodies directed against the mitochondrial isoenzyme

Mitochondrial aspartate aminotransferase (m-AST) was purified to homogeneity from human liver and used to prepare antisera in rabbits, and the antibodies were partially purified. Two convenient immuno-techniques were developed using these antibodies to estimate AST isoenzyme activity in human sera and tissue extracts: (i) A homogeneous immunoinhibition assay requires a 1-h incubation in the presence of anti-m-AST; residual soluble enzyme (s-AST) activity is then determined. Average coefficient of variation (CV) for this assay system was 9.6% for sera with residual s-AST within the normal range and 4.5% for specimens with elevated activities. A small portion (ca. 2%) of m-AST was not inhibited by this technique. (ii) An immunoprecipitation assay requires incubation of the specimen with antibody in the presence of polyethylene glycol; the mixture is then centrifuged, and supernatant s-AST is determined. Average CV for this assay was 6.4% at 22 U/l and 1.2% at 90 U/l of s-AST. Average CV for estimation of 140 U/l of m-AST was 3.6%. Precipitation of m-AST was complete at ≤ 500 U/l. The average contribution of m-AST to total activity in serum was 12% (2.0 ± 1.5 U/l). Isoenzyme patterns after cardiac surgery suggested that the appearance of m-AST in serum is delayed and that it does not merely follow the pattern of total AST activity.

Column chromatography and immunoassay compared for measuring the isoenzymes of aspartate aminotransferase in serum.

We compare a column-chromatographic method and a homogeneous immunoassay method for separately measuring the mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase. Analytical recovery for the two methods averaged 102% (SD, 2%) and 103% (SD, 4%), respectively, for 11 pools prepared by adding the purified isoenzymes to serum and 102% (SD 8.9%) and 89% (SD, 8.1%) for 26 unaltered specimens of human serum. In comparing the results of the immunoassay method (y) to the chromatographic method (x), our measurements agreed closely for the mitochondrial (y = 0.947 X + 7, r = 0.9991, standard error of estimate = 2.9 U/L) and cytoplasmic (y = 0.92x-6, r = 0.9995, standard error of estimate = 2.1 U/L) isoenzymes in pools prepared from the purified isoenzymes. Similar measurements of the 26 human serum specimens yielded the following results for least-squares evaluation; cytoplasmic isoenzyme y = 1.03x-11, r = 0.994, and standard error of estimate = 6.0 U/L; mitochondrial isoenzyme y = 0.75x+0, r = 0.927, and standard error of estimate = 3.9 U/L.

Apoenzyme of aspartate aminotransferase isozymes in serum and its diagnostic usefullness for hepatic diseases

Aspartate aminotransferase in the sera of normal subjects and of patients with hepatic diseases has been immunologically separated into two isoenzymes, cytosolic aspartate aminotransferase and mitochondrial aspartate aminotransferase. The activity of the isoenzymes was measured in three different buffer solutions with or without pyridoxal 5'-phosphate. To attain maximal activation, the apoenzyme of mitochondrial fraction must be preincubated with pyridoxal 5'-phosphate longer than that of the cytosolic fraction in either of the three reaction mixtures. In most sera the activity of both isoenzymes increased substantially in the presence of pyridoxal 5'-phosphate regardless of the type of buffer solutions. Both the apoenzymatic activity and the ratio of apo- to holo-enzymatic activity of each of the isoenzymes varied among samples from the patients with hepatic diseases. However, significantly high ratios of apo- to holo-enzymatic activity of both isoenzymes were observed in the patients with hepatoma in contrast with those with other hepatic diseases. These findings suggest that the simultaneous measurement of both apo- and holo-enzyme activities of aspartate aminotransferase isoenzymes may be useful in the clinical assessment of hepatic diseases.

Relative stabilities of purified human mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in lyophilized materials.

Eight different pools of purified human mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase were prepared, to examine the effects of the following matrix variables: the matrix support material (bovine serum albumin and polyvinylpyrrolidone), endogenous pyridoxal concentration, and azide as an antimicrobial preservation. Storage temperatures of 25 and 37 degrees C were used as a rapid and convenient means of accelerating the degradation process. Activity of the enzyme was measured with and without pyridoxal in the reaction solution. We found that the mitochondrial isoenzyme was consistently more labile than the cytoplasmic isoenzyme under identical storage conditions. Both isoenzymes were more stable in matrixes containing bovine serum albumin than in those containing polyvinylpyrrolidone. No apparent difference in the stability of either isoenzyme was observed at matrix pyridoxal concentrations of 15 micromol/L and 150 micromol/L. Only the mitochondrial isoenzyme in matrixes containing bovine serum albumin and 15 micromol of pyridoxal per liter had increased activity (about 9%) when pyridoxal was added to the enzymatic reagent. The amount of activity in reconstituted specimens did not apparently change after 72 h at 4 degrees C.

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.

Accurate determination of serum ASAT isoenzymes

An improved electrophoretic modification for measuring aspartate aminotransferase (ASAT) isoenzymes is presented. This method fulfils the clinical requirements for sensitivity and allows the detection of 1 U/l mitochondria ASAT activity at 25 degree C. The procedure is relatively simple, requiring about one hour for a series of 8 determinations. Mitochondrial ASAT activity was found in all patients suffering from acute myocardial infarction pathological activity was observed for several days longer than that of total serum ASAT enzyme. None of the 25 healthy people studied had mitochondrial ASAT in their serum.

80] Investigation of crystalline enzyme—Substrate complexes of pyridoxal phosphate-dependent enzymes

Publisher Summary Many pyridoxal phosphate (pyridoxal-P) dependent enzymes have been crystallized, but few of the crystals have been studied either by X-ray crystallography or by other physical techniques. Recently, three groups have initiated crystallographic studies on aspartate aminotransferases. The cytosolic enzyme from chicken hearts and from pig hearts has been prepared in orthorhombic forms, while the mitochondrial isoenzyme of chicken heart has been crystallized in a triclinic form. Many pyridoxal-P enzymes form very small and relatively insoluble crystals. However, if the solubility of these enzymes is increased by changing the pH or ionic composition of the buffer, it may be possible to use the polyethyleneglycol method to obtain larger crystals. This chapter describes the preparation of crystals of the cytosolic isoenzyme of aspartate aminotransferase from pig heart and crystals of enzyme-substrate or enzyme-inhibitor complexes. Crystals of enzyme containing analogs of pyridoxal-P can be prepared by reconstituting the apoenzyme and then treating it as above for the native enzyme.

Mitochondrial and cytosolic aspartate aminotransferase from chicken: Activity toward aromatic amino acids

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase from chicken heart accept as substrates L-phenylalanine, L-tyrosine and L-tryptophan. The specific activities of the mitochondrial isoenzyme toward these substrates are between 0.1 to 0.5% of that toward aspartate and two orders of magnitude higher than that toward alanine. The specific activities of the cytosolic isoenzyme toward the aromatic substrates are 10 to 70% of the respective values of the mitochondrial isoenzyme. The activities of both isoenzymes toward aromatic amino acids are increased two- to threefold by 1 M formate. Larger increases by formate were observed for the alanine aminotransferase activity of both isoenzymes whereas their aspartate aminotransferase activity was inhibited by formate. The opposite effects of formate on the activities toward the aromatic and aliphatic monocarboxylic substrates on the one hand and the dicarboxylic substrate on the other are consonant with the notion of formate occupying the binding site of the distal carboxylate group of the substrate (Morino Y., Osman A.M., and Okamoto M. (1974) J. Biol. Chem. 249 , 6684–6692). Apparently, in the ternary complex of aspartate aminotransferase with formate and aromatic amino acids, the aromatic rings of the latter bind to a site which does not overlap with the binding site for the distal carboxylate.

Column-chromatographic separation of isoenzymes of aspartate aminotransferase.

We describe a column-chromatographic method for separating the mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in human serum. Bed height of the ion exchanger, pH, and salt concentrations in the eluting buffers are shown to be variables affecting the separation of the isoenzymes. Under the optimized conditions selected for this study, a 30% increase in volume was observed in one fraction, associated with changing the salt concentration of the eluting buffer and attributed to a contraction of the DEAE-Sephadex A-50. Elution profiles (enzyme activity vs. fraction number) were examined with highly purified mitochondrial and cytoplasmic isoenzymes of human origin in bovine serum albumin and human serum. Recovery of the enzyme in the eluted fractions averaged 102% (SD, 2.0%) for specimens prepared from the purified isoenzymes and 104% (SD, 10.7%) for 38 human serum specimens. The separation technique showed linearity to catalytic concentrations in excess of 200 U/liter (reaction temperature 30 degrees C) for each isoenzyme. Additional information is presented regarding among-day precision and the effect of specimen dilution.

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.

Aminooxyacetate is not an adequate differential inhibitor of aspartate aminotransferase isoenzymes.

Aminooxyacetate is not an adequate differential inhibitor of aspartate aminotransferase isoenzymes.

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.

Aspartate Aminotransferase Isoenzymes in the Polyploid Wheats and their Diploid Relatives. On the Origin of Tetraploid Wheats

Polyacrylamide gel electrophoresis resolves three major and two minor aspartate aminotransferase isoenzymes from seedling extracts of wheats and diploid goatgrasses. The three major isoenzymes designated AAT-A, AAT-B, and AAT-C (in order of their decreasing anodal electrophoretic mobility) are genetically controlled by three separate gene loci and differ in their intracellular localization and solubility properties. Various extent of interspecific, intraspecific or intrapopulational variation in all the three AAT isoenzymes has been observed among diploid goatgrasses, while in wheats the variation was mostly correlated with the ploidy level. The origin of tetraploid wheats has resulted in fixation of heterozygosity at the AAT-C locus, and the origin of the hexaploid T. aestivum L. —in an additional fixation of heterozygosity at the AAT-B locus. Dimeric quarternary structure of the two isoenzymes, AAT-B and AAT-C, can be assumed from the appearance of triplet electrophoretic phenotypes with a hybrid heterodimer and two parental homodimers in polyploid wheats and in heterozygous individuals of some goatgrasses. Among the contemporary diploid goatgrasses, only Aegilops speltoides Tausch proved suitable for the role of the wheat B -genome donor with respect to AAT-B and AAT-C electrophoretic variants.

Mitochondria as a site of C 4 acid decarboxylation in C 4-pathway photosynthesis

One group of C 4, species utilize a NAD-malic enzyme to decarboxylate C 4 acids. This enzyme, together with a major isoenzyme of aspartate aminotransferase and a NAD-malate dehydrogenase, is localized in the mitochondria of the bundle sheath cells and the following pathway for C 4, acid decarboxylation has been proposed: aspartate → oxaloacetate → malate → CO 2 + pyruvate. The present study reports that mitochondria isolated from the bundle sheath cells of one of these species, Atriplex spongiosa, are capable of decarboxylating C 4, acids at rates between 5 and 8 μmol/min/mg chlorophyll. For maximum decarboxylating activities, these particles required aspartate, 2-oxoglutarate and phosphate as well as malate; in the absence of any one of these compounds, activity was reduced to 0.3–0.8 μmol/min/mg chlorophyll. Rates for C 4 acid decarboxylation were much greater than the respiratory activities of these particles, including the capacity to form citrate or to oxidize malate, succinate, pyruvate or 2-oxoglutarate (0.03–0.6 μmol/min/mg chlorophyll). A comparison of mitochondria prepared from leaves of various C 4, and C 3, species showed that only the mitochondria from the bundle sheath cells of plants with high NAD-malic enzyme have capacities for rapid C 4 acid decarboxylation. The effects of a variety of experimental conditions on C 4 acid decarboxylating activities are also reported. The role of these mitochondria in C 4 photosynthesis is discussed.

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.

Azide as a Preservative in Assays of Aspartate Aminotransferase Activity

Abstract Azide at an effective antimicrobial concentration, 7.7 mmol/liter, inhibits neither the cytoplasmic or the mitochondrial isoenzymes of human aspartate aminotransferase (EC 2.6.1.1) or porcine malate dehydrogenase (EC 1.1.1.37). It markedly prolongs the usefulness of substrates in assays for estimating aspartate aminotransferase activity (aspartate substrate without azide deteriorates in less than 24 h at 25 °C) and does not affect results obtained in assays in which activity is continuously monitored. It also has no effect on chromophore development or absorption spectra in 2,4-dinitrophenylhydrazine-coupled assays, but it eliminates oxalacetate-diazonium salt chromophore formation, thus making it unsuitable for use in this assay. At concentrations greater than 100 mmol/liter it inhibits activities of both enzymes. Azide also catalyzes the ketoenol tautomerization of oxalacetate.

Activity, location, and role of asparate aminotransferase and alanine aminotransferase isoenzymes in leaves with C 4 pathway photosynthesis

Further evidence has been provided that C 4-pathway species characterized by having low malic enzyme activity contain exceptionally high activities of aspartate and alanine aminotransferases. The total activity of both enzymes is distributed about equally between mesophyll and bundle sheath cells. However, the activity in the two cell types is due to different isoenzymes. In addition to the one quantitatively major isoenzyme associated with each cell type there were at least two additional isozymes of each aminotransferase detectable in the different species examined. Increases in activity of both aminotransferases of ten-fold or more were observed during greening of leaves of dark-grown plants. This increased activity was due specifically to the two quantitatively major isoenzymes associated, respectively, with the mesophyll and bundle sheath cells of green leaves, providing further evidence for their specific role in photosynthesis. Apparently, neither the aspartate nor alanine aminotransferases of mesophyll cells was associated with chloroplasts or other subcellular organelles. However, the major aspartate aminotransferase isoenzyme of bundle sheath cells was associated with mitochondria. These findings are discussed in relation to the probable role of aspartate and alanine aminotransferases in C 4-pathway photosynthesis.