The primary structure of mitochondrial aspartate aminotransferase from human heart

The complete amino acid sequence of the mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained.     

Organ specificity of glucocorticoid-sensitive tyrosine aminotransferase. Separation from aspartate aminotransferase isoenzymes

In order to study whether hormone-sensitive tyrosine aminotransferase exists in tissues other than liver, we have devised means to separate the liver-specific enzyme from other enzymes that transaminate tyrosine and to distinguish between the authentic enzyme and the principal "pseudotyrosine aminotransferases," which are the isoenzymes of aspartate aminotransferase. We accomplish this by suppressing proteolysis of the authentic enzyme using a buffer of pH 8.0 containing 0.1 M potassium chloride; enzyme extracted from liver in this buffer migrates as a single peak during chromatography on hydroxylapatite and represents the undegraded native form. A much smaller peak of tyrosine aminotransferase activity elutes at higher ionic strength and corresponds to a mixture of mitochondrial aspartate aminotransferase and partially degraded tyrosine aminotransferase. Cytosolic aspartate aminotransferase, in contrast, adsorbs weakly to the hydroxylapatite column and transaminates tyrosine very poorly although it readily utilizes monoiodotyrosine. The aspartate aminotransferase isoenzymes separate completely from tyrosine aminotransferase during chromatography on DEAE-Sepharose CL-6B. By combining these techniques with the use of specific antibodies, we show that brain, heart, and kidney do not contain tyrosine aminotransferase. Furthermore, we locate both isoenzymes of aspartate aminotransferase on polyacrylamide gels and show that both react histochemically as tyrosine aminotransferases when monoiodotyrosine is used as substrate. Use of these techniques, therefore, permits unambiguous identification of tyrosine aminotransferase and its separation from the background of nonspecific transamination.     

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.     

Purification and characterization of aspartate aminotransferase isoenzymes from carrot suspension cultures

Three aspartate aminotransferase isoenzymes were identified from extracts of carrot (Daucus carota L.) cell suspension cultures. These isoenzymes were separated by DEAE chromatography and were analyzed on native gradient polyacrylamide gels. The relative molecular weights of the isoenzymes were 111,000 ± 5000, 105,000 ± 5000, and 94,000 ± 4000 daltons; they were designated forms I, II, and III, respectively. Form I, the predominant form, has been purified to apparent homogeneity (>300-fold) using immunoaffinity chromatography with rabbit anti-pig AAT antibodies. Form I has a subunit size of 43,000 M_r, as determined on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Isoelectric focusing (IEF)-PAGE has resolved three bands at a pl of approximately 5.2. Form I may be composed of subunits of similar molecular weight and different charges, and the three bands with AAT activity on the IEF-PAGE gel are a combination of hetero- and homodimers. Form I has a broad pH optimum of 7.5 to 10.0. K_m values of 23.6, 2.8, 0.05, and 0.22 millimolar were obtained for glutamate, aspartate, oxaloacetate, and α-ketoglutarate, respectively. The mode of action is a ping-pong-bi-bi mechanism.     

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.     

Quantitation of aspartate aminotransferase isoenzymes after electrophoretic separation

A scheme for the quantitative detection of aspartate aminotransferase isoenzymes and multiple forms after electrophoretic separation is described. Glutamate generated from the aminotransferase reaction is quantitated by using the glutamate dehydrogenase/diaphorase-coupled enzyme system to form a formazan dye. Product inhibition of aspartate aminotransferase by oxaloacetate is prevented by including oxaloacetate decarboxylase in the overlay reagent. Results compare favorably with those of an immunochemical precipitation procedure. The method can also be used to detect quantitatively subforms and atypical forms (genetic variants, immunoglobulin-enzyme complexes) of aspartate aminotransferase.     

Structure of the genes of two homologous intracellularly heterotopic isoenzymes. Cytosolic and mitochondrial aspartate aminotransferase of chicken

The genes of mitochondrial and cytosolic aspartate aminotransferase of chicken were cloned and sequenced. In both genes nine exons encode the mature enzyme. The additional exon for the N-terminal presequence that directs mitochondrial aspartate aminotransferase into the mitochondria is separated by the largest intron from the rest of the gene. A comparison of the two genes of chicken with the aspartate aminotransferase genes of mouse [Tsuzuki, T., Obaru, K., Setoyama, C. & Shimada, K. (1987) J. Mol. Biol. 198, 21-31; Obaru, K., Tsuzuki, T., Setoyama, C. & Shimada, K. (1988) J. Mol. Biol. 200, 13-22] reveals closely similar structures: in the gene of both the mitochondrial and the cytosolic isoenzyme all but one intron positions are conserved in the two species and five introns out of nine are placed at the same positions in all four genes indicating that the introns were in place before the genes of the two isoenzymes diverged. The variant consensus sequence (T/C)11 T(C/T)AG at the 3' splice site of the introns of the genes for nuclear-encoded mitochondrial proteins, which had been deduced from a total of 34 introns [Jureti?, N., Jaussi, R., Mattes, U. & Christen, P. (1987) Nucleic Acids Res. 15, 10,083-10,086], was confirmed by including an additional 22 introns into the comparison. The position -4 at the 3' splice site is occupied by base T in 43% of the total 56 introns and appears to be subject to a special evolutionary constraint in this particular group of genes. The following course of evolution of the aspartate aminotransferase genes is proposed. Originating from a common ancestor, the genes of the two isoenzymes intermediarily evolved in separate lineages, i.e. the ancestor eukaryotic and ancestor endosymbiontic cells. When endosymbiosis was established, part of the endosymbiontic genome, including the aspartate aminotransferase gene, was transferred to the nucleus. This process probably led to the conservation of certain splicing factors specific for nuclear-encoded mitochondrial proteins. The presequence for the mitochondrial isoenzyme was acquired by DNA rearrangement. In the eukaryotic lineage, the mitochondrial isoenzyme evolved more slowly than its cytosolic counterpart.     

Primary structure of cytoplasmic aspartate aminotransferase from pit heart muscle. Amino acid sequence of thermolytic peptides]

Amino acid sequences of 128 thermolytic peptides from carboxymethylated aspartate aminotransferase were determined. These peptides contain a total of 515 amino acid residues and account for a sequence of 384 amino acid residues in the aspartate aminotransferase.     

A kinetic method for quantification of aspartate aminotransferase isoenzymes

A spectrophotometric assay is proposed to determine the levels of aspartate aminotransferase (AAT) isoenzymes from chicken liver by a steady-state kinetic method which depends on the differential inhibition of these isoenzyme forms by high concentrations of substrate 2-oxoglutarate at pH 6.2. The use of a standard curve permits the determination of the percentage of chicken liver c-AAT and m-AAT isoenzymes. This method yields results in good correlation with those achieved by different extent adipate inhibition and by differential centrifugation.     

Primary structure of cytoplasmic aspartate aminotransferase from pig heart muscle. Amino acid sequence of peptides from cyanogen bromide hydrolysate]

Amino acid sequences were determined for the six peptides from cyanogen bromide hydrolysis of cytoplasmic aspartate aminotransferase. These peptides accounted for 177 amino acid residues of the enzyme. Partial sequence of N-terminal peptide accounting for 212 amino acid residues of enzyme was also determined.     

Cloning and sequence analysis of mRNA for mouse aspartate aminotransferase isoenzymes

The nucleotide sequences of mRNAs for the mouse mitochondrial and cytosolic aspartate aminotransferase isoenzymes (mAspAT and cAspAT) (EC 2.6.1.1) were determined from complementary DNAs. The mAspAT mRNA comprises minimally 2460 nucleotides and codes for a polypeptide of 430 amino acid residues corresponding to the precursor form of the mAspAT (pre-mAspAT). The cAspAT mRNA comprises minimally 2086 nucleotides and codes for a polypeptide of 413 amino acid residues. The region coding for the mature mAspAT and that for the cAspAT show about 53% overall homology. The former shares 49% and the latter 48% of homology, respectively, with that of the Escherichia coli aspC gene, which has been shown to code for the E. coli AspAT (Kuramitsu, S., Okuno, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 1259-1262). When the deduced amino acid sequence of the mouse pre-mAspAT was compared with that of the pig pre-mAspAT polypeptide, we found that they share a 94% homology and that the mouse pre-mAspAT yields a presequence consisting of 29 amino acid residues and a mature mAspAT, consisting of 401 amino acid residues. These numbers and the amino acid residues present at the putative cleavage site are all in complete agreement in these two species. The deduced amino acid sequence of the mouse cAspAT shares 91% homology with that of the pig cAspAT. Comparisons of the nucleotide and deduced amino acid sequences between the mouse and E. coli AspATs suggest that the mammalian mAspAT gene is more closely related to the E. coli aspC gene than is the mammalian cAspAT gene.     

Genetic control of aspartate aminotransferase isoenzymes in Aegilops and Triticum species

Zymograms of the aspartate aminotransferase (AAT, EC 2.6.1.1) activity in leaf extracts from Aegilops and Triticum species revealed three AAT zones, denoted according to the decreasing electrophoretic mobility towards the anode as AAT-1, AAT-2 and AAT-3. The AAT activity zymograms of subcellular fractions isolated from T. aestivum seedlings made it possible to establish that the AAT-1 zone is located in the mitochondria, AAT-2 in the chloroplasts and AAT-3 in the cytoplasm. Most of the total AAT activity from wheat leaves arises from the chloroplasts and cytoplasm. The AAT-3 zone exhibited the lowest electrophoretic mobility, but 3 isoenzymes occurring within were the most visibly separated. The occurrence of a single band in this zone at the AAT-3a position (closest to the anode) for the aneuploid CS3ASDt AABBDD line (the absence of long arms of the 3rd pair of homologous chromosomes in the A genome) and at the AAT-3c position for Ae. umbellulata (genome UU), as well as three bands in the whole zone for T. durum (AABB) and T. aestivum (AABBDD) each, made it possible to evaluate the subunit composition of isoenzymes in the AAT-3 zone. The band at the AAT-3a position in the zymogram is formed from bb dimers, AAT-3b from ab and AAT-3c from aa. By comparing the distribution of isoenzyme bands intensities (the result of enzymatic activity) with the mathematical models, the frequencies of the occurrence of the a and b subunits within AAT-3 zone were evaluated. In AAT-3 from T. durum, a and b occurred at the ratio of 0.54:0.46, and in that from T. aestivum - 0.62:0.38, respectively.