Primary structure of mitochondrial glutamic oxaloacetic transaminase from rat liver: Comparison with that of the pig heart isozyme

The complete amino acid sequence of the mitochondrial glutamic oxaloacetic transaminase isozyme from rat liver is presented. The sequence contained 401 amino acid residues, 10 of which are methionine. Cyanogen bromide cleavage of mitochondrial glutamic oxaloacetic transaminase produced 12 peptides, one of which contained an internal homoserine residue resulting from incomplete cleavage by cyanogen bromide. The calculated molecular weight was 44,358. The sequence showed 94% homology with that of the corresponding isozyme from pig heart. These findings support the conclusion that the rate of evolution of the mitochondrial isozymes is lower than that of their cytosolic isozymes.     

Import of a putative precursor of rat liver mitochondrial glutamic oxaloacetic transaminase into mitochondria

A putative precursor of rat liver mitochondrial glutamic oxaloacetic transaminase which was about 2,000 daltons larger than the subunits of the mature enzyme synthesized in vitro was sensitive to proteases (trypsin and chymotrypsin). When this precursor was incubated with isolated mitochondria in the absence of protein synthesis, it was processed to the mature form; the mature form co-sedimented with mitochondria and was resistant to externally added proteases. Mature enzyme did not compete with this transport.     

Aldehyde as fixative for histochemical study of glutamic oxaloacetic transaminase

Summary Several compounds of the aldehyde group have been tested as fixatives for histochemical localization of GOT activity in the rat kidney. A brief fixation of cryostat tissue sections in 1% glutaraldehyde results in well localized enzymatic activity which is not greatly further affected by an additional treatment in 4% formaldehyde.This work was supported by grant (NB-04161-04) from the National Institute of Neurological Diseases and Blindness of the National Institutes of Health.     

Determination of the activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase in a microfluidic system

A microfluidic system for the analysis of the activities of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) was fabricated. The device consists of a glass chip with a micro-electrochemical L-glutamate sensor and a polydimethylsiloxane (PDMS) sheet with a Y-shaped micro-flow channel. A sample solution and a substrate solution for the enzymes were introduced from two injection ports at the end of the flow channel. When the flows were stopped, substrates in a solution mixed immediately with either of the enzymes by diffusion in a mixing channel. L-glutamate produced by the enzymatic reaction of GOT or GPT in the flow channel was detected by using the L-glutamate sensor. A distinct current increase was observed immediately after mixing, and the initial slope of the response curve varied in proportion to the activity of GOT or GPT. The relation between the slope of the response curve and the enzyme activity was linear between 7 and 228 U l-1 for GOT and 9 and 250 U l-1 for GPT. The quality of the response curve was improved with an increase in the channel height. The measurement based on the rate analysis in the micro-flow channel facilitated the reduction of the influence of interferents. The influence of the viscosity of the sample solution was also checked for the analysis of real samples. The determination of the enzyme activities was also conducted in a system with micropumps fabricated for a sample injection. Two solutions could be mixed in the mixing channel, and the activity of the enzymes could be measured as in the experiments using microsyringe pumps.     

Synthesis of glutamic oxaloacetic transaminase isozymes in rat liver cells

The synthesis of glutamic oxaloacetic transaminase isozymes in rat liver explants was studied using specific antisera against the cytosolic and mitochondrial isozymes. The pulse-labeled cytosolic isozyme was detected in the cytosolic fraction and remained there in pulse-chase experiments. On the other hand, the pulse-labeled mitochondrial isozyme was detected as a larger precursor in the cytosolic fraction. During chase, the amount of pulse-labeled precursor of the mitochondrial isozyme decreased and labeled mature mitochondrial isozyme appeared in the mitochondrial fraction.     

The membrane fatty acid-binding protein is not identical to mitochondrial glutamic oxaloacetic transaminase (mGOT)

Summary For evaluation whether the membrane fatty acid-binding protein is related to mGOT, studies on the structure and function of both purified proteins were performed. Physicochemical characterization revealed that both proteins are different: the membrane fatty acid-binding protein has a molecular weight of 40 kD and a pI of 8.5–9.0, whereas rat mGOT has a molecular weight of 44 kD and a pI of 9.5–10.0. According to this distinct differences, they migrated separately on 2-dimensional electrophoresis. Furthermore, monospecific antibodies against the membrane fatty acid binding protein did not react with rat mGOT. In co-chromatography studies only the membrane fatty acid-binding protein showed affinity for long chain fatty acids, but not mGOT. Moreover, membrane binding studies were performed with the monospecific antibody to the membrane fatty acid binding protein. The inhibitory effect of this antibody on plasma membrane binding of oleate was reversed after preabsorption of the antibody with the membrane fatty acid binding protein, but was not affected after preabsorption with mGOT. These results indicate that the membrane fatty acid binding protein and mGOT are structurally and functionally not related. The data also support the significance of this membrane protein in the plasma membrane binding process of long chain fatty acids.     

Molecular cloning of rat mitochondrial glutamic oxaloacetic transaminase mRNA and regulation of its expression in regenerating liver

cDNA clones for rat mitochondrial glutamic oxaloacetic transaminase (mGOT) have been isolated from a rat liver cDNA library. One of the clones, designated p501, contained a cDNA insert of 1.4 kilobase pairs in length and hybridized to a mRNA of 2.4 kilobases from rat liver.We measured mGOT mRNA content in a regenerating rat liver. In a regenerating rat liver, mGOT activity was increased and reached maximum (170% of control activity) at about 48 h following the operation. Using the cDNA of mGOT, it was revealed that the increase of mGOT in the regenerating rat liver depended on its mRNA content.     

Gene dosage effect: intraband mapping of human soluble glutamic oxaloacetic transaminase.

Quantitative assays of soluble GOT were performed on fibroblasts (from two individuals) with duplications of the long arm of chromosome 10 (10q24 leads to qter). Improved karyologic technique demonstrated that the duplications differ by half a band. Correlation of GOTS activity with the karyologic data indicates that the structural locus for GOTS maps within band 10q24. This study demonstrates the utility of this approach to fine structural mapping of the human genome.     

Polymorphism of mitochondrial glutamic oxalo-acetic transaminase in a German population

Summary 204 placental samples from a German population were screened for variants of mitochondrial glutamic oxalo-acetic transaminase. Three variants were found occurring at a frequency of 1.47%. This is comparable with frequencies reported in other Caucasian populations. No variants of the soluble form were found.

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Zusammenfassung 204 Placentaproben einer deutschen Stichprobe wurden auf Varianten der Mitochondrien-Glutamat-Oxalat-Transaminase (M-GOT) untersucht. Drei Varianten wurden gefunden, was einer Häufigkeit von 1,47% entspricht. Dieser Wert steht im Einklang mit den Ergebnissen von Untersuchungen anderer europider Populationen. Varianten der löslichen Form (S-GOT) konnten nicht gefunden werden.

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Alexander von Humboldt Fellow.     

Ultrastructural localization of glutamic oxaloacetic transaminase in mitochondria of the flight muscle of Simuliidae

Glutamate oxaloacetic transaminase was detected histochemically in mitochondria of flight muscles of adult female simuliids. The reaction product was mainly deposited in mitochondrial cristae and to a lesser extent in the inner membrane of the mitochondria.     

Transspecific variability of glutamic oxaloacetic transaminase (E.C. 2.6.1.1.) in Primates

Summary The genetic polymorphism of soluble and mitochondrial glutamic oxaloacetic transaminase has been investigated in Primates. 3 variants of the soluble enzyme and 2 variants of the mitochondrial enzyme could be demonstrated. The distribution of the various phenotypes has been estimated.

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Zusammenfassung Die Glutamat-Oxalacetat-Transaminasen der Primaten, zeigen eine genetisch determinierte Variabilität. Es konnten 3 cytoplasmatische und 2 mitochondriale Enzymvarianten nachgewiesen werden. Die Verteilung der Phänotypen wurde ermittelt.

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Direktor: Prof. Dr. Dr. H. Ritter

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Supported by the Deutsche Forschungsgemeinschaft.