Inheritance and linkage relationships of glutamate oxaloacetate transaminase isoenzymes in apple

Summary Independent dimeric genes GOT-2 and GOT-4 determining activity for glutamate oxaloacetate transaminase (E.C.2.6.1.1; GOT) in zones GOT-II and GOT-IV respectively were identified. Three alleles were found for GOT-2 and two for GOT-4, including a null allele for GOT-2 which produced detectable heterodimeric bands but not homodimeric bands. Linkage studies with leucine aminopeptidase (E.C.3.4.11.1; LAP) genes suggested linkage of GOT-2 with LAP-2 (r=0.13±0.23) and GOT-4 with LAP-1 (r=0.10±0.40).The results reported in this paper are part of a London University PhD thesis by the first author     

A comparative study of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in the saliva of diabetic and normal patients

Diabetes has been reported to affect salivary glands adversely in humans and experimental models. Glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) are salivary enzymes that also are widely distributed in animal tissues. We determined GOT and GPT levels in saliva samples of 100 type 1 and 30 type 2 diabetic patients using reflectance spectrophotometry and compared them to 30 age and sex matched healthy controls. Statistically significant differences were observed in the mean values of GOT and GPT in type 1 diabetics compared to type 2 and control groups. Significantly higher GOT levels were found in the 1–20 year age group of type 1 diabetics. Our findings suggest that salivary gland damage is due to the same immunological attack that affects pancreatic β cells and results in type 1 diabetes.     

Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction.

Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that the active sites of glutamate aspartate transaminase function independently and a compulsory flip-flop mechanism is not involved.     

Inheritance and linkage relationships of glutamate oxaloacetate transaminase isoenzymes in apple

Summary Four zones of enzymatic activity for glutamate oxaloacetate transaminase (GOT) were found in apple tissue. A dimeric gene, GOT-1, determining the fastest migrating zone, was identified. Six alleles were found, including a near null allelle which produced detectable heterodimeric bands but not homodimeric bands. A marked deficit or absence of certain geno-types in all backcrosses and in some crosses between unrelated varieties was attributed to the close linkage (r=0.02±0.005) of GOT-1 with the incompatibility S locus. GOT-1 was also closely linked with the isocitrate dehydrogenase locus IDH-1 (0.03±0.01). Proposed incompatibility genotypes for four cultivars, and the linked GOT-1 alleles are Cox: S ₁ b/S ₂ d, Idared: S ₃ a/S ₄ c, Fiesta: S ₃ a/S ₂ d and Kent: S ₃ a/S ₁ b.The results reported in this paper are part of a PhD Thesis by the first author     

Genetic control and intracellular localization of glutamate oxaloacetic transaminase in maize

Glutamate oxaloacetic transaminase (L-aspertate: 2-oxoglutarate aminotransferase, E.C. 2.6.1.1; GOT) was found to occur in five distinct electrophoretic forms in different tissue extracts from a number of highly inbred strains of Zea mays L. No major qualitative differences were detected in the various tissues examined, and the isozyme patterns did not undergo changes during temporal development of any given inbred strain. Cell fractionation studies showed one isozyme to be associated with the mitochondria (mGOT), another to be exclusively associated with the soluble fraction (sGOT), and a third to be associated with the glyoxysomes (gGOT). The glyoxysomal form occurs as two electrophoretically distinct variants which exist in different inbred strains of maize. The gGOT variants are under the control of two codiminant alleles (Got1A and Got1B) at the Got1 locus (isozyme5, gGOT). The genetic data and gene dosage effects suggest that GOT in maize is functionally a dimer.     

Hydrazinopropionic acid: a new inhibitor of aminobutyrate transaminase and glutamate decarboxylase

This paper deals with the synthesis of 3-pyrazolidone and the biochemical action of hydrazinopropionic acid. The latter compound is formed upon alkaline hydrolysis of 3-pyrazolidone. Hydrazinopropionic acid was found in vitro to be a very potent inhibitor of bacterial aminobutyrate transaminase as well as of aminobutyrate transaminase and glutamate decarboxylase from mouse brain. This inhibition was shown to occur despite the presence of high concentrations of pyridoxal phosphate in the incubation media. Injections of 20 mg hydrazinopropionic acid/kg into mice resulted in complete inhibition of aminobutyrate transaminase in brain and approximately 20 per cent inactivation of glutamate decarboxylase. This inhibition could not be prevented or antagonized by administration of pyridoxine to the animals. Addition of pyridoxal phosphate to homogenates of brain from animals treated with hydrazinopropionic acid also failed to reactivate the enzymes. The tentative conclusion reached from these results is that hydrazinopropionic acid has inhibitory action because of its close similarity to GABA with respect to molecular size, structural configuration and molecular charge distribution. This can be demonstrated by comparing a Dreiding model of hydrazinopropionic acid with that representing GABA.     

Synteny between the pro+ marker and human glutamate oxaloacetate transaminase.

Chinese hamster ovary cells with a specific auxotrophy for proline were fused with human cells from a variety of sources and the resulting hybrids analyzed for human genetic markers. Of 63 hybrid clones examined, 27 possessed both proline and cytoplasmic glutamate oxaloacetate transaminase markers; 36 had neither; and no clones were found possessing one and not the other. These results constitute evidence that the proline and glutamate oxalocetate transaminase markers are syntenic. Evidence for absence of synteny between these and a variety of other human genes is presented. Biochemical tracer experiments established that the proline biosynthetic pathway through glutamate has been restored in the Pro+ hybrids.     

Immobilized glutamate oxaloacetate transaminase. Steady state kinetic analysis and stability studies.

1. Glutamate oxaloacetate transaminase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) was immobilized on amino ethyl cellulose using the bifunctional reagent diethyl adipimidate. 2. The steady state kinetic analysis was performed for the particulate and the free enzyme, and the Michaelis constants measured for the amino ethyl cellulose derivative were not greatly different from those measured for the free glutamate oxaloacetate transaminase, while the latter were in good agreement with values in the literature. 3. The amino ethyl cellulose-glutamate oxaloacetate transaminase was slightly more stable than the free enzyme at 65 degrees C, but was stabilised less by polyethylene glycol than the free enzyme.     

Characterization of aLycopersicon esculentum xSolanum lycopersicoides somatic hybrid lacking a glutamate oxaloacetate transaminase isozyme

Morphological, cytological, isozyme and chloroplast DNA analyses were used to determine possible mechanism(s) for the loss of glutamate oxaloacetate transaminase-4 (GOT-4) isozyme activity in a somatic hybrid. Plant 204-1, derived by cell fusion between tomato (Lycopersicon esculentum) andSolanum lycopersicoides, was characterized for bothGot-4 and acid phosphatase-2 (Aps-2), two isozyme loci which are closely linked (recombination 2.5 cM). This hybrid was determined to be chimeric for bothGot-4 andAps-2. TheS. lycopersicoides plant used to provide cells for the fusion was determined to be heterozygous for bothGot-4 andAps-2. Only oneS. lycopersicoides allelic form ofAps-2 andGot-4 was found in plant 204-1. This observation indicated that either the alternative copy of theS. lycopersicoides chromosome region encodingGot-4 andAps-2 is deleted or the entire chromosome is absent. Plant 204-1 was cytologically determined to be aneuploid with approximately 62 chromosomes. Sixty-two somatic hybrids of separate callus origin were analysed for GOT-4 and a high proportion (27%) lacked theS. lycopersicoides form ofGot-4. The loss of this allele and the linkedAps allele most likely occurred in the suspension culture ofS. lycopersicoides used to provide cells for fusion.     

Determination of the chromosomal location of a glutamate oxaloacetate transaminase structural gene using triticum-agropyron translocations

The glutamate oxaloacetate transaminase (GOT) zymogram phenotypes of a series of 15 translocation lines, a chromosome addition line and a chromosome substitution line were determined. In each of the translocation lines a segment of the long arm of Triticum aestivum chromosome 3D has been replaced by a portion of an Agropyron elongatum homoeologue. Evidence was obtained that the products of the T. aestivum GOT-3 triplicate structural gene set randomly dimerize with the product of the homoeologous A. elongatum gene. Each translocation chromosome was found to carry either Got-D3 or Got-Ag3. By correlating the zymogram phenotype expressed by each translocation line with the observed frequency of meiotic pairing of each 3D/3Ag translocation chromosome with telocentric-3DL, it was shown that Got-D3 is located in the proximal portion of 3DL, slightly more than 4.3 crossover units from the centromere. The results of this genetic study confirm and extend earlier conclusions derived from cytogenetic studies as to the physical nature of the various 3D/3Ag chromosomes.     

The distribution of glutamate decarboxylase and aspartate transaminase in subcellular fractions of rat and guinea-pig brain

1. The subcellular distributions of glutamate decarboxylase and aspartate transaminase were studied in rat and guinea-pig brain. 2. Glutamate decarboxylase is localized in the synaptosome fraction. The mean density of the particles containing the enzyme is slightly greater than those derived from cholinergic neurones, though overlap is substantial. 3. The enzyme is readily released from synaptosomes by hypo-osmotic treatment, but in the presence of Ca(2+), Na(+) and K(+) it sediments with particulate material. 4. The release and binding of the enzyme to membrane fractions by Ca(2+) were investigated. 5. Aspartate transaminase is present in brain as two isoenzymes with different kinetic properties. One isoenzyme is associated with the cytoplasm and the other with mitochondria.