Identity of rat liver mitochondrial asparagine-pyruvate transaminase with phenylalanine-pyruvate transaminase

Identification of rat liver mitochondrial asparagine-pyruvate transaminase with phenylalanine-pyruvate transaminase has been done. When a mitochondria extract was subjected to isoelectric focusing, the two enzyme activities were identically focused. This procedure and DEAE-Sepharose chromatography revealed multiple forms of the enzyme, in which the main form was purified. In the various purification steps the two enzyme activities appeared in the same fraction. The enzyme of the final preparation step gave a single band in polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. During the purification, a similar increase of the specific activity and yield were obtained in the two activities. Phenylalanine was found to be a competitive inhibitor of asparagine transaminase. These results suggest the identity of the two enzymes.     

Red cell glutamate-pyruvate transaminase gene frequencies in Gambia, West Africa.

The red cell GPT phenotypes have been determined in two village populations in Gambia, West Africa. A total of 887 people have been investigated. The results confirm the previous observations that the frequency of the GPT gene is far higher in African populations than Caucasian populations.     

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 In each of the translocation lines a segment of the long arm of Triticum aestivum chromosome 3D has been replaced by a portion of an Agropyron elongatum homoeologue.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.     

Molecular cloning of the L-phenylalanine transaminase gene from Paracoccus denitrificans in Escherichia coli K-12

The L-phenylalanine transaminase gene of Paracoccus denitrificans was cloned by a shotgun method using the Escherichia coli K-12 mutant DG30, which lacks three distinct transaminase genes. Plasmid pPAP142 was constructed by inserting a 2.2-kb fragment carrying the transaminase gene into pUC18. Strain E. coli K-12 HB101 cells harboring the plasmid produced 20-fold to 30-fold more transaminase than wild type P. denitrificans cells. The nucleotide sequence of the 2.2-kb fragment was determined, revealing that the deduced amino acid sequence of the transaminase of P. denitrificans is similar to that of other transaminases.     

Occurrence of hydroxypyruvate-L-glutamate transaminase in Escherichia coli and its separation from hydroxypyruvate-phosphate-L-glutamate transaminase

Blatt, L. (University of Wisconsin, Madison), F. E. Dorer, and H. J. Sallach. Occurrence of hydroxypyruvate-l-glutamate transaminase in Escherichia coli and its separation from hydroxypyruvate-phosphate-l-glutamate transaminase. J. Bacteriol. 92:668-675. 1966.-The formation of l-serine from hydroxypyruvate by a transamination reaction with l-glutamate has been demonstrated in extracts of Escherichia coli. The level of activity with hydroxypyruvate is approximately one-tenth that observed with hydroxypyruvate-phosphate in cell-free extracts. The transamination of hydroxypyruvate, but not hydroxypyruvate-phosphate, is inhibited by inorganic phosphate. No marked differences in the levels of activity with hydroxypyruvate were observed in extracts from bacteria grown under different conditions. Heat treatment of enzyme preparations at 65 C rapidly destroys the activity with hydroxypyruvate-phosphate, but not that with hydroxypyruvate. Fractionation of extracts with lithium sulfate and alumina Cgamma resulted not only in a 10-fold purification, but also in a complete separation of the two activities, thereby establishing that two different enzymes are involved in the transamination of hydroxypyruvate and hydroxypyruvate-phosphate. Hydroxypyruvate transaminase is present in two mutants that require serine for growth. The inability of hydroxypyruvate to replace the growth requirement for serine, even to a limited extent, was shown to be due to the inability of the bacteria to accumulate this compound actively.     

The branched-chain amino acid transaminase gene family in Arabidopsis encodes plastid and mitochondrial proteins

Branched-chain amino acid transaminases (BCATs) play a crucial role in the metabolism of leucine, isoleucine, and valine. They catalyze the last step of the synthesis and/or the initial step of the degradation of this class of amino acids. In Arabidopsis, seven putative BCAT genes are identified by their similarity to their counterparts from other organisms. We have now cloned the respective cDNA sequences of six of these genes. The deduced amino acid sequences show between 47.5% and 84.1% identity to each other and about 30% to the homologous enzymes from yeast (Saccharomyces cerevisiae) and mammals. In addition, many amino acids in crucial positions as determined by crystallographic analyses of BCATs from Escherichia coli and human (Homo sapiens) are conserved in the AtBCATs. Complementation of a yeast Deltabat1/Deltabat2 double knockout strain revealed that five AtBCATs can function as BCATs in vivo. Transient expression of BCAT:green fluorescent protein fusion proteins in tobacco (Nicotiana tabacum) protoplasts shows that three isoenzymes are imported into chloroplasts (AtBCAT-2, -3, and -5), whereas a single enzyme is directed into mitochondria (AtBCAT-1).     

Separation of L-phenylalanine: pyruvate transaminase and L-phenylalanine: alpha-ketoglutarate transaminase by sephadex-electrophoresis.

By means of a Sephadex-electrophoresis column, L-phenylalanine: pyruvate transaminase (PPT) was separated from L-phenylalanine: α-ketoglutarate transaminase (PKT) from rat liver. These enzymes differed in heat lability $\text{in vitro}$ and in their inducibility by glucagon $\text{in vivo}$. PPT was heat-stable and was induced by chronic glucagon injection. On the other hand, PKT was heat-labile and was not induced by glucagon under the experimental conditions used. These studies provide evidence that distinct enzymes catalyze the transamination of phenylalanine with pyruvate or with α-ketoglutarate as the amino acceptor.