cDNA cloning, genomic structure, chromosomal mapping, and functional expression of a novel human alanine aminotransferase

Alanine aminotransferase (ALT) catalyzes the reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate, and thereby has a key role in the intermediary metabolism of glucose and amino acids. Two ALT isoenzymes are known to exist, but only one ALT gene has been cloned, GPT. In this study, we cloned a homolog of GPT and named it GPT2, and the corresponding protein ALT2. GPT2 shares 69% identity and 78% similarity at the protein level to the previously cloned GPT. The human gene GPT2 encodes a 3.9-kb mRNA, consists of 12 exons, spanning approximately 50 kb of the genome, and maps to chromosome 16q12.1. GPT2 and GPT differ in mRNA expression in that GPT2 is highly expressed in muscle, fat, and kidney, whereas GPT is mainly expressed in kidney, liver, and heart. In addition, GPT2 seems to be the predominant form of GPT at the mRNA level in these tissues. Expression of ALT2 protein in Escherichia coli produced a functional recombinant enzyme that catalyzes alanine transamination, confirming that the enzyme is an ALT. The more abundant expression of GPT2 than GPT, especially in muscle and fat, suggests a unique and previously unrecognized role of this gene product in glucose, amino acid, and fatty acid metabolism and homeostasis.     

Absence of red cell glutamic-pyruvate transaminase: discovery of a "silent" allele homozygote.

An individual with complete absence of red blood cell glutamic-pyruvate transaminase (GPT) activity has been discovered in a South African family of Lebanese origin. The subject, who also shows a low level of serum GPT, appears to be perfectly healthy. His children, all obligatory heterozygotes for the GPT0 allele, have lower than average levels of the red cell enzyme. An apparent instance of anomalous segregation of red cell GPT resulting from the inheritance of the GPT0 allele was recorded in one of the proband''s grandchildren.     

Comparative studies on the human glutamate-pyruvate transaminase phenotypes--GPT 1, GPT 2-1, GPT 2.

The quantitative differences between the activity of the 3 common phenotypes of human red cell GPT has been confirmed. In addition, the activity of red cell GPT 1 was found to be greater in young children than in adults. No such difference was found for the GPT 2 phenotype. The activity of the red cell GPT 1 was found to decrease with age, reaching the adult level at the age of 10 to 12 years. Red cell GPT of all the 3 common phenotypes in both adults and children was found to show a similar response to the addition of excess pyridoxal phosphate. A method has been devised for the partial purification of human GPT (cytoplasmic) from liver. GPT 1 and GPT 2 have been purified, and very few significant differences were found amongst the physical and kinetic parameters tested.     

Use of recombinant retroviruses to study the regulation of integrated adenovirus early promoters.

Adenovirus E1A gene products are capable of modulating the expression of a variety of integrated genes. To study the mechanisms by which this regulation occurs, recombinant retroviruses have been utilized to establish cell lines containing an integrated copy of either the adenovirus E2 or E3 promoter adjacent to the bacterial guanine phosphoribosyl transferase (GPT) gene. These cell lines have been characterized with respect to both basal and E1A-induced levels of GPT gene expression. Cell lines with low levels of GPT gene expression showed increased expression in the presence of E1A, whereas cell lines with high basal levels of GPT gene expression had decreased GPT RNA levels in the presence of E1A. Further characterization of these cell lines revealed E1A modulation of the accumulation of RNA initiating at a retrovirus promoter adjacent to the E2 or E3 promoter. The use of the GPT gene as a marker of E2 or E3 promoter activity has allowed the isolation of cell lines which have spontaneously increased their levels of GPT RNA. A preliminary characterization of four of these cell lines has indicated that GPT gene expression is increased as a result of cis activation of the E2 promoter.     

Studies on glutamic-oxalacetic (GOT) and glutamic-pyruvic (GPT) transaminases of swine kidney worm Stephanurus dentatus (Diesing, 1839). I. Assay and general properties.

Biochemical studies on the two transaminases GOT and GPT of swine kidney worm Stephanurus dentatus have been made. GOT has been found much more active than GPT. Enzyme activities are based on the formation of oxaloacetate (GOT) or pyruvate (GPT) from aspartic acid and alanine respectively with oxoglutarate. A linear relationship is observed between the enzyme concentration and activity. GOT shows a maximum activity at pH 8.0 and Michaelis constant 9 X 10(-3) M for male and 2.9 X 10(-3) M for female. GPT has an optimum pH of 7.5 and a Michaelis constant 19 X 10(-3) M for male and 8 X 10(-3) M for female. The optimum temperature for both GOT and GPT was 60 degrees C.     

Two previously undetected variants of glutamic-pyruvic transaminase found by acidic polyacrylamide gel electrophoresis.

Two new electrophoretic variants of glutamic-pyruvic transaminase (GPT) have been found by polyacrylamide gel electrophoresis at acidic pH. They appeared to represent a single allele, GPT 2, by the standard method of starch gel electrophoresis. Studies in families show that they are inherited as codominant alleles at the GPT locus. Population frequencies are about the same as those of other rare GPT variants. Their behavior on gels is consistent with both of them having substitutions of histidines in place of uncharged amino acids.     

Genetic polymorphism of GPT and GLO-I in Galicia (northwest Spain): a comparative study

GPT and GLO-I phenotypes were determined by means of isoelectric focusing and starch gel electrophoresis, respectively, in a sample of the Galician population (Northwest Spain); GPT: n = 302, GLO-I: n = 500. The gene frequencies come to: GPT1 = 0.5099, GPT2 = 0.4901; GLO1 = 0.4930, GLO2 = 0.5070. No rare variants were found. The Galician gene frequencies are compared with those obtained on other populations from different parts of the world.     

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.     

Cloning, sequence, and expression of a cDNA encoding hamster UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase

UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT) catalyzes the initial reaction required for synthesis of dolichol-P-P-oligosaccharides. We report here on the sequence and expression of a full-length cDNA clone encoding hamster GPT. The cDNA predicts a protein of 408 amino acid residues including 10 hydrophobic segments. Several portions of the hamster GPT sequence constituting one-third of the protein have 60% or greater identity with yeast GPT, and one-half of the conserved sequence falls within the hydrophobic segments. In addition, hamster GPT has two copies of a putative dolichol recognition sequence recently identified in three yeast enzymes that interact with dolichol. The protein lacks KDEL or DEKKMP-type carboxyl-terminal ER sorting sequences. When expressed in COS-1 cells, the cDNA causes a 5-7-fold increase of GPT activity in membrane fractions. The activity was completely inhibitable by tunicamycin, and the primary product was shown to be GlcNAc-pyrophosphoryldolichol. This cDNA represents the first enzyme of the dolichol-oligosaccharide biosynthetic pathway to be cloned from a vertebrate source and demonstrates structural homology between the enzymes of the yeast and mammalian pathways.     

Expression of a plastid-localized sugar transporter in the suspensor is critical to embryogenesis

Plant growth and development rely on sugar transport between source and sink cells and between different organelles. The plastid-localized sugar transporter GLUCOSE-6-PHOSPHATE TRANSLOCATER1 (GPT1) is an essential gene in Arabidopsis (Arabidopsis thaliana). Using a partially rescued gpt1 mutant and cell-specific RNAi suppression of GPT1, we demonstrated that GPT1 is essential to the function of the embryo suspensor and the development of the embryo. GPT1 showed a dynamic expression/accumulation pattern during embryogenesis. Inhibition of GPT1 accumulation via RNAi using a suspensor-specific promoter resulted in embryos and seedlings with defects similar to auxin mutants. Loss of function of GPT1 in the suspensor also led to abnormal/ectopic cell division in the lower part of the suspensor, which gave rise to an ectopic embryo, resulting in twin embryos in some seeds. Furthermore, loss of function of GPT1 resulted in vacuolar localization of PIN-FORMED1 (PIN1) and altered DR5 auxin activity. Proper localization of PIN1 on the plasma membrane is essential to polar auxin transport and distribution, a key determinant of pattern formation during embryogenesis. Our findings suggest that the function of GPT1 in the embryo suspensor is linked to sugar and/or hormone distribution between the embryo proper and the maternal tissues, and is important for maintenance of suspensor identity and function during embryogenesis.

Specific expression of a sugar transporter that localizes to the plastids of cells in the embryo suspensor affects auxin activity and embryo development.     

Enzymatic degradation protects neurons from glutamate excitotoxicity

Several enzymes with the capacity to degrade glutamate have been suggested as possible neuroprotectants. We initially evaluated the kinetic properties of glutamate pyruvate transaminase (GPT; also known as alanine aminotransferase), glutamine synthetase, and glutamate dehydrogenase under physiologic conditions to degrade neurotoxic concentrations of glutamate. Although all three enzymes initially degraded glutamate rapidly, only GPT was able to reduce toxic (500 microM) levels of glutamate into the physiologic (<20 microM) range. Primary cultures of fetal murine cortical neurons were subjected to paradigms of either exogenous or endogenous glutamate toxicity to evaluate the neuroprotective value of GPT. Neuronal survival after exposure to added glutamate ranging from 100 to 500 microM was improved significantly in the presence of GPT (> or =1 U/ml). Cultures were also exposed to the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), which produces neuronal injury by elevating extracellular glutamate. GPT significantly reduced the toxicity of PDC. This reduction was associated with a reduction in the PDC-dependent rise in the medium concentration of glutamate. These results suggest that enzymatic degradation of glutamate by GPT can be an alternative to glutamate receptor blockade as a strategy to protect neurons from excitotoxic injury.     

Human red cell glutamic-pyruvic transaminase polymorphism in Serbia, Yugoslavia

The polymorphism of red cell glutamic-pyruvic transaminase (GPT) was studied in 277 unrelated voluntary blood donors from the population of Serbia (Yugoslavia). The following phenotype frequencies were observed: GPT 1 0.309, GPT 2-1 0.454 and GPT 2 0.206, while gene frequencies were: GPT1 0.556 and GPT2 0.454.     

Glutamate-pyruvate transaminase subtypes in Singapore ethnic groups

Autopsy liver samples from 244 Chinese, 119 Malays and 136 Indians were screened for glutamate-pyruvate transaminase (GPT) subtypes by starch-gel electrophoresis and isoelectric focusing at pH 5-7. Altogether, ten phenotypes controlled by four alleles (GPT1, GPT2A, GPT2B and GPT3) were identified. There was no significant difference in the frequency of GPT alleles between the ethnic groups. The distribution of GPT types was in agreement with the Hardy-Weinberg equilibrium in all the ethnic groups.     

Evidence that the hamster tunicamycin resistance gene encodes UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase.

A cDNA clone isolated from Chinese hamster ovary cells conferred elevated GlcNAc-1-P-transferase (GPT) activity and resistance to tunicamycin in transfected cells (Zhu, X., and Lehrman, M. A. (1990) J. Biol. Chem. 265, 14250-14255). It had been assumed that this cDNA, termed TRG for tunicamycin resistance gene, encoded GPT enzyme. However, other functions were not ruled out. Thus, by one of several mechanisms, the TRG protein could have instead functioned by activation of the transfected host's endogenous GPT enzyme. To analyze the biochemical function of the TRG protein, hamster TRG cDNA was stably expressed at high levels in Chinese hamster ovary cells. In addition, several antipeptide polyclonal antibodies directed against the predicted TRG protein were obtained. With these tools in hand, experiments were performed to test the hypothesis that the TRG encodes GPT enzyme, as well as to rule out other possible functions for the TRG protein. These experiments included examination of the effects of solubilization of membranes on TRG-dependent GPT activity, the apparent binding of tunicamycin to the TRG protein, and the immunoadsorption of GPT activity with TRG protein-specific antibodies. From these results, we conclude that the hamster TRG most likely encodes GPT enzyme.     

Gene frequencies of the enzymes ALADH, GOT2, GPT, PGM3, SAHH and UMPK in a Swiss population

The polymorphisms of the enzymes ALADH, GOT2, GPT, PGM3, SAHH, and UMPK have been studied in a population of Northern Switzerland (Zürich). The results are compared with those of other European populations.     

Plasma ornithine carbamyl transferase level as an indicator of ischaemic injury of rat liver

The activity of ornithine carbamyl transferase (OCT) and glutamate pyruvate transaminase (GPT) in serum has been correlated with the extent of necrosis 24 h after different periods of ischaemia in rat liver. The extent of necrosis has been quantified as the volume density of necrosis in the total ischaemic liver lobes using tetranitro BT. The GPT-activity in serum is maximal between 1 and 5 h after different periods of ischaemia, whereas OCT reaches its maximum between 5 and 12 h after ischaemia. The total amount of leaked enzyme-activity as well as the peak value give a linear correlation with the extent of necrosis for OCT and GPT. There is a difference between the character of these two enzymes in that a small leakage of GPT does not indicate liver cell necrosis later on. However, the appearance of OCT in the blood, an enzyme localized in the mitochondrial matrix, has a predictive value for the extent of necrosis, likely to occur later on. GPT, an enzyme from the cytoplasm, can also occur in the blood during the reversible stage of liver cell damage.     

Unexpected basis for impaired Glc3Man9GlcNAc2-P-P-dolichol biosynthesis by elevated expression of GlcNAc-1-P transferase

GlcNAc-1-P transferase (GPT) transfers GlcNAc-1-P from UDP-GlcNAc to dolichol-P (Dol-P), forming GlcNAc-P-PDol to initiate synthesis of the lipid-linked oligosaccharide Glc3Man9GlcNAc2-P-P-dolichol (G3M9Gn2-P-P-Dol). Elevated expression of GPT in CHO-K1 cells is known to cause accumulation of the intermediate M5Gn2-P-P-Dol, presumably by excessively consuming Dol-P and thereby hindering Dol-P-dependent synthesis of Man-P-Dol (MPD) and Glc-P-Dol (GPD), which provide the residues for extending M5Gn2-P-P-Dol to G3M9Gn2-P-P-Dol. If so, elevated GPT expression should increase oligosaccharide-P-P-Dol quantities and reduce monosaccharide-P-Dol quantities, while requiring GPT enzymatic activity. Here we report that elevated GPT expression failed to appreciably alter the quantities of the two classes of dolichol-linked saccharide, and that neither a GPT inhibitor nor introduction of an inactivating mutation into GPT prevented M5Gn2-P-P-Dol accumulation,arguing against excessive Dol-P consumption. Unexpectedly,we noticed similarities between the phenotypes of GPT overexpressers and of CHO-K1 cells lacking Lec35p (encoded by MPDU1, the congenital disorder of glycosylation(CDG)-If locus), which is required for utilization of MPD and GPD. By compensatory overexpression of Lec35p, G3M9Gn2-P-P-Dol synthesis in GPT overexpressers could be restored. However, GPT overexpression did not affect the levels of Lec35 mRNA or protein. These results suggest that GPT may impair Lec35p function, and imply that upper as well as lower limits on GPT expression exist in normal cells. Since the mammalian GPT gene can undergo spontaneous amplification, the data also indicate a potential basis for forms of pseudo-CDG-If.     

Genetic polymorphism of red cell glutamate-pyruvate transaminase in Japanese

Six hundred and eight red cell hemolysates were screened for glutamate-pyruvate transaminase (GPT) by means of isoelectric focusing. Two new variant phenotypes were detected, neither of which could be distinguished from GPT2-1 and GPT2 by conventional starch gel electrophoresis. The two types were considered to correspond to GPT2B-1 and GPT2A-2C reported previously in samples of European origin.     

Complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) determined by a combination of conventional and mass spectral methods

The complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. Isolated peptides were analyzed with a protein sequencer or with a plasma desorption time of flight mass spectrometer and placed in the sequence on the basis of their molecular mass and homology to the sequence of rat GPT. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The Mr of the subunit was calculated to be 54,479, which was in good agreement with a Mr of 55,000 estimated by SDS-PAGE, and also indicated that the active enzyme with a Mr of 114,000 was a homodimer composed of two identical subunits. The amino acid sequence is highly homologous to that of rat GPT (87.9% identity) recently determined [Ishiguro, M., Suzuki, M., Takio, K., Matsuzawa, T., & Titani, K. (1991) Biochemistry 30, 6048-6053]. All of the crucial amino acid residues are conserved in human GPT, which seem to be hydrogen bonding to pyridoxal 5'-phosphate in rat GPT by the sequence homology to other alpha-aminotransferases with known tertiary structures.     

Finger dexterity measured by the Grooved Pegboard test indexes Parkinson’s motor severity in a tremor-independent manner

Fine motor impairments are frequent complaints in people with Parkinson’s disease (PD). While they may develop at an early stage of the disease, they become more problematic as the disease progresses. Tremors and fine motor symptoms may seem related, but evidence suggests two distinct phenomena. The purpose of our study was to investigate the relationships between fine motor skills and clinical characteristics of PD patients. We hypothesized worse fine motor skills to be associated with greater motor severity that is independent of tremor. We measured fine motor abilities using the Grooved Pegboard test (GPT) in each hand separately and collected clinical and demographics data in a cohort of 82 persons with PD. We performed regression analyses between GPT scores and a range of outcomes: motor severity, time from diagnosis, age and tremors. We also explored similar associations using finger and hand dexterity scores from a standardized PD rating scale. Our results indicate that scores on the GPT for each hand, as measures of manual dexterity, are associated with motor severity and time from diagnosis. The presence of tremors was not a confounding factor, as hypothesized, but age was associated with GPT scores for the dominant hand. Motor severity was also associated with hand and finger dexterity as measured by single items from the clinical Parkinson’s rating scale. These findings suggest that the GPT to be useful tool for motor severity assessments of people with PD.