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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage

The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.     

Red cell glutamic-pyruvic transaminase polymorphism in a sample of the Italian population a new variant allele: GPT8.

247 individuals from Northern Italy have been tested for red cell glutamic-pyruvic transaminase (GPT) polymorphism. An abnormal phenotype has been detected. Family data support the hypothesis of the existence of a new variant allele, GPT8, at the GPT locus.     

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.     

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.     

Whole-genome scan for guttural pouch tympany in Arabian and German warmblood horses

Equine guttural pouch tympany (GPT) is a hereditary disease in foals of several breeds, including thoroughbreds, Arabian, Quarter and warmblood horses. We performed a whole-genome scan for GPT in 143 horses from five Arabian and five German warmblood families and genotyped 257 microsatellites. Chromosome-wide significant linkage was detected on ECA2 and ECA15 using multipoint non-parametric linkage analyses. Analyses stratified by sex revealed chromosome-wide significant linkage on ECA2 for fillies and chromosome-wide significant linkage on ECA15 for colts. For Arabian colts, the quantitative trait locus (QTL) on ECA15 was genome-wide significant. Haplotypes including two to four microsatellites within the QTL on ECA2 and 15 in fillies and colts, respectively, were significantly associated with GPT for both breeds. Thus, our analysis indicated sex-specific QTL, a fact which is in agreement with a two- to fourfold higher incidence of GPT in females. This is the first report of QTL for equine GPT and a first step towards identifying genes responsible for GPT.     

Application of the GPT system in paternity cases.

Experiments show that the GPT starch gel pattern of any given blood sample is fully reproducible, and that an individual's GPT type is constant at least after the age of 1 month. GPT isoenzyme patterns are influenced and may be changed during storage of unfrozen blood. The interval between sampling and preparation of haemolysate should therefore not exceed 4 days. In haemolysates kept at -25 degrees C the isoenzyme patterns remain unchanged for at least many months. The GPT system forms a valuable means for statistical information in paternity cases. Thus, the overall chance is 18.7% for paternity exclusion or strong evidence against paternity for a falsely alleged father. Based on a material of 1,316 paternity cases, it is concluded that the GPT system is a valuable supplement to other systems of genetic markers in cases of disputed paternity.     

Glutamic-pyruvic transaminase (GPT) in non-human primates,I. Its activities and electrophoretic variation in red blood cells

Glutamic-pyruvic transaminase (GPT) in red cells of 25 species of non-human primates was investigated. There were significant differences in red cell GPT activities among species. Some species in the Prosimiae and the Ceboidea have high red cell GPT activities, while the others of these families examined have low activities. In contrast, red cell GPT activities were too low to be detected in the Cercopithecoidea and the Pongidea. The intraspecific variation of GPT zymograms was observed in Aotes trivirgatus by starch gel electrophoresis.     

Sporophytic control of anther development and male fertility by glucose-6-phosphate/phosphate translocator 1(OsGPT1) in rice

Coordination between the sporophytic tissue and the gametic pollen within anthers is tightly controlled to achieve the optimal pollen fitness. Glucose-6-phosphate/phosphate translocator(GPT) transports glucose-6-phosphate, a key precursor of starch and/or fatty acid biosynthesis, into plastids. Here, we report the functional characterization of Os GPT1 in the rice anther development and pollen fertility. Pollen grains from homozygous osgpt1 mutant plants fail to accumulate starch granules, resulting in pollen sterility. Genetic analyses reveal a sporophytic effect for this mutation. Os GPT1 is highly expressed in the tapetal layer of rice anther. Degeneration of the tapetum, an important process to provide cellular contents to support pollen development, is impeded in osgpt1 plants. In addition, defective intine and exine are observed in the pollen from osgpt1 plants. Expression levels of multiple genes that are important to tapetum degeneration or pollen wall formation are significantly decreased in osgpt1 anthers. Previously, we reported that At GPT1 plays a gametic function in the accumulation of lipid bodies in Arabidopsis pollen. This report highlights a sporophytic role of Os GPT1 in the tapetum degeneration and pollen development. The divergent functions of Os GPT1 and At GPT1 in pollen development might be a result of their independent evolution after monocots and dicots diverged.     

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.     

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.     

The polymorphism of alanine aminotransferase (EC:2.6.1.2): Densitometrical assay

Summary The variable differences in catalytic activity corresponding to identical charges of the gene products are particularly striking within the heterozygous phenotypes. By means of densitometry the portions of the gene products GPT 1 and GPT 2 are allowed to be described, quantitatively.Moreover, variants are described, which differ from the normal electrophoretic pattern of the heterozygotes showing different remainder activities of the gene product GPT 1.

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Zusammenfassung Die Variabilität der Aktivität der Genprodukte mit gleicher Ladung ist besonders auffällig bei den heterozygoten Phänotypen. Mit Hilfe der Densitometrie lassen sich die Anteile der Genprodukte GPT 1 und GPT 2 quantitativ beschreiben. So verhalten sich in der Regel die Intensitäten der Banden 1:2–1:2 wie 4:3:2.Außerdem existieren Varianten, die vom normalen Heterozygotenmuster abweichen und unterschiedliche Restaktivitäten für das Genprodukt GPT 1 aufweisen. Bei einer Variante GPT 2-1 verhalten sich die Proportionen der Bandenintensitäten gleich, bei der Variante GPT 2-1 M (Marburg) ist die Aktivität des Genproduktes GPT 1 um 83% gemindert gegenüber dem intakten Genprodukt GPT 1.

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Supported by the Deutsche Forschungsgemeinschaft.