Polymorphism of alanine aminotransferase (E.C.2.7.6.1): Common and rare alleles

Summary Gene frequencies of common and rare GPT alleles derived from an investigation of 1139 unrelated, healthy individuals from southwestern Germany are given. GPT typing was performed by means of horizontal starch gel electrophoresis in a Tris-histidinexHCl buffer system. In addition, a new electrophoretic variant, GPT ⁹, is described.The frequencies of the GPT alleles observed were calculated as: GPT ¹ 0.4987; GPT ², 0.4686; GPT ^1M, 0.022; GPT ⁰, 0.005; GPT ³, 0.0022; GPT ⁴, 0.0025; GPT ⁸, 0.0005; GPT ⁹, 0.0005.     

Determination of isozymes of phosphoglycerate-mutase (E.C.2.7.5.3) and enolase (E.C.4.2.1.11) in human erythrocytes.

525 human hemolysates were tested for the isozymic patterns of phosphoglycerate mutase and enolase. Genetic models for interpreting the pherograms are suggested.     

Assignment to chromosome 16 of a gene necessary for the expression of human mitochondrial glutamate oxaloacetate transaminase (aspartate aminotransferase) (E.C. 2.6.1.1.)

A gene necessary for the expression of human mitochondrial glutamate oxaloacetate transaminase (GOT-2) has been assigned to chromosome 16 on the basis of an immunochemical analysis of human-mouse somatic cell hybrids. Mitochondrial GOT cosegregates with adenine phosphoribosyl transferase (E.C. 2.4.2.7.).     

Testicular angiotensin I-converting enzyme (E.C. 3.4.15.1)

In the two mammalian species (i.e., rabbit and rat) in which it has been studied to date, testicular angiotensin I-converting enzyme possesses distinct physicochemical and immunological properties, and a susceptibility to hormonal regulation that makes it a unique isozyme of the converting enzyme ordinarily distributed throughout the body. The testicular isozyme appears to be a lower molecular weight version of the pulmonary enzyme, with similar, although not identical, catalytic properties. The testicular isozyme is under androgenic control and is associated with germinal cells. Although its function has yet to be elaborated, the testicular isozyme provides an excellent model for the study of tissue-specific regulation of carboxypeptidases.     

Some Properties of Potato Tuber UDPGd-fructose-2-glucosyltransferase (E.C. 2.4.1.14) and UDPGd-fructose-6-phosphate-2-glucosyltransferase (E.C. 2.4.1.13)

Sucrose and sucrose 6-phosphate synthetase were isolated from potato tubers, partially purified and their properties studied. The sucrose synthetase showed optimum activity at 45° and was inhibited competitively by ADP and some phenolic glucosides. The Ki′s for these inhibitors were determined. Mg²⁺ was found to activate this enzyme. Activity toward UDP-glucose or ADP-glucose formation was measured. The optimum conditions for sucrose and UDP-glucose formation were found to differ. The specificity for the glucosyl donor and acceptor were determined.

The optimum conditions for sucrose 6-phosphate synthetase activity were studied. This enzyme was not inhibited by either ADP or phenolic glucosides; UDP-glucose was the only glucosyl donor for sucrose 6-phosphate formation.

     

Aminolevulinate dehydratase (E.C. 4.2.1.24): Linkage analysis

Summary Linkage data on aminolevulinate dehydratase (ALADH, E.C. 4.2.1.24) and a series of other human genetic markers are presented. One hundred and two families (25 of them being informative) from southwestern Germany were tested. Close linkage (=0.05) between ALADH and the following markers could be excluded: Rh, PGM₁, Fy, ACP1, MNSs, HLA, Bf, GLO, PGM₃, Jk, Pi, PGP, K, GPT. There is some evidence of possible linkage with HPA.     

First determination of the isozyme patterns of phosphoglycerate mutases (E.C.2.7.5.3) and phosphoglycerate kinases (E.C.2.7.2.3) in human tissues.

We present in this paper the first report about identification of several fractions of phosphoglycerate mutase (PGlyM) activity using starch gel electrophoresis and two different buffer systems. A typical muscle form of PGlyM was detected. It is also shown that isozymes of phosphoglycerate kinase (PGK) can be separated through the buffer system used by Spencer et al; (1964) for the phosphogluco mutase.     

Site-directed mutagenesis of aspartate aminotransferase from E. coli

The gene for aspartate aminotransferase from E. coli (aspC) was subcloned into M13 phage and sequenced using the Sanger dideoxy method with synthetic oligonucleotide primers. A mutant gene was constructed using site-directed mutagenesis techniques in which the codon for the lysine that forms the Schiffs base with pyridoxal phosphate was replaced with one coding for alanine. The mutant gene was expressed under control of the Tac promoter to overproduce a mutant protein lacking enzymatic activity.     

Aldehyde oxidase isoenzymes (E.C. 1.2.3.1) in potato tubers (Solanum tuberosum)

Two isoenzymes of aldehyde oxidase (E.C. 1.2.3.1 [EC] ) can be separatedfrom potato tubers (Solanum tuberosum) by polyacrylamide gelelectrophoresis. The pH optima of these two isoenzymes werepH 7.5. Both enzymes can oxidize different aldehydes, e.g. crotonaldehyde,Propionaldehyde, acetaldehyde, formaldehyde, glyoxal and benzyl-aldehyde.The isoenzymes could not use xanthine as a substrate. Formaldehydewas oxidized only in the presence of phosphate ions. A substratedependent inhibition of the enzyme activity is possible throughchloral hydrate. PMS, FMN, riboflavine, cytochrome c and O₂ serve as electronacceptors. (Received December 12, 1973; )     

The determination of the red cell isoenzymes 6-phosphogluconate dehydrogenase (E.C.1.1.1.44) and acid phosphatase (E.C.3.1.3.2) by means of agarosegel thinlayer electrophoresis (author's transl)

Methods for the determination of the red cell isoenzymes 6-PGD and acP by means of agarosegel thinlayer electrophoresis are referred. Using these relatively simple techniques, results can be obtained after at least two hours. The quality of separation in both systems is higher than that obtained after starch gel electrophoresis.     

Daten zur Populationsgenetik der Adenosin-Desaminase (E.C. 3.5.4.4) aus Schleswig-Holstein

Zusammenfassung Es wird über die Häufigkeitsverteilung des Isoenzyms Adenosin-Desaminase (ADA) in Schleswig-Holstein unter Berücksichtigung geographischer Räume berichtet. Dabei konnte zwischen dem geographischen Raum Schleswig-Holstein-West und dem Raum Schleswig-Holstein-Ost ein signifikanter Unterschied in der Genfrequenzverteilung festgestellt werden. Die Auswertung von 516 Mutter-Kind-Paaren ergab keine Abweichung von der Erbhypothese. 62 Nabelschnurblute zeigten keine qualitativen und keine erkennbar quantitativen Unterschiede zu Vergleichsbluten im Stärkegelzymogramm.

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Data on population genetics of adenosine deaminase (E.C. 3.5.4.4) in Schleswig-Holstein

Summary There have been reports on the frequency of appearance of the isoenzyme adenosine deaminase in different parts of Schleswig-Holstein (Northern Germany). A significant difference in the distribution of the gene was found between the West and the East of Schleswig-Holstein. Examination of 516 mother-child pairs revealed no deviations from the hereditary hypothesis.

     

Human phosphoglycolate phosphatase (PGP) E.C.3.1.3.18: Linkage analysis

Summary Linkage data on phosphoglycolate phosphatase (PGP) E.C.3.1.3.18 and 26 other human genetic markers are presented. One hundred and one families from the southwestern area of Germany were tested. Close linkage between PGP and the following markers could be ruled out: AB0, acP, ADA, GPT, PGM₁, GLO, HLA, and PGM₃. There is some evidence for possible linkage with MNSs, Rh, Gm and EsD. Family segregation data confirm the hypothesis formerly established by Barker and Hopkinson: three common alleles PGP¹, PGP² and PGP³ at an autosomal locus PGP.Supported by the Deutsche ForschungsgemeinschaftSupported by DAAD and Portuguese Inst. for Scientific Research (INIC)     

Syntheses of (Z)-and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid and their inactivation of gamma-aminobutyric acid aminotransferase.

(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid (4 and 5, respectively) were synthesized and investigated as potential mechanism-based inactivators of gamma-aminobutyric acid aminotransferase (GABA-AT) in a continuing effort to map the active site of this enzyme. The core alpha-trifluoromethyl-alpha,beta-unsaturated ester moiety was prepared via a Reformatsky/reductive elimination coupling of the key intermediates tert-butyl 2,2-dichloro-3,3,3-trifluoropropionate and N,N-bis(tert-butoxy-carbonyl)glycinal. Both 4 and 5 inhibited GABA-AT in a time-dependent manner, but displayed non-pseudo-first-order inactivation kinetics; initially, the inactivation rate increased with time. Further investigation demonstrated that the actual inactivator is generated enzymatically from 4 or 5. This inactivating species is released from the active site prior to inactivation, and as a result, 4 and 5 cannot be defined as mechanism-based inactivators. Furthermore, 4 and 5 are alternate substrates for GABA-AT, transaminated by the enzyme with Km values of 0.74 and 20.5 mM, respectively. Transamination occurs approximately 276 and 305 times per inactivation event for 4 and 5, respectively. The enzyme also catalyzes the elimination of the fluoride ion from 4 and 5. A mechanism to account for these observations is proposed.     

Genetic polymorphism of glycerol-3-phosphate dehydrogenase (E.C.: 1.1.1.8)

Summary By means of starchgel electrophoresis several distinct proteins with G-3-PD activity can be detected in Primates. The relative activities of these isoenzymes are found to vary markedly from tissue to tissue. It is presumed that the G-3-PD proteins are dimers composed of two nonidentical polypeptide subunits (chain A and B), which are determined by two separate gene loci (G-3-PD _A and G-3-PD _B). In liver, kidney and skeletal muscle the subunit B is in great excess, while in heart and brain both subunits A and B are present in almost equal proportions. It is concluded, that the various isozyme patterns are the consequence of random combinations of different polypeptide chains. The results obtained so far indicate, that in Primates 2 alleles occur at the G-3-PD _A locus and 5 alleles at the G-3-PD _B locus. Formal notations are given, and a study on population genetics is reported.

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Zusammenfassung Bei den Primaten können mit der Stärkegelelektrophorese verschiedene G-3-PD-aktive Proteine nachgewiesen werden. Die transspezifische Variabilität ist beträchtlich. Für eine formalgenetische Interpretation ist das Modell zu unterlegen: zwie Cistrons G-3-PD _A und G-3-PD _B mit Information für G-3-PD-Polypeptidketten. Homozygote Individuen besitzen 3 Isoenzymbanden, da die beiden Polypeptidketten zu Dimermolekülen frei assoziieren. Heterozygote Individuen für das Cistron G-3-PD _A bzw. G-3-PD _B besitzen jeweils 6 Isoenzymbanden. Bei doppelt heterozygoten Individuen (sowohl für das Cistron G-3-PD _A als auch für G-3-PD _B) sind insgesamt 10 Isoenzymbanden zu erwarten. Unterschiede in den Syntheseraten für A- und B-Polypeptidketten bedingen eine stark ausgeprägte organspezifische Variabilität. In Leber, Niere und skeletmuskel überwiegt die Synthese für B-Ketten, im Herzmuskel und Gehirn werden A- und B-Ketten in annähernd gleicher Menge gebildet. Auf Grund der bisher vorliegenden Ergebnisse ist bei den Primaten mit 2 allelischen Varianten für das Cistron G-3-PD _A und mit 5 allelischen Varianten für das Cistron G-3-PD _B zu rechnen.

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(Director: Prof. Dr. Dr. H. Ritter)

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Supported by the Deutsche Forschungsgemeinschaft.