Transspecific variability of glutamic oxaloacetic transaminase (E.C. 2.6.1.1.) in Primates

Summary The genetic polymorphism of soluble and mitochondrial glutamic oxaloacetic transaminase has been investigated in Primates. 3 variants of the soluble enzyme and 2 variants of the mitochondrial enzyme could be demonstrated. The distribution of the various phenotypes has been estimated.

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Zusammenfassung Die Glutamat-Oxalacetat-Transaminasen der Primaten, zeigen eine genetisch determinierte Variabilität. Es konnten 3 cytoplasmatische und 2 mitochondriale Enzymvarianten nachgewiesen werden. Die Verteilung der Phänotypen wurde ermittelt.

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Direktor: Prof. Dr. Dr. H. Ritter

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Supported by the Deutsche Forschungsgemeinschaft.     

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.).     

Effect of metal ions in the culture medium on the stearoyl-coenzyme A desaturase activity of Mycobacterium phlei.

A particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome C reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system.     

Allelic and tissue variation of rat fumarate hydratase (E.C. 4.2.1.2.)

• 1.1. The polymorphism (two alleles at a single autosomal locus) of rat fumarate hydratase is described. 2. It provides a confirmatory genetic evidence for the tetrameric structure of the enzyme. 3. It is shown that fumarase may exist into two electrophoretically different (tissue-specific) forms.

     

Chromatin changes related to dedifferentiation and differentiation in tobacco tissue culture (Nicotiana tabacum L.)

Non-histone chromosomal proteins (NHP) were isolated from different stages of Nicotiana tabacum L. pith dedifferentiation to callus and callus redifferentiation. The NHP were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis on slab gels and analyzed by densitometry. Simultaneous histological changes are reported. In both processes, some high molecular weight protein (HMWP) bands increase drastically in an induction period, previous to cell proliferation, and decrease when cell division declines. Some low molecular weight protein bands, intense in pith tissue, decrease early when callus is forming and increase when cells differentiate. chromatin template activity is high when cells proliferate, coinciding with maximum HMWP-bands intensity.Abbreviations HMWP high molecular weight proteins - IAA indole-3-acetic acid - LMWP low molecular weight proteins - NHP non-histone proteins - TA template activity     

Effect of metal ions on the activity of the catalytic domain of calcineurin

Calcineurin (CN) is a heterodimer, composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). There are four functional domains present in CNA, which are catalytic domain (CNa), CNB-binding domain (BBH), CaM-binding domain (CBH) and autoinhibitory domain (AI). It has been shown previously that the in vitro activity of calcineurin is relied primarily on the binding of metal ions. Mn2+ and Ni2+ are the most crucial cation-activators for this enzyme. In order to determine which domain(s) in CN is functionally regulated by metal ions, the rat CNA alpha subunit and its catalytic domain (CNa) were cloned and expressed in E. coli. The effects of Mn2+, Ni2+ and Mg2+ on the catalytic activity of these purified proteins were examined. Our results demonstrate that all the metal ions tested in this study activated either CNA or CNa. However, the activation degree of CNa by the metal ions was much higher than that of CNA. In term of different metal ions, the activating extents to CNA and CNa were different. To CNA, the activating order from high to low was Mg2+ > > Ni2+ > Mn2+, but Mn2+ > Ni2+ > > Mg2+ to CNa. No effect of CaM/Ca2+ and CNB/Ca2+ on the activity of CNa was observed in our experiments. Moreover, a weak interaction (or untight coordination binding) between metal ions and the enzyme molecule was also identified. These results suggest that the activation of these enzymes by the exogenous metal ions might be via both regulating fragment of CNA (including BBH, CBH and AI) and catalytic domain (CNa), and mainly via regulating fragment to CNA and mainly via catalytic domain to CNa. The activating extents of metal ions via catalytic domain were higher than that via regulating fragment. The results obtained in this study should be very useful for understanding the molecular mechanism underlying the interaction between calcineurin and metal ions, especially Mn2+, Ni2+ and Mg2+.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Effects of mercury on acetylcholinesterase (E.C. 3.1.1.7.) activity of erythrocytes and bone marrow in rats

The studies were carried out on male Wistar rats, where the activity of acetylcholinesterase (AchE-E.C. 3.1.1.7) was determined in red cells and bone marrow under the influence of organic and anorganic mercury compounds. A marked decline in the activity of the enzyme could be noted, along with a more pronounced effect of the organic mercury compound on AchE.     

Effect of metal ions on the structure and activity of calcium-activated neutral protease (CANP)

To clarify the mechanism of activation of calcium activated neutral protease (CANP, or mCANP: active at mM Ca2+), the structure of mCANP was examined by measuring CD spectra and by titration of SH groups in the presence of Mn2+. Mn2+ significantly increases the sensitivity of CANP to Ca2+ but CANP is not active with Mn2+ alone. The structural changes induced by Mn2+ were compared with those induced by Ca2+, and the structure of muCANP, which is active at microM Ca2+, was also examined for comparison. Mn2+ and Ca2+ induced the same structural changes of CANP. However, specific activation of the active site SH group by Ca2+ was not observed with Mn2+. Six moles of calcium bound to mCANP and the average dissociation constant of Ca2+ was 150 microM. The structure of muCANP was similar to that of mCANP in terms of the CD spectra. The titration of SH groups of muCANP indicated that the structure of muCANP was looser or SH groups were more exposed than in the case of mCANP. A model which can explain the activation of mCANP is proposed and the mechanism of activation is discussed based on the proposed model. The role of Ca2+ can be explained in terms of conformational change and activation of the active-site SH group of CANP.     

Reaction of the aminic form of aspartate transaminase with difluoro-oxaloacetate.

Addition of difluoro-oxaloacetate to the aminic form of aspartate transaminase causes a rapid shift of absorbance maximum of the enzyme from 332 nm to 328 nm, followed by a much slower shift to 360 nm corresponding to complete conversion of the aminic form of the enzyme into the aldimine form or a species with similar spectral parameters in rapid equilibrium with it. Kinetic analysis of both the initial fast reaction and the overall slow reaction by using repeated spectral scanning and stopped-flow techniques allows formulation of a basic reaction mechanism involving at least two intermediate enzyme complexes. Computer simulation of the progress curves of the initial fast reaction based on the suggested reaction mechanism gives kinetic parameters that are consistent with all the data obtained by other methods. A molecular reaction scheme involving a ketimine Schiff-base intermediate is proposed.