HIBADH Plays an Important Role in the Course of Liver Cell Necrosis

Objective To observe the biological function of human 3-hydroxyisobutyrate dehydrogenase (HIBADH). Methods Human 3-hydroxyisobutyrate dehydrogenase (HIBADH, 3-hydroxy-2-methyl propanoate: NAD+ oxidoreductase) recombinant protein was expressed in E. coli BL21, and purified by Ni+ column. The special antisera was obtained from rabbits immunized by this purified antigen. On the distribution of HIBADH, it was found that HIBADH over-expressed in the injured liver cells when serious hepatitis occurred. The phenomenon was confirmed in the animal models of SD rats with acute liver cell injury induced by CCl4, but this phenomenon did not exist in the models induced by endotoxin combined with galactosamine. Further more, HIBADH’s overexpression in liver cells will induce cell necrosis through the pathway of oxidative stress. Results When the liver cells injured by drug or other chemical materials, HIBADH will be compensationally over-expressed for the deficiency of energy, so liver cells can make enough ATP through brand-chain amino acid catabolism. However, the overexpression of HIBADH will be harmful for liver cells through the product of much more active oxygens which will induce the cell necrosis. Conclusions HIBADH over-expression is a signal of the liver cell metabolism injury, and it can aggravate the liver cell injury through oxidative stress.

INFLUÊNCIA DA TEMPERATURA E DO pH NAS PROPRIEDADES CINÉTICAS DA LACTATO DESIDROGENASE DO MÚSCULO EPAXIAL DE Prochilodus scropha e Notothenia neglecta

Foi levado a efeito um estudo comparativo das propriedades cinéticas da lactico desidrogenase (Llactato NAD+ oxidorreductase, E.C.1.1.1.27) purificada do músuclo epaxial do peixe tropical Prochilodus scropha e do peixe antártico Notothenia neglecta. Os seguintes parâmetros foram estudados: a. Efeito do pH; efeito da temperatura e valores para a energia de ativação; efeito da concentração de substrato e o efeito da temperatura sobre os valores de Km. Abstract It has been carried out a comparative study on the kinetic properties of the lactate dehydrogenase (L-lactate NAD+ oxidoreductase, E.C.1.1.1.27) purified from the epaxial muscle of the tropical fish Prochilodus scropha and the Antarctic fish Notothenia neglecta. The following parameters were studied: a. Effect of pH; effect of temperature and values for energy of activation; effect of substrate concentration and the effect of temperature on the Km values.

PURIFICAÇÃO E ISOENZIMAS DA LACTATO DESIDROGENASE DO MÚSCULO EPAXIAL DE Prochilodus scropha e Notothenia neglecta

Foi levado a efeito um estudo comparativo de purificação da lactico desidrogenase (L-lactato NAD+ oxidorreductase, E.C.1.1.1.27) do músuclo epaxial do peixe tropical Prochilodus scropha e do peixe antártico Notothenia neglecta. A purificação da LDH de ambas as fontes foi procedida por cromatografia de afinidade em oxamato-agarose, tendo revelado uma única banda de proteína em eletroforese em acetato de celulose, para ambas as preparações. Contudo, foi verificado que em eletroforese em tampão barbital pH 8,6, a LDH de P. scropha migra para o cátodo enquanto que a de N. neglecta migra para o ânodo. Abstract It has been carried out a comparative study on the purification of the lactate dehydrogenase (Llactate NAD+ oxidoreductase, E.C.1.1.1.27) from the epaxial muscle of the tropical fish Prochilodus scropha and the Antarctic fish Notothenia neglecta. Purification of LDH from both sources was carried out by oxamateagarose affinity chromatography. The eletrophorectic profile in cellulose acetate showed a single band in both preparations. However it has been observed that in barbital buffer pH 8.6, the LDH from P. scropha migrates towards the cathode while the LDH from N. neglecta, migrates towards the anode.

Production of OH-Radical-Type Oxidant by Lucigenin

In the presence of NADH- reductases (dihydrolipoamide: NAD oxidoreductase E. C.l.8.1.4 from pig heart or from Clostridium kluyveri; frequently also addressed as “diaphorases”) and NADH lucigenin strongly increases ethylene production from a-ketomethylthiobutyrate (KMB) as an indicator for strong oxidants of the OH-radical type. These reactions are further stimulated in the presence of Fe3+ ions. With these NADH- “diaphorases”, the structurally similar poison, paraquat, in the absence or presence of Fe3+ has no effect. With ferredoxin-NADP reductase (E. C.1.18.1.2.), however, paraquat reacts quasi identical to lucigenin. Superoxide dismutase, catalase, free radical- or OH - scavengers such as mannitol, propylgallate, DABCO, and desferal inhibit the reaction whereas EDTA (in the presence or absence of added Fe3+) is stimulatory. From these data we conclude that the superoxide - indicator LUC is redox-active after unspecific coupling to several almost ubiquitory NAD(P)H- reductases catalyzing monovalent oxygen reduction. Lucigenin thus should no longer be used as a “specific” superoxide indicator. This report is in agreement with very recent results by Liochev and Fridovich (Arch. Biochem. Biophys. 337, 115 [1997]) and Vasquez-Vivar et al. (FEBS Lett. 403, 127 (1997).

IFCC methods for the measurement of catalytic concentration of enzymes. Part 8. IFCC method for lactate dehydrogenase (L-lactate: NAD+oxidoreductase, EC 1.1.1.27)

Human lactate dehydrogenase is a tetramer made up of two types of subunits, either H (heart) or M (muscle). Combination of these subunits gives rise to the five isoenzymes of lactate dehydrogenase which are found in mammalian tissues. The relative proportions of the individual isoenzymes found in serum of patients is related to the severity of the lesion in the organ or tissue from which they originate and the half-life of the individual tissue-specific enzymes. Thus, one cannot predict the relative proportions of the different isoenzymes in any one patient sample.Lactate dehydrogenase catalyses the reversible oxidation of lactate to pyruvate and either reaction can be measured readily. However, in this method, the lactate to pyruvate reaction has been selected because of the following reasons; the time-course of the reaction is more linear, the reaction results in an increase in absorbance and optimization of substrates is possible (see appendix A).The principles applied in the selection of the conditions of measurement are those stated in previous publications by the IFCC’s Committee on Enzymes [1]. Human serum and tissue extracts have been used as the sources of enzymes. The final concentration of substrates and the pH have been selected on the basis of experiments and empirical optimization techniques and have been confirmed by calculation from rate equations. The catalytic and physical properties of the isoenzymes differ, but because of the importance of the heart specific isoenzyme (LD1) in the assessment of coronary heart disease and as a tumour marker, this method has been optimized for this isoenzyme. However, the method is also suitable, although less optimally, for the determination of the other isoenzymes of lactate dehydrogenase which may be present in serum.

Origin of the human alcohol dehydrogenase system: implications from the structure and properties of the octopus protein

In contrast to the multiplicity of alcohol dehydrogenase in vertebrates, a class III type of the enzyme [i.e., a glutathione-dependent formaldehyde dehydrogenase; formaldehyde; NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1.] is the only form detectable in appreciable yield in octopus. It is enzymatically and structurally highly similar to the human class III enzyme, with limited overall residue differences (26%) and only a few conservative residue exchanges at the substrate and coenzyme pockets, reflecting "constant" characteristics of this class over wide time periods. It is distinct from the ethanol-active "variable" class I type of the enzyme (i.e., classical liver alcohol dehydrogenase; alcohol:NAD+ oxidoreductase, EC 1.1.1.1). The residue conservation of class III is also spaced differently from that of class I but is typical of that of proteins in general, emphasizing that class I, with divergence at three functional segments, is the form with deviating properties. In spite of the conservation in class III, surface charges differ considerably. The apparent absence of a class I enzyme in octopus and the constant nature of the class III enzyme support the concept of a duplicative origin of the class I line from the ancient class III form. Still more distant relationships define further enzyme lines that have subunits with other properties.