Voltammetric determination of lactate dehydrogenase using a carbon paste electrode.

A voltammetric study was performed by linear sweep voltammetry using a carbon paste electrode, in -0.1 to +1.3 V potential range, with the view of elaborating an assay for lactate dehydrogenase (LDH) in different enzymatic preparations and biological fluids. There have been performed studies concerning pH influence upon the enzymatic reaction, as well as the electrochemical behavior of LDH in the presence of modified carbon paste electrodes saturated with sodium pyruvate and/or NADH.

Influence of negative air ions on liver regeneration.

Partial hepatectomy led to an increased oxygen consumption over the whole respiratory cycle. Association of partial hepatectomy with negative aeroionotherapy determined a decrease of oxygen consumption as compared with the partially hepatectomized animals. Negative air ionization in controls determined a decrease of oxygen consumption at the level of the NAD+ dependent enzymes. On the other hand, neither partial hepatectomy, nor negative air ionization altered the oxidative phosphorylation of mitochondria.

Ion-exchange properties of Cibacron Blue 3G-A Sepharose (Blue Sepharose) and the interaction of proteins with Cibacron Blue 3G-A.

The affinity for Blue Sepharose of several proteins of known structure showed a pH dependence governed by their isoelectric points; Blue Sepharose behaved like a strong cationic ion exchanger because of the negative charges of its dye ligand, Cibacron Blue. A study of the protein-Cibacron Blue interactions by phase partition and equilibrium dialysis revealed the presence of high-affinity binding sites both in the case of the (di)nucleotide-dependent enzymes that possess the structural domain known as "dinucleotide fold", and in the case of other proteins consisting almost entirely of alpha-helix (human haemoglobin, cytochrome c) or beta-sheet (human immunoglobulin G). The presence of additional sites of low affinity, probably situated at the protein surface, was also inferred from the equilibrium dialysis data. In some instances, in contrast with the Sepharose-immobilized dye, the interaction of free Cibacron Blue with proteins was not pH dependent. Steric factors could be responsible for such a differential behaviour. It is suggested that certain nucleotide-dependent enzymes might also bind to Blue Sepharose by ion exchange. Preparative applications of these findings are illustrated and discussed in terms of the optimization of affinity chromatography experiments.

Cuvette and flow system for simultaneous determination of optical density and oxygen concentration.

The major components of the device for semiautomatic measurement of oxygen consumption and optical density changes are a thermostated glass reservoir of 50-250 ml for the reaction mixture, a roller pump to transport the reaction mixture from the reservoir into the measuring cuvette, and a stoppered glass cuvette equipped with a Clark type electrode. The glass cuvette (0.525 ml) is square shaped (the interior section 6 X 6 mm). The lower part housing the magnetic stirrer has a side aperture for the oxygen electrode. The upper part of the cuvette is cylinder shaped. The stopper is fitted in this part. The stopper made of lucite protrudes 15 mm into the cuvette. The access and the removal of the reaction mixture to/from the cuvette is ensured by three different channels machinated into the body of the stopper. The device adaptable to Eppendorf spectralline photometer may be used in the study of oxidative phosphorylation on intact mitochondria or submitochondrial particles, or of any oxygen consuming system in which one reagent or reaction product can be monitored photometrically.

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation.

Various analogs of adenosine 5'-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles. It is shown that the fluorophosphate analog ATP(gamma F) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(gamma F) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions. The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(gamma F), it is strong inhibitor of both soluble and membrane-bound mitochondrial ATPases. The biological implication of the complementary effects of ATP(gamma F) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.