Activity In Cell-free System Research Articles

Studies on the mode of action of diphtheria toxin. V. Inhibition of peptide bond formation by toxin and NAD in cell-free systems and its reversal by nicotinamide.

Inhibition of soluble transferase II activity in cell-free systems by diphtheria toxin and NAD can be prevented or reversed in the presence of a sufficient concentration of nicotinamide. Quantitative studies on inhibition of peptide bond formation in cell-free extracts by toxin and NAD have indicated that two successive reversible reactions are involved. First, toxin and NAD interact mole for mole to form a relatively dissociable complex. This toxin-NAD complex then reacts with transferase II to form an enzymatically inactive product that is but slightly dissociated. In the presence of sufficient nicotinamide, however, the latter complex can be broken down to yield active transferase II once more. Based on the above model, an equation has been derived that accurately predicts the per cent inhibition of amino acid incorporation in cell-free systems at any given toxin and NAD level. The observed inhibition appears to be independent of the sensitivity to toxin of the cell species from which the extracts were derived, and depends only on the toxin and NAD concentrations. Although the model satisfactorily explains inhibition of peptide bond formation by toxin in cell-free systems, further assumptions are needed to explain how still lower concentrations of toxin are able to arrest protein synthesis completely in the living cell.

INHIBITION OF GLYCOLYSIS BY PYRUVATE IN RELATION TO THE ACCUMULATION OF CITRIC ACID CYCLE INTERMEDIATES IN THE PERFUSED RAT HEART.

RECENT investigations have established that the addition of metabolic fuels such as acetoacetate, fatty acids, lactate or pyruvate to hearts perfused with glucose and insulin inhibits the rate of glucose uptake and oxidation1–4. It has been proposed that during respiration of these substrates, glycolysis is inhibited by a common mechanism; namely, an indirect inhibition of phosphofructokinase as indicated by increased levels of hexose monophosphates and decreased levels of fructose-1,6-diphosphate5. Furthermore, phosphofructokinase activity has been shown to be depressed in hearts from diabetic rats which also have a decreased rate of glucose utilization6. It has recently been reported that citrate not only inhibits phosphofructokinase activity in cell-free system, but also accumulates in hearts from diabetic rats, and in isolated hearts perfused with long- and short-chain fatty acids7–9. These results have led to the suggestion that the rate of glycolysis may in some measure be controlled by the activity of the citric acid cycle. Williamson10 has reported that the rate-limiting step of glycolysis in perfused hearts with pyruvate as substrate occurred not at the phosphofructokinase step, as proposed by Newsholme et al.5, but at a site located between glyceraldehyde-3-phosphate and pyruvate. It was therefore considered of interest to examine the kinetics of the changes of the glycolytic intermediates after addition of pyruvate, in conjunction with the levels of certain citric acid cycle intermediates in both the intact heart and isolated heart mitochondria.

Antibody Activity in Cell-free Systems

Antibody Activity in Cell-free Systems