Robert S. Horn

The role of sulfhydryl groups in oxidative phosphorylation and ion transport by rat liver mitochondria

Abstract The influence of the sulfhydryl reagent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DNTB) on metabolism of liver mitochondria was studied under different experimental conditions. 1. 1. DTNB prevented the stimulation of respiration produced by ADP and inorganic phosphate in the presence of glutamate or succinate but had little effect on 2,4-dinitrophenol-stimulated respiration. 2. 2. The formation of ATP from ADP and phosphate was depressed by DTNB. The inhibition could be reversed by dithiothreitol. 3. 3. The increase in mitochondrial oxygen uptake produced by calcium and phosphate was depressed by DTNB. 4. 4. The uptake of calcium by mitochondria in the presence of ATP and glumate was only partially inhibited by DTNB. In contrast, the stimulatory effect of inorganic phosphate on calcium transport was completely prevented. 5. 5. In the presence of glutamate and no added ATP the uptake of calcium was associated with an entrance of phosphate. DTNB almost abolished the uptake of phosphate and inhibited calcium uptake by about 60%. The decrease in calcium uptake produced by DTNB was equal, mole for mole, to the decrease in phosphate uptake. 6. 6. DTNB completely prevented the extrusion of calcium exhibited by calcium-loaded mitochondria incubated in the presence of inorganic phosphate and a low concentration of magnesium. 7. 7. N-Ethylmaleimide had effects similar to DTNB but in addition severely inhibited 2,4-dinitrophenol-stimulated respiration with glutamate as substrate. 8. 8. It was concluded that a reactive site involving a sulfhydryl group is intimately involved in either the entrance of inorganic phosphate into the mitochondrion or in the formation of a phosphorylated intermediate essential for oxidative phosphorylation and ion transport.

Mitochondrial calcium uptake in the perfused contracting rat heart and the influence of epinephrine on calcium exchange

Abstract Perfused rat hearts were exposed to solutions containing 45 Ca 2+ with and without epinephrine. They were subjected to differential centrifugation and the distribution of Ca and 45 Ca in mitochondria and microsomes was determined. It was found that the mitochondria contain most of the calcium of the intact heart and that the exchange of mitochondrial calcium with extracellular calcium was extremely rapid. This process was accelerated in hearts stimulated by epinephrine.

The influence of oligomycin on the actions of epinephrine and theophylline upon the perfused rat heart

Abstract Rat hearts were perfused with media containing pyruvate and iodoacetate with and without oligomycin. In the absence of oligomycin, both heart rate and force of contraction were well maintained. When oligomycin was included in the perfusion medium, there was a negative inotropic effect, a marked atrioventricular (A-V) block and a slowly developing contracture. In the oligomycin-treated heart, epinephrine or theophylline increased pacemaker activity, reduced the extent of A-V blockade and greatly accelerated the development of contracture. Rapid occurrence of contracture also followed electrical stimulation. Epinephrine was capable of exerting a positive inotropic effect in electrically driven hearts perfused with iodoacetate, pyruvate and oligomycin. Measurements of adenine nucleotides and creatine-phosphate showed that at the time when heart rate was markedly depressed by oligomycin ATP concentrations were not greatly reduced, although creatine-phosphate levels had decreased by 40 per cent. Administration of epinephrine or theophylline to these hearts greatly diminished the levels of high-energy phosphate. It was concluded that formation of new ATP from glycolysis or the respiratory chain is not required for the initial inotropic action of epinephrine or theophylline. The data allow one to speculate that the primary effects of these compounds or of 3′,5′ cyclic AMP may be concerned with calcium movements in the myocardial cell.

ACETYL DISULFIDE, (CH3COS)2, A MAJOR ACTIVE COMPONENT IN THE THIOLACETIC ACID HISTOCHEMICAL METHOD FOR ACETYLCHOLINESTERASE

Triple distilled thiolacetic acid (TA) was found to be much less active as a histochemical substrate for acetylcholinesterase (AChE) of mouse intercostal muscle than either the original, impure preparation, thiolacetic acid (practical, Eastman) or the residue from the first distillation. Iodimetric titration showed that the crude preparation had only 92-94% of its theoretical TA content, which fell to 70-72% with storage. Findings from column and thin layer chromatography and ether-water extraction indicated that the active material is relatively polar and acidic, and contains an S—S group. The addition of 0.0015-0.006 M acetyl disulfide (AcDiS) to 0.03 M triple distilled TA markedly enhanced the substrate activity of the latter so that it matched that of 0.03 M thiolacetic acid (practical); treatment with the reducing agents, sodium ascorbate and sodium borohydride, reduced the substrate activity of both the mixtures and thiolacetic acid (practical) but not that of triple distilled TA alone. Mass spectrometric examination of thiolacetic acid (practical), the distillation residue and a benzene extract of extemporaneously prepared AcDiS showed the presence in each of a peak at mass 150 (the molecular weight of AcDiS), which was not present in triple distilled TA. All except the AcDiS extract showed a peak also at mass 76 (the molecular weight of TA). It was concluded that AcDiS is the major component of thiolacetic acid (practical) that is responsible for its enhanced activity as a histochemical substrate for AChE.

The effect of metabolic inhibitors on the response of the perfused rat heart to epinephrine

Abstract The effects of epinephrine on contractility and metabolism were measured in the isolated, perfused rat heart. Epinephrine produced an initial increase in force of contraction followed by a lowering of tension and finally a sustained increase in contractility. When fluoroacetate or iodoacetate was present in the perfusion fluid, the mechanical response to epinephrine was markedly altered. The initial response to epinephrine was depressed in the presence of either inhibitor and the final increase in force of contraction was poorly maintained during metabolic blockade. When the perfusion fluid contained both pyruvate and iodoacetate, the mechanical response of the heart to epinephrine was restored. Results of determinations of metabolites in the myocardium demonstrated that stimulation of glycogenolysis is not essential for the contractile response to epinephrine when adequate substrate is provided for the reactions of the tricarboxylic acid cycle.

COMPLEX FORMATION BETWEEN HEAVY METALS AND THIOLACETIC ACID IN ELECTRON MICROSCOPIC HISTOCHEMISTRY

Evidence for the formations of complexes of aurous and lead ions with thiolacetic acid was obtained by two methods. (1) It was demonstrated that the addition of the metal ions to solutions of thiolacetic acid at pH 6.2 caused a bathochromic shift of the ultraviolet spectrum of this compound characteristic of those observed on combination of metal ions with an organic resonator. The experiments indicated that aurous ions form a coordinate complex with thiolacetic acid containing one metal ion and two thiolacetate residues. (2) It was observed that aurous and lead ions inhibited the reaction of thiolacetic acid with N-ethylmaleimide. Complex formation of the heavy metals with thiolacetic acid may be of importance in preventing their binding at nonspecific sites and in facilitating their penetration into tissue sections in the adaptation for electron microscopy of the thiolacetic acid method for cholinesterases.

The mechanism of inhibition of glycolysis by quinidine in heart tissue in vitro

Abstract Quinidine was shown to inhibit glycolysis and adenosine triphosphatase activity in centrifugea rat heart extracts at concentrations as low as 4.8 × 10−5M. Inhibition of phosphofructokinase was found to be the main cause of the reduction of glycolytic flux produced by the drug. Inhibitory effects on pyruvate kinase and lactic dehydrogenase were also noted. These glycolytic actions of quinidine were clearly shown to be secondary to increases in ATP and concurrent decreases in AMP and ADP.