Clinton D. Stoner

Effects of Acute Asphyxia and Deep Hypothermia on the State of Binding of Lysosomal Acid Hydrolases in Canine Cardiac Muscle

The amount and state of binding of three lysosomal acid hydrolases, acid phosphatase, cathepsin, and β-glucuronidase, were studied in canine cardiac muscle following severe asphyxia and deep hypothermia. The studies were done on adult mongrel dogs anesthetized with sodium pentobarbital. Asphyxia was produced by tracheal occlusion, and was maintained until the onset of cardiac arrest (6 to 10 min). Hypothermic conditions were achieved by placing the heart on partial bypass through a heat exchanger. The heart was cooled to 6 to 10°C and held at this temperature for 30 min while circulation and respiration were maintained by artificial means. Control dogs were subjected to similar surgical procedures. Immediately following the experiments, the whole hearts were removed, cooled to 0 to 4°C, homogenized, and fractionated into supernatant and lysosomal fractions. The levels of bound and free acid hydrolases were estimated in the whole homogenates and fractions. Asphyxia produced a large shift of acid hydrolase activity from a bound form to a free form, as evidenced by elevated ratios of free to bound activity in the whole homogenates and elevated ratios of supernatant to lysosomal activity with respect to the fractions. Hypothermia did not alter the binding status of the lysosomal enzymes. These findings suggest that lysosomal enzymes play a major role in asphyxic damage to the heart.

Oxidative Phosphorylation in Mitochondria Isolated from Chronically Stressed Dog Hearts

Oxidative and phosphorylative activities were measured polarographically in mitochondria isolated from the right and left ventricles of normal and chronically stressed dog hearts. Chronic myocardial stress was produced experimentally by surgical procedures (combined tricuspid insufficiency and pulmonary stenosis, pulmonary insufficiency, aortic stenosis, aortic insufficiency, Pottss anastomosis) and by inducing thyrotoxicosis. Experimental stress periods ranged from 332 to 608 days. Some of the dogs had overt symptoms of congestive heart failure at the time they were killed. Mitochondria isolated from the stressed hearts had abnormally high values for oxidative activity and respiratory control ratios when incubated in the presence of malate-pyruvate. In the presence of succinate, they had either normal or slightly elevated values for oxidative activity and respiratory control ratios. No differences were found between mitochondria from normal and stressed hearts with regard to the efficiency (ADP/O) of oxidative phosphorylation. Estimates of mitochondrial protein per gram of myocardial tissue indicated that the stressed hearts contained normal amounts of mitochondria. The results of this study suggest that the mitochondrial oxidative and phosphorylative capabilities of the chronically stressed myocardium are not impaired.

Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria. Determination of the coupling relationships between the respiratory reactions and miscellaneous observations concerning rate-limiting steps

The linear sequence of steps involved in the oxidation of extramitochondrial succinate by O2 in bovine heart mitochondria was examined by a steady-state kinetic method to determine whether or not freely diffusible intermediates occur between the various inhibitor-sensitive steps. The kinetic method is based on the facts (1) that if two inhibitor-sensitive steps within a sequence are linked by a freely diffusible intermediate, inhibition of one will make the other less rate limiting in the overall reaction and thus will increase the amount of inhibitor of the other step required for half-maximal inhibition of the overall reaction, and (2) that if the two steps are not linked in this manner, inhibition of one will make the other more rate limiting and thus will decrease the amount of inhibitor of the other required for half-maximal inhibition. These two types of “coupling relationships” between steps were designated as “sequential” and “fixed,” respectively. The results indicate the existence of freely diffusible intermediates (sequential coupling relationships) between the succinate transport and succinate dehydrogenase reactions, between the succinate dehydrogenase and cytochromebc1 reactions, and between the cytochromesbc1 andaa3 reactions. Uncoupling respiration from phosphorylation results in the coupling relationship between thebc1 andaa3 reactions becoming partially fixed. This change is accompanied by marked decreases in the degrees to which thebc1 andaa3 reactions limit the overall reaction and appears to account for the large uncoupler-induced releases of inhibition at the levels of thebc1 andaa3 reactions observed previously by others. It is suggested that cytochromec is the freely diffusible intermediate between thebc1 andaa3 reactions and that the uncoupler-induced changes occur as a result of formation of functional and highly efficient supercomplexes between cytochromec and the cytochromesbc1 andaa3 complexes.

Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria

The steady-state velocity dependence of the overall mitochondrial oxidative phosphorylation reaction on the concentrations of extramitochondrial ADP and P1 and of several of the catalytic components was investigated, using the O2 uptake step as the indicator reaction and conditions of saturation with O2, malate, and pyruvate. The studies were carried out with tightly coupled bovine heart mitochondria incubated in the presence of hexokinase, glucose, and Mg2+. The data were corrected to conditions of hexokinase saturation with factors determined in hexokinase dependence studies. The concentrations of catalytic components were varied, in effect, by application of highly specific, tight-binding inactivators of the components. The principal objectives were (a) to distinguish individual reactions coupled by freely diffusible intermediate reactants, (b) to determine the relationships (coupling relationships) between these reactions in regard to how a change in the degrees to which one limits the rate of the overall reaction affects the degree to which the others limit the rate, and (c) to use the findings to determine how the individual reactions are coupled. The feasibility of achieving these objectives was suggested by the observations (a) that the initial steady-state velocity of the overall reaction varies in fairly close accord with a rectangular hyperbola (i.e., with Michaelis-Menten kinetics) whether it is a catalytic component or a substrate that is varied, (b) that apparent Michaelis constants of the substrates and catalytic components may be used as indicators of the coupling relationships between the individual reactions, and (c) that two types of coupling relationships exist between the individual reactions: “sequential” (characteristic of reactions linked in simple sequence) and “nonsequential” (mechanism uncertain), in which a change in the degree to which one individual reaction of a pair is rate limiting results in an inverse change and in no change, respectively, in the degree to which the other is rate limiting. Six individual reactions were distinguished: the energy-yielding rotenone-, antimycin-, and cyanide-sensitive steps of the respiratory chain and the energy-consuming Pi transport, phosphorylation, and AdN (adenine nucleotide) transport reactions. The results indicate (a) that the coupling relationship is sequential between the Pi transport and rotenone-sensitive reactions, the Pi transport and cyanide-sensitive reactions, the AdN transport and rotenone-sensitive reactions, the AdN transport and cyanide-sensitive reactions, and the AdN transport and phosphorylation reactions, and (b) that the coupling relationship is nonsequential between the AdN and Pi transport reactions, the Pi transport and phosphorylation reactions, the Pi transport and antimycin-sensitive reactions, and the AdN transport and antimycin-sensitive reactions. In the sequential group of individual reaction pairs, the individual reactions of all but the AdN transport-phosphorylation reaction pair appear to be linked in a partially nonsequential manner. It is proposed that the nonsequential and partially nonsequential coupling relationships come about as a result of one individual reaction of a pair removing freely diffusible intermediate reactants at two or more points which are situated symmetrically and unsymmetrically, respectively, about the other.

Passive induction of the “energized-twisted” conformational state in bovine heart mitochondria☆

Abstract It is demonstrated that hypotonically-swollen bovine heart mitochondria undergo transformation into the “energized-twisted” conformational state ( Green et al. Arch. Biochem. Biophys. 125:684, 1968 ) when simultaneously exposed to glutaraldehyde fixative and induced to contract by passive osmotic means. It is suggested that this passively-induced transformation and energy-dependent transformation of mitochondria into the “energized-twisted” state occur by similar mechanisms.

Normal lysosomal enzyme levels in hypertrophied and failing dog hearts

Abstract The activity levels of three lysosomal acid hydrolases, acid phosphatase, acid ribonuclease, and cathepsin, were determined in whole homogenates of right and left ventricular myocardium from normal and chronically stressed dog hearts. Stressed hearts were from four dogs with right ventricular hypertrophy and congestive failure induced by progressive constriction of the main pulmonary artery and from four dogs with acquired right ventricular hypertrophy secondary to pulmonary hypertension resulting from infestation with heartworms ( Dirofilaria immitis ). Control hearts were from four unoperated and four sham operated normal dogs. No significant differences were found that could be related to myocardial stress.

Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria. Evidence for linkage of the energy-yielding and energy-consuming steps by freely diffusible intermediates and for an allosteric mechanism of respiratory control at coupling site 2.

The three coupling segments of the respiratory chain of bovine heart mito-chondria were examined individually by steady-state kinetic methods to determine whether or not freely diffusible intermediates occur between the energy-yielding and energy-consuming steps involved in the oxidative phosphorylation of extramitochondrial ADP. The principal method employed was the dual inhibitor technique, for which an appropriate model is provided. The results indicate that in accordance with the chemiosmotic theory the intermediate reactants that link the energy-yielding rotenone-sensitive (Site 1), cytochromebc1 (Site 2), and cytochromeaa3 (Site 3) reactions of the respiratory chain to the energy-consuming ATP synthetase, AdN transport, and Pi transport reactions are freely diffusible (delocalized). Site 2 was found to differ from the others in regard to the mechanism by which the energy-linked respiratory chain reaction is controlled by the energy-consuming steps. Whereas the Site 1 and Site 3 respiratory chain reactions are controlled primarily by the thermodynamic mechanism of reaction reversal, the Site 2 respiratory reaction is controlled primarily by a kinetic mechanism in which an intermediate that links it to the energy-consuming steps inhibits it allosterically. From the effects of nigericin and valinomycin the allosteric intermediate appears to be the electrical component of the protonmotive force.

Swelling and contraction of heart mitochondria suspended in ammonium phosphate.

Bovine heart mitochondria which have been allowed to swell in isotonic NH4+ phosphate contract in response to initiation of oxidative phosphorylation. The contraction occurs optimally at pH 6.0 and appears from inhibition studies to result from Pi uptake being slower than removal of internal Pi via phosphorylation of external ADP. Similar results are obtained when K+ + nigericin is substituted for NH4+. Mersalyl inhibition of Pi transport in respiring, nonphosphorylating mitochondria which have been allowed to swell in NH4+ phosphate reveals a contractile process having an alkaline pH optimum. This contraction resembles closely the contraction observed in salts of strong acids and presumably occurs by electrophoretic ejection of Pi anions driven by electrogenic H+ ejection.

Prevention and reversal of morphological changes in heart mitochondria associated with isolation in sucrose

Abstract Morphological transformation of heart mitochondria from elongate to spherical forms during isolation in buffered sucrose can be prevented and reversed by including in the suspending medium a low concentration (0.5 m m ) of a high molecular weight solute which is incapable of penetrating the outer mitochondrial membrane. In view of this observation and of the fact that mitochondria in their normal, intracellular milieu are suspended in the presence of high molecular weight solutes which are incapable of penetrating the outer membrane, it is considered likely that the elongate-to-spherical transformation of mitochondria during isolation into media containing only sucrose and/or other solutes capable of penetrating the outer membrane is due to removal of extramitochondrial solutes of high molecular weight which normally prevent expansion of the outer mitochondrial compartment by exerting osmotic pressure on the outer membrane.

Inquiries into the Nature of Free Energy and Entropy in Respect to Biochemical Thermodynamics

Abstract: Free energy and entropy are examined in detail from the standpoint of classical thermodynamics. The approach is logically based on the fact that thermodynamic work is mediated by thermal energy through the tendency for nonthermal energy to convert sponta-neously into thermal energy and for thermal energy to distribute spontaneously and uni-formly within the accessible space. The fact that free energy is a Second-Law, expendable energy that makes it possible for thermodynamic work to be done at finite rates is empha-sized. Entropy, as originally defined, is pointed out to be the capacity factor for thermal en-ergy that is hidden with respect to temperature; it serves to evaluate the practical quality of thermal energy and to account for changes in the amounts of latent thermal energies in sys-tems maintained at constant temperature. With entropy thus operationally defined, it is pos-sible to see that T ∆ S° of the Gibbs standard free energy relation ∆ G° = ∆ H° − T ∆ S° serves to account for differences or changes in nonthermal energies that do not contribute to ∆