Non-local thermodynamic effects and efficiency of oxidative phosphorylation

A non-equilibrium thermodynamic model of oxidative phosphorylation is formulated, which allows us to take into account some non-local effects. In this way, we compute the influence of the tangential resistivity of the inner mitochondrial membrane to proton current, as well as that of the distance between active sites, on the stoichiometry and efficiency of energy conversion.     

Amphetamine effects on acetylcholine release and mammalian neuromuscular transmission

The mechanisms by which (+)-amphetamine biphasically modifies neuromuscular transmission were studied in the rat phrenic nervediaphragm preparation. Low to moderate amphetamine concentrations (30–300 μM) enhanced twitch height and potentiated the nerve stimulated release of acetylcholine (ACh) by up to 4.8-fold from the phrenic nerve. Higher amphetamine concentrations depressed muscle twitch and ACh release. Using a cannulated diaphragm preparation, amphetamine enhanced the twitch response to nerve stimulation but markedly depressed the contractions elicited by a pulsed injection of ACh. Amphetamine-induced enhancement of ACh release was prevented by pretreatment of animals with α-methyl-p-tyrosine, suggesting that amphetamine may be acting indirectly by releasing catecholamines. These results support the hypothesis that amphetamine enhancement results from a presynaptic increase in ACh release and the blocking actions are mediated by a postsynaptic inhibitory effect.     

The effects of ionophores and metabolic inhibitors on methanogenesis and energy-related properties of Methanobacterium bryantii

The effects of numerous ionophores and inhibitors were tested on methane synthesis, intracellular ATP and potassium concentrations, and the proton motive force of the methanogenic archaebacterium Methanobacterium bryantii. M. bryantii had an internal pH near 6.8 (and hence little ΔpH during growth) with an electrical potential of ?127 mV in growth medium and ?105 mV in a pH 6.5 buffer. The study has identified agents which, in M. bryantii, can effectively cause a decline of intracellular ATP (gramicidin, acetylene) and potassium concentrations (gramicidin, nigericin), inhibit methane synthesis (acetylene, gramicidin, nigericin, triphenylmethylphosphonium bromide), eliminate the electrical potential (high extracellular potassium ion concentrations), and dissipate artificially imposed, inside alkaline, pH gradients (monensin, nigericin, carbonyl cyanide m-chlorophenylhydrazone). Carbonyl cyanide m-chlorophenylhydrazone was generally ineffective in media or buffers reduced with cysteine-sulfide but could be effective in cysteine-free solutions reduced with hydrogen sulfide.     

Rabbit muscle glycogen-bound phosphoprotein phosphatases: substrate specificities and effects of inhibitor-1

A phosphoprotein phosphatase which has an apparent molecular weight of 240,000 was partially purified (500-fold) from the glycogen-protein complex of rabbit skeletal muscle. The enzyme exhibited broad substrate specificity as it dephosphorylated phosphorylase, phosphohistones, glycogen synthase, phosphorylase kinase, regulatory subunit of cAMP-dependent protein kinase, and phosphatase inhibitor 1. The phosphatase showed high specificity towards dephosphorylation of the beta-subunit of phosphorylase kinase and site 2 of glycogen synthase. With the latter substrate, the presence of phosphate in sites 1a and 1b decreased the apparent Vmax, perhaps by inhibiting the dephosphorylation of site 2. The phosphorylated form of inhibitor 1 did not significantly inhibit this high-molecular-weight phosphatase. However, an inhibitor 1-sensitive phosphatase activity could be derived from this preparation by limited trypsinization. Furthermore, greater than 70% of the phosphatase activity in skeletal muscle extracts and in the glycogen-protein complex was insensitive to inhibitor 1. Limited trypsinization of each fraction obtained from the phosphatase purification increased the total activity (1.5- to 2-fold) and converted the enzyme into a form which was inhibited by inhibitor 1. The results suggest that inhibitor 1-sensitive phosphatase may be a proteolyzed enzyme.     

Collagen-induced platelet aggregation and release. I effects of side-chain modifications and role of arginyl residues

To investigate the mechanisms governing collagen interaction with blood platelets, the effects of side-chain modifications on collagen-induced platelet aggregation and release of serotonin were studied. Since many chemical modifications alter the ability of collagen to form fibers that, according to current theory, may complicate interpretation of data, we eliminated this possibility by using collagen stabilized in a native-type fibrillar structure by treatment with either glutaraldehyde or ultraviolet irradiation. Acetylation, methylation, succinylation, treatment with 2,4-dinitrofluorobenzene, 2,4,6-trinitrobenzene sulfonic acid or 1,2-cyclohexanedione, and deguanidination with hypobromite were used to modify collagen side-chain reactive groups: amino, carboxyl, hydroxyl and guanidino. Both unmodified monomeric dispersed and fibrillar collagen preparations initiated platelet aggregation and release, although the kinetics and magnitude of the response were different. Monomeric collagen which had been modified by deguanidination, methylation or succinylation, failed to polymerize in physiological conditions and did not induce platelet aggregation and release. However, none of the chemical modifications of stabilized native-type collagen fibers, except treatment with hypobromite or cyclohexanedione, had an effect on collagen-induced platelet aggregation and release. Both hypobromite and cyclohexanedione modified guanidino groups of arginyl residues. Results showed that the ability of a collagen sample to induce platelet aggregation and release of serotonin is dependent on the arginine content of fibrillar collagen.These data demonstrate that manipulation of amino, carboxyl and hydroxyl groups is unimportant as long as the native-type fibrillar structure is maintained, and that arginyl residues are directly involved in collagen-platelet interaction. Moreover, the data suggest that only the arginyl residues in the Y position of the tripeptide unit Gly-X-Y of collagen are responsible.     

Shape and elasticity effects on erythrocyte electrostatic repulsion

It is shown that the double-layer interaction between two symmetrically parallel red blood cells depends largely on their shape and elasticity. Various shapes are examined, including the realistic shape of a biconcave discocyte with an elastic membrane. It is found that changes in the shape and the elasticity of the erythrocyte may result in orders of magnitude differences in the forces and energies of pair interaction.     

The effects of ketone bodies,bicarbonate, and calcium on hepatic mitochondrial ketogenesis

The effect of various factors on hepatic mitochondrial ketogenesis was investigated in the rat. A comparison of three different incubation media revealed that bicarbonate ion inhibited the rate of ketone body production and decreased the ratio of 3-hydroxybutyrate/acetoacetate. The addition of 0.8 mm calcium caused significant inhibition of ketogenesis from both octanoate (40–50%) and palmitate (25–30%) and no change in the ratio of 3-hydroxybutyrate/acetoacetate. In the presence of components of the malate/aspartate shuttle, the inhibition by calcium was 80% or more with both substrates. Experimental alteration of the respiratory state of the mitochondria from state 3 to state 4 was associated with an enhanced rate of ketogenesis. The addition of ketone bodies themselves had marked effects on the rate of ketone body production. Increasing amounts of exogenously added acetoacetate were accompanied by increasing rates of total ketone body production reflecting enhanced 3-hydroxybutyrate synthesis. In the presence of added 3-hydroxybutyrate, there was striking inhibition of ketogenesis. Rotenone, which prevents oxidation of NADH₂ via the electron transport chain, almost completely inhibited ketone body synthesis. This inhibition was partially overcome by the addition of acetoacetate which regenerates NAD⁺ from NADH₂ during conversion to 3-hydroxybutyrate. These observations provide evidence for additional sites of metabolic control over hepatic ketogenesis.     

The hypothermic effect of 4-(5,6-dimethyl-2-benzofuranyl) piperidine HCl (CGP 6085 A) in Wistar Kyoto rats

CGP 6085 A [4-(5,6-dimethyl-2-benzofuranyl) piperidine HCl], a reported serotonin uptake and MAO (16) inhibitor, is a potent hypothermic agent. The hypothermic action of CGP 6085 A is dose dependent with a maximal reduction in rectal core temperature of greater than 1 degree C within one hour after drug administration. Fluoxetine and citalopram elicit a similar response at equal doses. These results suggest that inhibition of serotonin uptake may produce the hypothermic effect. To assess the in vivo action of CGP 6085 A in inhibiting hypothalamic serotonin uptake, CGP 6085 A (10 mg/kg) was injected one hour prior to injection of 3-hydroxy-4-methyl-alpha-ethyl-phenylethylamine (H75/12), a serotonin depletor. The ability of CGP 6085 A to block the uptake of H75/12 by the 5HT uptake system was indicative of its ability to block serotonin uptake. Pretreatment with p-chlorophenylalanine (pCPA), an inhibitor of serotonin synthesis, resulted in the loss of the hypothermic response to CGP 6085 A. Thus, these data are consistent with the idea that CGP 6085 A may produce its hypothermic response by inhibiting serotonin uptake.     

Oviduct dolichyl phosphate phosphatase: estrogen effects and a possible biosynthetic role

Estrogen-induced chick oviduct differentiation is accompanied by an increased capacity for protein glycosylation. A portion of this increase has been attributed to increased levels of dolichyl phosphate (Dol-P). Hormone withdrawal leads to an apparent decrease in Dol-P. Dol-P metabolism in the oviduct has been studied, and one of the enzymes having a direct effect on Dol-P, Dol-P phosphatase is herein described. Dol-P phosphatase has a pH optimum of 6.0, does not require a metal ion, and is inhibited by Mn2+ at concentrations greater than 5 mM. Inhibitor studies indicate that Dol-P hydrolysis is inhibited by polyprenyl phosphates having both saturated and unsaturated alpha-isoprene residues, but not by the corresponding alcohols. The enzyme is also inhibited by phosphatidic acid unless 2 mM Mn2+ is included in the incubations. Under these conditions Dol-P hydrolysis is only slightly inhibited (less than 10%), but phosphatidate inhibition is totally eliminated. Oviduct membranes also possess phosphatidate phosphatase, but this enzyme is distinct from Dol-P phosphatase based on thermolability, metal ion sensitivity, and sulfhydryl reagent sensitivity. Studies of enzyme activity in response to estrogen treatment reveal that both Dol-P phosphatase and phosphatidate phosphatase have maximal specific activity early in the differentiation process (peaking after 3 days of treatment), and low specific activity in fully differentiated oviducts, including laying hen oviduct. Hormone withdrawal elicits a small increase in specific activity of both phosphatases. The hormone effects suggest that Dol-P phosphatase may be a biosynthetic enzyme.     

The effects of insulin and tunicamycin on insulin receptors of cultured fibroblasts

The effect of down-regulation on the intracellular pool of insulin receptors and the role of glycosylation in recovery from down-regulation have been studied in fibroblastic cultures from the skin of non-diabetic mice. In control cultures, 55% of the total specific [¹²⁵I]insulin-binding activity was in the intracellular compartment. Insulin caused a time- and concentration-dependent decrease in the number of cell surface insulin receptors, with no significant change in total insulin receptors. This decrease in surface receptors was accompanied by an increase in the specific binding of [¹²⁵I]insulin in the intracellular compartment. Removal of insulin from down-regulated cells resulted in a time-dependent increase in the binding of [¹²⁵I]insulin to surface receptors, reaching 90% of that in controls by 12 h. The recovery of surface insulin receptors after removal of insulin was blocked by incubation of cultures with tunicamycin, but not by cycloheximide. These results indicate that down-regulation of surface insulin receptors by insulin is associated with translocation of receptors into the intracellular pool and suggest that protein glycosylation is important in insulin receptor recycling and externalization.     

The effects of cell proliferation on the lipid composition and fluidity of hepatocyte plasma membranes.

The fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, has been used to investigate the effects of controlled and uncontrolled growth on the dynamic properties of the lipid regions of hepatocyte plasma membranes. DPH was incubated with plasma membranes derived from quiescent and regenerating liver and Morris hepatoma 7777, and the resulting systems were studied by fluorescence polarization spectroscopy. Membranes from the rapidly growing hepatoma exhibited a significantly lower fluorescence polarization than observed in quiescent liver, suggesting the presence of a more fluid membrane lipid domain. Membranes from regenerating liver exhibited a time-dependent increase in membrane fluidity, reaching a maximum 12 h after growth stimulation. A close correspondence between membrane fluidity and the cholesterol-phospholipid ratio was also observed where a decrease in this ratio resulted in a more fluid lipid matrix. These results suggest that cell cycling, as observed in regenerating liver and Morris hepatoma 7777, results in significant increases in membrane fluidity, a property which may play an important regulatory role in various cell functions.     

Adenylate cyclase from guinea pig lung: further characterization and inhibitory effects of substrate analogs and cyclic nucleotides

A potent (K_i = 0.01 mM), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′O-palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N⁶,2′O-dibutyryl cyclic AMP at concentrations of up to 1 mm or more. The possibility that 2′O-palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 mm Mg²⁺ in the presence of 1.2 mm ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 mm isoproterenol is dependent on the Mg²⁺ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E₁, E₂, and F_2α, histamine, and glucagon.     

Differential effects of N-acetylglutamate on citrulline synthesis by coupled and uncoupled mitochondria

When rats were placed on a low-protein (5%) diet for 24 h or less, liver mitochondrial acetylglutamate decreased rapidly, carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase decreased little, and carbamyl phosphate synthesis (measured as citrulline) by isolated mitochondria occurred at very low rates. The matrix acetylglutamate content of these mitochondria, whether coupled or uncoupled, was increased similarly by preincubating them with added acetylglutamate, but citrulline synthesis increased from less than 1 to 2.3 nmol min-1 mg-1 in the coupled state, and from less than 1 to 35 nmol min-1 mg-1 in the uncoupled state. However, when coupled mitochondria were incubated with the substrates required for the synthesis of acetylglutamate in the matrix, citrulline synthesis increased to 48 nmol min-1 mg-1; this rate was similar to that of mitochondria from control rats (fed a normal diet). When mitochondria from controls were incubated with up to 5mM acetylglutamate, citrulline synthesis by coupled mitochondria was increased by 10 to 40%, while synthesis by uncoupled mitochondria was 1.5 to 4 times higher than that observed with the coupled mitochondria; matrix acetylglutamate in both conditions rose to levels similar to those in the medium. The reason for the different behavior of carbamyl phosphate synthetase (ammonia) in coupled and uncoupled mitochondria was not apparent; neither oxidative phosphorylation nor ornithine transport were limiting in the coupled system. These observations are an example of the restrictions imposed upon enzymatic systems by the conditions existing in the mitochondrial matrix, and of the different behavior of carbamyl phosphate synthetase in situ and in solution. In addition, they show that conclusions about the characteristics of the enzyme in coupled mitochondria based on observations made in uncoupled mitochondria are not necessarily justified.     

Biological effects of D2O administration to Coturnix japonica

Levels of 7.8, 18.5 and 26 mole % deuterium oxide were administered sequentially to $\text{Coturnix japonica}$ (Japanese quail) via the drinking water. The primary effect observed was on egg frequency, which decreased from a normal level of 0.89 for 7.8 mole % D₂O to a low of 0.38 during the administration of 26 mole % D₂O. Adverse symptoms, such as hyperexcitability, convulsions, skin ulcerations, comatosity, weight loss, or death, which have been associated with deuterium toxicity in other animals, were not observed in these experiments. The amount of deuterium deposited in the water of the egg was 6.9, 13.98, and 19.83 mole % when 7.8, 18.5 and 26 mole % deuterium respectively was administered. For each period, the deuterium content of egg water rapidly reached a maximum concentration after which the concentration decreased slightly. This dilution effect has not been noted previously in body fluids from other animals.     

The effects of ketamine, phencyclidine and lidocaine on catecholamine secretion from cultured bovine adrenal chromaffin cells

The ability of ketamine, phencyclidine and analogues to alter catecholamine secretion from cultured bovine adrenal chromaffin cells was investigated. Both ketamine and phencyclidine specifically inhibited nicotinic agonist-induced secretion at concentrations which did not alter secretion induced by elevated K+ depolarization. The inhibition of nicotinic agonist-induced secretion was not overcome by increasing concentrations of nicotinic agonist. The effects of stereoisomer pairs of phencyclidine-like drugs - dexoxadrol, levoxadrol and (+)PCMP, (-)PCMP - did not reveal stereospecificity for the inhibition, in contrast to the stereospecific behavioral effects of the drugs. The local anesthetic lidocaine (0.3 mM) also noncompetitively inhibited nicotinic agonist-induced secretion without inhibiting elevated K+-induced secretion. The data indicate that ketamine and phencyclidine at clinically relevant concentrations specifically inhibit the adrenal chromaffin cell nicotinic receptor at a site similar to or identical with the site of action of local anesthetic. Although the nicotinic receptor inhibition is probably not related to the anesthetic and behavioral effects of ketamine and phencyclidine, it is likely that the centrally mediated increase in sympathetic nervous system activity which is characteristic of these drugs is moderated by the peripheral blocking effects on catecholamine secretion from the adrenal medulla.     

Biochemical effects of PR toxin on rat liver mitochondrial respiration and oxidative phosphorylation

The in vitro effects of PR toxin, a toxic secondary metabolite produced by certain strains of Penicillium roqueforti, on the membrane structure and function of rat liver mitochondria were investigated. It was found that the respiratory control and oxidative phosphorylation of the isolated mitochondria decreased concomitantly when the toxin was added to the assay system. The respiratory control ratio decreased about 60% and the ADP/O ratio decreased about 40% upon addition of 3.1 X 10(-5) M PR toxin to the highly coupled mitochondria. These findings suggest that PR toxin impairs the structural integrity of mitochondrial membranes. On the other hand, the toxin inhibited mitochondrial respiratory functions. It exhibited noncompetitive inhibitions to succinate oxidase, succinate-cytochrome c reductase, and succinate dehydrogenase activities of the mitochondrial respiratory chain. The inhibitory constants of PR toxin to these three enzyme systems were estimated to be 5.1 X 10(-6), 2.4 X 10(-5), and 5.2 X 10(-5) M, respectively. Moreover, PR toxin was found to change the spectral features of succinate-reduced cytochrome b and cytochrome c1 in succinate-cytochrome c reductase and inhibited the electron transfer between the two cytochromes. These observations indicate that the electron transfer function of succinate-cytochrome c reductase was perturbed by the toxin. However, PR toxin did not show significant inhibition of either cytochrome oxidase or NADH dehydrogenase activity of the mitochondria. It is thus concluded that PR toxin exerts its effect on the mitochondrial respiration and oxidative phosphorylation through action on the membrane and the succinate-cytochrome c reductase complex of the mitochondria.     

Hormonal regulation of key gluconeogenic enzymes and glucose release in cultured hepatocytes: effects of dexamethasone and gastrointestinal hormones on glucagon action

Hormonal regulation of key gluconeogenic enzymes and glucose release by glucagon, dexamethasone, secretin and somatostatin was evaluated in maintenance cultured rat hepatocytes. (i) Phosphoenolpyruvate (PEP)-carboxykinase activity declined rapidly during the first 24 h in serum- and hormone-free culture with a further slight decay during the following 2 days. Dexamethasone and glucagon independently increased PEP-carboxykinase and acted synergistically when added in combination. Glucose-6-phosphatase activity declining linearly during hormone-free culture was stimulated by glucagon. Dexamethasone itself was without significant effects but completely abolished glucagon action. Fructose-1,6-diphosphatase was maintained at its initial level during the first day under control conditions and declined thereafter. Neither glucagon nor dexamethasone affected total activity or substrate (fructose-1,6-diphosphate) affinity of this enzyme. In short-term experiments on cells cultured under control conditions, protein synthesis-dependent stimulation of PEP-carboxykinase by glucagon and the permissive action of dexamethasone was demonstrated. Glucose-6-phosphatase and fructose-1,6-diphosphatase were not altered by hormones within this period. (ii) Stimulation by glucagon of gluconeogenesis was independent of its action on PEP-carboxykinase. Dexamethasone inhibited glycogenolysis but maintained glucose release at control levels probably by stimulation of gluconeogenesis. When added in combination, the glycogen-preserving action of dexamethasone acutely reduced the glucose release in response to glucagon. Glucagon sensitivity remained unchanged. (iii) The gastrointestinal hormones secretin and somatostatin were ineffective in modulating basal or glucagon-stimulated glucose release and gluconeogenic key enzymes. They are therefore unlikely to play a physiological role in hepatic glucose metabolism.     

pH-dependent leaving group effects on hydrolysis reactions of phosphate and phophonate esters catalyzed by wheat germ acid phosphatase.

The substrate specificity of carefully purified wheat germ acid phosphatase was examined and the Michaelis constants for substrates having widely varying leaving groups were determined at pH values 4.6, 8.0, and 9.2. The pH-dependent leaving group effects were consistent with the formation of a covalent phosphoryl histidine intermediate in the reaction process catalyzed by this enzyme. In addition, the enzyme was found to hydrolyze nitrophenyl esters of methyl-, chloromethyl-, and phenylphosphonic acids at rates comparable to those observed for phosphomonoester hydrolysis. The data are most simply interpreted on the basis of a nucleophilic displacement by an active-site histidine residue to form an intermediate N′-phosphonyl histidine species, followed by decomposition of this intermediate by nucleophilic attack by water, analogous to the decomposition process of the N′-phosphoryl enzyme species.