The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.     

Glomerular filtration rate and blood pressure monitoring in awake baboons

Minimally invasive techniques were used to collect urine with an external catheter together with automated intermittent monitoring of arterial blood pressure in awake male baboons. Using endogenous creatinine, 24-hour creatinine clearances were measured for 2 to 3 consecutive days in four intact and in four uninephrectomized baboons. Despite large differences in urinary volume and sodium excretion, reproducibility of 24-hour creatinine clearances was within 15% in 15 of 19 studies obtained from 6 of 8 animals. Arterial blood pressure was monitored intermittently at 30 to 60 minute intervals over 24 hours with a Dinamap monitor and recorder. Mean blood pressure averaged 71 +/- 4.4 to 89 +/- 5.5 mm Hg in different animals. Blood pressure tended to be lower at night than during the day. In separate studies using 15 to 60 minute urine collection periods, inulin clearance was compared in awake and in anesthetized animals with endogenous or exogenous creatinine clearance measured simultaneously. The clearance of creatinine systematically exceeded the clearance of inulin, even in intact animals with a normal serum creatinine. The creatinine-to-inulin clearance ratio averaged 1.16 +/- 0.03 at a serum concentration of 0.7 to 0.8 mg/dl; 1.27 +/- 0.03 at a serum creatinine of 1.0 to 1.1 mg/dl and 1.56 +/- 0.04 at a serum creatinine greater than 10 mg/dl. All values exceed unity significantly (p less than 0.001). Thus, renal function, including inulin clearance, can be measured in awake baboons. Duplicate or triplicate 24-hour urine collections are needed to assess the reliability of creatinine excretion. However, creatinine clearance overestimates glomerular filtration rate, as it does in humans.     

Cadmium uptake and toxicity via voltage-sensitive calcium channels

The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.     

Studies on the electron transfer pathway, topography of iron-sulfur centers, and site of coupling in NADH-Q oxidoreductase

Electron transfer activities and steady state reduction levels of Fe-S centers of NADH-Q oxidoreductase were measured in mitochondria, submitochondrial particles (ETPH), and complex I after treatment with various reagents. p-Chloromercuribenzenesulfonate destroyed the signal from center N-4 (gx = 1.88) in ETPH but not in mitochondria, showing that N-4 is accessible only from the matrix side of the inner membrane. N-Bromosuccinimide also destroyed the signal from N-4 but without inhibiting rotenone-sensitive electron transfer to quinone, suggesting a branched pathway for electron transfer. Diethylpyrocarbonate caused oxidation of N-3 and N-4 in the steady state without changing N-1, suggesting N-1 is before N-3 and N-4. Difluorodinitrobenzene and dicyclohexylcarbodiimide inhibited oxidation of all Fe-S centers and tetranitromethane inhibited reduction of all Fe-S centers. Titrations of the rate of superoxide (O2-) generation in rotenone-treated submitochondrial particles were similar with the ratio [NADH]/[NAD] and that of 3-acetyl pyridine adenine nucleotide in spite of different midpoint potentials of the two couples. On reaction with inhibitors the inhibition of O2- formation was similar to that of ferricyanide reductase rather than quinone reductase. The rate of O2- formation during ATP-driven reverse electron transfer was 16% of the rate observed with NADH. The presence of NAD increased the rate to 83%. The results suggest that bound, reduced nucleotide, probably E-NAD., is the main source of O2- in NADH dehydrogenase. The effect of ATP on the reduction levels of Fe-S centers in well-coupled ETPH was measured by equilibrating with either NADH/NAD or succinate/fumarate redox couples. With NADH/NAD none of the Fe-S centers showed ATP induced changes, but with succinate/fumarate all centers showed ATP-driven reduction with or without NAD present. The effect on N-2 was smaller than that on N-1, N-3, and N-4. These observations indicate that the major coupling interaction is between N-2 and the low potential centers, N-1, N-3, and N-4. Possible schemes of coupling in this segment are discussed.     

Non-ohmic proton conductance of mitochondria and liposomes

Direct measurements of the proton/hydroxyl ion flux across rat liver mitochondria and liposome membranes are reported. H+/OH- fluxes driven by membrane potential (delta psi) showed nonlinear dependence on delta psi both in mitochondria and in liposomes whereas delta pH-driven H+/OH- flux shows linear dependence on delta pH in liposomes. In the presence of low concentrations of a protonophore the H+/OH- flux was linearly dependent on delta psi and showed complex dependence on delta pH. The nonlinearity of H+/OH- permeability without protonophore is described by an integrated Nernst- Plank equation with trapezoidal energy barrier. Permeability coefficients depended on the driving force but were in the range 10(-3) cm/s for mitochondria and 10(-4)-10(-6) cm/s for liposomes. The nonlinear dependence of H+/OH- flux on delta psi explains the nonlinear dependence of electrochemical proton gradient on the rate of electron transport in energy coupling systems.     

Pituitary thyrotropin-releasing hormone receptors: local anesthetic effects on binding and responses

Pharmacological agents are widely used to probe the mechanism of action of TRH. A number of these drugs behave as local anesthetics at high concentrations. The effect of local anesthetics on the binding of [3H]Me-TRH to specific receptors was studied using the GH4C1 line of rat pituitary tumor cells. [3H]Me-TRH binding was inhibited by classical local anesthetics with the order of potency (IC50 values): dibucaine (0.37 mM) greater than tetracaine (1.2 mM) greater than lidocaine (3.3 mM) greater than procaine and benzocaine (greater than 10 mM). IC50 values for other drugs with local anesthetic properties that inhibited [3H]Me-TRH were: 100 microM trifluoperazine, 100 microM imipramine, 170 microM chlorpromazine, 300 microM verapamil, and 700 microM propranolol. Inhibition by tetracaine and verapamil increased as the pH was raised from 6 to 8.5, indicating that the free base form of the amine drugs was the inhibitory species, and the local anesthetic effect was greater at 37 C than at 24 C or 0 C. [3H]Me-TRH binding to receptors in isolated membranes was inhibited to the same extent as binding to receptors on intact cells. Local anesthetics were 3- to 20-fold less potent at inhibiting [3H]Me-TRH to digitonin-solubilized receptors than binding to intact cells. In contrast, the potency of chlordiazepoxide, a putative TRH antagonist, to inhibit [3H]Me-TRH binding was equal using cells and solubilized receptors (IC50 = 10 microM). Local anesthetics inhibited TRH-stimulated PRL release and also inhibited basal PRL secretion and secretion stimulated by two nonhormonal secretagogues, (Bu)2cAMP and a phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

Egg production, larval development, and adult longevity of cat fleas (Siphonaptera: Pulicidae) exposed to ultrasound

Adult cat fleas, Ctenocephalides felis felis (Bouché), on cats (Felis catus) were exposed to emissions from an ultrasonic flea collar worn by the cat. No significant differences were found in total numbers of eggs produced per day (mean = 524 control, 614 treatment), in length of larval development time (mean = 7.7 d control, 7.7 d treatment), or in total daily pupal production (mean = 485 control, 445 treatment) between the treatment and the control groups. Tests off the host were conducted to determine whether ultrasonic exposure caused mortality in adult fleas; no significant differences were found in daily mortality between the treated and control fleas during 1 wk of exposure.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities.

The yeast Saccharomyces cerevisiae catalytic DNA polymerase I 180-kDa subunit and the tightly associated 86-kDa polypeptide have been purified using immunoaffinity chromatography, permitting further characterization of the DNA polymerase activity of the DNA primase-DNA polymerase protein complex. The subunits were purified to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit. When the individual subunits were recombined in vitro a p86p180 physical complex formed spontaneously, as judged by immunoprecipitation of 180-kDa polypeptide and DNA polymerase activity with the anti-86-kDa monoclonal antibody. The 86-kDa subunit stabilized the DNA polymerase activity of the 180-kDa catalytic subunit at 30 degrees C, the physiological temperature. The apparent DNA polymerase processivity of 50-60 nucleotides on poly(dA).oligo(dT)12 or poly(dT).oligo(A)8-12 template-primer was not affected by the presence of the 86-kDa subunit but was reduced by increased Mg2+ concentration. The Km of the catalytic 180-kDa subunit for dATP or DNA primer terminus was unaffected by the presence of the 86-kDa subunit. The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis that had been observed previously in the DNA primase-DNA polymerase protein complex. The 180-kDa subunit possessed a 3'----5'-exonuclease activity that catalyzed degradation of polynucleotides, but degradation of oligonucleotide substrates of chain lengths up to 50 was not detected. This exonuclease activity was unaffected by the presence of the 86-kDa subunit. Despite the striking physical similarity of the DNA primase-DNA polymerase protein complex in all eukaryotes examined, the data presented here indicate differences in the enzymatic properties detected in preparations of the DNA polymerase subunits isolated from S. cerevisiae as compared with the properties of preparations from Drosophila cells. In particular, the 3'----5'-exonuclease activity associated with the yeast catalytic DNA polymerase subunit was not masked by the 86-kDa subunit.