A sensitive double antibody RIA has been developed for the 28,000 mol wt rat renal vitamin D-dependent calcium-binding protein. Using this assay, concentrations of calcium-binding protein (CaBP) as low as 30 ng can be measured. The assay is precise (intraassay variability, 5.0%) and reproductible (interassay variability, 8.2%). Measurements of renal CaBP by RIA showed a good correlation with measurements of CaBP by the chelex resin assay and by polyacrylamide gel analysis by densitometric tracing using a purified CaBP marker. The concentration of CaBP in the vitamin D-replete rat kidney is 7.3 +/- 1.0 (mean +/- SEM) micrograms/mg protein. In vitamin D-deficient rats the level of renal CaBP is 2.6 +/- 0.3 micrograms/mg protein. Tissue distribution of immunoreactive rat renal CaBP showed the highest concentration of CaBP in the rat cerebellum (38.3 +/- 5.1 micrograms/mg protein). Lower concentrations of immunoreactive CaBP were detected in several other rat tissues. No immunoreactive CaBP was detected in rat or human serum. In necropsy human kidney and cerebellum, high levels of immunoreactive CaBP were also detected (1.5 +/- 0.1 and 27.3 +/- 2.1 micrograms/mg protein, respectively). When extracts of rat kidney and brain and human cerebellum and kidney were assayed at several dilutions, immunodisplacement curves parallel to that of pure renal CaBP were observed, indicating immunochemical similarity. Fractionation of extracts of rat cerebellum, human kidney, and human cerebellum on Sephadex G-100 revealed immunoreactivity and calcium-binding activity in the 28,000 mol wt region similar to rat kidney.
Proton conduction in submitochondrial particles with various degrees of resolution of the H '--ATPase has been studied.Proton conduction was analyzed by following the kinetics of the anaerobic decay of protons taken up by submitochondrial particles during respiratory pulses.1. In EDTA submitochondrial particles proton release exhibited biphasic kinetics; both phases were depressed by collapsing aerobic A$.2. The slow phase of proton diffusion was inhibited by oligomycin and N,N'-dicyclohexylcarbodiimide as well as by FI ligands like adenylyl5'-imidophosphate and alkyl cations. Inhibition of proton conduction by F1 ligands exhibited a sigmoidal titration curve and was synergistic with inhibition by oligomycin. Removal of the ATPase inhibitor markedly enhanced the kinetic constant of the slow proton diffusion process.3. The initial rapid phase of proton diffusion was inhibited by N,N'-dicyclohexylcarbodiimide and oligomycin but was unaffected by FI ligands.4. When the FI moiety was removed from the particles, proton conduction became monophasic and was not any more inhibited by F1 ligands.5. It is concluded that the H'-ATPase contributes to both phases of proton conduction and that energy-linked cooperative interaction of the F1 with the FO moiety controls proton conductivity.Evidence is also provided for involvement of negatively charged groups in hydrophobic environments of the FI moiety in proton translocation by the H+-ATPase.The HA-ATPase of mitochondria consists of two moieties (see for review [l -61). The ATPase proper, or the F1 moiety [l -3,5], and a proteolipid complex, the FO moiety, which is thought to function as an oligomycin-sensitive and a N,N'-dicyclohexylcarbodiimidesensitive proton conductor [4,7,8].In the intact H+-ATPase complex, normally arranged in the membrane, downhill proton diffusion through the FO moiety has to be strictly coupled to ATP synthesis, otherwise uncoupling of oxidative phosphorylation occurs.In loosely coupled submitochondrial particles [9], where displacement of the F1 moiety might have occurred [lo], or in particles from which the F1 moiety and a protein component of the H+-ATPase known as oligomycin-sensitivity-conferral protein [l 11, have been removed [12], a proton leakage occurs, which
8-Bromoadenine nucleotides were tested as potential substrates and/or inhibitors of mitochondrial processes in intact or disrupted organelles, as substrates of various phosphotransferases, and as allosteric effectors in the reactions catalyzed by phosphofructokinase, isocitrate dehydrogenase, glutamate dehydrogenase, and fructose-1,6-bisphosphatase. 8-BrATP and 8-BrADP are not recognized by the translocase system located in the inner mitochondrial membrane and cannot be used as usbstrates in oxidative phosphorylation and related reactions catalyzed be beef heart submitochondrial membranes. This confirms the high specificity for adenine nucleotides of the mammalian systems involved in energy-yielding and energy-requiring reactions. However, 8-BrATP and 8-BrADP are able to substitute for the natural adenine nucleotides in reactions catalyzed by many phosphotransferases, although their capacity as phosphate donors and acceptors is generally much reduced. On the other hand, in almost all investigated cases, the 8-bromoadenine nucleotides have lost the capability of the natural adenine nucleotides to act as allosteric effectors, indicating that the structural requirements for allosteric activity are more stringent than those for catalytic activity.
A study is presented of the effect of acidic uncouplers and oligomycin on energy-linked and passive proton translocation, oxidative phosphorylation, and energy-linked nicotinamide-adenine-nucleotide transhydrogenase in EDTA submitochondrial particles from beef-heart. A flow potentiometric technique has been applied to resolve the kinetics of the initial rapid phase of the redox proton pump. Rapid kinetics analysis shows that carbonyl-cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) does not exert any direct effect on redox-linked active proton transport. The uncoupling action of FCCP on oxidative phosphorylation and energy-linked transhydrogenase is shown to be quantitatively accounted for by its promoting effect of passive proton-diffusion across the mitochondrial membrane. Oligomycin depresses passive proton diffusion in EDTA sonic particles and this effect accounts for the coupling action exerted by the antibiotic on oxidative phosphorylation and energy-linked transhydrogenase. In fact, rapid kinetic analysis demonstrates that oligomycin does not directly affect the redox-linked proton pump. The present results show that there does not exist any labile intermediate in the redox-linked proton pump which is sensitive to acidic uncouplers.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.