The cytoplasmic surface of bacteriorhodopsin is characterized by a group of carboxylates that function as a proton attractive domain [Checover, S., Nachliel, E., Dencher, N. A., and Gutman, M. (1997) Biochemistry 36, 13919−13928]. To identify these carboxylates, we selectively mutated them into cysteine residues and monitored the effects of the dynamics of proton transfer between the bulk and the surface of the protein. The measurements were carried out without attachment of a pH-sensor to the cysteine residue, thus avoiding any structural perturbation and change in the surface charge caused by the attachment of a reporter group, and the protein was in its BR state. The purple membranes were suspended in an unbuffered solution of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) and exposed to a train of 1000 laser pulses (2.1 mJ/pulse, λ = 355 nm, at 10 Hz). The excitation of the dye ejected the hydroxyl's proton, and a few nanoseconds later, a pair of free protons and ground-state pyranine anion was formed. The experimental observation was the dynamics of the relaxation of the system to the prepulse state. The observed signals were reconstructed by a numeric method that replicates the chemical reactions proceeding in the perturbed space. The detailed reconstruction of the measured signal assigned the various proton-binding sites with rate constants for proton binding and proton exchange and the pK values. Comparison of the results obtained by the various mutants indicates that the dominant proton-binding cluster of the wild-type protein consists of D104, E161, and E234. The replacement of D104 or E161 with cysteine lowered the proton binding capacity of the cluster to ∼60% of that of the native protein. The replacement of E234 with cysteine disrupted the structure of the cluster, causing the two remaining carboxylates to function as isolated residues that do not interact with each other. The possibility of proton transfer between monomers is discussed.
1. In the presence of KCN and a saturating concentration of antimycin the reduction of the b-type cytochromes in submitochondrial particles is biphasic. This phenomenon was explained by suggesting the existence of two kinetic forms of cytochrome b:bA-the active form which was reduced in the rapid phase, and bS-the sluggish form which was reduced in the slow phase. The ratio between these forms and the transformation from one to other was controlled by the redox state of an unknown component, names "y", located between cytochromes b and c1. Pre-treatment with ascorbate plus N,N,N1,N1-tetramethyl-p-phenylenediamine transforms all the b-type cytochromes to their sluggish form, and the reduction by succinate follows slow monophasic kinetics. The name "dynamic control mechanism" was given to this mechanism [Eisenbach, M. & Gutman, M. (1975) Eur. J. Biochem. 52, 107-116] 2. Increasing concentrations of antimycin (0-2 nmol/mg) in the presence of KCN increased the fraction of the rapid phase of the reduction but did not affect the calculated absolute rates of the reduction. It is concluded that antimycin delays the reduction of "y" and thus permits the observation of the biphasic phenomen, but that it is not essential for the operation of this dynamic control mechanism.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe pH jump: kinetic analysis and determination of the diffusion-controlled rate constantsMenachem Gutman, Esther Nachliel, Eli Gershon, Rina Giniger, and Ehud PinesCite this: J. Am. Chem. Soc. 1983, 105, 8, 2210–2216Publication Date (Print):April 1, 1983Publication History Published online1 May 2002Published inissue 1 April 1983https://pubs.acs.org/doi/10.1021/ja00346a018https://doi.org/10.1021/ja00346a018research-articleACS PublicationsRequest reuse permissionsArticle Views329Altmetric-Citations24LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Abstract An improved estimate from Scatchard plots of the number of specific binding sites of the electron transport particle for 14C-piericidin A gives a value of 2 moles of inhibitor bound per mole of calculated NADH dehydrogenase content. The affinity of these two specific binding sites for piericidin A is very similar, since the binding curve is a smooth hyperbola and the Scatchard plot is linear. The two sites may be distinguished, however, by their unequal contribution to the inhibition of NADH oxidation. This is shown by the sigmoidicity of the curves relating inhibition of NADH oxidase, NADH-coenzyme Q reductase, and energy-linked NAD reductase activities to piericidin concentration. The inhibition of NADH oxidase and energy-linked NAD reductase activities but not of NADH-coenzyme Q reductase activity are partially reversed by washing of inhibited particles with bovine serum albumin. Hence both specifically and unspecifically bound piericidin contribute to the inhibition of the first two activities while only specifically bound inhibitor is involved in the block of external coenzyme Q reduction. The titration curve for inhibition of NADH oxidase by rotenone is more complex, apparently because of the contribution of inhibition at unspecific sites. The number of specific binding sites for piericidin may be reduced to near 1 per mole of NADH dehydrogenase by treatment of the electron transport particle with mercurials and by dissociation into the component complexes with bile salts. The results are discussed in terms of the location of the binding and inhibition sites of rotenone and piericidin in the respiratory chain and of the possible participation of NADH dehydrogenase itself and other membrane components in the binding of these inhibitors.
Ca 2+ regulates multiple processes in nerve terminals, including synaptic vesicle recruitment, priming, and fusion. Munc13s, the mammalian homologs of Caenorhabditis elegans Unc13, are essential vesicle-priming proteins and contain multiple regulatory domains that bind second messengers such as diacylglycerol and Ca 2+ /calmodulin (Ca 2+ /CaM). Binding of Ca 2+ /CaM is necessary for the regulatory effect that allows Munc13-1 and ubMunc13-2 to promote short-term synaptic plasticity. However, the relative contributions of Ca 2+ and Ca 2+ /CaM to vesicle priming and recruitment by Munc13 are not known. Here, we investigated the effect of Ca 2+ /CaM binding on ubMunc13-2 activity in chromaffin cells via membrane-capacitance measurements and a detailed simulation of the exocytotic machinery. Stimulating secretion under various basal Ca 2+ concentrations from cells overexpressing either ubMunc13-2 or a ubMunc13-2 mutant deficient in CaM binding enabled a distinction between the effects of Ca 2+ and Ca 2+ /CaM. We show that vesicle priming by ubMunc13-2 is Ca 2+ dependent but independent of CaM binding to ubMunc13-2. However, Ca 2+ /CaM binding to ubMunc13-2 specifically promotes vesicle recruitment during ongoing stimulation. Based on the experimental data and our simulation, we propose that ubMunc13-2 is activated by two Ca 2+ -dependent processes: a slow activation mode operating at low Ca 2+ concentrations, in which ubMunc13-2 acts as a priming switch, and a fast mode at high Ca 2+ concentrations, in which ubMunc13-2 is activated in a Ca 2+ /CaM-dependent manner and accelerates vesicle recruitment and maturation during stimulation. These different Ca 2+ activation steps determine the kinetic properties of exocytosis and vesicle recruitment and can thus alter plasticity and efficacy of transmitter release.
