RESUMEN
We have proposed that phosphorylated and dephosphorylated forms of the mitochondrial sulfurtransferase, rhodanese, function as converter enzymes that interact with membrane-bound iron-sulfur centers of the electron transport chain to modulate the rate of mitochondrial respiration (Ogata, K., Dai, X., and Volini, M. (1989) J. Biol. Chem. 204, 2718-2725). In the present studies, we have explored some structural aspects of the mitochondrial rhodanese system. By sequential extraction of lysed mitochondria with phosphate buffer and phosphate buffer containing 20 mM cholate, we have shown that 30% of the rhodanese activity of bovine liver is membrane-bound. Resolution of cholate extracts on Sephadex G-100 indicates that part of the bound rhodanese is complexed with other mitochondrial proteins. Tests with the complex show that it forms iron-sulfur centers when incubated with the rhodanese sulfur-donor substrate thiosulfate, iron ions, and a reducing agent. Experiments on the rhodanese activity of rat liver mitochondria give similar results. Taken together, the findings indicate that liver rhodanese is in part bound to the mitochondrial membrane as a component of a multiprotein complex that forms iron-sulfur centers. The findings are consistent with the role we propose for rhodanese in the modulation of mitochondrial respiratory activity.
Asunto(s)
Membranas Intracelulares/enzimología , Mitocondrias Hepáticas/enzimología , Proteínas/metabolismo , Sulfurtransferasas/metabolismo , Tiosulfato Azufretransferasa/metabolismo , Animales , Bovinos , Ácido Cólico , Ácidos Cólicos , Cromatografía en Gel , Citosol/enzimología , Concentración de Iones de Hidrógeno , Proteínas Hierro-Azufre/metabolismo , Cinética , Peso Molecular , Fosfatos , Unión ProteicaRESUMEN
The mitochondrial sulfurtransferase, rhodanese, has been analyzed for phosphate content. Significant amounts of protein-bound phosphate (30-40%) were measured in the six rhodanese preparations examined. Chromatographic experiments followed by phosphate analyses done on two of the preparations indicated that rhodanese A and rhodanese B, two enzyme forms that were previously resolved on DEAE-Sephadex by Blumenthal and Heinrikson (Blumenthal, K., and Heinrikson, R. L. (1971) J. Biol. Chem. 240, 2430-2437), correspond to dephospho- and phosphorhodanese, respectively. The phosphorylation of rhodanese by [gamma-32P]ATP is catalyzed by cAMP-dependent protein kinase. The stoichiometry of 32P incorporation based on the amount of dephosphorhodanese in the enzyme preparation approaches 1.0. The phosphorylation site is accessible in rhodanese that is free of substrate sulfur but not in the covalent enzyme-sulfur intermediate which is formed as an obligatory step during the course of catalysis. Because the cellular localization of cAMP-dependent protein kinase makes it unlikely as the physiologic modulator of rhodanese activity, liver extracts have been tested for a rhodanese kinase that does not require cAMP. Rhodanese kinase activity which is independent of cAMP is observed in extract fractions resolved by Affi-Gel Blue chromatography and freed from endogenous rhodanese by chromatography on Sephadex G-100. These results together with previous findings from this and other laboratories have led to a working model of a bicyclic cascade system that can modulate the rate of mitochondrial respiration. The essence of the model is a transduction and amplification of cellular signals into the altered covalent phosphorylation of rhodanese. Rhodanese, in turn, serves as a converter enzyme which directly alters the rate of the respiratory chain and, thus, ATP production by the reversible sulfuration of key iron-sulfur centers. The model, when expanded to include signal pathways initiated by hormones or neurotransmitters, represents a mechanism by which mitochondria can recognize and meet changing energy demands.
Asunto(s)
Isoenzimas/metabolismo , Mitocondrias Hepáticas/enzimología , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Sulfurtransferasas/metabolismo , Tiosulfato Azufretransferasa/metabolismo , Animales , Bovinos , Glucógeno Sintasa/metabolismo , Isoenzimas/aislamiento & purificación , Cinética , Modelos Teóricos , Fosfoproteínas/aislamiento & purificación , Fosforilación , Tiosulfato Azufretransferasa/aislamiento & purificaciónRESUMEN
A rhodanese enzyme of less than 20,000 molecular weight has been purified from Escherichia coli. The enzyme is accessible to substrates upon addition of whole cells to standard assay mixtures. This rhodanese has a Stokes radius of 17 A which for a globular protein corresponds to a molecular weight close to 14,000. It undergoes autoxidation to a polymeric form which is probably an inert dimer. Enzyme inactivated by oxidation can be reactivated by millimolar concentrations of cysteine. Steady-state initial velocity measurements indicate that the enzyme catalyzes the transfer of sulfane sulfur by way of a double displacement mechanism with formation of a covalent enzyme-sulfur intermediate. The turnover number for the enzyme-catalyzed reaction, with thiosulfate as donor substrate and cyanide ion as the sulfur acceptor, is 260 s-1. This value corresponds to a catalytic efficiency 60% of that measured for a previously characterized bovine liver enzyme of more than twice the molecular weight. Furthermore, KmCN is 24 mM which is 2 orders of magnitude higher than the value observed previously for the bovine enzyme. Evidence from chemical inactivation studies implicates an essential sulfhydryl group in the enzyme activity. It is proposed that this group is the site of substrate-sulfur binding in the obligatory enzyme-sulfur intermediate. Furthermore, a cationic site important for binding of the donor thiosulfate is tentatively identified from anion inhibition studies. Tests of alternate acceptor substrates indicate that the physiological dithiol, dihydrolipoate, is a more efficient acceptor than cyanide ion for the enzyme-bound sulfur. Of possibly greater physiological significance, it has been found that the enzyme catalyzes the formation of iron-sulfur centers. Other work indicates the E. coli rhodanese is subject to catabolite repression and suggests a physiological role for the enzyme in aerobic energy metabolism.
Asunto(s)
Escherichia coli/enzimología , Sulfurtransferasas/metabolismo , Tiosulfato Azufretransferasa/metabolismo , Aniones , Cisteína/farmacología , Cinética , Peso Molecular , Oxidación-Reducción , Especificidad de la Especie , Tiosulfato Azufretransferasa/aislamiento & purificaciónAsunto(s)
Cianuros/metabolismo , Animales , Artrópodos/metabolismo , Bacterias/metabolismo , Compartimento Celular , Cianuros/biosíntesis , Eucariontes/metabolismo , Hongos/metabolismo , Glicósidos/metabolismo , Leucocitos/fisiología , Fagocitosis , Plantas/metabolismo , Ratas , Selenio/toxicidad , Tiosulfato Azufretransferasa/metabolismoRESUMEN
Sedimentation equilibrium studies show that there are two forms of bovine liver rhodanese in crystalline enzyme preparations of full specific activity. One form dissociates to a species with a limiting molecular weight close to 19,000. The second form is nondissociable under the same experimental conditions. It exhibits a molecular weight of approximately 33,000. These conclusions are augmented by data from polyacrylamide gel electrophoresis and molecular exclusion chromatography. They serve to explain apparent inconsistencies in previous reports on this enzyme.