Two membrane-bound b-type cytochromes in Nitrosomonas europaea

Abstract Membranes from Nitrosomonas europaea were found to contain two b -type cytochromes. One had an α-band centred at 562 nm and E _m,7=+ 155 mV; the other had an α-band maximum close to 558 nm and E _m,7=+ 40 mV. A b -type cytochrome ran at an apparent M _r of 32000 on lithium dodecyl sulphate/polyacrylamide gels at 4°C.     

Purification of creatine kinase from beef heart mitochondria]

53-fold purified creatine kinase is isolated from beef heart mitochondria by phosphate buffer extraction followed by chromatography on DEAE-cellulose and KM-cellulose and preparative electrophoresis in phosphate buffer density gradient. The purified enzyme was homogenous under electrophoresis in agarose gel and moved to cathode. The enzyme did not enter into separating gel under disc electrophoresis in conditions for the separation of neutral anc acid proteins, while under conditions for separating alkaline proteins it produced five fractions. The stability of creatine kinase under storage considerably decreased after the purification.     

Four different b-type cytochromes in the halophilic archaebacterium, Halobacterium halobium

The halophilic archaebacterium, Halobacterium halobium has been found to contain four different b-type cytochromes. The four components were recognized by their potentiometric characteristics in situ in their functional environment in the membrane of H. halobium. Oxidation-reduction midpoint potentials of these four b-type cytochromes were determined to be +261, +160, +30, and -153 mV, respectively. We also demonstrate that the pathway involved in the transport of reducing equivalents from succinate to oxygen proceeds through the b-type cytochromes with oxidation-reduction midpoint potentials of +261 and +161 mV. The cytochrome with oxidation-reduction midpoint potential of -153 mV was not substrate reducible by NADH but was chemically reducible by dithionite. Antimycin inhibits reduction of b-type cytochrome in the succinate pathway, but has no effect on b-type cytochrome reduction when reducing equivalents are provided by NADH. The carbon monoxide difference spectrum of H. halobium membranes shows at least one carbon monoxide-binding b-type cytochrome, indicating a terminal oxidase. A scheme for electron transport in H.halobium involving the b-type cytochromes and terminal oxidase is suggested.     

Microwave Hall mobility measurements on heavy beef heart mitochondria

The observed initial microwave Hall mobility values at 1·21 tesla of heavy beef heart mitochondria is at least six times greater than that observed for bovine serum albumin at similar resistivity values. The respiratory inhibitor cyanide significantly reduces the initial Hall mobility values for HBHM and for a preparation of HBHM cytochrome oxidase.The four enzymic complexes of the respiratory chain were partially or completely separated. Of these complexes cytochrome oxidase exhibits the largest microwave Hall mobility.The maximum hydration content of loosely bound water for freezedried preparations of cytochrome oxidase is 5% by weight; 60% of this hydration content is driven off by microwave power. Since the effective ac resistivity of the samples of cytochrome oxidase did not appreciably vary with changes in hydration content, the true resistivity of cytochrome oxidase has a value of the order 5×10³ ohm cm and possibly much lower.The electron transport pathway (as measured by Hall signal) of cytochrome oxidase is irreversibly damaged by prolonged exposure to microwave irradiation at 9·2 GHz. This is accompanied by the complete loss of capacity to oxidise ferrocytochromec. Such changes do not occur with HBHM or with the other respiratory complexes.There appears to be a direct relationship between observed Hall signals and the capacity of cytochrome oxidase to oxidize ferrocytochromec. There is a background signal which is not directly related to electron transport but which is dependent on the conformation of the cytochrome oxidase.The observed electronic parameters of cytochrome oxidase do not depend appreciably on its redox state.Acid denaturation of cytochrome oxidase drastically reduces the Hall signal, to include almost complete removal of the background signal. It also more than doubles ac resistivity.An electron tunnelling model is outlined.     

Quantities of ubiquinone bound to proteins in beef heart mitochondria

Ubiquinone-binding proteins were isolated and purified from heavy beef heart mitochondria. 35% of the total ubiquinone in the mitochondria was associated with the purified proteins. About 83% of the associated ubiquinone could be released from the proteins by proteolytic treatment showing that at least 29% (0.87 nmol/mg) of the total ubiquinone (3.0 nmol ubiquinone/mg) in the mitochondria is in the bound form. The purified ubiquinone-binding proteins were resolved into 5 polypeptides with the molecular weights of 17.4, 12.9, 12.6, 9.8 and 8.6 kD on sodium dodecyl sulfate-gel electrophoresis.