Audrey A. Painter

Phosphorylation coupled to oxidation of thiol groups (GSH) by cytochrome c with disulfide (GSSG) as an essential catalyst II. Demonstration of ATP formation from ADP and HPO42

Abstract The oxidation of GSH by cytochrome c in buffered solution in the presence of EDTA requires GSSG as a catalyst. It appears that an intermediate complex between GSSG and GSH is more active in reducing cytochrome c . During this electron transfer reaction ADP can be generated from AMP and inorganic phosphate. AMP and inorganic pyrophosphate yield ATP. This reaction, which uses common biological materials and occurs in aqueous medium, is a most interesting model for oxidative phosphorylation. It may involve the basic mechanism for coupling of phosphorylation to electron transport in mitochondria.

Phosphorylation coupled to oxidation of thiol groups (GSH) by cytochrome c with disulfide (GSSG) as an essential catalyst IV. Stability of intermediates and possible mechanism of reaction

Abstract The primary intermediates responsible for energy conservation during GSSG catalyzed electron transfer from GSH to cytochrome c under anaerobic conditions are labile but have a life time of a few minutes. The presence of phosphate results in relatively stable intermediates, presumably by forming a more stable charge transfer complex or phosphorylated form. The formation of 1 ∼ P per cytochrome c reduced requires postulation of a one electron transfer mechanism or a 2 electron transfer mechanism with an unrecognized acceptor for the second electron.

Phosphorylation coupled to oxidation of thiol groups (GSH) by cytochrome c with disulfide (GSSG) as an essential catalyst III. Uncoupling and uncoupler-dependent ATPase

Abstract Phosphorylation coupled to electron transfer from GSH to cytochrome c is uncoupled by 10 μM 2,4-dinitrophenol, 10 μM dicoumarol, 3 μ,M pentachlorophenol, and 1 μM carbonyl-cyanide-m chlorophenylhydrazone under anaerobic conditions, and by oxygen. When the uncouplers are added 2 minutes before the enzymatic trapping system, all of the ATP disappears. Added ATP is hydrolyzed within 2 minutes in uncoupler-dependent ATPase reversal of the phosphorylation reaction. These characteristics of the system emphasize the fact that oxidative phosphorylation in mitochondria may depend on the same fundamental properties of thiol and disulfide.

Phosphorylation Coupled to the Transfer of Electrons from Glutathione to Cytochrome c

Formation of adenosine diphosphate from adenosine monophosphate and inorganic phosphate can be coupled to the oxidation of reduced glutathione by cytochrome c in a reaction which requires oxidized glutathione as a catalyst. The reaction occurs with purified materials in tris(hydroxymethyl)aminomethane buffer and may represent the type reaction for one or more oxidative phosphorylations.

Antigenic differences between corresponding DNA-dependent RNA polymerases from both phases of Histoplasma capsulatum

Abstract Reaction of antisera generated against DNA-dependent RNA polymerase III from the yeast phase of the dimorphic fungusHistoplasma capsulatum with the purified RNA polymerases of the organism indicated that there were major antigenic differences in the corresponding enzymes from the yeast and mycelial phases. These differences were most marked when the enzymes were reacted against antisera obtained early in the immunization and less after hyperimmunization. This extends our earlier demonstration of extreme differences in the corresponding RNA polymerases from two differentiated states of the same organism.

Quantification of creative kinase isoenzymes by a radioimmunoassay

SummaryIt is not known whether loss of enzyme activity from the circulation is due to denaturation, inactivation or removal of intact enzyme molecules. This is in part due to the lack of an assay to measure enzyme protein concentration since available assays measure only enzyme activity. Radioimmunoassays for plasma enzymes and isoenzymes have not been possible because of oxidation in radioactive labelling by conventional methods and the problem of subunit dissociation. In the present study, antibodies specific to the B and M subunits of creatine kinase isoenzymes were obtained by immunization of rabbits with canine BB and MM creatine kinase. Anitgens (MM and BB) were radioactively labelled with 125I by acylation, avoiding the problem of oxidation and subunit stabilized by mercaptoethanol (0.020 m) and Trisbuffer (1.6 m). A radioimmunoassay capable of detecting picogram amounts of CK isoenzymes was developed which measures the concentration of enzyme protein rather than activity. The method was shown to provide a sensitive quantitative method for analysis of plasma CK isoenzymes in dogs after myocardial infarction produced by coronary occlusion. This technique may provide a prototype for the development of radioimmunoassays for other plasma isoenzymes and should help to elucidate the nature of the disappearance of isoenzymes from the circulation.