Analysis of human erythrocyte membrane vesicles produced by shearing

Shearing of ghosts in a French pressure cell produces three classes of microvesicles that differ from endocytic vacuoles, exocytic vacuoles, and inside-out vesicles. It was thought that an analysis of these vesicles might provide some clues about the assembly of proteins within the human erythrocyte membrane. The microvesicles were separated into three visible bands, labeled top, middle, and bottom, and assayed for activity of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase, acetylcholinesterase, glyceraldehyde-phosphate dehydrogense, and NADH oxidoreductase. Their proteins were also characterized by polyacrylamide gel electrophoresis with both Coomassie blue staining, to assess total protein content and distribution, and PAS-staining, to characterize sialoglycopeptides. In order to minimize problems inherent in ghost preparation, Dodge or hypotonic ghosts and glycol or isotonic ghosts were used in all studies. Middle membrane vesicles most resembled intact ghosts. Top vesicles had reduced levels of NADH oxidoreductase and more PAS-2 at the expense of PAS-1. The bottom vesicle class was very much enriched with PAS-1 at the expense of PAS-2, and PAS-3 was completely absent. In addition bottom vesicles had highest NADH oxidoreductase activity but lowest activity of all the other enzymes measured. These vesicle classes could not have been produced by tangential shearing through the membrane, nor could radial shearing through a membrane in which all proteins were free to move laterally have accounted for the three discrete vesicle classes or for their different patterns of enzymes and proteins. The analysis of the microvesicles produced by shearing is most consistent with radial shearing through membranes where there may be fixed domains superimposed on the basic fluid-mosaic structure.     

Comparison of androgen biosynthesis in isolated leydig cells from rat and mouse testis: incubation and superfusion studies

Production of testosterone by highly purified Leydig cells prepared from rat and mouse testes is compared. Testosterone formation is improved to a higher degree in rat (2.7-fold) than in mouse (1.7-fold) cells by collagenase treatment of the testis compared with mechanical isolation. Mouse Leydig cells respond to exogenous stimuli (choriogonadotropin, dibutyryl cyclic AMP) with 2.4-fold higher testosterone secretion than rat cells. A 1.7-fold increased conversion of androgen precursors to testosterone by mouse compared with rat Leydig cells is demonstrated in static incubations as well as in steady-state superfusion experiments and can be derived from enhanced androstenedione reduction and a less inhibitory effect of progesterone on this process in mouse Leydig cells.     

The heme domain of cellobiose oxidoreductase: a one-electron reducing system

Phanerochaete chrysosporium cellobiose oxidoreductase (CBOR) comprises two redox domains, one containing flavin adenine dinucleotide (FAD) and the other protoheme. It reduces both two-electron acceptors, including molecular oxygen, and one-electron acceptors, including transition metal complexes and cytochrome c. If the latter reacts with the flavin, the reduced heme b acts merely as a redox buffer, but if with the b heme, enzyme action involves a true electron transfer chain. Intact CBOR fully reduced with cellobiose, CBOR partially reduced by ascorbate, and isolated ascorbate-reduced heme domain, all transfer electrons at similar rates to cytochrome c. Reduction of cationic one-electron acceptors via the heme group supports an electron transfer chain model. Analogous reactions with natural one-electron acceptors can promote Fenton chemistry, which may explain evolutionary retention of the heme domain and the enzyme's unique character among secreted sugar dehydrogenases.     

Semiquinones derived from anthraquinone-containing antitumor drugs can partition into phosphatidylcholine bilayers

Semiquinones derived from anthraquinone-containing antitumor drugs (doxorubicin, daunorobucin and 4′-epidoxorubicin) were generated by the hypoxanthine/xanthine oxidase system in argon-saturated phosphate buffer (pH 7.4) in the presence of egg-yolk phosphatidylcholine multilamellar vesicles (MLVs) containing 1 mon% of a doxylstearic acid (DSA)_isomer. The destruction of the electron spin resonance signal corresponding to 5-, 12- and 15-DSA included in the MLVs follows pseudo-first-order kinetics. Higher rates of destruction are obtained for the 12-DSA isomer which indicates that these semiquinones can localize preferentially about the depth of the 12^th position of stearic acid in membranes. It is demonstrated that DSA destruction is due to a reversible reduction of DSA to the hydroxylamine species. This work shows that anthracycline semiquinones can partition into phosphatidylcholine bilayers under anoxic conditions which may imply another pathway on their cytotoxic action.     

Artificial Electron Carriers for Preparative Biocatalytic Redox Reactions Forming Reversibly Carbon Hydrogen Bonds with Enzymes Present in Strict or Facultative Anaerobes

The application of various artificial electron mediators in combination with redox enzymes present in anaerobically grown cells is described. Examples include viologens of different redox potential, cobalt complexes, anthraquinones, phenoxazines, phenazines and others. The regeneration of the reduced or oxidised mediators by various methods is discussed, with hydrogen gas, carbon monoxide, formate or a cathode as electron donors, and dimethylsulphoxide, quinones, oxygen or an anode as electron acceptors. The enzymes used, usually in the form of resting cells or crude cell extracts of clostridia or Proteus species are mostly reversible. Examples for preparative reductions as well as for dehydrogenations are given.     

Exploring the molecular nature of alternative oxidase regulation and catalysis

Plant mitochondria contain a non-protonmotive alternative oxidase (AOX) that couples the oxidation of ubiquinol to the complete reduction of oxygen to water. In this paper we review theoretical and experimental studies that have contributed to our current structural and mechanistic understanding of the oxidase and to the clarification of the molecular nature of post-translational regulatory phenomena. Furthermore, we suggest a catalytic cycle for AOX that involves at least one transient protein-derived radical. The model is based on the reviewed information and on recent insights into the mechanisms of cytochrome c oxidase and the hydroxylase component of methane monooxygenase.