Ezzatollah Keyhani

Energy conservation capacity and morphological integrity of mitochondria in hypotonically treated rabbit epididymal spermatozoa

Abstract Hypotonic treatment of rabbit epididymal spermatozoa in 10 mM phosphate buffer disrupts the plasma membrane and removes the cytoplasmic droplet from those cells to which it is still attached. There is, however, no effect on the mitochondria, which retain their helical configuration around the axial filament complex, have intact inner and outer membranes, and show the same cristal morphology as do the mitochondria in untreated cells. Hypotonically treated spermatozoa respire with added malate-pyruvate, succinate, and ascorbate plus N , N ′-tetramethyl- p -phenylenediamine, but not with added fructose or NADH. Respiration is inhibited by oligomycin and stimulated by uncoupler, showing that the mitochondria have retained their capacity for energy conservation. The uncoupled respiration rate is not further stimulated by added cytochrome c . Reduced- minus -oxidized difference spectra obtained at −196 °C of the hypotonically treated cells show a full complement of cytochromes, including cytochrome c . This result implies that the cytochrome c lost from mammalian spermatozoa on storage or chilling [Mann, T. (1951) Biochem. J. 48, 386–388] is cytoplasmic cytochrome c not yet incorporated into the mitochondria. The mitochondrial cytochrome c remains firmly held inside the intact outer membrane.

Epr study of the effect of formate on cytochrome c oxidase

Abstract The addition of formate to oxidized cytochrome c oxidase (ferrocytochrome c : oxygen oxidoreductase, EC 1.9.3.1) causes the appearance of a high spin heme signal at g = 6 and a splitting of g = 3 signal to g = 2.98 and 3.07. When formate-cytochrome c oxidase is reduced, the g = 2.98 signal decreases significantly. The spectrophotometric studies showed that formate is a specific ligand to cytochrome a 3. Data suggest that binding of formate to oxidized cytochrome c oxidase produces a ligand- a 3 interaction leading to the splitting of g = 3 signal hitherto considered as due to cytochrome a . Thus both cytochrome a and a 3 contribute to the resonance of g = 3 signal of cytochrome c oxidase.

Cytochrome biosynthesis under copper-limited conditions in Candida utilis

Yeasts grown strictly under anaerobic conditions in the presence of a fermentable substrate lose their ability to respire and become devoid of the typical cytochrome system [ 1,2] . The mitochondria of these cells differ in ultrastructure from normal mitochondria obtained in aerobically grown yeast and are termed pro-mitochondria [3,4], During aeration, the cells rapidly recover the cytochrome system and acquire typical mitochondria exhibiting normal respiratory activity. These changes in cell structure and function and the synthesis of cytochrome oxidase are initiated by oxygen [ 1,2] . However, the cell in anaerobic conditions exhibits a change in all the cytochromes rather than a specific alteration of cytochrome oxidase. In contrast, when yeast is grown in a copper deficient medium, the synthesis of cytochrome oxidase is considered to be specifically inhibited with no change in the other cytochromes PI. The present work describes the induction of cytochrome oxidase by copper and the behavior of the other cytochromes during the recovery phase of cytochrome oxidase.

Fractionation by sucrose density gradient centrifugation of membrane fragments derived by sonic disruption of beef heart mitochondria

Abstract 1. 1. Fractionation of beef heart submitochondrial membrane fragments derived by sonic disruption was achieved by linear sucrose density gradient centrifugation. Three distinct membrane fractions were obtained: a pellet (P), a middle (X) and an upper (Y) zone. 2. 2. Electron micrographs revealed that the pellet contained large single pieces and aggregates of membrane fragments. The X zone contained rough membrane fragments with protruding subunits (F1) and the Y zone contained relatively small fragments without subunits. 3. 3. In comparison with the Y zone, the X zone exhibited higher activities of NADH oxidase and ATPase, higher oligomycin-induced respiratory control and cytochrome content, but lower activities of NADH:cytochrome c reductase (rote-none-sensitive) and cytochrome oxidase with externally-added cytochrome c as the substrate. Lubrol greatly enhanced the cytochrome oxidase activity in the X zone but not in the Y zone. NADH oxidase was greatly stimulated by externally added cytochrome c in the Y zone but not in the X zone. In general, the pellet exhibited varying activities falling between those of the X zone and the Y zone. 4. 4. The relationship between these membrane fractions and the conventional EDTA particle preparation is discussed.

Interaction of calcium ion with the mitochondria of rabbit spermatozoa.

Mitochondria isolated from many mammalian tissues accumulate calcium ions in a rapid reaction which is energy linked competes successfully for the high-energy intermediates that drive oxidative phosphorylation [ 1,2] , and is inhibited by lanthanide ions and Ruthenium red [3,4], Lehninger [5] has proposed that the biological function of this reaction is to produce a high local concentration of calcium and phosphate ions which then react to form amorphous tricalcium phosphate in ‘micropackets’. These are then excreted by the mitochondrion and eventually by the cell, from whence they are transported to a target collagen structure and deposited. A plausible mechanism for the formation of calcified tissue is provided by this proposal. The liver, heart, kidney, brain, and spleen of the rabbit all yield mitochondria that show the calcium accumulation reaction [6] , which raises the question: to what extent is this reaction genetically programmed into the mitochondria of all differentiated tissues of the mammal? The recent demonstration that the mitochondria of rabbit epididymal spermatozoa become accessible to reagents in the suspending medium when the spermatozoa are treated with hypotonic buffer, but themselves remain

Membrane structure and function: I. An electron microscope study of the alteration induce by laser irradiation on the chloroplast lamellar membranes

Abstract Negative-staining and thin-section techniques were used to study the ultrastructural change produced in isolated chloroplasts by high energies of the ruby laser (6943 A). Various degress of alterations were observed as a function of laser energy input. Two to four pulses of the ruby laser (2.34 J/pulse) mainly caused a decrease in granularity and an increase in vacuolization of the membrane. After 8–16 pulses, fragmentation and complete destruction of the chloroplast membrane were observed. This damage to the chloroplast membrane is a result of the large absorption of the laser light by chlorophyll; however, the chlorophyll itself is not changed. Correlation of ultrastructural damage with the biochemical alterations suggest that the main effect of the laser is to block energy coupling, whereas the photosystems remain intact. We conclude that ultrastructural damage to the chloroplast membranes by laser treatment is much more drastic than biochemical alterations.

Membrane structure and function: II. Alterations in the photo-induced absorption changes after treatment of isolated chloroplasts with large pulses of the ruby laser☆

Abstract Treatment of isolated chloroplasts with high-energy pulses of the ruby laser causes graded structural changes in the chloroplast membranes and is here correlated with the biochemical changes produced. The laser treatment caused decreases in the photoinducible absorption changes of cytochromes b559, b563, and P520 (the carotenoid shift), but smaller decreases in cytochrome f. The decreases correlated with the quantum efficiency alterations produced by the laser treatment. Ferricyanide photoreduction and O2 evolution was only slightly affected by the laser treatment. The slow phase of the dark recovery kinetics of P520 was increased maximally by the lowest laser input energies and NADP+ photoreduction induced by carbonylcyanide-P-trifluoromethoxyphenylhydrazone (FCCP) was decreased maximally by the lowest energies, suggesting that uncoupling of the chloroplasts was the most sensitive parameter. This was corroborated by our previous observation (5) that chloroplast membrane bound surface particles (coupling factor) was the ultrastructural change most sensitive to the laser pulses. Electron flow from photosystem II to photosystem I was not altered by the laser treatment. The laser treatments did not cause a detectable decrease in total chlorophyll in the chloroplasts, however, approximately 10% of the total chlorophyll was present in the solution phase after the treatment, whereas no detectable cytochromes were present in the solution phase.

Increased resistance to detergent in Enterococcus faecalis

Enteroccocus faecalis, a gram-positive bacterium ubiquitous in the environment and normal inhabitant of the human gastrointestinal tract, has emerged as a major health problem exacerbated by its ability at surviving adverse conditions, including prolonged stay on environmental surfaces. Here its tolerance to detergents that are routine household chemicals was investigated and compared to that of E. coli. A clinical isolate of E. faecalis with plasmid-mediated antibiotics resistance grown in the presence of increasing concentrations of sodium Nlauroylsarcosinate (sarkosyl) and sodium dodecyl sulphate (SDS) exhibited a sarkosyl minimum inhibitory concentration (MIC) of 0.1% (10% for E. coli) and a SDS MIC of 0.05% (6% for E. coli). A mutant with accrued sarkosyl resistance (MIC = 0.6%) was isolated after growth in the presence of 0.06% sarkosyl. While there was no cross-resistance to SDS, other mutant characteristics gave it advantages over the original strain, emphasizing the potential emergence of E. faecalis with increasing tolerance to common household detergents.

Oxidation-reduction potential of cytochrome C oxidase in mitochondria of yeast grown under various copper concentrations.

Abstract Growth of copper deficient yeast cells in copper supplemented medium led to an increase in the amount of cytochrome c oxidase and cytochromes b and c . Potentiometric titration of cytochrome c oxidase showed that the oxidation-reduction midpoint potentials of cytochromes a and a 3 were, respectively, for cells grown at 56μg copper/liter: 205 ± 10, 325 ± 10; for cells grown at 140μg copper/liter, 215 ± 10, 360 ± 10; and for cells grown at 330μg copper/liter, 215 ± 10, and 345 ± 10. Data showed that the midpoint potential of cytochromes a and a 3 in yeast mitochondria was close to the value reported for rat liver and beef heart mitochondria. In addition the stimulation of cytochrome c oxidase biosynthesis in yeast by copper does not alter significantly the redox properties of the enzyme.

Energy Metabolism of Spermatozoa: III. Energy-Linked Uptake of Calcium Ion by the Mitochondria of Rabbit Epididymal Spermatozoa*†*Presented at the 30th Annual Meeting of The American Fertility Society, Hollywood, Florida, April 1974.†Supported by Public Health Service grants HD-06274 and GM-12202 and National Science Foundation grant GB-23063.

n A study of energy-linked calcium (Ca++) uptake by rabbit sperm mitochondria is reported. The respiration rate of hypotonically treated rabbit epididymal spermatozoa with succinate in the presence of oligomycin and rotenone was increased by 50% upon addition of Ca++ and an additional 30% by the introduction of KPi. Compared with mitochondria from other tissues, the uptake with sperm mitochondria is quite inefficient, evidenced by a Ca++ ratio of .3 compared with values of 1.7 to 2.0 with liver mitochondria. This is probably due to an ineffeciency in the Ca++ carrier, during maturation of the spermatozoa, to utilize high energy intermediates generated by substrate oxidation. The uptake of Ca++ was not inhibited by La+++ nor by concentrations of ruthenium red which prevent Ca++ uptake in other mitochondria. The Ca++ uptake reaction is known to be genetically determined in all mammalian mitochondria such that modifications in the Ca++ carrier provide a specific biochemical correlate for the study of alterations in rabbit spermatozoa mitochondria.n