Characterization of dicyclohexylcarbodiimide binding sites in beef-heart mitochondria.

Binding studies with [¹⁴C]-dicyclohexylcarbodiimide showed the presence of binding sites in the beef-heart mitochondrial membrane at a concentration of 1.8 nmol/mg protein (1.4 sites per cytochrome a+a₃). Saturation of these sites correlated with the inhibition of the ATPase activity. The maximum binding capacity could be related with the amount of F₁-ATPase in mitochondria from various tissues.     

Evidence for a function of core protein in complex III from beef-heart mitochondria.

Puried complex III ) ubiquinol-cytochrome c reductase) from beef heart mitochondria was alkylated with iodol [1-14C]acetamide. After 6-8 h of incubation with iodo[1-14C]acetamide, duroquinol and ubiquinol-2-cytochrome c reductase activites were inhibited approximately 50%. During this time 4.5 +/- 1.6 nmol of iodo[1-14C]acetamide reacted per mg of complex III protein. Experiments carried out over 24 h indicated that enzyme activity could be inhibited to 70% and the alkylation of complex III was proportional to inhibition. The rates of cytochrome b and c1 reduction by duroquinol are also decreased upon treatment of complex III with iodoacetamide. Separation of the peptides of complex III by electrophoresis in sodium dodecylsulfate shows that all of the radioactivity is located in a single peptide of 50 000 molecular weight, which has been identified as one of the two core proteins. The possible functions of core protein are discussed.     

The hedgehog uterus. A comparative transmission electron-microscopic study

The ultrastructure of the luminal epithelium of the hedgehog uterus is described on the basis of material taken from 11 animals in three different hormonal situations: castrated, active and hibernating animals. The whole uterine epithelium is composed of microvillous cells. Its appearance is very similar in ovariectomized and hibernating animals. It differs from that observed in active animals where the epithelium is taller, microvilli are more numerous and longer, and where nuclei and cytoplasm display a very active ultrastructural appearance. The results now available indicate that ultrastructural changes occurring within the cells are certainly correlated with plasma sex steroid hormone concentrations. The present paper also reports the regular occurrence of nuclear bodies in uterine cells.     

Innervation of the rabbit cornea. A histochemical and electron-microscopic study

The innervation of the rabbit cornea was investigated histochemically and electron-microscopically with special reference to the autonomic nerves. Both formaldehyde- and glyoxylic-acid-induced fluorescence methods revealed adrenergic nerves in the stroma; a few fibres were also observed between the basal epithelial cells near the limbus. Acetylcholinesterase- (AChE-) positive nerves were found both in the stroma and in the epithelium, whereas nonspecific cholinesterase (NsChE) activity appeared only in the stromal nerves. Under the electron microscope, both AChE and NsChE activities were observed to be located in the axon membranes. A weak NsChE reaction also appeared in the Schwann cells. When the specimens fixed with KMnO4 were examined under the electron microscope, most nerve fibres did not contain any special axoplasmic structures, although several axons contained mitochondria. Moreover, two vesicle-containing axon types were found in the stromal nerves; axons with small granular vesicles and axons containing small agranular vesicles. In the epithelium, two types of fibres were observed; one type containing only mitochondria while the other showed both agranular vesicles and mitochondria.     

An electron microscopic study of structural changes during the large amplitude swelling and contraction of isolated beef-heart mitochondria

Summary The structural changes of isolated beef-heart mitochondria undergoing hypotonic swelling and ATP-induced contraction are described. During swelling the mitochondria take up water, the matrix space enlarges, the inner membrane becomes greatly extended with material which is apparently derived from the cristae (the rest of which fragment) and the outer membrane is ruptured. On addition of ATP, Mn⁺⁺ and Ca⁺⁺ water is extruded as the extended inner membrane contracts around the remnants of the cristae to give compact bundles of vesicles which bear little resemblance to the original organized structure.On leave of absence from Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge, England.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

Reducibility of cytochromes b in aerobic beef-heart mitochondria treated with antimycin

Under anaerobic conditions cytochrome b in beef-heart mitochondria is partially reduced in the presence of NADH, whereas other cytochromes are completely reduced. Addition of antimycin together with oxygen under these conditions causes an immediate reduction of cytochromes b-558, b-562 and b-566 and oxidation of cytochrome c. During the subsequent transient aerobic steady state cytochromes b-558 and b-566 are rapidly re-oxidized without changes in redox state of cytochrome c, but cytochrome b-562 remains reduced. When oxygen is consumed by the leak through or around the antimycin-inhibition site, cytochrome b-562 becomes oxidized with concomitant reduction of cytochrome c.

The cytochromes b in lyophilized beef-heart mitochondria are more readily accessible to electrons from NADH, and in the presence of antimycin and NADH a complete and stable reduction is obtained under both aerobic and anaerobic conditions. Gradual addition of rotenone under these conditions causes re-oxidation of cytochromes b in which oxidation of cytochromes b-558 and b-566 precedes that of cytochrome b-562.

It is concluded that (1) the effect of antimycin in the presence of oxygen involves all three cytochromes b, (2) the reducibility of the cytochromes b in the aerobic steady state of antimycin-treated mitochondria is dependent upon the potential of the substrate redox couple registered on the cytochromes, and (3) the midpoint potential of cytochrome b-562 in the presence of antimycin is higher than that of cytochrome b-558 or b-566.     

Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria.

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O₂^? at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H₂O₂ at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H₂O₂ was essentially produced by disproportionation of O₂^?, which constitutes the precursor of H₂O₂. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O₂^? and 0.63 mol of H₂O₂/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O₂ to O₂^?, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H₂O₂ generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H₂O₂ generation. The efficiency of added quinones as peroxide generators decreased in the order Q₁ > Q₀ > Q₂ > Q₆ = Q₁₀, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymatically by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O₂^? and H₂O₂ during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q₀H₂), benzoquinol, duroquinol and menadiol generated O₂^? with k₃ values of 0.1 to 1.4 m^? · s^?1 and H₂O₂ with k₄ values of 0.009 to 4.3 m^?1 · s^?1.     

Postnatal development of the rabbit pineal gland. A light- and electron-microscopic study.

The development of the rabbit pineal gland has been studied by light and electron microscopy from the 1st to the 120th postnatal day. After 24 h of postnatal life, the pineal parenchyma is highly cellular, showing two identifiable cell types: pinealocytes I and II. Immature type II pinealocytes arrange either in cellular cords or clusters or forme rosette-like structures. At the 5th postnatal day, corticomedullar differentiation is established. Rosette-like structures and cellular cords are absent from the cortex. Along the postnatal period, nuclei of pinealocytes are set apart due to cytoplasmic widening and development of cell processes. These structures pervade the cellular cords and rosette-like structures formed by immature type II pinealocytes. Rosette-like structures are no longer seen beyond the 30th postnatal day, and cords of type II pinealocytes from the 90th postnatal day on. At this time, the rabbit pineal gland is considered to be histologically mature.     

Differential localization of annexins in ram germ cells: a biochemical and immunocytochemical study.

We used antibodies that specifically bind annexins on Western blots to determine the distribution and abundance of these proteins in ram spermatids and sperm by immunogold electron microscopy. Annexins I and II were found essentially within the entire acrosome of spermatids. During epididymal maturation, they concentrated in the postacrosomal region or the acrosomal equatorial segment, respectively. They were also present in sperm flagellum, on the surface of the coarse fibers and fibrous sheath. These findings show that during ram germ cell maturation, annexins I and II are exported from the spermatid acrosome towards structurally and functionally defined parts of the sperm. Annexins III, IV, and V were not found in ram germ cells. Annexin VI was isolated from testis and sperm. In spermatids, it was found to be associated with endoplasmic reticulum and the mitochondria but was absent from the acrosome. In sperm, it was confined to the flagellum, the mitochondria, and on the coarse fibers and fibrous sheath. The presence of three annexins, in addition to calmodulin, in functional areas may indicate differential ways for sperm to control and regulate events that are known to be calcium dependent, such as flagellar motility, acrosome reaction, and fertilization.