Sea urchin sperm head plasma membranes: characteristics and egg jelly induced Ca2+ and Na+ uptake

Sea urchin sperm respond to egg factors with changes in the ionic permeability of their plasma membrane. It has been previously shown that plasma membranes isolated preferentially from sea urchin sperm flagella respond to egg jelly increasing their Ca2+ and Na+ uptake (Darszon et al. (1984) Eur. J. Biochem. 144, 515-522). However, the egg jelly induced acrosome reaction occurs in the sperm head, and there is evidence for an heterogeneous distribution of plasma membrane components within the various regions of this cell. We here report a method for purifying sperm head membranes using positively charged beads according to Jacobson (1977) Biochim. Biophys. Acta 471, 331-335). Under the transmission electron microscope these membranes appeared homogeneous and apparently free of internal membranes. The yield of the preparation was 0.9% of the total protein in the sperm homogenate. The preparation contained less than 5% of the mitochondrial marker cytochrome oxidase, and 10% of the total DNA/mg protein. Surface labeling with 125I indicated a 2.5-3-fold enrichment in specific activity of the head membranes with respect to whole sperm. The SDS band pattern and the lipid composition of this preparation were different from those of isolated flagellar membranes. Phosphatidylcholine was higher in the head membranes, while phosphatidylserine and phosphatidylethanolamine were lower. The head membranes displayed a 1.7-2.3-fold higher Ca2+-ATPase activity and a 2.5-fold lower Na+/K+-ATPase activity, than the flagellar membranes. These results are consistent with a heterogeneous distribution of membrane components along the sea urchin sperm plasma membranes. Isolated head membranes sonicated in the presence of soybean phospholipid liposomes responded to egg jelly with a species-specific increase in Ca2+ and Na+ uptake. As in whole sperm, Ca2+ uptake was inhibited by the Ca2+ channel blocker nisoldipine. A close analog of this compound, [3H]nitrendipine, binds with high affinity to head membranes in a saturable, reversible manner, showing a Kd and Bmax of 31 nM and 5.3 pmol/mg protein, respectively.     

Gene activity of polytene chromosomes in Drosophila species of the obscura group

The polytene chromosome puffing patterns of Drosophila guanche were established and compared with those of Drosophila subobscura. A total of 150 loci, active in some of the 17 developmental stages studied, were described and 23 of them were found to form the characteristic puffing pattern of D. guanche. Taking into account the number of puffs as well as the gene activity of each chromosome and the total gene activity, D. guanche seems to be less active than D. subobscura. Although both species show a degree of homology in their puffing patterns lower than that found for sibling species, the degree of homology is stronger than that between species belonging to the same group but to different subgroups. Thus, D. guanche and D. subobscura must be considered as phylogenetically closely related species, belonging to the same subgroup.     

The site and mechanism of dioxygen reduction in bovine heart cytochrome c oxidase

The site and mechanism of dioxygen reduction in cytochrome c oxidase from bovine heart muscle have been investigated. The rate of cytochrome c2+ oxidation by O2 is shown to be affected by several factors: 1) pH, with optima at 5.65 and 6.0, 2) temperature between 0 and 29 degrees C, with E alpha = 13 kcal mol-1, 3) D2O exchange, with a reduction in rate of 50% or more at the pH optima, and 4) the addition of ethylene glycol or glycerol, which significantly lowers the rate. The extremely narrow (delta vCO approximately 4 cm-1) infrared stretch bands at approximately 1964 and approximately 1959 cm-1 for liganded CO are only slightly affected by factors 1-4 or by changes in the oxidation state of metals other than the heme alpha 3 iron. These results indicate a stable, unusually immobile O2 reduction site well-isolated from the external medium, a characteristic expected to be important for oxidase function. Precise stereochemical positioning of hydrogen donors adjacent to O2 liganded to heme alpha 3 iron can be expected in order to achieve the optimization of the time/distance relationships required for enzyme catalysis. These findings support a novel mechanism of O2 reduction via a hydroperoxide intermediate within a reaction pocket that experiences little change in conformation during the hydrogen and electron transfer steps.     

Leiurus quinquestriatus venom inhibits different kinds of Ca2+-dependent K+ channels

A minor protein component of Leiurus quinquestriatus venom has been reported to inhibit selectively the apamin-insensitive Ca2+-dependent K+ channels of mammalian skeletal muscle (Miller, C., Moczydlowski, E., Latorre, R. and Phillips, M. (1985) Nature 313, 316-318). We report the effect of the venom on both the apamin-insensitive channels of the human erythrocyte, the Ehrlich cell and the rat thymocyte and the apamin-sensitive channel of the guinea pig hepatocyte. The venom inhibited Ca2+-dependent K+ transport in all the cases with a Ki value within the range of 1 to 10 micrograms/ml, similar to that reported previously in muscle. Valinomycin-induced K+ transport was also antagonized by the venom but its sensitivity was about 1/10 as much as that of the Ca2+-dependent K+ channel.     

The effect of pH on the phase transition temperature of dipalmitoylphosphatidylcholine-palmitic acid liposomes

The shift in the gel-liquid crystal phase transition temperature (tm) of dipalmitoylphosphatidylcholine liposomes induced by incorporation of 10 mol% palmitic acid, was measured by 90 degrees light scattering at different bulk pH values. It has been found that the tm shift decreases sigmoidally from 4.7 to -0.3 degrees C as the bulk pH is raised from 5 to 11. Since it is in this range that the carboxyl group of a membrane-bound fatty acid should ionize, our results can be interpreted to mean that there is relationship between the tm shift and the degree of dissociation of palmitic acid, the uncharged fatty acid increasing tm and its conjugate, anionic form, slightly decreasing the transition temperature of dipalmitoylphosphatidylcholine liposomes. The experimental results are fitted by a modified form of the Henderson-Hasselbach equilibrium expression which takes into account the effect of the anionic fatty acid on the surface potential and hence, on the surface pH of liposomes, according to Gouy-Chapman and Boltzmann equations, respectively. Best fit between theory and experiments is found when the intrinsic interfacial pK of palmitic acid is set equal to 7.7. This high pK value can be explained as due to the effect of the lower dielectric constant of the interfacial region, as compared to bulk water, on the acid-base dissociation of the carboxyl group. The results presented here show that upon incorporation of palmitic acid, the phase transition of dipalmitoylphosphatidylcholine bilayers becomes extremely sensitive to changes of pH in the vicinity of the physiological range. This property is not shown by the pure phospholipid bilayers in the same pH range.     

Cold exposure as a potentiating factor of pineal actions in nonhibernating mammals

Twenty-eight-day-old male rats were used in three experiments to study whether cold exposure potentiates pineal actions in nonhibernating mammals. The following questions were considered: (a) Can cold exposure increase the antigonadal effects of light deprivation? (b) Are the effects induced by blindness plus cold exposure pineal dependent? (c) Can cold exposure modify the response of the endocrine-reproductive axis to exogenously administered melatonin? Blind cold-exposed rats showed a significant loss in body weight as well as in weights of pituitary and reproductive tract organs compared with either intact or blind animals kept at 22 degrees C, or intact rats exposed to cold; serum testosterone levels were also lowest in blind cold-exposed rats. These effects were not present in blind cold-exposed animals that were pinealectomized at the beginning of the experiment. When intact animals placed at 22 or 10 degrees C were treated with daily injections of melatonin (50 micrograms) there was a reduction of body weight and weights of the hypophyso-gonadal axis organs. Those effects of melatonin were, however, significantly greater in cold-exposed rats than in rats placed at 22 degrees C. These results suggest that cold exposure should be considered as another state which potentiates the pineal-dependent actions of light deprivation. Cold exposure probably acts by increasing the sensitivity of sites at which pineal melatonin exerts its actions.     

Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases. Role of H+ gradient

A comparative study of the orthophosphate-pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane-bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed. In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO4(2-) or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7-8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were included in the assay medium. In the presence of 6.7-8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO4(2-) and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24-0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total Pi, HPO4(2-) and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5-7.5 mM free Mg2+ was very high and could not be measured. In the presence of 0.24-0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate measured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5-8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used.     

Specific inhibition of human granulocyte elastase with peptide aldehydes

The kinetic features of human granulocyte elastase, chymotrypsin, porcine pancreatic elastase and elastomucoproteinase were compared. Amino acyl ester substrates were assayed and Km and kcat values were defined. Aldehyde analogues of the p-nitroanilide substrates designed for granulocyte elastase as optimal for Km appeared to be potent inhibitors. Suc-D-Phe-Pro-valinal (Ki = 40 microM) was found to inhibit granulocyte elastase competitively and specifically when measured with synthetic substrates, and the Ki was 3 microM with the natural protein substrate, elastin.     

Thermostability of membrane systems in organic solvents

Two multisubunit enzymes of the inner mitochondrial membrane, cytochrome oxidase and the H+-ATPase may be transferred into highly apolar solvents as protein-lipid complexes. At 70 degrees C and an initial water concentration of 13 microliters per ml organic solvent (toluene), the half-life of the ATPase was approx. 11 h, whereas that of cytochrome oxidase was about 100 s. Thermostability of cytochrome oxidase could be increased more than 100-times by decreasing the water concentration to 3 microliters per ml toluene. At this latter concentration of water the half-life of the ATPase at 90, 80 and 70 degrees C was 5, 48 and 96 h, respectively.     

The Pleistocene lake deposits of the NE Baza Basin (Spain): salinity variations and ostracod succession

The Guadix-Baza Basin in Spain covers an area of approximately 3000 km² and yields a sedimentary sequence ranging from Lower Miocene to Pleistocene. Twenty five meters of Lower Pleistocene lacustrine sediments have been located in the NE part of the Basin at about 1000 meters in altitude. This sequence which overlies dolomitic mud flat deposits consists of limestones, calcareous and dolomitic mudstones, dolostones, silty clays, sands and gravels. Salinity fluctuations and short dry episodes, related to lake level oscillations, have been recorded by textural, mineralogical and faunal changes throughout the sequence. Ostracods, which are the most commonly encountered fossils, permit to detect recurrent changes in water salinity and regime, and solute composition. The faunal changes indicate an alternation of slightly saline and bicarbonate-rich water (when ostracods and gastropods occur) with a saline NaCl-dominant water (in which ostracods, Cerastoderma bivalves and non-marine foraminifers are found).The frequent and recurrent hydrochemical changes in the Baza Basin in the Early Pleistocene point to a climate of high contrast like in the Mediterranean region today but with a greater availability of water within the system compared to the present situation in the area.