USE OF ARRHENIUS PLOTS OF Na-K ATPase and ACETYLCHOLINESTERASE AS A TOOL FOR STUDYING CHANGES IN LIPID-PROTEIN INTERACTIONS IN NEURONAL MEMBRANES DURING BRAIN DEVELOPMENT

Abstract— The activities of Na-K ATPase and acetylcholinesterase in the rat brain cortex were measured at different postnatal ages as a function of temperature. It was found that compared to acetylcholinesterase, Na-K ATPase is more strongly affected by the rise in temperature and that this response is further enhanced with age. Arrhenius plots of the data were prepared and the apparent energies of activation were computed for each plot. It was observed that all plots were biphasic except that for Na-K ATPase of the immature (5-day-old) brain which showed no transition temperature, with an apparent energy of activation of 15.5 kcal/mol. The enzyme from the mature brain (25-day-old) showed an average transition temperature of 22.6°C, with average apparent energies of activation of 15.3 and 27.2 kcal/mol above and below the transition temperature respectively. The cortex of 1-day-old rat showed no Na-K ATPase activity. Arrhenius plots of acetylcholinesterase studied at ages 1, 5 and 25 days postnatally all showed transition temperatures which increased from an average of 16.1°C for 1-day-old to 17 and 21.5°C for 5- and 25-day-old animals respectively. The average apparent energies of activation for acetylcholinesterase below the transition temperature changed from 8.3 kcal/mole at day 1 to 8.7 and 7.2 kcal/mol at days 5 and 25, while above the transition temperature they were 4.3, 5.2 and 4.1 at days 1, 5 and 25 respectively. The results are discussed in terms of the differences and changes in the interactions of Na-K ATPase and acetylcholinesterase with membrane lipids during the postnatal phase of brain development.     

Temperature dependence of the apparent affinity and the maximum velocity of the membrane-bound monosaccharide transport system in the yeast Rhodotorula gracilis

Analysis of the temperature dependence of the monosaccharide transport system in the yeast Rhodotorula gracilis (ATCC 26194, CBS 6681), as tested with D-xylose, revealed that the apparent affinity of the transport system, measured as the reciprocal of the half-saturation constant KT, increased when transport velocity was stimulated by temperature (15--30 degrees C) and decreased when the rate of uptake was reduced at temperatures aboce 30 degrees C. Breaks in Arrhenius plots were accompanied by corresponding breaks in van't Hoff plots. Whereas untreated cells exhibited in the van't Hoff plot a discontinuity at 28--30 degrees C this was not observed in heat-treated cells (at either 37 or 45 degrees C). In heat-treated cells the maximum transport velocity was always lower and the apparent affinity higher than in untreated cells at the same temperature; the optimum temperature for both transport velocity and apparent affinity was shifted to higher values. The data are interpreted in terms of a reversible phase transition of membrane lipids effecting an irreversible alteration of membrane structure. The temperature-induced reversible alkalinization of unbuffered yeast suspensions supports this interpretation.     

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.     

Changes in (Na+ + K+)-ATPase activity of Ehrlich ascites tumor cells produced by alteration of membrane fatty acid composition.

The fatty acid composition of plasma membrane derived from Ehrlich ascites tumor cells was altered in vivo by changing the dietary lipid of the tumor-bearing mice. The activity of (sodium + potassium)-adenosinetriphosphatase ((Na+ + K+ATPase), in partially purified plasma membranes, was measured ass a function of temperature. Arrhenius plots of the data were biphasic. Striking differences, dependent on the membrane fatty acid composition, were observed in the transition temperature and in the energies of activation below the transition temperature. The transition temperatures for the (Na+ + K+)-ATPase of plasma membrane derived from tumor cells grown in mice fed a regular chow diet containing a mixture of fatty acids (PMC), a 16% sunflower oil diet (PMSU), or a 4% tristearin diet (PMTS) were 20, 21, and 13.5 degrees C, respectively...     

Change of synaptic membrane lipid composition and fluidity by chronic administration of lithium

The effect of chronic administration of lithium salts on the lipid composition and physical properties of the synaptosomal plasma membrane was examined in rat brain. The effect of lithium treatment has been studied on the fluorescence polarization of synaptosomal plasma membrane and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from lithium-treated animals. Altered DPH polarization was due to a decrease in the order parameter of the probe. The lithium-treatment also changed the fluorescence of 1-anilino-8-naphthalene sulfonate (ANS), a probe that binds to the polar head group of the phospholipids and to proteins on the membrane surface. Synaptic plasma membranes from treated rats presented no significant changes on the cholesterol-to-phospholipid ratio, although the phospholipid class distribution was altered and the membrane phospholipid unsaturation increased. In summary, the neural plasma membranes became disorder after chronic lithium administration at therapeutic levels. This structural change may be due to changes in plasma membrane phospholipid distribution and to the degree of unsaturation of phospholipid fatty acids.     

Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma-membrane enzymes occurring on hibernation.

1. Arrhenius plots of the glucagon-stimulated adenylate cyclase, 5'-nucleotidase, (Na+ + K+)-stimulated adenosine triphosphatase and Mg2+-dependent adenosine triphosphatase activities of control hamster liver plasma membranes exhibited two break points at around 25 and 13 degrees C, whereas Arrhenius plots of their activities in hibernating hamster liver plasma membranes exhibited two break points at around 25 and 4 degrees C. 2. A single break occurring between 25 and 26 degrees C was observed in Arrhenius plots of the activities of fluoride-stimulated adenylate cyclase, basal adenylate cyclase and cyclic AMP phosphodiesterase of liver plasma membranes from both control and hibernating animals. 3. Arrhenius plots of phosphodiesterase I activity showed a single break at 13 degrees C for membranes from control animals, and a single break at around 4 degrees C for liver plasma membranes from hibernating animals. 4. The temperature at which break points occurred in Arrhenius plots of glucagon- and fluoride-stimulated adenylate cyclase activity were decreased by about 7--8 degrees C by addition of 40 mm-benzyl alcohol to the assays. 5. Discontinuities in the Arrhenius plots of 4-anilinonaphthalene-1-sulphonic acid fluorescence occurred at around 24 and 13 degrees C for liver plasma membranes from control animals, and at around 25 and 4 degrees C for membranes from hibernating animals. 6. We suggest that in hamster liver plasma membranes from control animals a lipid phase separation occurs at around 25 degrees C in the inner half of the bilayer and at around 13 degrees C in the outer half of the bilayer. On hibernation a change in bilayer asymmetry occurs, which is expressed by a decrease in the temperature at which the lipid phase separation occurs in the outer half of the bilayer to around 4 degrees C. The assumption made is that enzymes expressing both lipid phase separations penetrate both halves of the bilayer, whereas those experiencing a single break penetrate one half of the bilayer only.     

Phase transitions in yeast mitochondrial membranes. The effect of temperature on the energies of activation of the respiratory enzymes of Saccharomyces cerevisiae.

The effect of temperature on the activation energies of mitochondrial enzymes of the yeast Saccharomyces cerevisiae was examined. Non-linear Arrhenius plots with discontinuities in the temperature range 14-19 degrees C and 19-22 degrees C were observed for the respiratory enzymes and mitochondrial ATPase (adenosine triphosphatase) respectively. A straight-line Arrhenius plot was observed for the matrix enzyme, malate dehydrogenase. The activation energies of the enzymes associated with succinate oxidation, namely, succinate oxidase, succinate dehydrogenase and succinate-cytochrome c oxidoreductase, were in the range 60-85kJ/mol above the transition temperature and 90-160kJ/mol below the transition temperature. In contrast, the corresponding enzymes associated with NADH oxidation showed significantly lower activation energies, 20-35kJ/mol above and 40-85kJ/mol below the transition temperature. The discontinuities in the Arrhenius plots were still observed after sonication, treatment with non-ionic detergents or freezing and thawing of the mitochondrial membranes. Discontinuities for cytochrome c oxidase activity were only observed in freshly isolated mitochondria, and no distinct breaks were observed after storage at -20 degrees C. Mitochondrial ATPase activity still showed discontinuities after sonication and freezing and thawing, but a linear plot was observed after treatment with non-ionic detergents. The results indicate that the various enzymes of the respiratory chain are located in a similar lipid macroenvironment within the mitochondrial membrane.     

Effect of bilirubin on the activity and thermokinetic characteristics of brain (synaptosomal) membrane NO synthase

NO synthase activity was found in the plasma (synaptosomal) membrane particles isolated from the homogenate of adult rat brain (without cerebellum) under conditions preventing the protease attack and formation of reactive oxygen species. The NO synthase discovered exhibited some properties of a neuronal constitutive integral membrane enzyme and was inhibited by N-nitro-L-arginine. NO synthase activity decreased when bilirubin entered the synaptosomal membrane in vitro. Bilirubin caused the shift of the transition temperature in the temperature dependence of NO synthase activity in Arrhenius plots. The incorporation of bilirubin into synaptosomal membranes resulted in an increase in the apparent activation energy for NO synthase within a temperature range of 10-30 degrees C. The membrane NO synthase was susceptible to the photodynamic effect of membrane-bound bilirubin molecules. Monomeric human serum albumin without organophilic ligands exerted a protective effect on NO synthase in bilirubin-containing membrane particles.     

Mechanism of stimulation of renal phosphate transport by 1,25-dihydroxycholecalciferol

Vitamin D has been shown to stimulate renal phosphate transport and to alter membrane phospholipid composition. The present studies examine the possibility that the effects of 1,25(OH)2D3 on phosphate transport are related to its effects on membrane lipids. Arrhenius plots, which relate maximum rates of sodium dependent phosphate uptake into brush-border membrane vesicles to temperature were constructed. Phosphate transport was studied using brush-border membrane vesicles from normal, vitamin D-deficient, and physiologically replete (15 pmol/100 g body weight per 24 h) rats. These plots were triphasic with characteristic, lipid-dependent, slopes (M1,M2,M3) representing activation energies and transition temperatures (T1,T2). Physiologic 1,25(OH)2D3 repletion normalized these plots by stimulating phosphate transport at all temperatures, increasing T2 from 18 +/- 0.7 to 23.5 +/- 0.9 degrees C and decreasing M2 and M3 from -5.8 +/- 0.2 and -10.2 +/- 0.4 to -4.5 +/- 0.4 and -7.7 +/- 0.3, respectively. Pharmacologic (1.2 nmol/100 g per 3 h) 1,25(OH)2D3 treatment resulted in a change in the Arrhenius plot of phosphate transport to a biphasic one with a transition temperature of 30 degrees C. This effect was not blocked by cycloheximide. The Arrhenius plots of glucose transport were triphasic and unchanged with vitamin D repletion. These data support a liponomic mechanism of action for 1,25(OH)2D3 on phosphate transport.     

Effects of temperature and benzyl alcohol on the structure and adenylate cyclase activity of plasma membranes from bovine adrenal cortex

Adenylate cyclase activation by corticotropin (ACTH), fluoride and forskolin was studied as a function of membrane structure in plasma membranes from bovine adrenal cortex. The composition of these membranes was characterized by a very low cholesterol and sphingomyelin content and a high protein content. The fluorescent probes 1,6-diphenylhexa-1,3,5-triene (DPH) and a cationic analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) were, respectively, used to probe the hydrophobic and polar head regions of the bilayer. When both probes were embedded either in the plasma membranes or in liposomes obtained from their lipid extracts, they exhibited lifetime heterogeneity, and in terms of the order parameter S, hindered motion. Under all the experimental conditions tested, S was higher for TMA-DPH than for DPH but both S values decreased linearly with temperature within the range of 10 to 40 degrees C, in the plasma membranes and the liposomes. This indicated the absence of lipid phase transition and phase separation. Addition to the membranes of up to 100 mM benzyl alcohol at 20 degrees C also resulted in a linear decrease in S values. Membrane perturbations by temperature changes or benzyl alcohol treatment made it possible to distinguish between the characteristics of adenylate cyclase activation with each of the three effectors used. Linear Arrhenius plots showed that when adenylate cyclase activity was stimulated by forskolin or NaF, the activation energy was similar (70 kJ.mol-1). Fluidification of the membrane with benzyl alcohol concentrations of up to 100 mM at 12 or 24 degrees C produced a linear decrease in the forskolin-stimulated activity, that led to its inhibition by 50%. By contrast, NaF stabilized adenylate cyclase activity against the perturbations induced by benzyl alcohol at both temperatures. In the presence of ACTH, biphasic Arrhenius plots were characterized by a well-defined break at 18 degrees C, which shifted at 12.5 degrees C in the presence of 40 mM benzyl alcohol. These plots suggested that ACTH-sensitive adenylate cyclase exists in two different states. This hypothesis was supported by the striking difference in the effects of benzyl alcohol perturbation when experiments were performed below and above the break temperature. The present results are consistent with the possibility that clusters of ACTH receptors form in the membrane as a function of temperature and/or lipid phase fluidity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of ethinyl estradiol on intestinal membrane structure and function in the rabbit

Structural and functional properties of the small intestinal microvillus membrane were evaluated in the rabbit after administration of ethinyl estradiol, a synthetic estrogen with a demonstrated propensity to alter hepatic membrane lipid fluidity, and promote cholestasis. In the jejunum, no estrogen-induced changes in microvillus membrane total lipid, cholesterol or phospholipid content were observed. However, the ileal microvillus membrane in estradiol-treated animals demonstrates significant reductions vs. controls (per mg protein) in total lipid (0.55 milligrams vs. 0.89 milligrams) [corrected] and phospholipid (206.7 micrograms vs. 304.91 micrograms) (p less than 0.001) content, as well as modifications in specific phospholipid species. The increase in the ileal microvillus membrane cholesterol: phospholipid molar ratio (0.65 vs. 0.51, p less than 0.05) was associated with a significant decrease in membrane lipid fluidity reflected by an increase in fluorescence anisotropy measurements utilizing diphenyl hexatriene as the fluorophore (r at 25 degrees C = 0.306 vs. 0.282, p less than 0.05). Thermotropic lipid phase transitions, assessed by Arrhenius plots of both fluorescence data and ileal microvillus membrane p-nitrophenylphosphatase activity demonstrate that phase changes occur between and 24 and 28 degrees C in both treated and untreated groups. Within the temperature range studied (40-10 degrees C) no differences from control were observed in microvillus membrane alkaline phosphatase activity following estrogen treatment. These data therefore indicate that ethinyl estradiol-induced effects on microvillus membrane lipid composition and physical properties occur predominantly in the ileum and appear to be related, in part, to specific alterations in the availability of phospholipid following estrogen treatment.     

Dexamethasone-induced alterations in lipid composition and fluidity of rat proximal-small-intestinal brush-border membranes.

A series of experiments were conducted to examine the possible effects of subcutaneous administration of the synthetic glucocorticoid dexamethasone (100 micrograms/day per 100 g body wt.) on the lipid fluidity and lipid composition of rat proximal-small-intestinal brush-border membranes. After 4 days of treatment, membranes and their liposomes prepared from treated animals possessed a greater fluidity than did their control (diluent, 0.9% NaCl) counterparts, as assessed by steady-state fluorescence-polarization techniques using several different fluorophores. Examination of the effects of temperature on the anisotropy values of 1,6-diphenylhexa-1,3,5-triene, using Arrhenius plots, moreover, revealed that the mean break-point temperatures of the treated preparations were approx. 3-4 degrees C lower than those of their control-preparation counterparts. Changes in the sphingomyelin/phosphatidylcholine (PC) molar ratio as well as in certain of the fatty acids of the PC fraction of treated membranes, secondary to alterations in membrane PC levels and in lysophosphatidylcholine acyltransferase activities respectively, were also noted after dexamethasone administration. These compositional alterations appeared to be responsible, at least in part, for the differences in fluidity noted between treated and control plasma membranes. These results therefore demonstrate that dexamethasone administration can modulate the lipid fluidity and lipid composition of rat proximal-small-intestinal brush-border membranes.     

Subcellular localization of binding sites for cytochalasin D: evidence from activation energies.

The activation energies for binding of tritiated cytochalasin D to HEp-2 cells and isolated plasma membrane were determined by Arrhenius plots. The higher value for intact cells (24 kcal/mol) compared to the plasma membrane fraction (4 kcal/mol at greater than 11.5 degrees C, 18 kcal/mol at less than 11.5 degrees C) was taken as evidence that [3H]cytochalasin D must penetrate the plasma membrane in order to reach its binding sites. The data support the conclusion that binding sites for [3H]cytochalasin D are intracellular, on the cytoplasmic face of the plasma membrane (rather than within the lipid bilayer), and on microsomes (endomembranes).     

Calorimetric and spectroscopic studies of lipid thermotropic phase behavior in liver inner mitochondrial membranes from a mammalian hibernator

Arrhenius plots of various enzyme and transport systems associated with the liver mitochondrial inner membranes of ground squirrels exhibit changes in slope at temperatures of 20-25 degrees C in nonhibernating but not in hibernating animals. It has been proposed that the Arrhenius breaks observed in nonhibernating animals are the result of a gel to liquid-crystalline phase transition of the mitochondrial membrane lipids, which also occurs at 20-25 degrees C, and that the absence of such breaks in hibernating animals is due to a major depression of this lipid phase transition to temperatures below 4 degrees C. In order to test this hypothesis, we have examined the thermotropic phase behavior of liver inner mitochondrial membranes from hibernating and nonhibernating Richardson's ground squirrels, Spermophilus richardsonii, by differential scanning calorimetry and by 19F nuclear magnetic resonance and fluorescence polarization spectroscopy. Each of these techniques indicates that no lipid phase transition occurs in the membranes of either hibernating or nonhibernating ground squirrels within the physiological temperature range of this animal (4-37 degrees C). Moreover, differential scanning calorimetric measurements indicate that only a small depression of the lipid gel to liquid-crystalline phase transition, which is centered at about -5 degrees C in nonhibernating animals and at about -9 degrees C in hibernators, occurs. We thus conclude that the Arrhenius plot breaks observed in some membrane-associated enzymatic and transport activities of nonhibernating animals are not the result of a lipid phase transition and that a major shift in the gel to liquid-crystalline lipid phase transition temperature is not responsible for seasonal changes in the thermal behavior of these inner mitochondrial membrane proteins.     

Temperature dependence of the microsomal oxidation of ethanol by cytochrome P450 and hydroxyl radical-dependent reactions

The temperature dependence and activation energies for the oxidation of ethanol by microsomes from controls and from rats treated with pyrazole was evaluated to determine whether the overall mechanism for ethanol oxidation by microsomes was altered by the pyrazole treatment. Arrhenius plots of the temperature dependence of ethanol oxidation by pyrazole microsomes were linear and exhibited no transition breaks, whereas a slight break was observed at about 20 +/- 2.5 degrees C with control microsomes. Energies of activation (about 15-17 kcal/mol) were identical for the two microsomal preparations. Although transition breaks were noted for the oxidation of substrates such as dimethylnitrosamine and benzphetamine, activation energies for these two substrates were similar for control microsomes and microsomes from the pyrazole-treated rats. The addition of ferric-EDTA to the microsomes increased the rate of ethanol oxidation by a hydroxyl radical (.OH)-dependent pathway. Arrhenius plots of the .OH-dependent oxidation of ethanol by both microsomal preparations were linear with energies of activation (about 7 kcal/mol) that were considerably lower than values found for the P450-dependent pathway. These results suggest that, at least in terms of activation energy, the increase in microsomal ethanol oxidation by pyrazole treatment is not associated with any apparent change in the overall mechanism or rate-limiting step for ethanol oxidation but likely reflects induction of a P450 isozyme with increased activity toward ethanol. The lower activation energy for the .OH-dependent oxidation of ethanol suggests that different steps are rate limiting for oxidation of ethanol by .OH and by P450, which may reflect the different enzyme components of the microsomal electron transfer system involved in these reactions.     

Temperature dependence of beta receptor, adenosine receptor, and sodium fluoride stimulated adenylate cyclase from turkey erythrocytes

The individual temperature dependencies of the process which control the activity of turkey erythrocyte adenylate cyclase have been determined. The temperature dependence of the fraction of activable cyclase units experiences a thermal transition at 24 degrees C for all three modes of enzyme activation: l-epinephrine, adenosine, and NaF. This thermal transition probably reflects the phase transition in the inner monolayer of the membrane which influences the behavior of the GTP regulatory unit which is involved in all three modes of enzyme activation. The "rate constant" of enzyme activation by adenosine reflects two thermal transitions, at 24 and at 35 degrees C; the apparent rate constant of cyclase activation by NaF activation experiences a transition only at 24 degrees C whereas the rate constant of the beta-receptor-bound agonist decreases monotonously with no "breaks" on the Arrhenium plot. Following the temperature dependence of the fluorescence intensity of dansylphosphatidylethanolamine embedded in both sides of the membrane and exclusively in the outer monolayer, one can assign the thermal transition of 24 degrees C to the inner monolayer and the other two transitions to the outer monolayer (10 and 35 degrees C). We interpret these results as follows. (a) The monomolecular rate constant characterizing the activation of cyclase by the precoupled adenosine receptor experiences both the transition at 24 and 35 degrees C, indicating that the latter may span the bilayer. (b) The bata receptor activates the cyclase units only in fluid areas since it can diffuse exclusively in the fluid areas of the membrane and is unable to interact with cyclase units in "frozen" areas. the linear dependence of the logarithm of the rate constant on 1/T for the bata receptor reflects the change of membrane fluidity as a function of temperature.     

Development of the gastrointestinal mucosal barrier. Evidence for structural differences in microvillus membranes from newborn and adult rabbits

Electron spin resonance (ESR) and spin label methods with 5-doxylstearic acid as a probe were used to investigate the structure of microvillus membrane from the small intestine of adult and newborn rabbits. The spin label in microvillus membrane of newborns appeared to be in a more disordered environment than spin label in microvillus membrane of adult animals in the temperature range from 4 to 56 degrees C. In addition, a temperature transition at 39.6 +/- 0.3 degrees C was observed in the temperature dependence of the hyperfine splitting parameter for microvillus membrane from adult animals whereas a linear temperature dependence of the hyperfine splitting parameter was found for microvillus membrane from newborns. Cholera toxin was used as an external stimulus to test for the structural response in these two membrane preparations. Cholera toxin at 6 pM caused a decrease in the hyperfine splitting parameter at temperatures below 40 degrees C and a shift in the temperature break from 39.6 degrees C to 30.7 degrees C in microvillus membrane from adults. Using microvillus membrane from newborns, the temperature dependence of the hyperfine splitting parameter remained linear with a cholera toxin stimulus and the disordering effect of cholera toxin was only observed below 30 degrees C. These studies suggested that microvillus membrane from newborns were inherently more disordered than microvillus membrane from adult animals and that this difference in membrane organization might in part account for the increased attachment and penetration of macromolecules noted during the perinatal period.     

Alterations of oxygen uptake and the redox state of ubiquinone in rabbit sperm exposed to a variety of physiologic treatments

The rate of TEMPONE reduction by electrons originating from ubiquinone in intact rabbit spermatozoa was observed for control, high ionic strength (HIS) medium-treated, and HIS-seminal plasma-treated (HIS-SP) samples. The presence of TEMPONE in the incubation medium had no effect on oxygen consumption, demonstrating the utility of TEMPONE as a nonperturbing probe of the ubiquinol redox state. The rate of TEMPONE reduction was significantly increased over control levels for sperm incubated in hypertonic medium and was correlated to a decrease in oxygen consumption and a relative increase in ATP in the total adenine nucleotide pool. This increase in TEMPONE reduction in HIS sperm was reversed by treatment of sperm with seminal plasma, but seminal plasma had no effect on oxygen consumption or relative amounts of ATP in the adenine nucleotide pool. These observations are consistent with state 3 respiration in control sperm and state 4 respiration in HIS- and HIS-SP-treated sperm. Arrhenius data were obtained for ejaculated and epididymal sperm subjected to a variety of treatments. Lines fitted to plots of Arrhenius data revealed that each treatment affected the activation energy and intercept relative to controls. Evidence is presented for a phase transition occurring at 13 degrees C based on changes in the rate of TEMPONE reduction by ubiquinol. It was noted that, above the phase transition, rate constants for the reaction were dependent upon both treatment and temperature, but below the transition the differential effects of treatment were no longer apparent. The present study has demonstrated that events taking place in the respiratory chain can be closely monitored by measuring oxygen uptake and TEMPONE reduction, and that these events are affected by alterations in the sperm environment.     

Effect of ethanol on synaptosomal (Na+ + K+)-ATPase in control and ethanol-dependent rats

Rats were made dependent upon ethanol by feeding them liquid diets containing ethanol. Synaptosomal plasma membranes (SPM) were isolated from cerebral cortex and midbrain regions of isocaloric-fed control and ethanol-dependent rats. No major alcohol-induced alteration in in vitro (Na+ + K+)-ATPase activity was found in SPM of either brain area. At 37 degrees C, ethanol (0.10 to 0.98 M) added to incubations caused a dose-dependent inhibition of (Na+ + K+)-ATPase activity. The degree of inhibition found was independent of the diet administered or whether ethanol was present in the diet. At temperatures between 14 and 22 degrees C, 0.48 M ethanol caused a temperature-dependent decrease in activity. Arrhenius plots for SPM (Na+ + K+)-ATPase showed that in control and ethanol-dependent rats fed the Lieber de Carli diet, 0.48 M ethanol did not alter the transition temperature of this enzyme. Activation energies both above and below the transition temperature were decreased by the addition of ethanol to incubations. These results indicate that (Na+ + K+)-ATPase, a membrane-bound enzyme that is sensitive to its lipid environment and to the presence of ethanol, is not altered by the chronic administration of ethanol to rats.     

Low levels of the pesticide, delta-hexachlorocyclohexane, lyses human erythrocytes and alters the organization of membrane lipids and proteins as revealed by Raman spectroscopy.

We studied the nature of the interaction of delta-hexachlorocyclohexane (delta-HCCH), a pesticide having a stereoisomeric structure similar to inositol, with red blood cells. Cell survival data, measured as percent of hemoglobin released by delta-HCCH, show that the cell lysis increases with post exposure time. delta-HCCH at 55-60 micrograms/ml causes about 70% cell lysis after 24 h of exposure. The nature of interaction of delta-HCCH with membrane components was evaluated by studying the thermotropic transitions and protein structure of ghosts using Raman spectroscopy. Control ghosts show transitions with onset/completion temperatures 30 degrees C/38 degrees C (high temperature transition) and 3 degrees C/10 degrees C (middle temperature transition) when monitored by the I2935/I2850 ratio. The interaction of delta-HCCH drastically broadens the high temperature transition and shifts it to the temperature range of 10-29 degrees C. The plots of (I2880-90/I2850) vs. temperature show two transitions for control ghosts, one extending from -10 degrees C to 3 degrees C (lower temperature transition) and the other from about 7 degrees C to about 15 degrees C (middle temperature transition). Ghosts lysed with delta-HCCH shows only a single and a very broad transition in the range of about -3 degrees C to about 15 degrees C. These changes in the thermal transition properties suggest that delta-HCCH alters lipid and lipid-protein phases of erythrocyte membranes. The comparison of Raman spectra in the amide I and III regions of erythrocyte ghosts and purified band 3 with several amidated compounds reveals that cytoskeleton proteins contain highly amidated residues (probably glutamine and asparagine). The interaction of delta-HCCH with erythrocytes drastically alters the environment of these amidated residues indicating the involvement of cytoskeleton proteins. We conclude that the interaction of delta-HCCH with red blood cells disrupt membrane structure and change the environment of cytoskeleton proteins that could cause cell lysis.