Evidence for glucocorticoid transport into AtT-20/D-1 cells.

Glucocorticoid uptake by AtT-20/D-1 mouse pituitary adenocarcinoma cells grown in tissue culture was examined. The binding of triamcinolone acetonide, a potent synthetic glucocorticoid, by intact cells and by cell cytosol was studied at both 4 and 25 degrees. Specific binding of [3H]triamcinolone acetonide by intact cells was markedly different from cell-free cytosol binding at 4 degrees. Intact cells bound a relatively small amount of labeled steroid within 2 min, after which no further binding was observed. In contrast, the receptor in a cell-free cytosol preparation was capable of binding steroid progressively at 4 degrees, indicating that the limited binding by intact cells was not a consequence of receptor characteristics. At 25 degrees, uptake by intact cells and cytosol was nearly identical and appeared to be limited only by the binding kinetics of the cytosol receptor. Estradiol-17 beta, a nonglucocorticoid steroid, was not bound by the AtT-20/D-1 cell at 4 degrees. Triamcinolone was not bound significantly at 4 or 25 degrees by an adrenal carcinoma cell that does not appear to be a glucocorticoid target cell. An Arrhenius plot of cell steroid uptake vs. the reciprocal of absolute temperature revealed an abrupt change in slope at 16 degrees, which is compatible with the temperature-dependent mechanism involved in glucocortidoid uptake being associated with lipid constituents of the cell membrane. These data suggest that glucocorticoid uptake by this target cell involves a mechanism of specific, temperature-dependent transport through the cell membrane.     

Effect of temperature on alanine uptake by membrane vesicles isolated from a psychrophilic marine bacterium.

The effect of temperature on the membranes of Ant-300, a psychrophilic marine bacterium, was studied by measuring alanine uptake by isolated membrane vesicles. Uptake was observed from 0 to 35 degrees C. The maximum initial rate of uptake occurred at 25 degrees C although more alanine was ultimately taken up at temperatures from 10 to 20 degrees C. An ARRHENIUS plot of these data shows a single infection point at 7.8 degrees C. Within 10 min, over 50% of the alpha-aminoisobutyric acid taken up by whole cells at 5 degrees C was lost after a temperature shift to 25 degrees C. Vesicles preloaded with alanine at 5 degrees C did not become leaky when shifted to 25 degrees C. In addition, exposure of the vesicles to 25 degrees C for 30 min did not affect subsequent alanine uptake at 5 degrees C. The data obtained suggest that the loss of the uptake and permeability control functions of membranes from psychrophilic bacteria at elevated temperatures is not due to degeneration of the membrane itself, but rather to a control or regulatory mechanism associated with whole cells.     

Uptake of L-tri-iodothyronine by isolated rat liver cells. A process partially inhibited by metabolic inhibitors; attempts to distinguish between uptake and binding to intracellular proteins.

1. Rat liver cells obtained by dispersion with collagenase were used to investigate the mode of entry of L-tri-iodothyronine into the cell. 2. The hormone was taken up very rapidly at 23 degrees C; the linear phase of uptake lasted for up to approx. 20 s. 3. A plot of the initial rates of uptake against different concentrations of L-tri-iodothyronine yielded a sigmoidal curve. The Eadie--Hofstee plot (v/[S]2 versus v) yielded two straight lines. The uptake component with an apparent Kt value of 86 +/- 15 pM was designated as system I, and the second uptake component with an apparent Kt of 726 +/- 11 pM as system II. The Hill plot for system I was not linear; the apparent Hill coefficient for system II was calculated to be 2.1.4. Uptake of L-tri-iodothyronine by system I was higher at pH 6.4 than at pH 7.4; system II was relatively insensitive to changes in the pH of the external medium. 5. Both systems exhibited a transition temperature at about 16 degrees C in the Arrhenius plot. The activation energies of the two systems below and above 16 degrees C were 72.8 and 47.7 and 54.4 and 33.1 J/mol respectively. 6. Inhibitors of cellular energy reduced the uptake by system I to a larger extent than that by system II. 7. Replacement of Na+ in the external medium by either K+ or choline led to uptake that followed normal Michaelis--Menten kinetics. 8. Thiol-group-blocking agents reduced the uptake of the hormone by both systems. 9. Treatment of liver cells with beta-glucosidase, Pronase and neuraminidase led to a decrease in the uptake of L-tri-iodothyronine by system I, whereas uptake by system II was decreased after treatment with phospholipase A2, beta-galactosidase. Pronase and neuraminidase. 10. The stereoisomer D-tri-iodothyronine (100--3000 pM) did not affect system I, but uptake by system II decreased with increasing concentration of D-tri-iodothyronine. Reverse L-tri-iodothyronine (2--100 pM) and L-thyroxine (100--3000 pM) did not influence uptake by either system. 11. Under identical conditions of incubation, the uptake of L-tri-iodothyronine was 3.7 times higher than binding to cytosol proteins. The binding was insensitive to metabolic inhibitors. The results suggest that cytosol proteins are not directly involved in the uptake of L-tri-iodothyronine. 12. Plasma-membrane vesicles also take up the hormone rapidly at 23 degrees C. Increasing the osmolarity of the external medium led to a decrease in the uptake of L-tri-iodothyronine by vesicles. 13. Uptake as a function of L-tri-iodothyronine concentration exhibited a sigmoidal curve. The Eadie--Hofstee plot showed two uptake components with apparent Kt values of 96.8 and 1581 pM. 14. The results of our study are consistent with a carrier-mediated translocation of the hormone into the cell.     

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.     

Temperature dependence of solute transport and enzyme activities in hog renal brush border membrane vesicles

The temperature dependence of sodium-dependent and sodium-independent d-glucose and phosphate uptake by renal brush border membrane vesicles has been studied under tracer exchange conditions. For sodium-dependent d-glucose and phosphate uptake, discontinuities in the Arrhenius plot were observed. The apparent activation energy for both processes increased at least 4-fold with decreasing temperature. The most striking change in the slope of the Arrhenius plot occurred between 12 and 15°C. The sodium-independent uptake of d-glucose and phosphate showed a linear Arrhenius plot over the temperature range tested (35–5°C). The behavior of the transport processes was compared to the temperature dependence of typical brush border membrane enzymes. Alkaline phosphatase as intrinsic membrane protein showed a nonlinear Arrhenius plot with a transition temperature at 12.4°C. Aminopeptidase M, an extrinsic membrane protein exhibited a linear Arrhenius plot. These data indicate that the sodium-glucose and sodium-phosphate cotransport systems are intrinsic brush border membrane proteins, and that a change in membrane organization alters the activity of a variety of intrinsic membrane proteins simultaneously.     

Effects of calcium on hepatocyte iron uptake from transferrin, iron-pyrophosphate and iron-ascorbate.

Calcium stimulates hepatocyte iron uptake from transferrin, ferric-iron-pyrophosphate and ferrous-iron-ascorbate. Maximal stimulation of iron uptake is observed at 1-1.5 mM of extra-cellular calcium and the effect is reversible and immediate. Neither the receptor affinity for transferrin, nor the total amounts of transferrin associated with the cells or the rate of transferrin endocytosis are significantly affected by calcium. In the presence of calcium the rate of iron uptake of non-transferrin bound iron increases abruptly at approximate 17 degrees C and 27 degrees C and as assessed by Arrhenius plots, the activation energy is reduced in a calcium dependent manner at approx. 27 degrees C. At a similar temperature, i.e., between 25 degrees C and 28 degrees C, calcium increases the rates of cellular iron uptake from transferrin in a way that is not reflected in the rate of transferrin endocytosis. By the results of this study it is concluded that calcium increases iron transport across the plasma membrane by a mechanism dependent on membrane fluidity.     

The temperature dependence of State IV respiration, the calcium uptake system, and the activity of the calcium ionophore A23187 in mitochondria from endo- and ectothermic animals.

1. Arrhenius plots of State IV respiratory activity of liver mitochondria from both rainbow trout and rat were linear over the temperature range 5-35 degrees C. 2. Calcium uptake was monitored by stimulation of oxygen consumption and by calcium electrode recording, with quite comparable results. Rainbow trout gave the usual linear Arrhenius plot but this plot for rat mitochondria exhibited two well-defined inflections or discontinuities. 3. The temperature dependence of the activity of the ionophore A23187 was investigated by measuring the increase in oxygen uptake following a sub-maximal dose of this drug. Again a linear relation was found for rainbow trout, but in this case the rat curves showed only a single inflection point. 4. These results are discussed in relation to other work on the effects of lipid phase transitions on mitochondrial membrane-associated systems.     

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.     

Studies on the special properties of actomyosin in the gel form. I. Unusual features of the Mg2+-ATPase activity

The hydrolysis of MgATP by actomyosin gel at low ionic strength is known to show two unusual features: (1) an Arrhenius plot with a shallow slope in the higher temperature range (35-16 degrees C) and a steep slope in the lower temperature range (16-0 degrees C); (2) a rate curve of hydrolysis that begins with a 'burst' and falls to a lower steady-state level. Both of these can now be interpreted in terms of a specific, relatively slow transformation in the gel (t 1/2 = 9 s at 25 degrees C), induced by the binding of MgATP to the active sites of the myosin filaments. In the rate curves, this transformation is reflected in the transition from the burst rate (catalyzed by the original gel) to the steady-state rate (catalyzed by the modified gel). Importantly, this transition does not occur to a significant extent at low temperatures. Thus, in the typical nonlinear Arrhenius plot, where steady-state rates are used, the shallow slope in the high temperature range is a property of the modified gel, whereas the steep slope at low temperatures is a property of the original gel. Consistent with this interpretation, when the burst rates (presumably due to the original gel) were used in the high temperature range (and when substrate inhibition of hydrolysis by high levels of MgATP was avoided), the Arrhenius plot was linear over the entire temperature range (40-0 degrees C); the steep slope of this plot gives a high apparent heat of activation (25-30 kcal), similar to that reported for actin-activated hydrolysis by the soluble subfragment, heavy meromyosin. It is the steady-state form of the gel at high temperatures that gives a low apparent heat of activation (6-10 kcal). It was found that the regulatory proteins with calcium activate hydrolysis by the original form but have no effect on the steady-state form of the gel. Oxygen exchange measurements made during the burst and steady state at 25 degrees C indicate that the mechanism of hydrolysis is essentially the same for both, but that there is a higher effective actin concentration around the myosin sites in the original form.     

Factors involved in the uptake of corticosterone by rat liver cells

Isolated rat liver cells take up corticosterone rapidly; the initial rates increase with increasing temperature. A plot of the initial rates against the concentration of corticosterone indicated the presence of saturable and nonsaturable uptake systems. The Eadie-Hofstee plot showed the presence of two saturable and one nonsaturable uptake components. The apparent Kt values of the saturable systems were 64 +/- 40 nM (n = 3) and 1085 +/- 313 nM (n = 12). The nonsaturable system, probably diffusion, contributed 12% to the total uptake between 15 and 72 nM corticosterone, the physiological concentration of the free corticosterone in rat serum. Metabolic inhibitors did not influence the uptake of corticosterone. N-Ethylmaleimide, 1-fluoro-2,4-dinitrobenzene and sodium ethyl mercurithiosalicylate (1 mM each) decreased the uptake by 40%. Iodoacetate did not have any influence. Treatment of cells with phospholipase A inhibited the uptake 35--45%. In the presence of cortisone, cortisol, dexamethasone, aldosterone, testosterone, estradiol-17beta and estrone (2 muM each) the uptake decreased 30--50%. The presence of serum proteins in the external medium inhibits the uptake of corticosterone. These results suggest that corticosterone is transported into the cell and is accumulated. Only the free hormone is available for uptake which in turn may be regulated by protein and lipid components in the plasma membrane of the liver cell.     

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.     

A freeze-fracture study of the aggregation state of Ca2+,Mg2+-ATPase of sarcoplasmic reticulum in reconstituted vesicles at low and high temperature

Since it was possible for Ca2+,Mg2+-ATPase of sarcoplasmic reticulum (SR) to change its aggregation state in the membrane depending on temperature, and since the change could be the cause of the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity, the aggregation state of Ca2+,Mg2+-ATPase at 0 degrees C in the membrane was compared with that at 35 degrees C by freeze-fracture electron microscopy. These temperatures are below and above the break in the Arrhenius plot (about 18 degrees C), respectively. Two kinds of samples were used; fragmented SR vesicles and egg PC-ATPase vesicles, a reconstituted preparation from purified Ca2+,Mg2+-ATPase and egg yolk phosphatidylcholine (egg PC). For both the appearance of particles in the fracture faces of the samples fixed at 0 degrees C was similar to that at 35 degrees C, and phase separation between protein and lipid was not observed even at 0 degrees C. The size of the particles was measured and histograms of the sizes at 0 degrees C and 35 degrees C were made. The histogram at 0 degrees C was similar to that at 35 degrees C with a peak at 7.1 nm, which is 1-2 nm smaller than the value reported so far. The number of the particles per unit area of the membrane was also counted. The value at 0 degrees C was similar to that at 35 degrees C. These results indicate that Ca2+,Mg2+-ATPase of SR exists in the same aggregation state (estimated as oligomer based on the values obtained in this experiment) between 0 degrees C and 35 degrees C. Based on the results of this study we think that the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity in SR is not caused by the change in the aggregation state of Ca2+,Mg2+-ATPase.     

Influence of temperature on enzyme activity determination in serum : L-aspartate aminotransferase isoenzymes]

The influence of temperature on activity assays of the isoenzymes of L-aspartic aminotransferase in described. For this purpose, isolated human isoenzymes were added to inactivated serum. Half-saturation constants were determined at 17.8 degrees C, 25 degrees C, 30 degrees C, and 37 degrees C, and the substrate saturation and pH curves were recorded. The cytoplasmatic (c) and mitochondrial (m) GOT showed temperature-dependent differences in the half-saturation constants for the substrates L-aspartate and 2-oxoglutarate. For both isoenzymes pH 7.4 is considered the optimum regardless of the temperature of measurement, and Tris-HCl is the optimal buffer. In the Arrhenius plot there is a bent at 27 degrees C for both isoenzymes. Thermal denaturation as a possible reason for this deviation from the linearity in the Arrhenius plot could be ruled out.     

Hepatocellular uptake of aflatoxin B1 by non-ionic diffusion. Inhibition of bile acid transport by interference with membrane lipids

Aflatoxin B1 permeates isolated rat hepatocytes by non-ionic diffusion. Its uptake is neither saturable nor influenced by metabolic energy and not inhibited by treatment of cells with proteases. The initial rate of aflatoxin B1 uptake measured at 7 degrees C is between 40 and 50% compared to that at 37 degrees C. However, after an incubation period of 7 minutes identical equilibrium uptake is reached at both temperatures. The apparent activation energies, calculated for aflatoxin B1 uptake by Arrhenius diagrams ranged between 1.69 and 4.5 kcal/mol. A Q10 value of 1.34 was calculated for a temperature interval of 7-17 degrees C but decreased to 1.05 for the interval of 27-37 degrees C. Liposomes or lipoproteins added to the cell suspension inhibited the aflatoxin B1 uptake into hepatocytes. Liposomes mainly composed of unsaturated fatty acids bind twice as much aflatoxin B1 as those composed of saturated ones, indicating that the lipophilicity of the mycotoxin is crucial in the determination of its uptake into liver cells. At concentrations above 5 micrograms/ml, aflatoxin B1 inhibited the carrier-mediated uptake of cholic acid and of phalloidin into hepatocytes. This effect was reversible and abolished by washing the cells after preincubation with aflatoxin. In concentrations below 5 micrograms/ml the uptake of phallotoxin and cholic acid was however stimulated by 15-25%. These results indicate, that a carrier-mediated uptake into hepatocytes via the multispecific bile salt transporter is not responsible for the organoselective clearance of aflatoxins by the liver. On the other hand, the cholestatic effect of aflatoxin B1 results at least partially from the inhibition of the multispecific bile acid transport system. This inhibition may arise from affinity of aflatoxins to lipid domains of the cell membrane.     

The kinetic and structural changes of the mitochondrial F1-ATPase with temperature

Mitochondrial F1-ATPase shows a break in the Arrhenius plot with an increase of the activation energy below 17 degrees C, this may imply that the F1-ATPase undergoes a conformational change at this temperature. Further, a structural change of the F1-ATPase is indicated by analysis of the intrinsic fluorescence at 307 nm between 33 and 11 degrees C and also by evaluation of the circular dichroism spectra of the enzyme at temperatures below and above the temperature corresponding to the discontinuity of the Arrhenius plot. It is therefore suggested that F1-ATPase exists in two temperature dependent conformational states to which different catalytic properties may be assigned.     

Alanine transport by Chinese hamster ovary cells with altered phospholipid acyl chain composition

The Na+-dependent transport of alanine has been examined in Chinese hamster ovary (CHO) cells as a function of the fatty acid composition of their membrane lipids. Significant changes in the fatty acid composition of the CHO cell phospholipids were achieved by supplementation of the growth medium with specific saturated (palmitate) or monoenoic (oleate) free fatty acids. Arrhenius plots of the temperature-dependent uptake of alanine were constructed for cells of altered fatty acid composition. Alanine uptake was characterized by a single discontinuity in the Arrhenius plot. The temperature of this break was observed to be dependent upon the fatty acid composition of the cell phospholipids, ranging from 16 degrees C for cells enriched with oleate to 32 degrees C for cells enriched in palmitate. Calculation of the Km value for the uptake process showed no significant change with temperature or fatty acid supplementation. Correlations are made between the physical state of the membrane lipids and the temperature-dependence for alanine transport. The results are discussed in terms of membrane fatty acid composition, ordered in equilibrium fluid phase transitions and amino acid transport.     

Functional interactions of lipids and proteins in rat intestinal microvillus membranes.

Interactions of lipids and proteins in isolated rat intestinal microvillus membranes were examined by studying the temperature dependence of enzyme activities and of D-glucose transport in relation to the membrane lipid thermotropic transition observed by fluorescence polarization (26 +/- 2 degrees C) and differential scanning calorimetry (23--39 degrees C). Two groups of activities were defined. Enzymes of the first group, comprising lactase, maltase, sucrase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, all yielded a single slope on the Arrhenius plot in the range 10--40 degrees C and did not appear to experience functionally the effects of the lipid thermotropic transition. Each activity of the second group, comprising calcium- and magnesium-dependent adenosine triphosphatases, p-nitrophenylphosphatase, and D-glucose transport, showed a change in the slope of the Arrhenius plot in the range 25--30 degrees C, corresponding to the lower region of the lipid transition. The terms "extrinsic" and "intrinsic" activities could be applied to these groups. Delipidation of the particulate p-nitrophenylphosphatase removed the discontinuity in the Arrhenius plot. Subsequent relipidation with a variety of lipids restored a break point, but the temperature corresponded to the original discontinuity (25--29 degrees C) rather than to the phase transition temperature of the exogenous lipid added.     

Temperature and Ca(2+)-dependence of the sarcoplasmic reticulum Ca(2+)-ATPase in haddock,salmon, rainbow trout and zebra cichlid

Temperature dependence of Ca(2+)-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degrees C. Whether the break point arises as a result of temperature dependent changes in the enzyme or its membrane lipid environment is still a matter of discussion. In this study we compared the temperature dependence and Ca(2+)-dependence of SR Ca(2+)-ATPase in haddock (Melanogrammus aeglefinus), salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and zebra cichlid (Cichlasoma nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degrees C, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the temperature area, 6-14 degrees C. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca(2+)-ATPase activity. The temperature range of the plateau was 14-21 and 18-25 degrees C in salmon and rainbow trout, respectively. Ca(2+)-dependence in the four different fish species investigated was very similar with half maximal activation (K(0.5)) between 0.2 and 0.6 micro M and half maximal inhibition (I(0.5)) between 60 and 250 micro M. Results indicated that interaction between SR Ca(2+)-ATPase and its lipid environment may play an important role for the different Arrhenius plot of the different types of fish species investigated.     

Temperature-dependencies of various catalytic activities of membrane-bound Na+/K+-ATPase from ox brain, ox kidney and shark rectal gland and of C12E8-solubilized shark Na+/K+-ATPase

The temperature dependence of ouabain-sensitive ATPase and phosphatase activities of membrane fragments containing the Na+/K+-ATPase were investigated in tissue from ox kidney, ox brain and from shark rectal glands. The shark enzyme was also tested in solubilized form. Arrhenius plots of the Na+/K+-ATPase activity seem to be linear up to about 20 degrees C, and non-linear above this temperature. The Arrhenius plots of mammalian enzyme (ox brain and kidney) were steeper, especially at temperatures below 20-30 degrees C, than that of shark enzyme. The Na+-ATPase activity showed a weaker temperature-dependence than the Na+/K+-ATPase activity. The phosphatase reactions measured, K+-stimulated, Na+/K+-stimulated and Na+/K+/ATP-stimulated, also showed a weaker temperature-dependence than the overall Na+/K+-ATPase activity. Among the phosphatase reactions, the largest change in slope of the Arrhenius plot was observed with the Na+/K+/ATP)-stimulated phosphatase reaction. The Arrhenius plots of the partial reactions were all non-linear. Solubilization of shark enzyme in C12E8 did not change the curvature of Arrhenius plots of the Na+/K+-ATPase activity or the K+-phosphatase activity. Since solubilization involves a disruption of the membrane and an 80% delipidation, the observed curvature of the Arrhenius plot can not be attributed to a property of the membrane as such.     

Enhanced activity of the tricarboxylate carrier and modification of lipids in hepatic mitochondria from hyperthyroid rats

The effect of hyperthyroidism on the activity of the mitochondrial tricarboxylate carrier has been studied. The activity of this transporting system in liver mitochondria was quantitatively determined by the rate of malate-[14C]citrate exchange using the 1,2,3-benzene-tricarboxylate inhibitor stop technique. It has been found that the rate of citrate uptake is significantly enhanced in liver mitochondria from hyperthyroid rats as compared to that obtained in mitochondria from control rats. Kinetic analysis of the malate-citrate exchange reaction indicates that only the Vmax of this transporting process is enhanced, while there is practically no change in the Km values. Inhibitor titrations with the inhibitor palmitoyl-CoA show that mitochondria from hyperthyroid rats require the same concentrations of inhibitor to produce 100% inhibition of citrate uptake as control mitochondria, suggesting that the amount of functional translocase enzyme present is unaffected. The Arrhenius plot characteristics differ for tricarboxylate carrier activity in mitochondria from hyperthyroid rats as compared with control rats in that the break point of the biphasic plot decreases from 18.1 +/- 1.4 degrees C in controls to 12.9 +/- 1.2 degrees C in hyperthyroid animals. The hepatic mitochondrial lipid composition is altered significantly in hyperthyroid rats; the total cholesterol decreases and the phospholipids increase. The liver mitochondrial phospholipid composition is altered significantly in hyperthyroid rats. In particular negatively charged phospholipid cardiolipin increases by more than 50%. Minor alterations were found in the pattern of fatty acids. The thyroid hormone induced change in the activity of the tricarboxylate carrier can be ascribed either to a general modification of membrane lipid composition which increases the membrane fluidity and in turn the mobility of the carrier or to a more localized change of lipid domain (cardiolipin content) surrounding the carrier molecule in the mitochondrial membrane.