Kinetics of internalization and degradation of asialo-glycoproteins in isolated rat hepatocytes

The rate constants for internalization of surface-bound asialo-orosomucoid by hepatocytes were 0.040 min-1 at 20 degrees C, 0.18 min-1 at 30 degrees C and 0.28 min-1 at 40 degrees C. At 40 degrees C, internalization accounted for most of the increase in cell-associated radioactivity. The activation energy over the temperature range 20 to 40 degrees C was 68 +/- 7 (S.D.) kJ/mol. At 10 degrees C, most of the cell-associated asialo-orosomucoid was bound to the cell surface in a reaction which followed ordinary chemical kinetics. Pre-incubation of hepatocytes with a large concentration of unlabelled asialo-orosomucoid did not influence the uptake of subsequently added 125I-asialofetuin; neither was degradation of 125I-asialo-fetuin affected in this experiment. The fractional rate of degradation (the fraction of cell-associated asialo-fetuin which was degraded per unit time) was constant over a twelve-fold range of intracellular asialo-fetuin concentrations. Increasing the temperature from 20 to 30 degrees C produced approximately a ten-fold increase in the rate of degradation of either asialo-fetuin or asialo-orosomucoid. The average activation energies of degradation over the range 20 to 40 degrees C were 125 kJ/mol for asialo-fetuin and 149 kJ/mol for asialo-orosomucoid; however, the Arrhenius plots were not straight lines over this temperature range.     

Temperature dependence of protein degradation, autophagic sequestration and mitochondrial sugar uptake in rat hepatocytes

Lysosomal (propylamine-sensitive) protein degradation as well as the energy-dependent (chymostatin-sensitive) part of the non-lysosomal protein degradation was found to be strongly affected by temperature in isolated rat hepatocytes, the activation energy (Ea) being about 25 kcal/mol for both processes. In contrast, the energy-independent (chymostatin-resistant) part of the non-lysosomal degradation had an Ea of approx. 10 kcal/mol only. Sequestration of electroinjected [14C]sucrose into sedimentable organelles showed a pronounced temperature dependence. By means of digitonin extraction it was possible to distinguish between a moderately temperature-sensitive mitochondrial sugar uptake (Ea approx. 12 kcal/mol) and a strongly temperature-dependent autophagic sequestration (Ea approx. 22 kcal/mol). There was no significant autophagic sequestration below 20 degrees C. The sequestration process is more temperature-sensitive than, for example, the early steps of endocytosis, and is likely to represent the major controlling step in the overall autophagic-lysosomal pathway.     

Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes

Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,     

Evidence that the hepatic asialoglycoprotein receptor is internalized during endocytosis and that receptor recycling can be uncoupled from endocytosis at low temperature

Rat hepatocytes in the continuous presence of [³H]asialo-orosomucoid quickly establish a steady state number of free and occupied surface receptors and rate of endocytosis. These values do not change even though many times more glycoprotein is internalized than there are surface receptors per cell. However, when cells endocytose only one round of surface bound [³H]asialo-orosomucoid at 37°C the internalization of glycoprotein is about 5 times faster than the increase of functional receptors on the cell surface. At 18°C new surface receptors appear at only 6% of the rate of internalization of pre-bound asialoglycoprotein. The results suggest that reutilization of asialoglycoprotein receptors is preferentially inhibited at low temperature and that receptor-ligand complexes enter the cell.     

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.     

The pathways for fluid phase and receptor mediated endocytosis in rat hepatocytes are different but thermodynamically equivalent

In isolated rat hepatocytes fluid phase endocytosis, determined by the uptake of the fluorescent dye lucifer yellow (LY), and receptor mediated endocytosis, determined using a ligand for the asialoglycoprotein receptor (asialo-orosomucoid; ASOR), are different pathways based on their different sensitivities to hyperosmolarity induced by sucrose (Oka and Weigel, J. Cell. Biol. 105, 311a, 1987). LY uptake was unaffected by 0.2 M sucrose at all temperatures tested between 12 degrees and 37 degrees C whereas the uptake of 125I-ASOR was completely inhibited at any temperature. Since the two probes are taken up by different pathways it was possible to determine independently the activation energies (Ea) for the fluid phase versus the receptor mediated coated pit endocytic process. The Ea was 26.4 +/- 3.5 and 25.8 +/- 1.9 kcal/mole for, respectively, receptor mediated and fluid phase endocytosis. These values are not significantly different, and we conclude that the fluid phase and receptor mediated pathways are thermodynamically equivalent even though they are independent.     

Elementary steps in the formation of horseradish peroxidase compound I: direct observation of compound 0, a new intermediate with a hyperporphyrin spectrum

The reaction of horseradish peroxidase (HRP) with H2O2 has been studied in 50% v/v methanol/water over the 25.0 to -36.0 degrees C temperature range by using the low-temperature stopped-flow technique. All reactions were carried out under pseudo-first-order conditions with [H2O2] much greater than [HRP]. Arrhenius plots for the pseudo-first-order rate constant kobs were linear over the 17.6 to -36.0 degrees C temperature range studied with an activation energy of 4.8 +/- 0.5 kcal/mol. Above 0 degrees C, kobs varies linearly with peroxide concentration. However, saturation kinetics are observed below -16.0 degrees C, indicating that there is at least one reversible elementary step in this reaction. Double-reciprocal plots at -26.0 degrees C at pH* 7.3 for the reaction give kappa max(obs) = 163 s-1 and KM = 0.190 mM. Rapid-scan optical studies carried out at -35.0 degrees C with [H2O2] much greater than KM reveal the presence of a transient intermediate referred to as compound 0 whose conversion to compound I is rate limiting. The Soret region of the optical spectrum of compound 0 resembles that of a "hyperporphyrin" with prominent bands near 330 and 410 nm. The temperature dependencies of kappa max(obs) and KM have been measured over the -16.0 to -26.0 degrees C range and give an activation energy for kappa max(obs) of 1.6 +/- 0.7 kcal/mol and an enthalpy of formation for compound 0 of 4.0 +/- 0.7 kcal/mol.     

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).     

Temperature dependence of unitary properties of an ATP-dependent potassium channel in cardiac myocytes.

The temperature dependence of the properties of unitary currents in cultured rat ventricular myocytes has been studied. Currents flowing through an ATP-dependent K+ channel were recorded from inside-out patches with the bath temperature varied from 10 degrees to 30 degrees C. The channel conductance was 56 pS at room temperature (22 degrees C), and the amplitudes of unitary currents and the channel conductance exhibited a relatively weak (Q10 from 1.4 to 1.6) dependence on temperature. The temperature dependence of channel mean open times was biphasic with the low temperature (10-20 degrees C) range showing a relatively stronger temperature dependence (Q10 of 2.3) than the high temperature (20-30 degrees C) range (Q10 of 1.6). The activation energies for the two regions were determined from an Arrhenius plot with the activation energy, corresponding to the lower temperature range, near 16 kcal/mol. Thermodynamic analysis, using transition rate theory, indicated that the formation of a transition state prior to channel closure to be associated with a positive entropy component for the high Q10 region.     

The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes

The function of intracellular asialoglycoprotein receptors during the endocytosis of asialo-orosomucoid in isolated hepatocytes was assessed by following changes in the occupancy of intracellular receptors. Unoccupied total cellular (inside and surface) or surface receptors were quantified at 0 degrees C by the binding of 125I-asialo-orosomucoid in the presence or absence, respectively, of digitonin. Freshly isolated cells had about 17% of their total receptors on the surface. After incubation at 37 degrees C, the receptor distribution changed to 25 to 50% on the cell surface and 50 to 75% inside the cell. At 37 degrees C, the average total number of receptors/cell was 4.5 x 10(5). Dissociation constants, determined from equilibrium binding studies in the presence or absence of digitonin to assess total or surface receptors, were identical (5.4 +/- 1.4 and 5.6 +/- 1.1 x 10(-9) M, respectively). In the presence of asialo-orosomucoid at 37 degrees C, there was both a time- and a concentration-dependent decrease in surface and intracellular receptor activity. This receptor activity decrease was reversed by removing asialo-orosomucoid from the medium or by washing the digitonin-permeabilized cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prior to quantification of receptor activity. Within 1 to 2 h in the presence of excess asialo-orosomucoid, a steady state was attained in which approximately 70% of the intracellular receptors were occupied. The kinetics of receptor activity recovery on the cell surface after internalization of a pulse of ligand is different than the rate of recovery of internal receptor activity. The results suggest that all of the internal asialoglycoprotein receptors are functional and participate during endocytosis. Internal receptors may be functionally equivalent to those on the surface or they may serve a reservoir or routing function for internalized ligand.     

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.     

Effects of temperature on the mitochondrial malate dehydrogenase of adult muscle of Toxocara canis

Purified mitochondrial malate dehydrogenase isoenzyme (m-MDH) of Toxocara canis muscle presented maximum activity at 48 degrees C. A clear change in slope of the Arrhenius plot was observed. The energy of activation calculated for the catalytic process showed values of 3.2 kcal/mol and 10.5 kcal/mol. Thermal inactivation of m-MDH showed that it is more thermolabile than the s-isoenzyme. The inactivation of the enzyme by heat could be reduced at least in part by the addition of 0.1 mM NADH. The heat denaturation showed to be a first-order process. The rate constant (k) was calculated as being of the order of 5.28 X 10(-4) s-1 at 40 degrees C. The activation energy for the heat inactivation process was 16.45 kcal/mol between 30 degrees C and 40 degrees C and 13.79 kcal/mol between 40 degrees C and 48 degrees C.     

The surface content of asialoglycoprotein receptors on isolated hepatocytes is reversibly modulated by changes in temperature

We have investigated the effect of temperature on the content of surface asialoglycoprotein receptors on isolated rat hepatocytes. Receptor was determined by measuring the specific binding of 125I- or [3H] asialo-orosomucoid at 0 degrees C. As reported previously, the receptor number/cell increases 2-3-fold within 30-60 min when freshly isolated cells are warmed from 0-37 degrees C (Weigel, P. H. (1980) J. Biol. Chem. 255, 6111-6120). This increase in receptor number is not inhibited by cycloheximide and also occurs on cells which have first been treated with EDTA to expose a population of cryptic receptors on the cell surface. The rate and extent of the receptor number increase on the cell surface are proportional to the temperature above about 17 degrees C. If cells are first equilibrated at 37 degrees C and then transferred to a lower temperature, the surface receptor number decreases at a rate and to an extent dependent on the temperature. The surface receptor number can be modulated up and down by successive temperature change cycles between 25 and 37 degrees C. In this temperature range, the number of surface receptors/cell is dependent on the final temperature but independent of the pathway to that temperature and is, therefore, a function of state with respect to temperature. The results demonstrate that temperature changes reversibly modulate the number of receptors on the hepatocyte surface. We conclude that, in the absence of ligand, surface receptors can either recycle or can be reversibly internalized or sequestered to prevent access to ligand. The results may also explain why different laboratories have reported a wide range of values for the number of receptors per hepatocyte.     

Temperature dependence of the conductivity of individual potential-dependent K+-channels in mollusk neurons]

Using the patch-clamp method temperature dependences of the chord conductance of single potential--dependent slow and fast K+ channels in mollusk neurons were studied. Under control conditions (20 degrees C, 0 mV, [K+]o = 1.5 mM and [K+]i = 100 mM) the conductances of the fast and slow K+ channels were equal to 20-25 pS and 30-40 pS, respectively. Besides, the temperature dependences of the currents through the K+ channels of lesser conductance (5-20 pS) were studied. Some of these channels may be regarded as subtypes of the fast and slow K+ channels named above. It was found that for the channels of all types single channel currents arise with temperature. However, in the range of 10-20 degrees C an anomalous conductance decrease at temperature elevation was observed. For all channels except for the fast one at temperatures above 20 degrees C activation energy (delta Ea) calculated from the Arrhenius plots of the currents was about 4 kcal/mol. At the temperatures below 10 degrees C delta Ea was equal to about 12 kcal/mol. In this temperature range delta Ea had a pronounced potential dependency. Temperature dependences of the fast K+ channel conductance were opposite to those of the slow K+ channel to some extent.     

Solid phase catalytic hydrogen isotope exchange in dalargin

A [3H]Dalargin preparation with a molar radioactivity of 52 Ci/mmol was obtained by the high temperature solid-state catalytic isotope exchange (HSCIE) of tritium for hydrogen at 150 degrees C. This tritium-labeled peptide was shown to completely retain its biological activity in the test of binding to opioid receptors from rat brain. The dissociation constant of the Dalargin-opioid receptor complex was found to be 4.3 nM. The dependencies of the chemical yield and the molar radioactivity on the reaction time and temperature of HSCIE were determined. The activation energy of the HSCIE reaction for the peptide was calculated to be 32 kcal/mol. The amino acid analysis showed that tritium is distributed between all the amino acid residues of [3H]Dalargin at the HSCIE reaction, with the temperature growth significantly increasing the total tritium incorporation and, especially, enhancing the radioactivity incorporation into aromatic residues.     

Microtubule-depolymerizing agents inhibit asialo-orosomucoid delivery to lysosomes but not its endocytosis or degradation in isolated rat hepatocytes

Microtubule-depolymerizing drugs, such as colchicine, vinblastine sulfate, colcemide and podophyllotoxin, cause an apparent inhibition of the ability of rat hepatocytes to degrade asialo-orosomucoid. However, the binding of asialo-orosomucoid to the cell surface at 0 degrees C, the endocytosis of pre-bound glycoprotein at 37 degrees C, and the dissociation of internal receptor-glycoprotein complexes are unaffected by these microtubule drugs. Receptor recycling is slowed but still occurs, although degradation is blocked. The rate of degradation is decreased by low concentrations of drugs. (For example, 0.25 microM vinblastine sulfate, colchicine and colcemide inhibited 93%, 79% and 26%, respectively.) Neither beta- nor gamma-lumicolchicine affected any of the processes examined. The degree of inhibition with colchicine could be enhanced by a brief treatment of the cells at low temperature to depolymerize microtubules. However, if cells were allowed to endocytose asialo-orosomucoid at 37 degrees C prior to addition of the microtubule drug, then the inhibition of protein degradation was greatly reduced. The decrease in the inhibition of degradation was proportional to the amount of time that cells were exposed to asialoglycoprotein before addition of the drug. The results indicate that the segregation of protein from receptor after they dissociate and/or the subsequent translocation of internalized asialoglycoprotein from the cell perimeter to the lysosomal region requires intact microtubules.     

Characteristics of taurine transport in rat hepatocytes in primary culture

Characteristics of taurine transport in rat hepatocytes maintained in primary culture for 24 h (cultured hepatocytes) have been investigated. The uptake of [3H] taurine by cultured hepatocytes at 2 degrees C was unsaturable, whereas that at 37 degrees C consisted of unsaturable and saturable processes. The saturable transport system was sodium-dependent and consisted of two processes with low and with high affinities. The latter process (Km, 76.9 microM; Vmax, 0.256 nmole/mg protein/min; activation energy (EA), 37.8 kcal mol-1) was competitively inhibited by 2,4-dinitrophenol and ouabain, as well as by taurine analogues such as hypotaurine and guanidinoethyl sulphonate. The Vmax and EA values found in cultured hepatocytes at 37 degrees C were 6.0 and 6.8 times higher than those found in freshly isolated hepatocytes. These results indicate that taurine transport in hepatocytes in primary culture consisted of unsaturable, and saturable, sodium and energy-dependent carrier-mediated transport processes, respectively. The facilitation of the latter transport system by primary culture of hepatocytes is also suggested.     

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.     

Temperature dependence of mammalian muscle contractions and ATPase activities

Isolated rat and mouse extensor digitorum longus (EDL) and soleus muscles were studied under isometric and isotonic conditions at temperatures from approximately 8 degrees -38 degrees C. The rate constant for the exponential rise of tension during an isometric tetanus had a Q10 of approximately 2.5 for all muscles (corresponding to an enthalpy of activation, delta H = 66 kJ/mol, if the rate was determined by a single chemical reaction). The half-contraction time, contraction time, and maximum rate of rise for tension in an isometric twitch and the maximum shortening velocity in an isotonic contraction all had a similar temperature dependence (i.e., delta H approximately 66 kJ/mol). The Mg++ ATPase rates of myofibrils prepared from rat EDL and soleus muscles had a steeper temperature dependence (delta H = 130 kJ/mol), but absolute rates at 20 degrees C were lower than the rate of rise of tension. This suggests that the Mg++ ATPase cycle rate is not limiting for force generation. A substantial fraction of cross-bridges may exist in a resting state that converts to the force-producing state at a rate faster than required to complete the cycle and repopulate the resting state. The temperature dependence for the rate constant of the exponential decay of tension during an isometric twitch or short tetanus (and the half-fall time of a twitch) had a break point at approximately 20 degrees C, with apparent enthalpy values of delta H = 117 kJ/mol below 20 degrees C and delta H = 70 kJ/mol above 20 degrees C. The break point and the values of delta H at high and low temperatures agree closely with published values for the delta H of the sarcoplasmic reticulum (SR) Ca++ ATPase. Thus, the temperature dependence for the relaxation rate of a twitch or a short tetanus is consistent with that for the reabsorption rate of Ca++ into the SR.     

Fatty acid and sterol synthesis by hepatocytes of thermally acclimated rainbow trout (Salmo gairdneri).

Incorporation of tritium from tritiated water into lipid fractions was measured in isolated hepatocytes from rainbow trout (Salmo gairdneri) acclimated to 5 degrees C and 20 degrees C. Hepatocytes from cold-acclimated trout exhibited significantly higher rates of tritium incorporation into both fatty acid and sterol fractions at assay temperatures of 15 degrees C and 20 degrees C than did hepatocytes from warm-acclimated trout. Tritium incorporation into the fatty acid fraction was nearly temperature independent in hepatocytes from warm-acclimated trout (Q10 = 1.39) but markedly temperature dependent (Q10 = 2.63) in hepatocytes from cold-acclimated trout; in contrast, rates of sterol synthesis were more temperature dependent in warm-acclimated trout. At 5 degrees C, fatty acid lipogenesis comprised a significantly greater percentage of the total tritium incorporation in hepatocytes from warm-acclimated trout and the percentage of total lipogenesis attributable to fatty acids decreased significantly in warm-acclimated trout as the assay temperature increased; the opposite trends were observed in cold-acclimated trout.