Diacytosis of 125I-asialoorosomucoid by rat hepatocytes. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37 degrees C for 30 min or 18 degrees C for 2 h, washing free of cell surface receptor-bound tracer at 4 degrees C and then reincubating at 37 degrees C. The cells preloaded at 37 degrees C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min-1 (t1/2 = 39 min). When the preloaded cells were incubated in the presence of 100 micrograms/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 microM colchicine, 20 microM cytochalasin B, 20 microM chloroquine, 10 mM NH4Cl, 10 microM monensin or 20 microM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoorosomucoid were observed with cells preloaded at 18 degrees C. These results indicate that diacytosis of 125I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.     

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.     

Transferrin endocytosis and the mechanism of iron uptake by reticulocytes in the toad (Bufo marinus)

Reticulocytosis was induced in toads (Bufo marinus) by treatment with phenylhydrazine. Iron and transferrin uptake and transferrin endocytosis and exocytosis by these cells were measured. The mean number of transferrin receptors per cell was found to be 4.5 X 10(5) and the affinity constant of transferrin to receptors was 0.2 X 10(7) M-1. Iron and transferrin uptake were temp.-dependent processes. An inflection point occurred at 15-16 degrees C in the Arrhenius plots of endocytosis and iron uptake. The activation energies of these two processes above and below the inflection temperature were 31 and 71 kJ/mol. It is concluded that iron uptake by immature toad erythroid cells occurs by receptor-mediated endocytosis which still functions at temps as low as 5 degrees C.     

Endocytosis and subsequent processing of 125I-labelled immunoglobulin G by guinea pig yolk sac in vitro.

We have developed conditions for studying the binding, uptake, degradation and transport of 125I-labelled IgG by yolk sac in vitro. Specific binding to tissue at 4 degrees C and to paraformaldehyde-treated tissue at 37 degrees C was time- and temperature-dependent and showed saturation kinetics (Kd,4 degrees C = 2.9 X 10(-6) M, Kd,37 degrees C = 5.3 X 10(-6) M). Uptake was studied at 37 degrees C using untreated tissue (K uptake = 13.3 X 10(-6) M) and was inhibited by preincubation with metabolic poisons but not with cycloheximide. Tissue that had been incubated with 125I-labelled IgG at 37 degrees C released radiolabelled degradation products and intact 125I-labelled IgG into the medium. Experiments with paraformaldehyde-treated and untreated tissue showed that release of intact 125I-labelled IgG was mostly the result of ligand dissociation from surface binding sites. However, more 125I-labelled IgG was released from untreated tissue than could be accounted for solely by loss of surface-bound ligand and the difference was presumed to reflect uptake, transport and exocytosis of 125I-labelled IgG. Degradation of 125I-labelled IgG was inhibited by leupeptin and lysosomotropic amines. These drugs had no detectable effect on 125I-labelled IgG release. The results suggest that degradation and transport of IgG are not intimately related and are consistent with a previously proposed model for IgG transport via coated vesicles which do not fuse with lysosomes and for non-selective uptake into another class of vesicle which does fuse with lysosomes.     

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. Dissociation of internalized ligand from receptor occurs in two kinetically and thermally distinguishable compartments

We have examined the rate of dissociation of internalized 125I-asialo-orosomucoid-receptor complexes in freshly isolated rat hepatocytes. Cell suspensions were washed with ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 0 degrees C to remove surface-bound ligand and then assessed for the retention of radioactive glycoprotein in the presence of digitonin, which permeabilized the cells and released the internal soluble contents. In cells which initially contained only surface-bound ligand, about 50% of the internalized ligand dissociated from receptor very rapidly (t1/2 less than or equal to 2.5 min, k greater than or equal to 0.28 min-1), at 37 degrees C, whereas the other 50% dissociated more slowly with apparent first order kinetics (t1/2 = 50 min, k = 0.014 min-1). This equal distribution of internalized ligand into two compartments, from which dissociation occurred with very different kinetics, did not depend on the extent of surface receptor occupancy and also occurred under non-steady state conditions of continuous exposure to ligand. Ligand entering both the rapid and slow dissociation compartments was eventually degraded with apparent first order kinetics (k = 0.0047 min-1), suggesting that the intracellular routing of ligand to lysosomes after dissociation from either compartment was via the same pathway. The fast and slow dissociation of receptor-ligand complexes were also distinguished by different temperature sensitivities; the slow dissociation process ceased below 18 degrees C, whereas the fast dissociation process still proceeded. The equal partition of internalized complexes into the two kinetic compartments did not change as a function of temperature but did change as cells continued to endocytose asialo-orosomucoid at 37 degrees C. As the internal receptor pool approached a steady state level of occupancy, there was an increase in the average time for receptor recycling and an increase in the fraction of incoming receptor-ligand complexes which dissociated rapidly (approximately 75%). In addition, under steady state conditions, the rate of the slow dissociation process increased (k = 0.026 min-1, t1/2 = 27 min).     

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.     

Binding and processing of (125)I-ACTH by isolated rat splenic lymphocytes

The effect of incubation temperature and ligand competition was tested for (125)I-ACTH binding to isolated rat lymphocytes. AlphaMSH but not Agouti-like peptide was an effective competitive inhibitor for cell surface binding at 4 degrees C. Cells incubated with (125)I-ACTH at 37 degrees C rapidly associated ligand for 10 min and then gradually lost the radioactivity with time. Cells incubated with (125)I-ACTH at 4 degrees C accumulated ligand to only about half the maximal amount when compared to cells incubated at 37 degrees C for 10 min. Temperatures below 20 degrees C and toxins that block lysosomal degradation blocked the loss of cell-associated radioactivity. These results suggest the lymphocyte ACTH receptor is the Melanocortin 5 receptor and the receptor is internalized by endocytosis to deliver ligand to the lysosome.     

Intracellular transport of formaldehyde-treated serum albumin in liver endothelial cells after uptake via scavenger receptors.

Endocytosis of formaldehyde-treated serum albumin (FSA) mediated by the scavenger receptor was studied in rat liver endothelial cells. Suspended cells had about 8000 receptors/cell, whereas cultured cells had about 19,000 receptors/cell. Kd was 10(-8) M in both systems. Cell-surface scavenger receptors were found exclusively in coated pits by electron microscopy, by using ligand labelled with colloidal gold. Cell-surface-bound FSA could be released by decreasing the pH to 6.0; it was therefore possible to assess the rate of internalization of surface-bound ligand. This rate was very high: t1/2 for internalization of ligand prebound at 4 degrees C was 24 s. The endocytic rate constant at 37 degrees C, Ke, measured as described by Wiley & Cunningham [(1982) J. Biol. Chem. 257, 4222-4229], was 2.44 min-1, corresponding to t1/2 = 12 s. Uptake of FSA at 37 degrees C after destruction of one cell-surface pool of receptors by Pronase was decreased to 60%. This finding is compatible with a relatively large intracellular pool of receptors. The intracellular handling of 125I-tyramine-cellobiose-labelled FSA (125I-TC-FSA) was studied by subcellular fractionation in sucrose gradients, Nycodenz gradients or by differential centrifugation. The density distributions of degraded and undegraded 125I-TC-FSA after fractionation of isolated non-parenchymal cells and whole liver were similar, when studied in Nycodenz and sucrose gradients, suggesting that the subcellular distribution of the ligand was not influenced by the huge excess of non-endothelial material in a whole liver homogenate. Fractionation in sucrose gradients showed that the ligand was sequentially associated with organelles banding at 1.14, 1.17 and 1.21 g/ml. At 9-12 min after intravenous injection the ligand was in a degradative compartment, as indicated by the accumulation of acid-soluble radioactivity at 1.21 g/ml. A rapid transfer of ligand to the lysosomes was also indicated by the finding that a substantial proportion of the ligand could be degraded by incubating mitochondrial fractions prepared 12 min after intravenous injection of the ligand. The results indicate that FSA is very rapidly internalized and transferred through an endosomal compartment to the lysosomes. The endosomes are gradually converted into lysosomes between 9 and 12 min after injection of FSA. The rate-limiting step in the intracellular handling of 125I-TC-FSA is the degradation in the lysosomes.     

Degradation of asialoglycoproteins mediated by the galactosyl receptor system in isolated hepatocytes. Evidence for two parallel pathways

The ability of rat hepatocytes to degrade internalized surface-bound 125I-asialoorosomucoid (ASOR) was determined by measuring the appearance of acid-soluble radioactivity at 37 degrees C. The degradation kinetics were biphasic in cells previously equilibrated at 37 degrees C for 1 h or cultured for 24 h. Degradation began immediately and was linear for at least 20 min after which the rate increased to a steady state value 3-4 times greater than the initial rate. We previously showed that hepatocytes have two functionally distinct populations of galactosyl receptors that mediate ligand dissociation by two kinetically different pathways (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). The activity of one receptor population, designated State 2 galactosyl receptors, can be reversibly modulated by incubating cells between 22 and 37 degrees C and is not expressed on the surface of freshly isolated cells. When 125I-ASOR was prebound to freshly isolated cells at 4 degrees C and degradation was assessed subsequently at 37 degrees C, the kinetics were monophasic, not biphasic. Degradation of the surface-bound 125I-ASOR began immediately and was greater than 90% complete by 6 h. Freshly isolated cells were incubated at temperatures between 22 and 37 degrees C, chilled to 4 degrees C, allowed to pre-bind 125I-ASOR, and then incubated at 37 degrees C. As the State 2 galactosyl receptor population increased, the kinetics of degradation became progressively more biphasic and the rate of the delayed degradation process increased. This effect could be reversed in cells in culture or in suspension by down-modulating surface receptor activity at temperatures below 37 degrees C; only the degradation process appearing after a 20-min lag was affected. Degradation in both pathways is an apparent first order process with identical rate constants (kappa = 0.006 min-1, t1/2 = 116 min). We conclude that there are two separate pathways by which asialoglycoproteins are degraded. The major "classic" pathway mediated by State 2 galactosyl receptors occurs after a 20-min lag and the minor pathway mediated by State 1 galactosyl receptors begins immediately with no detectable lag.     

In vivo exocytosis of lysosomes by the endothelium of the venous sinuses of bone marrow and liver: visualization at normal and low body temperature

We have visualized the exocytosis of lysosomes into the peripheral circulation by the phagocytic endothelia of the venous sinuses of liver and bone marrow of rats. Perfusion fixation at normal body temperature produced images of the earliest stages of lysosomal exocytosis. After fixation at low body temperatures (7-12 degrees C), advanced stages of this process became evident, showing extrusion of lysosomes and their contents into the circulation. It is postulated that this form of exocytosis has escaped structural detection because of its rapidity and relative infrequency as compared to merocrine secretory exocytosis, and that fixation at low body temperatures arrests or slows down these exocytic events in sufficient measure for ultrastructural visualization. The possibility that this lysosomal exocytosis contributes to the presence of lysosomal enzymes detected in the peripheral blood should be considered. In addition, it is likely that lysosomal degradation products may be discharged by exocytosis into the circulation.     

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

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 and destruction of 125I-CSF-1 by peritoneal exudate macrophages

The binding and uptake of the colony-stimulating factor CSF-1 by peritoneal exudate macrophages (PEM) from lipopolysaccharide insensitive C3H/HeJ mice was examined at 2 degrees C, and at 37 degrees C. At 2 degrees C, 125I-CSF-1 was bound irreversibly to the cell surface. At 37 degrees C, 90% of the cell surface associated 125I-CSF-1 was rapidly internalized and subsequently degraded and the remaining 10% dissociated as intact 125I-CSF-1. Thus classical equilibrium or steady state methods could not be used to quantitatively analyze ligand-cell interactions at either temperature, and alternative approaches were developed. At 2 degrees C, the equilibrium constant (Kd less than or equal to 10(-13) M) was derived from estimates of the rate constants for the binding (kon congruent to 8 x 10(5) M-1 s-1) and dissociation (koff less than or equal to 2 x 10(-7) s-1) reactions. At 37 degrees C, the processes of dissociation and internalization of bound ligand were kinetically competitive, and the data was formally treated as a system of competing first order reactions, yielding first order rate constants for dissociation, koff = 0.7 min-1 (t1/2 = 10 min) and internalization, kin = 0.07 min-1 (t 1/2 = 1 min). Approximately 15 min after internalization, low-molecular weight 125I-labeled degradation products began to appear in the medium. Release of this degraded 125I-CSF-1 was kinetically first order over three half-lives (Kd = 4.3 x 10(-2) min-1, t1/2 = 16 min). Thus CSF-1 binds to a single class of receptors on PEM, is internalized with a single rate limiting step, and is rapidly destroyed without segregation into more slowly degrading intracellular compartments.     

Rabbit's ear in cold acclimation studied on the change in ear temperature.

The role of the rabbit's ear in cold acclimation was studied by varying the temperature of a climatic room in the range from -10 to +30 degrees C; The skin temperature in a nonanesthetized rabbit's ear showed a characteristic response to changes in ambient temperatures; plotting the ear temperature against the ambient temperature yielded an S-shaped curve. The mean ambient temperature corresponding to the inflection point on the S-shaped curve shifted significantly from about 13 degrees C to about 8 degrees C after cold acclimated of a group fed for 7 wk at -10 degrees C. The shift of the S-shaped curve after cold acclimation may not be due to the change in the norepinephrine sensitivity of the vascular beds of the ear: the effect of norepinephrine on the pressure-flow curve in the isolated rabbit's ear was almost unchanged between the control and the cold-acclimated groups. It is proposed that the shift of the inflection point gives a qualitative index of the acclimated state of the rabbit at a particular temperature.     

The effect of gibberellic acid on the response of leaf extension to low temperature

The effect of cooling on leaf extension rate (LER) and on relative elemental growth rate (REGR) was measured in both gibberellic acid (GA)-responsive dwarf barley and in the same barley variety treated with GA. Seedlings were maintained at 20 degrees C while their leaf extension zone (LEZ) temperature was reduced either in steps to -6 degrees C in short-term cooling experiments, or to 10 degrees C for 48 h in long-term cooling experiments. Short-term cooling resulted in a biphasic response in LER, with a clear inflection point identified. Below this point, the activation energy for leaf extension becomes higher. The short-term response of LER to cooling was altered by the application of GA, which resulted in a lower base temperature (Tb), inflection point temperature and activation energy for leaf extension. Both GA-treated and untreated seedlings were less sensitive to cooling maintained for a prolonged period, with LER making a partial recover over the initial 5 h. Although long-term cooling reduced maximum REGR, it resulted in a longer LEZ and an increase in the length of mature interstomatal cells in GA-treated and untreated seedlings. These changes in overall physiology appear to enhance the ability of the leaves to continue expansion at suboptimal temperatures. In both GA-treated and cold-acclimated tissue, the occurrence of a longer LEZ was associated with a lower temperature sensitivity in LER.     

Effects of temperature on the degradation and biliary secretion of asialoorosomucoid by the perfused rat liver: evidence for two intracellular pathways

We have utilized the in situ perfused rat liver under nonrecirculating conditions to examine the effect of temperature on the metabolism and biliary secretion of [125I]-asialoorosomucid (ASOR). In this manner we were able to follow the fate of a single round of internalized ligand. In control livers perfused at 37 degrees C, approximately 50% of [125I]-ASOR injected into the portal vein was extracted on first pass. Five minutes after the injection, radioactivity, which had been extracted initially, began to appear in the hepatic venous effluent. Within 25 min, 50% of the initially extracted radioactivity was released into the perfusion medium; the bulk of this radioactivity (greater than 95%) was soluble in trichloroacetic acid. In livers perfused at temperatures slightly less than 37 degrees C (30-35 degrees C), first-pass extraction of [125I]-ASOR was similar to that observed at 37 degrees C. However, a severalfold decrease in the rate of release of radioactivity from the liver into the perfusion medium was noted at the lower perfusion temperatures; whereas greater than 50% of the initially extracted radioactivity was released within 30 min from livers perfused at 37 degrees C, only 5% was released at 30 degrees C. At the lower perfusion temperature, a larger proportion of the released radioactivity was acid precipitable (24% vs. 5%). Some radioactivity also was recovered in the bile; of the total amount of radioactivity released from the liver in 30 min at 37 degrees C, approximately 5% was directed into the bile. At lower temperatures of perfusion, a greater fraction of the radioactivity that was released from the liver was directed into the bile (20% at 30 degrees C vs. 5% at 37 degrees C). The data imply that the endosomal pathway to the lysosome is highly sensitive to slight reductions in temperature while the transcytotic route into bile is less sensitive. Lower temperatures might prolong the residence time of ASOR in the prelysosomal endosomal compartments, and thereby increase the likelihood that undegraded ligand will be returned to the blood or be missorted into bile.     

Receptor-mediated endocytosis of tissue-type plasminogen activator by the human hepatoma cell line Hep G2

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) was characterized with the human hepatoma cell line Hep G2. At 4 degrees C binding of 125I-t-PA to Hep G2 cells is rapid, specific, saturable, and reflective of a homogeneous population of 76,000 high-affinity surface sites per cell (Kd = 3.7 nM). The kinetics of 125I-t-PA binding to its receptor are characterized by rate constants for association (k1 = 1.2 x 10(6) min-1 M-1) and dissociation (k-1 = 0.001 min-1). A specific glycosylation pattern does not appear to be required for binding. Binding does not appear to be mediated by other recognized hepatic receptor systems. At 37 degrees C a single cohort of bound 125I-t-PA molecules disappears rapidly from the cell surface. Ligand then accumulates intracellularly. Thereafter, the intracellular concentration of ligand declines simultaneously with the release of ligand degradation products into the media. In the continued presence of 125I-t-PA at 37 degrees C the concentration of cell-associated ligand plateaus after 30 min with the concomitant appearance of low molecular weight 125I-labeled fragments in the media. Cumulative degradation then increases linearly with time. Under steady state conditions half-maximal ligand uptake and degradation is 26.6 nM and maximal rate of catabolism is 1.2 pmol/10(6) cells/h. At saturating ligand concentrations uptake and degradation by Hep G2 cells continue linearly for at least 6 h even in the absence of protein synthesis. During this period the cumulative ligand uptake exceeds the total cellular capacity of binding sites, consistent with receptor recycling. We conclude that t-PA clearance in human Hep G2 cells involves ligand binding, uptake, and degradation mediated by a novel high-capacity, high-affinity specific receptor system.     

Fluorescent glucagon derivatives. II. The use of fluorescent glucagon derivatives for the study of receptor disposition in membranes

When monolayer cultured hepatocytes were incubated with 1 nM [125I]glucagon at 30 degrees C, equilibrium was reached after 10 min, whereas at 4 degrees C, equilibrium was reached after 60 min. At the higher temperature, 11.2% of the bound ligand was broken down after 60 min, at the lower temperature, the amount of degradation was negligible. At 30 degrees C, acid-washing did not remove specifically bound ligand; thus, it was assumed that the ligand was internalised at this temperature, since some of the specifically bound ligand could be washed off at lower temperatures. This was confirmed in experiments when monolayer cultures of hepatocytes were incubated with fluorescein-labelled derivatives of glucagon. The distribution of specific binding on the cell surface was studied at both 30 and 4 degrees C using video intensification microscopic techniques. In keeping with studies using radiolabelled glucagon, more fluorescence was detected following incubation at 4 degrees C than at 30 degrees C and it could be removed by washing the cells. Video intensification microscopy indicated that at the lower temperature, the bound ligand was distributed all over the cell surface. At the higher temperature, ligand-derived fluorescence could only be detected in mobile intracellular vesicles.     

Catalytic efficiency and some structural properties of cold-active protein-tyrosine-phosphatase

A procedure was established for expression and purification of abundant recombinant cold-active protein-tyrosine-phosphatase (RCPTPase), which showed identical enzymatic characteristics to the native enzyme (NCPTPase). The purified RCPTPase showed high catalytic activity at low temperature and maximal activity at 30 degrees C. RCPTPase has a thermodynamic characteristic in that its activation enthalpy was determined to be low, 4.3 kcal/mol, at temperatures below 19.3 degrees C, where the Arrhenius relationship exhibited an inflection point, in comparison with 20.3 kcal/mol above 19.3 degrees C. Also, the thermostability, DeltaG(water), of the catalytic site in the RCPTPase molecule was increased with a decrease in temperature. It was considered that cold-active protein-tyrosine-phosphatase could maintain its catalytic site in a stable conformation for eliciting high catalytic activity with low activation enthalpy at low temperature.     

Reduction of DNA-polymerase beta activity of CHO cells by single and combined heat treatments

The effect of single and combined heat treatments on the activity of DNA polymerase beta was studied in CHO cells. The activity of polymerase beta was determined by measuring the amount of [3H]TTP incorporated into activated calf thymus DNA in the presence of aphidicolin, a specific inhibitor of DNA polymerase alpha. Biphasic response curves were obtained for all temperatures tested (40-46 degrees C) showing the sensitivity to decrease during heating. A constant activation energy of Ea = 120 +/- 10 kcal/mole was found for the initial heat sensitivity, whereas the Arrhenius plot for the final sensitivity is characterized by an inflection point at 43 degrees C with Ea = 360 +/- 40 kcal/mole or Ea = 130 +/- 20 kcal/mole for temperatures below or above 43 degrees C, respectively. The observed decrease of the polymerase activity is not due to a decrease in the number of active enzyme molecules but to a change in its affinity, since the inhibition is reversible when increasing concentrations of TTP are applied. When acute or chronic thermo-tolerance was induced by a priming heat treatment at 43 degrees C for 45 min followed by a time interval at 37 degrees C for 16 h or by a preincubation at 40 degrees C for 16 h, respectively, the thermal sensitivity of polymerase beta was lowered by a factor of up to 5. By contrast, pretreatment at a higher temperature followed by a lower temperature (step-down heating) did not alter the sensitivity of polymerase beta to the second treatment. The results indicate that heat-induced cell death cannot be the consequence of the reduction of the polymerase beta activity, confirming earlier studies on this subject.