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.     

Transferrin uptake and release by reticulocytes treated with proteolytic enzymes and neuraminidase.

The mechanism of transferrin uptake by reticulocytes was investigated using rabbit transferrin labelled with 125I and 59Fe and rabbit reticulocytes which had been treated with trypsin, Pronase or neuraminidase. Low concentrations of the proteolytic enzymes produced a small increase in transferrin and iron uptake by the cells. However, higher concentrations or incubation of the cells with the enzymes for longer periods caused a marked fall in transferrin and iron uptake. This fall was associated with a reduction in the proportion of cellular transferrin which was bound to a cell membrane component solubilized with the non-ionic detergent, Teric 12A9. The effect of trypsin and Pronase on transferrin release from the cells was investigated in the absence and in the presence of N-ethylmaleimide which inhibits the normal process of transferrin release. It was found that only a small proportion of transferrin which had been taken up by reticulocytes at 37 degrees C but nearly all that taken up 4 degrees C was released when the cells were subsequently incubated with trypsin plus N-ethylmaleimide, despite the fact that about 80% of the 59Fe in the cells was released in both instances. Neuraminidase produced no change in transferrin and iron uptake by the cells. These experiments provide evidence that transferrin uptake by reticulocytes requires interaction with a receptor which is protein in nature and that following uptake at 37 degrees C, most of the transferrin is located at a site unavailable to the action of proteolytic enzymes. The results support the hypothesis that transferrin enters reticulocytes by endocytosis.     

Transport of AMP by Rickettsia prowazekii.

Rickettsia prowazekii possesses an exchange transport system for AMP. Chromatographic analysis of the rickettsiae demonstrated that transported AMP appeared intracellularly as AMP, ADP, and ATP, and no hydrolytic products appeared in either the intracellular or extracellular compartments. The phosphorylation of AMP to ADP and ATP was prevented by pretreatment of the cells with 1 mM N-ethylmaleimide without inhibiting the transport of AMP. Although no efflux was demonstrable in the absence of nucleotide in the medium, the intracellular adenine nucleotide pool could be exchanged with external unlabeled adenine nucleotides. Both ADP and ATP were as effective as AMP at inhibiting the uptake of [3H]AMP. Although this transport system was inhibited by low temperature (0 degrees C) and partially inhibited by the protonophore carbonyl cyanide-m-chlorophenyl hydrazone (1 mM), it was relatively insensitive to KCN (1 mM). The uptake of AMP at 34 degrees C had an apparent Kt for influx of 0.4 mM and a Vmax of 354 pmol min-1 per mg. At 0 degrees C there was a very rapid and unsaturable association of AMP with these organisms. Correction of the uptake data at 34 degrees C for the 0 degrees C component lowered the apparent Kt to 0.15 mM. Both magnesium and phosphate ions are required for optimal transport activity. Chemical measurements of the total intracellular nucleotide pools demonstrated that this system was not a net adenine nucleotide transport system, but that uptake of AMP was the result of an exchange with internal adenine nucleotides.     

Uptake of 4-toluene sulfonate by Comamonas testosteroni T-2.

The mechanism of transport of the xenobiotic 4-toluene sulfonate (TS) in Comamonas testosteroni T-2 was investigated. Rapid uptake of TS was observed only in cells grown with TS or 4-methylbenzoate as a carbon and energy source. Initial uptake rates under aerobic conditions showed substrate saturation kinetics, with an apparent affinity constant (Kt) of 88 microM and a maximal velocity (Vmax) of 26.5 nmol/min/mg of protein. Uptake of TS was inhibited completely by uncouplers and only marginally by ATPase inhibitors and the phosphate analogs arsenate and vanadate. TS uptake was also studied under anaerobic conditions, which prevented intracellular TS metabolism. TS was accumulated under anaerobic conditions in TS-grown cells upon imposition of an artificial transmembrane pH gradient (delta pH, inside alkaline). Uptake of TS was inhibited by structurally related methylated and chlorinated benzenesulfonates and benzoates. The results provide evidence that the first step in the degradation of TS by C. testosteroni T-2 is uptake by an inducible secondary proton symport system.     

Energy-dependent uptake of 4-chlorobenzoate in the coryneform bacterium NTB-1.

The uptake of 4-chlorobenzoate (4-CBA) in intact cells of the coryneform bacterium NTB-1 was investigated. Uptake and metabolism of 4-CBA were observed in cells grown in 4-CBA but not in glucose-grown cells. Under aerobic conditions, uptake of 4-CBA occurred with a high apparent affinity (apparent Kt, 1.7 microM) and a maximal velocity (Vmax) of 5.1 nmol min-1 mg of protein-1. At pH values below 7, the rate of 4-CBA uptake was greatly reduced by nigericin, an ionophore which dissipates the pH gradient across the membrane (delta pH). At higher pH values, inhibition was observed only with valinomycin, an ionophore which collapses the electrical potential across the membrane (delta psi). Under anaerobic conditions, no uptake of 4-CBA was observed unless an alternative electron acceptor was present. With nitrate as the terminal electron acceptor, 4-CBA was rapidly accumulated by the cells to a steady-state level, at which uptake of 4-CBA was balanced by excretion of 4-hydroxybenzoate. The mechanism of energy coupling to 4-CBA transport under anaerobic conditions was further examined by the imposition of an artificial delta psi, delta pH, or both. Uptake of 4-CBA was shown to be coupled to the proton motive force, suggesting a proton symport mechanism. Competition studies with various substrate analogs revealed a very narrow specificity of the 4-CBA uptake system. This is the first report of carrier-mediated transport of halogenated aromatic compounds in bacteria.     

Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Kt of 506 +/- 79 nM and a Vmax of 17.9 +/- 4.2 pmol min-1 (mg of total cellular protein)-1. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate (I50 = 1.0 microM). The parent compounds of the iodinated photoprobe inhibit [3H]methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a Ki of 66 +/- 21 nM. UV irradiation, at 4 degrees C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated. Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr 38K and 21K) derived from the cell homogenate supernatant.     

Binding sites for angiotensin II in human mononuclear leucocytes.

Angiotensin II binding sites were demonstrated in human mononuclear leucocytes by use of [125I]angiotensin II. The binding of [125I]angiotensin II to mononuclear leucocytes was rapid and reversible. The abilities of unlabeled compounds to displace [125I]angiotensin II were proportional to their abilities to displace labeled hormone in adrenal and smooth muscle membrane preparations. The Scatchard plot revealed two apparent orders of binding sites. The affinity constants were comparable with those for binding sites in other main target tissues of angiotensin II.     

Inhibitory effects of calcium channel blockers on thyroid hormone uptake in neonatal rat cardiomyocytes

The effects of the Ca2+ channel blockers verapamil, nifedipine, and diltiazem on triiodothyronine (T3) and thyroxine (T4) uptake were tested in cultured cardiomyocytes from 2-day-old rats. Experiments were performed at 37 degrees C in medium with 0.5% BSA for [125I]T3 (100 pM) or 0.1% BSA for [125I]T4 (350 pM). The 15-min uptake of [125I]T3 was 0.124 +/- 0.013 fmol/pM free T3 (n = 6); [125I]T4 uptake was 0.032 +/- 0.003 fmol/pM free T4 (n = 12). Neither T3 nor T4 uptake was affected by 1% DMSO (diluent for nifedipine and verapamil). Uptake of [125I]T3 but not of [125I]T4 was dose dependently reduced by incubation with 1-100 microM verapamil (49-87%, P < 0.05) or nifedipine (53-81%, P < 0.05). The relative decline in [125I]T3 uptake after 4 h of incubation with 10 microM verapamil or nifedipine was less than after 15 min or 1 h, indicating that the major inhibitory effect of the Ca2+ channel blockers occurred at the level of the plasma membrane. The reduction of nuclear [125I]T3 binding by 10 microM verapamil or nifedipine was proportional to the reduction of cellular [125I]T3 uptake. Diltiazem (1-100 microM) had no dose-dependent effect on [125I]T3 uptake but reduced [125I]T4 uptake by 45% (P < 0.05) at each concentration tested. Neither the presence of 20 mM K+ nor the presence of low Ca2+ in the medium affected [125I]T3 uptake. In conclusion, the inhibitory effects of Ca2+ channel blockers on T3 uptake in cardiomyocytes are not secondary to their effects on Ca2+ influx but, rather, reflect interference with the putative T3 carrier in the plasma membrane.     

Characterization of a binding protein-dependent glutamate transport system of Rhodobacter sphaeroides.

The mechanism of L-glutamate uptake was studied in Rhodobacter sphaeroides. Uptake of L-glutamate is mediated by a high-affinity (Kt of 1.2 microM), shock-sensitive transport system that is inhibited by vanadate and dependent on the internal pH. From the shock fluid, an L-glutamate-binding protein was isolated and purified. The protein binds L-glutamate (apparent Kd of 1.3 microM) and L-glutamine (Ki of 15 microM) with high affinity. The expression level of this binding protein is maximal at limiting concentrations of glutamine in the growth medium. The glutamate-binding protein restores the uptake of L-glutamate in spheroplasts. L-Aspartate is a strong competitive inhibitor of L-glutamate uptake (Ki of 3 microM) but competes only poorly with L-glutamate for binding to the binding protein (Ki of > 200 microM). The uptake of L-aspartate in R. sphaeroides also involves a binding protein which is distinct from the L-glutamate-binding protein. These data suggest that in R. sphaeroides, the L-glutamate- and L-aspartate-binding proteins interact with the same membrane transporter.     

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 copper by mouse hepatocytes

This study has investigated the uptake of copper by mouse hepatocytes. The cells gave similar results whether they were used right after isolation or maintained overnight on collagen-coated dishes. Uptake from cells in suspension followed two phases: an initial rapid binding followed by a linear uptake phase. The two phases were not so easily distinguishable in cells grown in culture where uptake was linear over the first hour. The uptake showed saturation but may not have followed simple kinetics. Histidine stimulated uptake in a concentration-dependent manner, as did some other amino acids, but copper had very little effect on histidine uptake. The process was not dependent on intracellular adenosine triphosphate (ATP), since inhibitors that substantially reduced ATP levels inside the cell did not alter copper uptake. The inhibitors, however, blocked histidine uptake to varying degrees, suggesting that copper and histidine are taken up by different pathways. The uptake was reduced markedly by N-ethyl maleimide, and preincubation of the cells with "Pronase" resulted in a decrease of uptake. A model for the uptake of copper by hepatocytes that incorporates the data presented in this paper with that produced by earlier workers is suggested.     

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

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

Enzymatic properties of neuraminidases from Arthrobacter ureafaciens.

Neuraminidase I and neuraminidase II from Arthrobacter ureafaciens were characterized. As determined by gel filtration on Ultrogel AcA 44, the molecular weights of neuraminidases I and II were 51,000 and 39,000, respectively. Neuraminidases I and II were similar to each other in their enzymatic properties except for the substrate specificities towards gangliosides and erythrocyte stroma. Their optimal pHs were between 5.0 and 5.5 with N-acetylneuraminosyl-lactose or bovine submaxillary mucin as substrates, but with colominic acid as a substrate, the pH optimum was between 4.3 and 4.5. They were most active around 53 degrees C, were stable between pH 6.0 and 9.0, and were thermostable up to 50 degrees C. They did not require Ca2+ for activity and were not inhibited by EDTA. They were inhibited only slightly or not at all by p-chloromercuribenzoic acid of Hg2+. Both neuraminidases I and II were able to hydrolyze the alpha-ketosidic linkage of N-glycolylneuraminic acid as well as that of N-acetylneuraminic acid, and were able to liberate substantially all of the sialic acid from various kinds of substrates. However, they cleaved only about 50% of the sialic acid from bovine submaxillary mucin. The saponification of bovine submaxillary mucin by mild alkali treatment, on the other hand, resulted in an increased susceptibility to the neuraminidases and brought about the complete liberation of sialic acid. Remarkable differences were observed between neuraminidases I and II as regards substrate specificities on gangliosides; the initial rate of hydrolysis by neuraminidase I was 74 times, and its maximum velocity constant was 91 times those of neuraminidase II. The addition of sodium cholate markedly stimulated the enzymatic hydrolysis of gangliosides, and increased the maximum velocity constant of neuraminidase I twofold and that of neuraminidase II 143-fold. Although neuraminidases I and II were able to hydrolyze (alpha,2-3), (alpha,2-6), and (alpha,2-8) linkages, the initial rate of hydrolysis of N-acetylneuraminosyl-alpha,2-6-lactose was greater than that of the alpha,2-3-isomer.     

Interaction of cations with phosphate uptake by Saccharomyces cerevisiae. Effects of surface potential.

The effect of bivalent cations on phosphate uptake by Saccharomyces cerevisiae was investigated. Phosphate uptake via the Na+-dependent transport system at pH 7.2 is stimulated by bivalent cations. The apparent affinity of phosphate for the transport mechanism is increased, but the apparent affinity for Na+ is decreased. Uptake of phosphate via the Na+-independent transport system is accompanied by a net proton influx of 2H+ and an efflux of 1 K+ for each phosphate ion taken up. At pH 4.5 phosphate uptake via the Na+-independent system is stimulated by bivalent cations, whereas at pH 7.2 uptake is inhibited. The effect of bivalent cations on phosphate uptake can be ascribed to a decrease in the surface potential.     

Transport of steroid hormones facilitated by serum proteins.

The affinities with steroid hormones (alpha-estradiol, ethynylestradiol, progesterone, androsterone, dehydroisoandrosterone and testosterone) were observed for Cohn's fraction IV-1 and V (albumin). It was estimated from the comparison with the binding coefficient K (protein-bound form/free form of hormone) in a 3.5% (w/v) bovine serum albumin (BSA) solution that 40-80% of bound hormone in bovine serum is the BSA-bound form. It becomes clear in a liquid membrane system consisting of a hexane source phase (I), a water phase and a hexane receiving phase (II) that the transport flux of hormone is governed primarily by the partition coefficients between the water/hexane phases. In the case of a hormone with a lower partition coefficient, the uptake process from the hexane phase (I) to the water phase is a rate-determining step in the transport system and the serum proteins accelerate the transport of hormones, while with an increase in the partition coefficient the rate-determining step changes from the uptake step to the release step from the water phase to the hexane phase (II) and the hormone transport is decelerated owing to the significant decrease of free hormone concentration in the aqueous phase by the associated with serum proteins for the system having the restricted amount of hormone in the hexane source phase.     

Effects of Naturally Occurring Aquatic Organic Fractions on Am Uptake by Scenedesmus obliquus (Chlorophyceae) and Aeromonas hydrophila (Pseudomonadaceae)

Naturally occurring organics were extracted from water collected from Skinface Pond near Aiken, S.C. Organics were separated into four nominal diameter size fractions (I, >0.0183; II, 0.0183 to 0.0032; III, 0.0032 to 0.0009; IV, <0.0009 mum) by membrane ultrafiltration and introduced into Scenedesmus obliquus and Aeromonas hydrophila cultures to determine their effects on Am availability for uptake. Effects on Am uptake were determined in actively growing S. obliquus cultures after 96 h of growth and in dense cultures of nongrowing cells after 4 h. Uptake by A. hydrophila was determined after 4 and 24 h in actively growing cultures. All organic fractions stimulated S. obliquus growth, with the most pronounced effects due to larger organic fractions, whereas no apparent growth stimulation of A. hydrophila was observed for any organic fraction. For both long-term and short-term studies, cellular Am concentration (picocuries/cell) increased with increasing Am concentration for S. obliquus and A. hydrophila. Fraction IV increased Am uptake by both S. obliquus and A. hydrophila during 4-h incubations. During 96-h incubations fraction I was flocculated and cosedimented, with S. obliquus and A. hydrophila cells causing an apparent increase in Am uptake. Fractions II and III reduced apparent Am uptake by S. obliquus as a result of biological dilution caused by increased algal growth due to the organics. Fraction IV caused a reduction in Am uptake by S. obliquus not attributable to biological dilution. Organics increased Am uptake by A. hydrophila during 4- and 24-h incubations. A. hydrophila also caused flocculation of fraction I during 96-h incubations.     

Cooperative interaction between factor VII and cell surface-expressed tissue factor

Assembly of the extrinsic pathway on cell surfaces was investigated by studying the binding and activity of factor VII on the bladder carcinoma cell line J82 which expressed 18,800 milliunits of tissue factor activity/10(6) cells. In binding studies, the association of factor VII to monolayers of cells was time-, temperature-, and calcium-dependent. The ligand binding was specific, reversible, and saturable. This interaction was inhibited by a monoclonal antibody to human brain tissue factor. Factor VII added to the cells was recovered as factor VII rather than factor VIIa when incubated in the presence of factor X neutralizing antibodies, suggesting that these cells produced factor X. Specific factor VII binding to the cell revealed a sigmoidal binding isotherm with half-maximal binding occurring at 314 +/- 145 pM to 38,300 +/- 14,300 sites/cell. Hill plots of the binding data indicated an average slope of 2.1. Binding parameters were also determined kinetically. At maximal factor VII-tissue factor complex formation the apparent Km for factor X was 274 nM, the Vmax was 4.15 nM/min, and the kcat was estimated to be 14 s-1. In the presence of excess tissue factor and factor X, increasing amounts of factor VII added to the J82 cells demonstrated a sigmoidal relationship with the rate of factor Xa formation. Hill plots indicated a slope of 2.0 at the lower factor VII concentrations which changed to 1.0 at the higher input amounts of factor VII. Hanes plots were used to determine the apparent dissociation constant of the interaction (222 +/- 85 pM). The Vmax was 5.54 +/- 1.04 nM/min for the cleavage of factor X. These data are consistent with factor VII binding to at least two sites on tissue factor (receptor) with positive cooperativity. Because at saturation the stoichiometry of the factor VII-tissue factor complex is 1:1, tissue factor must be expressed as a dimer on the surface of the J82 cells.     

Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

1. Influx and efflux of l-tri-[(125)I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23 degrees C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3-5pm) influx into cells was saturable; an apparent K(t) value of 8.6+/-1.6pm was obtained. 3. At 5pm-l-tri-[(125)I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23 degrees C) uptake of l-tri-[(125)I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a K(d) value of 6.1+/-1.3pm. 4. Efflux of l-tri-[(125)I]iodothyronine from cells equilibrated with the hormone (5-123pm) was constant up to 21 s; the amount that flowed out was 17.7+/-3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[(125)I]iodothyronine (556-1226pm) 66.8+/-5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[(125)I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18-22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[(125)I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[(125)I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process. Contribution of cytoplasmic proteins to uptake by plasma-membrane vesicles was negligible. The amount of l-tri-[(125)I]iodothyronine required to achieve half-maximal uptake agrees with that occurring in the free form in the blood, conferring physiological importance to the transporting system in the plasma membrane of the liver cell.     

Studies on a wide-spectrum intestinal dipeptide uptake system in the monkey and in the human.

1. The intestinal transport of glycine and leucine residues of glycyl-L-leucine was studied in the monkey and in the human in vitro. Uptake of both [14C]glycyl-L-leuine and glycyl-L-[14C]leucine show similar Kt values, but there is a marked difference in the Vmax. values. Preliminary studies suggest that this anomalous difference in the Vmax. values may be due to the greater efflux rate of glycine from the tissue. 2. Arrhenius plots of both [14C]glycyl-L-leucine uptake and glycyl-L-[14C]leucine uptake in the monkey intestine show a discontinuity at about 20 degrees C. The activation energies above and below the discontinuity are similar for both [14C]glycyl-L-leucine uptake and glycyl-L-[14C]leucine uptake. These similarities in uptake characteristics suggest that the dipeptide glycyl-L-leucine is transported as one unit. 3. In the monkey intestine, glycyl-L-leucine uptake is inhibited by a wide variety of dipeptides, including those containing acidic and basic amino acids. The inhibition was shown to be competitive by using four representative dipeptides namely: L-alanyl-L-alanine, L-alanyl-L-leucine, L-glutamyl-L-glutamic acid and L-lysyl-L-lysine. The results strongly suggest that in the monkey intestine there may be a dipeptide-uptake system with an extremely broad specificity. These results were also confirmed in the human in a limited way.     

Inositolphospholipid-accelerated activation of prekallikrein by activated factor XII and its inhibition by beta 2-glycoprotein I

Inositolphospholipid-accelerated activation of prekallikrein by alpha-factor XIIa was determined by measuring the appearance of kallikrein amidolytic activity towards the chromogenic substrate, D-prolyl-phenylalanyl-arginyl p-nitroanilide (S-2302). The activation reaction did not exhibit normal Michaelis-Menten kinetics. The Hill coefficient was found to be 1.6 indicating that the activation followed an allosteric reaction mechanism. The temperature dependence of the reaction showed a thermal transition at 30 degrees C, which in addition to the allosteric reaction mechanism is indicative of a conformational change of prekallikrein following binding to the inositolphospholipid. The reaction exhibited pH optimum at pH 7.2 and ionic strength optimum at 50 mM NaCl. At optimal conditions the apparent KA value and the kcat/KA value for factor XIIa on prekallikrein were calculated to be 73 nM and 9.3 x 10(6) s-1 M-1, respectively. Kinetic constants could not be calculated at salt concentrations higher than the optimal concentrations, as Lineweaver-Burk plots were curvilinear in agreement with the Hill coefficient greater than unity. The activation was inhibited competitively by beta 2-glycoprotein I with a Ki value of 77 nM as determined by the Dixon plot.