Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.     

Ornithine-σ-Aminotransferase Exhibits Different Kinetic Properties in Astrocytes, Cerebral Cortex Interneurons, and Cerebellar Granule Cells in Primary Culture

To evaluate a possible role of ornithine-delta-aminotransferase (EC 2.6.1.13; Orn-T) as a rate-limiting enzyme for the synthesis of transmitter glutamate and gamma-aminobutyric acid (GABA), respectively, its activity and kinetic properties were analyzed in cultured astrocytes as well as in neuronal cultures consisting mainly of glutamatergic neurons (cerebellar granule cells) or GABAergic neurons (cerebral cortex interneurons). For comparison the activity and kinetics of Orn-T were also assayed in mouse brain homogenates. The highest activity of Orn-T was found in astrocytes and in cerebral cortical neurons (5.3 +/- 0.5 and 5.3 +/- 0.4 nmol X mg-1 X min-1, respectively) whereas the activities of Orn-T in cerebellar granule cell cultures and in mouse brain were found to be about half of these values (3.1 +/- 0.3 and 2.8 +/- 0.1 nmol X min-1 X mg-1, respectively). From a kinetic study of Orn-T in the different preparations only a relatively low affinity for the enzyme with respect to ornithine was found in cerebellar granule cells, astrocytes, and whole brain [apparent Km values (at 0.5 mM alpha-ketoglutarate): 4.7 +/- 0.9, 4.3 +/- 2.2, and 6.8 +/- 2.2 mM, respectively] whereas the corresponding Km value for Orn-T in cerebral cortex interneurons was found to be significantly lower (apparent Km: 0.8 +/- 0.3 mM). The enzyme was not found to be inhibited by GABA (range 0.1 - 10 mM) in any of the preparations.     

Transport of glycyl-L-proline in intestinal brush-border membrane vesicles of the suckling rat: characteristics and maturation

Transport of the dipeptide glycine-L-proline (Gly-L-Pro) in the developing intestine of suckling rats and its subsequent maturation in adult rats was examined using the brush-border membrane vesicles (BBMV) technique. Uptake of Gly-L-Pro by BBMV was mainly the result of transport into the intravesicular space with little binding to membrane surfaces. Transport of Gly-L-Pro in BBMV of suckling rats was: (1) Na+ independent; (2) pH dependent with maximum uptake at an incubation buffer pH of 5.0; (3) saturable as a function of concentration (apparent Km = 21.5 +/- 7.9 mM, Vmax = 8.6 +/- 1.5 nmol/mg protein per 10 s); (4) inhibited by other di- and tripeptides; and (5) stimulated and inhibited by inducing a negative and positive intravesicular membrane electrical potential, respectively. Similarly, transport of Gly-L-Pro in intestinal BBMV of adult rats was saturable as a function of concentration (apparent Km = 17.4 +/- 8.6 mM, Vmax = 9.1 +/- 2.1 nmol/mg protein per 10 s) and was stimulated and inhibited by inducing a relatively negative and positive intravesicular membrane potential, respectively. No difference in the transport kinetic parameters of Gly-L-Pro was observed in suckling and adult rats, indicating a similar activity (and/or number) and affinity of the transport carrier in the two age groups. These results demonstrate that the transport of Gly-L-Pro is by a carrier-mediated process which is fully developed at the suckling period. Furthermore, the process is H+-dependent but not Na+-dependent, electrogenic and most probably occurs by a Gly-L-Pro/H+ cotransport mechanism.     

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.     

Anion/anion exchange in human neutrophils

Of the total one-way chloride fluxes (approximately 1.4 meq/liter cell water X min) in steady state human polymorphonuclear leukocytes bathed in 148 mM Cl media, approximately 70% behaves as self-exchange mediated by a nonselective anion carrier that is not inhibited by stilbene disulfonates. Five properties of this carrier-mediated exchange were investigated: substrate saturation is seen with respect to 36Cl influx as a function of the external Cl concentration [for normal-Cl cells, the apparent Km(Cl) is approximately 22 mM when Cl replaces para-amino- hippurate (PAH) and approximately 5 mM when Cl replaces glucuronate], and with respect to 36Cl efflux as a function of the concentration of internal Cl replacing PAH [apparent Km(Cl) congruent to 35 mM for cells bathed in 148 mM Cl]; there is trans stimulation of 36Cl influx by internal Cl (replacing PAH) with an apparent Km(Cl) congruent to 35 mM, and of 36Cl efflux by external Cl with an apparent Km(Cl) congruent to 22 mM (Cl replacing PAH) or approximately 5 mM (Cl replacing glucuronate); there is substrate competition between Cl and PAH, but the carrier appears devoid of affinity for glucuronate; influxes and effluxes mediated by the carrier are subject to competitive inhibition by extracellular alpha-cyano-4-hydroxycinnamate (CHC), with an apparent Ki congruent to 9 mM in Cl medium or approximately 1 mM in PAH medium (transport of the inhibitor itself is very slow); and internal Cl and external Cl or PAH undergo 1:1 countertransport, which is CHC sensitive. A simple equilibrium-competition model is proposed that accounts for all the extracellular ligand interactions presented for normal-Cl cells. Least-squares values of the carrier's true Michaelis constants for extracellular Cl, PAH, and CHC are 5.03 +/- 0.83, 50.3 +/- 14.9, and 0.29 +/- 0.09 mM, respectively.     

Distinction between the two basic mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.     

Kinetics of 3-O-methyl-D-glucose transport in isolated rat hepatocytes.

3-O-Methyl-D-glucose transport across the plasma membrane of isolated rat hepatocytes was followed for net entry of the sugar into sugar-free cells (zero trans entry), net exit of sugar into sugar-free medium (zero trans exit) and for unidirectional entry and exit fluxes when cells had been equilibrated with sugar in the extracellular medium (equilibrium exchange entry and exit). These measurements were performed at 20 degrees C and pH 7.4 by the use of simple manual methods. Initial rates of transport showed a Michaelis--Menten dependency on the sugar concentration at the cis side of the membrane over the range of concentrations tested (100 microM to 100 mM). Transport was found to be symmetrical with no evidence of substrate stimulation of transport from the trans side of the membrane. Parameters (mean values +/- S.E.M.) of transport were estimated as Vmax. 86.2 +/- 9.7 mmol/litre of cell water per min and Km 18.1 +/- 5.9 mM for exchange entry, Vmax. 78.8 +/- 5.3 mmol/litre of cell water per min and Km 17.6 +/- 3.5 mM for exchange exit, Vmax. 84.1 +/- 8.4 mmol/litre of cell water per min and Km 16.8 +/- 4.6 mM for zero trans exit.     

Purine nucleobase transport in human erythrocytes. Reinvestigation with a novel "inhibitor-stop" assay

A novel "inhibitor-stop" method for the determination of initial rates of purine nucleobase transport in human erythrocytes has been developed, based on the addition of seven assay volumes of cold 19 mM papaverine to terminate influx. In view of our finding that the initial velocities of adenine, guanine, and hypoxanthine influx into human erythrocytes were linear for only 4-6 s at 37 degrees C, the present method has been used to reexamine the kinetics of purine nucleobase transport in these cells. Initial influx rates of all three purine nucleobases were shown to be the result of concurrent facilitated and nonfacilitated diffusion. The nonfacilitated influx rates could be estimated either from the linear concentration dependence of nucleobase influx at high concentrations of permeant or from residual influx rates which were not inhibited by the presence of co-permeants. Appropriate corrections for nonfacilitated diffusion were made to the influx rates observed at low nucleobase concentrations. Kinetic analyses indicated that adenine (Km = 13 +/- 1 microM, n = 7), guanine (Km = 37 +/- 2 microM, n = 5), and hypoxanthine (Km = 180 +/- 12 microM, n = 6) were mutually competitive substrates for transport. The Ki values obtained with each nucleobase as an inhibitor of the influx of the other nucleobases were similar to their respective Km values for influx. Furthermore, the transport of the purine nucleobases was not inhibited by nucleosides (uridine, inosine) or by inhibitors of nucleoside transport (6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, dilazep, dipyridamole). It is concluded that all three purine nucleobases share a common facilitated transport system in human erythrocytes which is functionally distinct from the nucleoside transporter.     

Kinetics of D-glucose and 2-deoxy-D-glucose transport by Rhodotorula glutinis

The yeast Rhodotorula glutinis (Rhodosporidium toruloides) is capable of accumulative transport of a wide variety of monosaccharides. Initial velocity studies of the uptake of 2-deoxy-D-glucose were consistent with the presence of at least two carriers for this sugar in the Rhodotorula plasma membrane. Non-linear regression analysis of the data returned maximum velocities of 0.8 +/- 0.2 and 2.0 +/- 0.2 nmol/min per mg (wet weight) and Km values of 18 +/- 4 and 120 +/- 20 microM, respectively, for the two carriers. Kinetic studies of D-glucose transport also revealed two carriers with maximum velocities of 1.1 +/- 0.4 and 2.4 +/- 0.4 nmol/min per mg (wet weight) and Km values of 12 +/- 3 and 55 +/- 12 microM. As expected, 2-deoxy-D-glucose was a competitive inhibitor of D-glucose transport. Ki values for the inhibition were 16 +/- 8 and 110 +/- 40 microM. These Ki values were in good agreement with the Km values for 2-deoxy-D-glucose transport. D-Xylose, the 5-deoxymethyl analog of D-glucose, appears to utilize the D-glucose/2-deoxy-D-glucose carriers. This pentose was observed to be a competitive inhibitor of D-glucose (Ki values = 0.14 +/- 0.06 and 5.6 +/- 1.6 mM) and 2-deoxy-D-glucose (Ki values = 0.15 +/- 0.07 and 4.6 +/- 1.2 mM) transport.     

Insulin and phorbol esters affect the maximum velocity rather than the half-saturation constant of 3-O-methylglucose transport in rat adipocytes

The kinetics of the equilibrium exchange flux of 3-O-methylglucose (MeGlc) were examined in isolated rat adipocytes using a recently described technique (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899) in which the cells, under basal conditions, were reported to exhibit a high Km (35 mM) that was reduced (to 3 mM) upon treatment with insulin. When this technique was employed in the present study, the Km observed in basal adipocytes was 6.4 +/- 0.4 mM; insulin treatment did not affect this parameter (6.3 +/- 0.5 mM), although it increased the maximum velocity (phi max) 21-fold (from 3.0 +/- 0.3 to 63.7 +/- 1.1 nmol X min-1 X microliter of intracellular water-1). The large discrepancy in the basal Km values observed in the previous (35 mM) and the present (6.4 mM) studies is shown to be associated with relatively minor differences in basal MeGlc flux; these minor differences may reflect insufficient mixing of labeled MeGlc in the flux measurements of the previous study. In addition, the active phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, at a concentration of 0.3 microM, caused a 2.8-fold elevation of phi max, with no modulation of Km. These results indicate that phi max, not Km, is the major kinetic parameter of hexose transport affected by insulin and phorbol esters, leading to enhancement of hexose uptake by the isolated rat adipocyte.     

Brain uptake of pipecolic acid,amino acids,and amines following intracarotid injection in the mouse

The uptake of pipecolic acid by the mouse brain was compared to that of several amino acids and amines, following an injection of a double-labeled mixture into the carotid artery. In general, BUI (brain uptake index) values were lower in the mouse than those previously reported in the rat. The only exception was proline. Lysine, a precursor of pipecolic acid biosynthesis in brain, showed a higher BUI than pipecolic acid. The BUI ofD,l-[³H]pipecolic acid was found to be 3.39 (at 0.114 mM). This was saturable between a concentration of 0.114 and 3.44 mM. Kinetic analysis suggests the presence of two kinds of transport systems. Substances structurally related to pipecolic acid, such as nipecotic acid, isonipecotic acid,l-proline, and piperidine show a significant inhibitory effect. Among the amino acids tested, only GABA showed an inhibitory effect. Data are reported which, when considered with other findings (5), present evidence that pipecolic acid is (1) synthesized both in vitro and in vivo in the mouse brain, (2) actively transported in vivo into the brain, and (3) taken up in vitro by synaptosomal preparations.     

Uptake of GABA and putrescine by UGA4 on the vacuolar membrane in Saccharomyces cerevisiae

The product of the UGA4 gene in Saccharomyces cerevisiae, which catalyzes the transport of 4-aminobutyric acid (GABA), also catalyzed the transport of putrescine. The Km values for GABA and putrescine were 0.11 and 0.69 mM, respectively. The UGA4 protein was located on the vacuolar membrane as determined by the effects of bafilomycin A1 and by indirect immunofluorescence microscopy. Uptake of both GABA and putrescine was inhibited by spermidine and spermine, although these polyamines are not substrates of UGA4. The UGA4 mRNA was induced by exposure to GABA, but not putrescine over 12h. The growth of an ornithine decarboxylase-deficient strain was enhanced by putrescine, and both putrescine and spermidine contents increased, when the cells were expressing UGA4. The results suggest that a substantial conversion of putrescine to spermidine occurs in the cytoplasm even though UGA4 transporter exists on vacuolar membranes.     

Transport of Leucine-Enkephalin Across the Blood-Brain Barrier in the Perfused Guinea Pig Brain

Transport of [tyrosyl-3,5-3H]enkephalin-(5-L-leucine) [( 3H]Leu-enkephalin) across the blood-brain barrier was studied in the adult guinea pig, by means of vascular perfusion of the head in vivo. The unidirectional transfer constant (Kin) estimated from the multiple-time uptake data for [3H]Leu-enkephalin ranged from 3.62 X 10(-3) to 3.63 X 10(-3) ml min-1 g-1 in the parietal cortex, caudate nucleus, and hippocampus. Transport of [3H]Leu-enkephalin was not inhibited by unlabelled L-tyrosine (the N-terminal amino acid) at a concentration as high as 5 mM, or by the inhibitor of aminopeptidase activity bacitracin (2 mM), suggesting that there was no enzymatic degradation of peptide at the blood-brain barrier. By contrast, 2 mM unlabelled Leu-enkephalin strongly inhibited the unidirectional blood-to-brain transport of [3H]Leu-enkephalin by 74-78% in the parietal cortex, caudate nucleus, and hippocampus. The tetrapeptide tyrosyl-glycyl-glycyl-phenylalanine (without the C-terminal leucine of Leu-enkephalin), at a concentration of 5 mM, caused a moderate inhibition ranging from 15 to 29% in the brain regions studied, whereas the tetrapeptide glycyl-glycyl-phenylalanyl-leucine (without the N-terminal tyrosine) at 5 mM was without effect on Leu-enkephalin transport. Unidirectional brain uptake of Leu-enkephalin was not altered in the presence of naloxone at a concentration as high as 3 mM (1 mg/ml), suggesting that there is no binding of Leu-enkephalin to opioid receptors at the blood-brain barrier. It is concluded that there is a specific transport mechanism for Leu-enkephalin at the blood-brain barrier in the guinea pig.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of insulin-stimulated glucose transport in rat adipocytes by nucleoside transport inhibitors

In isolated rat adipocytes, basal as well as insulin-stimulated 3-O-methylglucose transport was inhibited nearly completely (maximal inhibition: 95%) by the nucleoside transport inhibitors dipyridamole (IC50 = 5 microM), nitrobenzylthioguanosine (20 microM), nitrobenzylthioinosine (35 microM) and papaverine (130 microM). Transport kinetics in the presence of 10 microM dipyridamole revealed a significant increase in the transport Km value of 3-O-methylglucose (3.45 +/- 0.6 vs 2.36 +/- 0.29 mM in the controls) as well as a decrease in the Vmax value (4.84 +/- 0.95 vs 9.03 +/- 1.19 pmol/s per microliter lipid in the controls). Half-maximally inhibiting concentrations of dipyridamole were one order of magnitude higher than those inhibiting nucleoside (thymidine) uptake (0.48 microM). The inhibitory effect of dipyridamole (5 microM) reached its maximum within 30 s. The agent failed to affect insulin's half-maximally stimulating concentration (0.075 nM) indicating that it did not interfere with the mechanism by which insulin stimulates glucose transport. Further, dipyridamole fully suppressed the glucose-inhibitable cytochalasin B binding (IC50 = 1.65 +/- 0.05 microM). The data indicate that nucleoside transport inhibitors reduce glucose transport by a direct interaction with the transporter or a closely related protein. It is suggested that glucose and nucleoside transporters share structural, and possibly functional, features.     

Effect of parathyroid hormone and dietary phosphate on phosphate transport in renal outer cortical and outer medullary brush-border membrane vesicles

Two distinctive sodium-dependent phosphate transport systems have been identified in early and late proximal tubules; a high-capacity process located only in outer cortical tissue, and a high affinity present in both outer cortical and outer medullary brush-border membranes (Km 0.1-0.25 mM). A third, sodium-independent, pH gradient-stimulated system (Vmax 4.7 +/- 0.3 nmol.mg-1.min-1, Km 0.15 +/- 0.002 mM) is present in the outer medulla, but absent in outer cortex. Brush-border vesicles were prepared from outer cortical and outer medullary tissue of pigs maintained on low (less than 0.05%), normal (0.4%), or high (4%) phosphate diets. Sodium-dependent phosphate uptake of the high-capacity system decreased (Vmax, 9.4 to 2.2 nmol.mg-1.min-1) from low to high phosphate diet, whereas uptake rates decreased about 50% in the high-affinity system. There were no changes in the respective Km values. The pH gradient-stimulated uptake also decreased (Vmax, 6.9 to 3.0 nmol.mg-1.min-1) with no change in mean Km value (0.15 +/- 0.001 mM) with dietary manipulation. Administration of 1 U parathyroid hormone prior to study resulted in a decrease in sodium-dependent uptake by 40-50% and in pH-dependent uptake (36%) with no change in the respective Km values. In conclusion, the antecedent dietary phosphate intake and parathyroid hormone administration appropriately alters phosphate uptake across the brush-border membrane of all three systems, sodium-dependent and pH gradient-stimulated phosphate transport.     

Interaction of "aza" and "deaza" analogs of adenosine cyclic 3', 5'-phosphate with some enzymes of adenosine cyclic 3', 5'-phosphate metabolism: evidence that the lone pair electrons of N-3 are involved in the binding of adenosine cyclic 3', 5'-phosphate to type II adenosine cyclic 3', 5'-phosphate-dependent protein kinase.

Five hetercyclic analogs of adenosine cyclic 3',5'-phosphate (cyclic AMP) were examined for their ability (1) to stimulate type II cyclic AMP-dependent kinases from bovine brain, bovine heart, and rat liver; (2) to serve as substrates for "high Km" (Km for cyclic AMP = 0.13-0.43 mM) cyclic nucleotide phosphodiesterases from bovine heart, rabbit kidney, and rat liver; and (3) to inhibit the hydrolysis of cyclic AMP catalyzed by "low Km" (Km for cAMP = 0.32-1.5 muM) cyclic nucleotide phosphodiesterases from bovine brain, bovine heart, dog heart, rabbit liver, rat brain and rat liver. The analogs all had a purine ring system which had been modified by replacement of a ring carbon with nitrogen or vice versa to yield 2-aza-cAMP (7-amino-4-beta-D-ribofuranosylimidazo [4,5-d] -v-triazine cyclic 3',5'-phosphate); 8-aza-cAMP (7-amino-3-beta-D-ribofuranosyl-v-triazolo-[4,5-d]-pyrimidine cyclic 3',5'-phosphate); 1 deaza-cAMP (7-amino-3-beta-D-ribofuranosylimidazo [4,5-b[pyridine cyclic 3',5'-phosphate); 3-deaza-cAMP (4-amino-1-beta-D-ribofuranosylimidazo[4,5-c]pyridine cyclic 3',5'-phosphate) and 7-deaza-cAMP (7-amino-4-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidine cyclic 3',5'-phosphate).     

Studies on the thermotropic behavior of aqueous phosphatidylethanolamines

Transport of phosphate has been studied in subconfluent monolayers of LLC-PK1 cells. It was found that this transport system shows similar characteristics to those observed in the kidney. Uptake of phosphate is mediated by a Na+-dependent, substrate-saturable process with an apparent Km value for phosphate of 96 +/- 15 mumol/l. Kinetic analysis of the effect of Na+ indicated that at (pH 7.4) two sodium ions are cotransported with one HOP4(2-) ion (Hill coefficient 1.5) with an apparent Km value for sodium of 56 mmol/l. Pi uptake is inhibited by metabolic inhibitors (ouabain and FCCP). In the pH range of 6.6 of 7.4 Pi uptake rate does not change significantly, indicating that both the monovalent and the divalent form of phosphate are accepted by the transport system. It is suggested that phosphate is transported by LLC-PK1 cells together with sodium (2 Na+:1 HPO4(2-) in an electroneutral manner down a favourable sodium gradient.     

The active transport of 2-keto-D-gluconate in vesicles prepared from Pseudomonas purida.

The transport of 2-keto-D-gluconate (alpha-D-arabino-2-hexulopyranosonic acid; 2KGA) in vesicles prepared from glucose-grown Pseudomonas putida occurs by a saturable process with a Km of 110.0 +/- 2.9 microM and a Vmax. of 0.55 +/- 0.04 nmol X min-1 X (mg of protein)-1. The provision of phenazine methosulphate/ascorbate or L-malate leads to an accumulation of intravescular 2KGA, a decrease in the Km value to 50 +/- 2.1 microM and 35 +/- 2.9 microM respectively and no change in the Vmax. In the presence of electron donors the transport of 2KGA is inhibited by the respiratory poisons antimycin A, rotenone and the uncoupler 2,4-dinitrophenol. 2KGA transport is also competitively inhibited by 4-deoxy-4-fluoro-2-keto- or 3-deoxy-3-fluoro-2-keto-D-gluconate with Ki values of 50 microM and 160 microM respectively. The carrier system for 2KGA is repressed in vesicles from cells grown on succinate. Such vesicles transport 2KGA by non-specific physical diffusion with a Km value of infinity in the absence or presence of electron donors. Vesicles from glucose or succinate grown cells, in the presence of phenazine methosulphate/ascorbate at pH 6.6, generate a proton-motive force (delta p) of approx. 140 mV. The delta p, composed of proton gradient (delta pH) and a membrane potential (delta psi), is collapsed in the presence of dinitrophenol. Based on the results obtained with valinomycin, nigericin and carbonyl cyanide m-chlorophenylhydrazone, the active transport of 2KGA at pH 6.6 is coupled predominately to the delta pH component of delta p.     

Inhibition of the intestinal transport of uracil by hexoses and amino acids

Various hexoses and amino acids were tested as potential inhibitors of the active mucosal to serosal transport of uracil across the everted rat jejunum. Uracil transport displayed Michaelis-Menten type kinetics with a Vmax of 10.4 +/- 0.2 mumol X g-1 X h-1 and an apparent Km of 0.047 +/- 0.002 mM (means +/- S.D.). Scilliroside, an inhibitor of the basolateral (Na+ + K+)-ATPase, dose-dependently inhibited the transport of uracil consistent with the Na+ dependency of uracil transport. Thymine was a full competitive inhibitor (Ki = 0.021 +/- 0.002 mM) of uracil transport. All actively transported substances tested including L-phenylalanine, L-leucine, D-galactose, D-glucose, and 3-O-methylglucose inhibited the transport of uracil. In contrast, L-glucose and fructose, substances which are not actively transported, were without effect on uracil transport. Further studies with D-galactose indicated that it acts as a partial noncompetitive inhibitor (Ki = 6.0 +/- 1.4 mM) of uracil transport. This Ki is in good agreement with the apparent Kt (5.8 +/- 1.1 mM) for D-galactose transport. Phlorizin (0.1 mM), an inhibitor of galactose transport, blocked the inhibitory effect of galactose on uracil transport. In the ileum D-galactose had no effect on uracil transport but thymine caused the same degree of inhibition as in the jejunum. The results demonstrate that heterologous inhibition is a more general phenomenon than had previously been realized.     

Characterization of leukotriene C4 synthetase in mouse peritoneal exudate cells

Cell lysates of mouse peritoneal macrophages, in the presence of reduced glutathione, converted leukotriene LTA4 to LTC4, and neither LTD4 nor LTE4 was detected. Therefore, like cultured rat basophilic leukemia cells (RBL cells), the peritoneal macrophage contains LTC4 synthetase and appears to contain little, if any, gamma-glutamyl transpeptidase. When LTA4 was added to subcellular fractions of mouse macrophage lysate, the highest specific activity of LTC4 synthetase (nmol LTC4/mg protein per 10 min) was associated with the particulate or membrane fractions (i.e., 10(4) and 10(5) X g pellets). The 10(5) X g supernatant contains approx. 1% of the specific activity and 6% of the total LTC4 synthetase activity compared with that of the 10(5) X g pellet. Conversely, the 10(5) X g supernatant had four-times more specific activity and 19-times more total GSH S-transferase activity than did the 10(5) X g pellet when evaluated using 1-chloro-2,4-dinitrobenzene (DNCB) as the substrate. LTA4 was converted to LTC4 by the membrane enzyme LTC4 synthetase in a dose-dependent manner at low LTA4 concentrations (3-50 microM) and reached a plateau of approx. 30 microM LTA4 using the macrophage 10(5) X g pellet as an enzyme source. The apparent Km value of LTC4 synthetase for LTA4 was estimated to be 5 microM based on Lineweaver-Burk plots. Enzyme in the 10(5) X g supernatant produced negligible quantities of LTC4 (1% or less of the particulate fractions) over a wide range of LTA4 concentrations. However, an enzyme in the 10(5) X g supernatant fraction presumed to be GSH S-transferase effectively catalyzes the conjugation of glutathione (GSH) with the aromatic compound DNCB. The apparent Km value of GSH S-transferase for DNCB was estimated to be 1.0-1.5 mM. On the other hand, enzyme from the membrane fraction (i.e., 10(5) X g pellet) catalyzed this reaction at a negligible rate over a wide range of DNCB concentrations. The apparent Km value of LTC4 synthetase for GSH was estimated to be 0.36 mM and the corresponding Km value estimated for the glutathione S-transferase was 0.25-0.76 mM. These values indicate similar kinetics for GSH utilization by both enzymes. These Km values are also significantly lower than the intracellular GSH levels of 2 to 5 mM. Therefore, it is suggested that the substrate limiting LTC4 synthetase activity is LTA4 and not GSH. Our results indicate that LTC4 synthetase from mouse peritoneal macrophages is a particulate or membrane-bound enzyme, as was reported by Bach et al.(ABSTRACT TRUNCATED AT 400 WORDS)