Sodium-alanine cotransport in renal proximal tubule cells investigated by whole-cell current recording

Sodium-alanine cotransport was investigated in single isolated proximal tubule cells from rabbit kidney with the whole-cell current recording technique. Addition of L-alanine at the extracellular side induced an inward-directed sodium current and a cell depolarization. The sodium-alanine cotransport current was stereospecific and sodium dependent. Competition experiments suggested a common cotransport system for L-alanine and L-phenylalanine. Sodium-alanine cotransport current followed simple Michaelis-Menten kinetics, with an apparent Km of 6.6 mM alanine and 11.6 mM sodium and a maximal cotransport current of 0.98 pA/pF at -60 mV clamp potential. Hill plots of cotransport current suggested a potential-independent coupling ratio of one sodium and one alanine. The apparent Km for sodium and the maximal cotransport current were potential dependent, whereas the apparent Km for L-alanine was not affected by transmembrane potential. The increase in Km for alanine with decreasing inward-directed sodium gradients suggested a simultaneous transport mechanism. These results are consistent with a cotransport model with potential-dependent binding or unbinding of sodium (high-field access channel) and a potential-dependent translocation step.     

Sodium dependence of neutral amino acid uptake into rabbit ileum

Alanine uses two mediated pathways to enter the rabbit ileal mucosa. Present results suggest that one of them (K_m = 4.1 mM) is fully dependent on sodium in the mucosal medium, while the other (K_m = 91 mM) is sodium-independent. Similar results are obtained for methionine and serine. Reinterpretation of previous alanine/sodium coupling coefficients suggests that two sodium ions per alanine molecule are transported via the high affinity system.     

Isolation and characterization of mitochondrial alanine aminotransferase from porcine tissue.

Mitochondrial alanine aminotransferase L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) has been isolated in homogeneous form from both porcine liver and kidney cortex, but in low yield. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate or 8 M urea gave a single band. An isoelectric point of 8.5 +/- 0.5 and a molecular weight of 75--80 000 were obtained. The enzyme is specific for L-alanine and is inhibited by D-alanine, aminooxyacetate and cyclosterine. The Km for pyruvate and glutamate is 0.4 mM and 32 mM, respectively. These values are similar to those determined for the cytoplasmic enzyme; however, at high concentrations, both compounds strongly inhibit the mitochondrial enzyme, an inhibition not observed with cytosolic alanine aminotransferase. These characteristics and the fact that the mitochondrial alanine aminotransferase was inactivated by procedures effective in the preparation of the cytosolic enzyme, clearly differentiate the two proteins and further support different roles for the two alanine aminotransferases in vivo.     

Investigations into enzymes of nitrogen metabolism of the ectomycorrhizal basidiomycete, Suillus bovinus

Axenic mycelia of the ectomycorrhizal basidiomycete, Suillus bovinus, were grown in liquid media under continuous aeration with compressed air at 25 degrees C in darkness. Provided with glucose as the only carbohydrate source, they produced similar amounts of dry weight with ammonia, with nitrate or with alanine, 60-80% more with glutamate or glutamine, but about 35% less with urea as the respectively only exogenous nitrogen source. In crude extracts of cells from NH4(+)-cultures, NADH-dependent glutamate dehydrogenase exhibited high aminating (688 nmol x mg protein(-1) x min(-1)) and low deaminating (21 nmol x mg protein(-1) x min(-1)) activities. Its Km-values for 2-oxoglutarate and for glutamate were 1.43 mM and 23.99 mM, respectively. pH-optimum for amination was about 7.2, that for deamination about 9.3. Glutamine synthetase activity was comparatively low (59 nmol x mg protein(-1) x min(-1)). Its affinity for glutamate was poor (Km = 23.7 mM), while that for the NH4+ replacing NH2OH was high (Km = 0.19 mM). pH-optimum was found at 7.0. Glutamate synthase (= GOGAT) revealed similar low activity (62 nmol x mg protein(-1) x min(-1)), Km-values for glutamine and for 2-oxoglutarate of 2.82 mM and 0.28 mM, respectively, and pH-optimum around 8.0. Aspartate transaminase (= GOT) exhibited similar affinities for aspartate (Km = 2.55 mM) and for glutamate (Km = 3.13 mM), but clearly different Km-values for 2-oxoglutarate (1.46 mM) and for oxaloacetate (0.13 mM). Activity at optimum pH of about 8.0 was 506 nmol x mg protein(-1) x min(-1) for aspartate conversion, but only 39 nmol x mg protein(-1) x min(-1) at optimum pH of about 7.0 for glutamate conversion. Activity (599 nmol x mg protein(-1) x min(-1)), substrate affinities (Km for alanine = 6.30 mM, for 2-oxoglutarate = 0.45 mM) and pH-optimum (6.5-7.5) proved alanine transaminase (= GPT) also important in distribution of intracellular nitrogen. There was comparatively low activity of the obviously constitutive enzyme, urease, (42 nmol x mg protein(-1) x min(-1)) whose substrate affinity was rather high (Km = 0.56 mM). Nitrate reductase proved substrate induced; activity could only be measured after exposure of the mycelia to exogenous nitrate. Routes of entry of exogenous nitrogen and tentative significance of the various enzymes in cell metabolism are discussed.     

Monkey liver microsomal glucose-6-phosphatase]

Kinetic studies indicate that glucose-6-phosphatase is a multifunctional enzyme. a) Phosphohydrolase activities. The mannose-6-phosphatase activity is low (Km = 8 mM, VM = 90 nmoles. min-1mg-1). The enzyme shows a strong affinity for glucose-6-phosphate (Km = 2.5 mM, VM = 220 nmoles.min-1mg-1). beta-glycerophosphate (K1 = 30 mM), D-glucose (Ki = 120 mM) are mixed type inhibitors; pyrophosphate (Ki = 2 mM) is a non competitive one. b) Phosphotransferase activities. Di and triphosphate adenylic nucleosides or phosphoenol pyruvate are not substrates. Carbamylphosphate serves as a phosphoryl donor with D-glucose as acceptor. The phosphate transfer is consisstent with a random mechanism in which the binding of one substrate increases the enzymes affinity for the second substrate. Apparent Km values for carbamyl-phosphate range from 5.2 mM (D-glucose concentration leads to infinity) to 8 mM (D-glucose concentration leads to 0). The corresponding apparent Km values for D-glucose are 59 mM (carbamyl-phosphate concentration leads to infinity) to 119 mM (carbamyl-phosphate concentration leads to 0). Maximal reaction velocity with infinite levels of both substrates is 270 nmoles.min-1.mg-1. Pyrophosphate is a poor phosphoryl donnor (Km = 55 mM with D-glucose concentration 250 mM). In addition we do not find any latency; detergents, namely sodium deoxycholate, Triton X 100 do not affect or inhibit glucose-6-phosphatase activity.     

Peptides reproducing the phosphoacceptor sites of pp60c-src as substrates for TPK-IIB, a splenic tyrosine kinase devoid of autophosphorylation activity.

TPK-IIB, a spleen tyrosine protein kinase devoid of autophosphorylation activity (Brunati, A. M., and Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457), has been purified to near homogeneity and assayed for its ability to phosphorylate the synthetic peptides EDNEYTA and EPQYQPA reproducing the two conserved phosphoacceptor sites of pp60c-src (Tyr-416 and Tyr-527). While EPQYQPA was phosphorylated with low efficiency (Km = 16.7 mM, Kcat = 14.4), EDNEYTA is an excellent substrate displaying a Km value of 58 microM and a Kcat value of 31.2. The single substitution, in the latter peptide, of the glutamic acid adjacent to the tyrosine by alanine to give EDNAYTA caused a 6-fold increase in the Km. The positive influence on the phosphorylation of the acidic residues at -3 and -4 relative to the tyrosine is indicated by comparison of the kinetic constants for peptides EDAAYAA (Kcat = 4.6, Km 0.325 mM) and QNAAYAA (Kcat 2.4, Km 1.7 mM). Furthermore, when residues in the peptide NEYTA were replaced by alanine, the phosphorylation of the peptides NAYTA and AAYAA, was almost negligible (in terms of Kcat/Km ratio). However, AEYTA, NEYAA and AEYAA were still phosphorylated, albeit less efficiently than NEYTA. The probability that these peptides will adopt a beta-turn is EDNAYTA = EDNEYTA, NAYTA greater than NEYTA, and no predicted beta-turn for AEYTA, NEYAA, and AEYAA. Therefore these results support the concept that an amino-terminal acidic residue(s) is strictly required by TPK-IIB, irrespective of peptide conformation, although a beta-turn may enhance the phosphorylation of those peptides that satisfy this requirement. Two other spleen tyrosine kinases, TPK-I/lyn and TPK-III, both related to the src family, also have a far greater preference for the peptide EDNEYTA over EPQYQPA. However, they can be distinguished from TPK-IIB by their lower affinity for the peptides EDNEYTA and NEYTA and by their different specificity towards the substituted derivatives of NEYTA. TPK-I/lyn, accepts most of the substitutions that are detrimental to TPK-IIB, the triply substituted peptide AAYAA being actually preferred over the parent peptide NEYTA. The substitution of glutamic acid by alanine is also tolerated by TPK-III, although, in contrast to TPK-IIB, the phosphorylation efficiency is drastically decreased by the substitution of the asparagine at position -2.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phosphoenol-pyruvate branchpoint in adult Hymenolepis diminuta (Cestoda): a study of pyruvate kinase and phosphoenol-pyruvate carboxykinase.

The properties of pyruvate kinase (PK) and phosphoenol pyruvate carboxykinase (PEP CK), two enzymes that determine the preferrential accumulation of either succinate or lactate as endproducts of carbohydrate metabolism, are described in adult Hymenolepis diminuta. PK activity at Vmax and Km levels of PEP was unaffected by ATP, alanine, FDP4, OR H+ ions, but was inhibited by 50% at 6.3 mM L-lactate and 30 mM HCO3. The addition of 30 mM HCO3 increased the Km(PEP) by 6-fold but did not alter the Vmax. The inhibition of PK by HCO3 cannot be explained entirely by an effect of ionic strength, but probably represents a specific modulator-enzyme interaction. Under similar conditions PEP CK was maximally activated. Although L-lactate inhibited PEP CK (Ki(lac) = 1.8 mM), this effector may play a minor role in regulation of PEP flux. These results implicate the poise of the HCO3-:CO2 system as a major determiner of endproduct accumulation in H. diminuta.     

Sugar transport in Coprinus cinereus.

Two transport systems for glucose were detected: a high affinity system with a Km of 27 muM, and a low affinity system with a Km of 3.3 mM. The high affinity system transported glucose, 2-deoxy-D-glucose (Km = 26 muM), 3-O-methylglucose (Km = 19 muM), D-glucosamine (Km = 652 muM), D-fructose (Km = 2.3 mM) and L-sorbose (Km = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45 degrees C). The low affinity system transported glucose, 2-deoxy-D-glucose (Km = 7.5 mM), and 3-O-methylglucose (Km = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30--50 degrees C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-D-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present insporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation inglucose-free medium. The half-ti me for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5--7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-D-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity. Analysis of transport defective mutants revealed defects in both transport systems although the mutants used were alleles of a single gene. It is concluded that this gene (the ftr cistron) is the structural gene for an allosteric molecule which serves both transport systems.     

The interaction of aromatic amino acids with rat liver phenylalanine hydroxylase

We have examined the interaction of hepatic phenylalanine hydroxylase with the phenylalanine analogs, tryptophan and the diastereomers of 3-phenylserine (beta-hydroxyphenylalanine). Both isomers of phenylserine are substrates for native phenylalanine hydroxylase at pH 6.8 and 25 degrees C, when activity is measured with the use of the dihydropteridine reductase assay coupled with NADH in the presence of the synthetic cofactor, 6-methyl-5,6,7,8-tetrahydropterin. However, while erythro-phenylserine exhibits simple Michaelis-Menten kinetics (Km = 1.2 mM, Vmax = 1.2 mumol/min X min) under these conditions, the threo isomer exhibits strong positive cooperativity (S0.5 = 4.8 mM Vmax = 1.4 mumol/min X mg, nH = 3). Tryptophan also exhibits cooperativity under these conditions (S0.5 = 5 mM, Vmax = 1 mumol/min X mg, nH = 3). The presence of 1 mM lysolecithin results in a hyperbolic response of phenylalanine hydroxylase to tryptophan (Km = 4 mM, Vmax = 1 mumol/min X mg) and threo-phenylserine (Km = 2 mM, Vmax = 1.4 mumol/min X mg). erythro-Phenylserine is a substrate for native phenylalanine hydroxylase in the presence of the natural cofactor, L-erythro-tetrahydrobiopterin (BH4) (Km = 2 mM, Vmax 0.05 mumol/min X mg, nH = 2). Preincubation of phenylalanine hydroxylase with erythro-phenylserine results in a 26-fold increase in activity upon subsequent assay with BH4 and erythro-phenylserine, and hyperbolic kinetic plots are observed. In contrast, both threo-phenylserine and tryptophan exhibit negligible activity in the presence of BH4 unless the enzyme has been activated. The product of the reaction of phenylalanine hydroxylase with either isomer of phenylserine was identified as the corresponding p-hydroxyphenylserine by reaction with sodium periodate and nitrosonaphthol. With erythro-phenylserine, the hydroxylation reaction is tightly coupled (i.e. 1 mol of hydroxyphenylserine is formed for every mole of tetrahydropterin cofactor consumed), while with threo-phenylserine and tryptophan the reaction is largely uncoupled (i.e. more cofactor consumed than product formed). Erythro-phenylserine is a good activator, when preincubated with phenylalanine hydroxylase (A0.5 = 0.2 mM), with a potency about one-third that of phenylalanine (A0.5 = 0.06 mM), while threo-phenylserine (A0.5 = 6 mM) and tryptophan (A0.5 approximately 10 mM) are very poor activators. Addition of 4 mM tryptophan or threo-phenylserine or 0.2 mM erythro-phenylserine to assay mixtures containing BH4 and phenylalanine results in a dramatic increase in the hydroxylation at low concentrations of phenylalanine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Difference in sodium requirement of branched chain amino acid carrier between Pseudomonas aeruginosa PAO and PML strains is due to substitution of an amino acid at position 292

Sodium dependence of leucine transport, a measure of the Na+-coupled leucine-isoleucine-valine II (LIV-II) transport system in Pseudomonas aeruginosa, was compared between two wild-type strains, PAO and PML. The leucine transport activity was saturated at 0.1 mM NaCl for PAO and at 5.0 mM for PML. From kinetics experiments, the apparent Km value for Na+ with respect to leucine transport was estimated to be 3 microM for PAO and 95 microM for PML. The Km value for leucine was 6 microM for PAO and 13 microM for PML. The LIV-II carrier gene (braB) of PML was isolated for comparison of its amino acid sequence with that of the PAO carrier cloned previously. The Km values for Na+ and leucine of the cloned LIV-II carriers of PAO and PML expressed in LIV-II defective mutants were similar to those in wild-type strains. Determination of the nucleotide and deduced amino acid sequences of the LIV-II carrier gene of PML showed an amino acid difference at position 292 between the PAO and PML carriers. The amino acid was threonine for PAO and alanine for PML. These results indicate that the substitution of the amino acid at position 292 of the LIV-II carrier causes a difference in the sodium requirement of the carriers of the PAO and PML strains.     

Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second beta-glucosidase.

A second extracellular beta-glucosidase (betalarge) of Aspergillus fumigatus was purified to homogeneity and shown to be a glycoprotein, as determined by polyacrylamide gel electrophoresis followed by staining for protein and for carbohydrate. Its molecular weight was approximately 340,000 by gel filtration, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave an apparent molecular weight of 170,000, suggesting that the enzyme has two subunits. The glucosidase contained covalently bound sugars consisting of about 2 mol of glucosamine and 16 mol of mannose per mol of protein. The carbohydrate was found to be attached to the peptide via glucosaminyl leads to peptide linkage, possibly to asparagine residues. At pH 4.5 this enzyme readily hydrolyzed p-nitrophenyl-beta-D-glucopyranoside (Km = 0.88 mM) and cleaved two glucose disaccharides: gentiobiose (beta,1 leads to 6; Km = 0.75 mM) and cellobiose (beta,1 leads to 4; Km = 0.84 mM). Although its activity is similar to that of a previously purified beta-glucosidase (betasmall), the two enzymes differ with respect to their pH activity profiles, substrate specificities, and molecular weights. Also double diffusion tests with anti-betasmall antiserum and both purified beta-glucosidases revealed a nonidentical cross-reaction. Microcomplement fixation of native and periodate-oxidized betasmall suggested that the oligosaccharide chain(s) was not a major antigenic site.     

A novel low molecular weight alanine aminotransferase from fasted rat liver

Alanine is the most effective precursor for gluconeogenesis among amino acids, and the initial reaction is catalyzed by alanine aminotransferase (AlaAT). Although the enzyme activity increases during fasting, this effect has not been studied extensively. The present study describes the purification and characterization of an isoform of AlaAT from rat liver under fasting. The molecular mass of the enzyme is 17.7 kD with an isoelectric point of 4.2; glutamine is the N-terminal residue. The enzyme showed narrow substrate specificity for L-alanine with Km values for alanine of 0.51 mM and for 2-oxoglutarate of 0.12 mM. The enzyme is a glycoprotein. Spectroscopic and inhibition studies showed that pyridoxal phosphate (PLP) and free -SH groups are involved in the enzymatic catalysis. PLP activated the enzyme with a Km of 0.057 mM.     

Purification and characterization of hydroxypyruvate reductase from the facultative methylotroph Methylobacterium extorquens AM1.

Hydroxypyruvate reductase was purified to homogeneity from the facultative methylotroph Methylobacterium extorquens AM1. It has a molecular mass of about 71 kDa, and it consists of two identical subunits with a molecular mass of about 37 kDa. This enzyme uses both NADH (Km = 0.04 mM) and NADPH (Km = 0.06 mM) as cofactors, uses hydroxypyruvate (Km = 0.1 mM) and glyoxylate (Km = 1.5 mM) as the only substrates for the forward reaction, and carries out the reverse reaction with glycerate (Km = 2.6 mM) only. It was not possible to detect the conversion of glycolate to glyoxylate, a proposed role for this enzyme. Kinetics and inhibitory studies of the enzyme from M. extorquens AM1 suggest that hydroxypyruvate reductase is not a site for regulation of the serine cycle at the level of enzyme activity.     

The utilization of alanine, glutamic acid, and serine as amino acid substrates for glycine N-acyltransferase

The conjugation of benzoyl-CoA with the aliphatic and acidic amino acids by glycine N-acyltransferase, as well as the amides of the latter group, was investigated. Bovine and human liver benzoyl-amino acid conjugation were investigated using electrospray ionization tandem mass spectrometry (ESI-MS-MS). Bovine glycine N-acyltransferase catalyzed conjugation of benzoyl-CoA with Gly (Km(Gly) = 6.2 mM), Asn (Km(Asn) = 129 mM), Gln (Km(Gln) = 353 mM), Ala (Km(Ala) = 1573 mM), Glu (Km(Glu) = 1148 mM) as well as Ser in a sequential mechanism. In the case of the human form, conjugation with Gly (Km(Gly) = 6.4 mM), Ala (Km(Ala) = 997 mM), and Glu was detected. The presence of these alternative conjugates did not inhibit bovine glycine N-acyltransferase activity significantly. Considering the relatively low levels at which these conjugates are formed, it is unlikely that they will have a significant contribution to acyl-amino acid conjugation under normal conditions in vivo. However, their cumulative contribution to acyl-amino acid conjugation under metabolic disease states may prove to have a useful contribution to detoxification of elevated acyl-CoAs.     

Importance of methionine residues in the enzymatic carboxylation of biotin-containing peptides representing the local biotinyl site of E. coli acetyl-CoA carboxylase

A biotin-containing hexapeptide Ac-Glu-Ala-Met-Bct-Met-Met (1) that represents the local biotin-containing site of Escherichia coli acetyl-CoA carboxylase has been prepared by the solid phase method. Peptide 1 is carboxylated by the biotin carboxylase subunit dimer of E. coli acetyl-CoA carboxylase with the following kinetic parameters; Km 12 mM, Vmax 2.8 microM X min-1. These compare with the parameters for biotin of Km 214 mM and Vmax 28 microM X min -1. Hence, the overall reactivity (Vmax/Km) of 1 is 1.8 times greater than that of free biotin. When all methionines in 1 are replaced by alanine, the resulting peptide (2) retains a similar binding ability but with a much decreased Vmax. It was also found that peptide 3, which carries an N epsilon-benzyloxycarbonyllysine in place of biocytin in 1, decreases the Km of biotin threefold.     

Cotransport of phosphate and sodium by yeast.

Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a Km of 30 micronM and is independent of sodium, and a sodium-dependent mechanism with a Km of 0.6 micronM, both Km values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na+, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Ribidium has no effect on the stimulation of phosphate uptake by sodium. Phosphate and arsenate enhance sodium uptake at pH 7.2. The Km of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake. The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed.     

Characterization of threonine transport into a kidney epithelial cell line (BSC-1). Evidence for the presence of Na(+)-independent system asc [corrected

The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.     

Kinetics of glucose transport in human erythrocytes: zero-trans efflux and infinite-trans efflux at 0 degree C

The kinetic features of glucose transport in human erythrocytes have been the subject of many studies, but no model is consistent with both the kinetic observations and the characteristics of the purified transporter. In order to reevaluate some of the kinetic features, initial rate measurements were performed at 0 degree C. The following kinetic parameters were obtained for fresh blood: zero-trans efflux Km = 3.4 mM, Vmax = 5.5 mM/min; infinite-trans efflux Km = 8.7 mM, Vmax = 28 mM/min. For outdated blood, somewhat different parameters were obtained: zero-trans efflux Km = 2.7 mM, Vmax = 2.4 mM/min; infinite-trans efflux Km = 19 mM, Vmax = 23 mM/min. The Km values for fresh blood differ from the previously reported values of 16 mM and 3.4 mM for zero-trans and infinite-trans efflux, respectively (Baker, G.F. and Naftalin, R.J. (1979) Biochim. Biophys. Acta 550, 474-484). The use of 50 mM galactose rather than 100 mM glucose as the infinite-trans sugar produced no change in the infinite-trans efflux Km values but somewhat lower Vmax values. Simulations indicate that initial rates were closely approximated by the experimental conditions. The observed time courses of efflux are inconsistent with a model involving rate-limiting dissociation of glucose from hemoglobin (Naftalin, R.J., Smith, P.M. and Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235-249). The results presented here support the adequacy of the carrier model to account for the kinetics.     

Inverse relationship between glucose metabolism and glucose-induced insulin secretion in rat insulinoma cells

Slowly growing X-ray-induced rat insulinomas and derived cell lines have been used as a model system for glucose-induced insulin release. During perfusions of tumors transplanted under the kidney capsule, the carbohydrates glucose and D-glyceraldehyde increased insulin secretion. These stimuli and the amino acids leucine and alanine also provoked insulin release in freshly isolated tumor cells. Under these conditions, glucose utilization had a Km of 4.6 mM and maximal velocity of 0.9 nmol/min/10(6) cells. A continuous cell line was established from such a preparation. In culture, glucose-induced insulin secretion was no longer detectable while responses to D-glyceraldehyde and amino acids were retained. Glucose metabolism in the cell line showed a decrease in Km to 0.7 mM glucose and an increased maximal velocity of 1.4 nmol/min/10(6) cells. Attempts to revert these alterations were undertaken using glucose-deficient culture medium to diminish glycolytic flux. Basal insulin release was lowered, while the growth pattern of the cells remained unchanged. Another approach involved the use of sodium butyrate which has been demonstrated to promote differentiation in other cell systems. Whereas sodium butyrate markedly increased cellular insulin content, the secretory responses were not improved. These results provide evidence that the loss of glucose-induced insulin secretion is paralleled by alterations in glucose metabolism.     

Investigations on the sodium dependence of bile acid fluxes in the isolated perfused rat liver.

At [Na+]o = 118 mM the concentrative transfer of cholic and taurocholic acid from the perfusate into the isolated rat liver displays saturation kinetics (taurocholate: V = 299 nmol-min-1-g-1, Km = 61 muM; Cholate: V=327 nmol-min-1-g-1, Km = 436 muM). Perfusion with an isotonic sodium-free medium did not change the feature of a carrier-mediated transport but did markedly reduce V without affecting Km (taurocholate: V = 65 nmol-min-1-g-1, Km = 78 muM; cholate: V = 104 nmol-min-1-g-1, Km = 354 muM). It was experimentally assured that the observed reduction of bile salt uptake was not a consequence of regurgitation of bile salts or due to an excessive intracellular accumulation during cholestasis in the sodium-free state. The rate of taurocholate efflux is very low when compared with the rapid rate of the uptake. A stimulatory action of extracellular sodium on this pathway was also observed. Inhibition of the (Na+ + K+)-ATPase by 1 mM ouabain resulted in a decrease of bile salt uptake. Activation of the enzyme by potassium readmission to a K+-deprived liver enhanced bile salt uptake. The immediate response to alteration of the enzyme activity suggests a close association of a fraction of bile acid active transport with the sodium pump.