Effect of natalizumab on clinical and radiological disease activity in a French cohort of patients with relapsing-remitting multiple sclerosis.

"Disease activity free" in relapsing-remitting multiple sclerosis (RRMS) is a new concept introduced by the results of the AFFIRM study. Our objective was to analyze the clinical and radiological efficacy of natalizumab treatment in actual clinical practice and compare it with the post hoc analysis of the AFFIRM study. All patients with RRMS who began treatment with natalizumab at our two French MS centres between April 2007 and May 2008 were included and followed-up for at least 2 years. No measurable disease activity ("disease activity free") was defined as no activity on clinical measures (no relapses and no sustained disability progression) and radiological measures (no gadolinium-enhancing lesions and no new T2-hyperintense lesions on cerebral MRI). A total of 193 patients were included. Natalizumab was discontinued in 25.9% of cases before the completion of 2 years of treatment. In our cohort, we observed patients with more severe disease than in the AFFIRM study. The proportion of patients remaining free of clinical activity during 2 years of treatment was lower than in the AFFIRM study (37.8% vs. 64.3%). The proportion of patients remaining free of radiological activity during 2 years of treatment was higher than in the AFFIRM study (68.9% vs. 57.7%), while the proportion of patients remaining free of disease activity during 2 years of treatment was comparable to the AFFIRM study (33.3% vs. 36.7%). Natalizumab seems to be as effective in a real-life setting as in pivotal and post hoc studies. The confirmation of such benefits is important because of the progressive multifocal leukoencephalopathy risk.

[Primitive leptomeningeal malignant melanoma: a rare etiology of pachymeningitis and leptomeningitis]. / Mélanome malin leptoméningé primitif : une étiologie rare de pachy- et leptoméningite.

INTRODUCTION: Leptomeningitis and pachymeningitis are known to occur consecutive to many causes. OBSERVATION: We report the case of a 24-year-old woman with symptoms of raised intracranial pressure and repeated switching transient hemiparesis. The magnetic resonance imaging (MRI) showed a pachyleptomeningitis. Search for a cause was negative. The pathology examination of meningeal tissue revealed a malignant melanoma, without any sign of cutaneous melanoma, leading to the diagnosis of primary leptomeningeal malignant melanoma. CONCLUSION: A meningeal biopsy can enable the rare diagnosis of primary leptomeningeal malignant melanoma in a patient with unexplained pachyleptomeningitis.

Kinetic mechanism of Na+ -glucose cotransport through the rabbit intestinal SGLT1 protein.

No consensus has yet been reached regarding the order of substrate addition to the high-affinity Na+ -D-glucose cotransporter (SGLT1). This problem was addressed by computer-assisted derivation of the steady-state velocity equations characterizing the eight-state Na+:Na+:substrate (NNS) and Na+:substrate:Na+ (NSN) mechanisms of cotransport. A notable difference was found in their denominator expressions and used to device a new strategy aimed at model discrimination in which the initial rate data are recorded at fixed S and analyzed relative to the N dependence of transport using a Hill equation. According to this protocol, the values of the Hill coefficient (n(H)) should be finite at all S (1.0 < n(H) < or =2.0) or decrease down to a limit value of 1.0 at high S in the case of the NNS and NSN models, respectively. These key experiments were performed in rabbit intestinal brush border membrane vesicles and demonstrated that a Hill equation with n(H) = 2.0 best describes the steady-state kinetics of Na+ -glucose cotransport at all S. We therefore propose a kinetic mechanism whereby Na+ binding should occur with very strong cooperativity within a rapid equilibrium segment of the transport cycle and be followed by a slow isomerization step before glucose addition.

Probing into the function of the gene product responsible for glycogen storage disease type Ib.

This study aimed at directly assessing glucose 6-phosphate (G6P) transport by intact rat liver microsomes. Tracer uptake from labeled G6P occurred with T(1/2) values that proved insensitive to unlabeled G6P or 100 microM vanadate, and could not be activated over background levels by intravesicular phosphate in the complete absence of G6P hydrolysis. [(32)P]Phosphate efflux was similarly unaffected by G6P or phosphate in the incubation medium. We conclude that the gene product responsible for glycogen storage disease type Ib is functionally distinct from the bacterial hexose phosphate transporter, which operates as an obligatory phosphate:phosphate or G6P:phosphate exchanger.

Two-step mechanism of phlorizin binding to the SGLT1 protein in the kidney.

The relationships between phlorizin binding and Na+-glucose cotransport were addressed in rabbit renal brush-border membrane vesicles. At pH 6.0 and 8.6, high affinity phlorizin binding followed single exponential kinetics. With regard to phlorizin concentrations, the binding data conformed to simple Scatchard kinetics with lower apparent affinities of onset binding (Kdi = 12-30 microM) compared to steady-state binding (Kde = 2-5 microM), and the first-order rate constants demonstrated a Michaelis-Menten type of dependence with Km values identical to Kdi. Phlorizin dissociation from its receptor sites also followed single exponential kinetics with time constants insensitive to saturating concentrations of unlabeled phlorizin or D-glucose, but directly proportional to Na+ concentrations. These results prove compatible with homogeneous binding to SGLT1 whereby fast Na+ and phlorizin addition on the protein is followed by a slow conformation change preceding further Na+ attachment, thus occluding part of the phlorizin-bound receptor complexes. This two-step mechanism of inhibitor binding invalidates the recruitment concept as a possible explanation of the fast-acting slow-binding paradigm of phlorizin, which can otherwise be resolved as follows: the rapid formation of an initial collision complex explains the fast-acting behavior of phlorizin with regard to its inhibition of glucose transport; however, because this complex also rapidly dissociates in a rapid filtration assay, the slow kinetics of phlorizin binding are only apparent and reflect its slow isomerization into more stable forms.

An integrated view of the kinetics of glucose and phosphate transport, and of glucose 6-phosphate transport and hydrolysis in intact rat liver microsomes.

The dynamics of the glucose 6-phosphatase system were investigated in intact rat liver microsomes using a fast-sampling, rapid-filtration apparatus. Glucose and phosphate transport followed single exponential kinetics, appeared to be homogeneous, was unaffected by unlabeled substrate concentrations up to 100 mM, proved insensitive to various potential inhibitors, and demonstrated similarly low energies of activation. The extent of tracer accumulation from glucose 6-phosphate depended on which of the glucose or phosphate moieties was the labeled species in the parent molecule. The rates of tracer equilibration reflected those of glucose or phosphate transport but similar initial rates of uptake were observed for the glucose and phosphate products of hydrolysis. However, the latter accounted for only 12-13% of the steady-state rate of total glucose production. It is concluded that tracer uptake cannot represent substrate transport, that labeled glucose 6-phosphate at best represents a tiny fraction of the intramicrosomal glucose or phosphate pools, and that glucose 6-phosphate transport is not an obligatory prerequisite to its hydrolysis. The latter conclusion invalidates a major postulate of the substrate transport-catalytic unit concept but proves compatible with a conformational model whereby glucose 6-phosphate transport and hydrolysis are tightly coupled processes while glucose and phosphate share, along with water and a variety of other organic and inorganic solutes, a common porelike structure for their transport through the microsomal membrane.

New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system.

The operation of glucose 6-phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46-kDa putative glucose 6-phosphate (Glc6P) translocase (P46). Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N-terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm. P36 gene expression is increased by glucose, fructose 2,6-bisphosphate (Fru-2,6-P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin. P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36. Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter. Mutations in P36 and P46 lead to glycogen storage disease (GSD) type-1a and type-1 non a (formerly 1b and 1c), respectively. Adenovirus-mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS-1 cells results in decreased glycolysis and glucose-induced insulin secretion. The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined. The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism.

Kinetic mechanisms of inhibitor binding: relevance to the fast-acting slow-binding paradigm.

Although phlorizin inhibition of Na+-glucose cotransport occurs within a few seconds, 3H-phlorizin binding to the sodium-coupled glucose transport protein(s) requires several minutes to reach equilibrium (the fast-acting slow-binding paradigm). Using kinetic models of arbitrary dimension that can be reduced to a two-state diagram according to Cha's formalism, we show that three basic mechanisms of inhibitor binding can be identified whereby the inhibitor binding step either (A) represents, (B) precedes, or (C) follows the rate-limiting step in a binding reaction. We demonstrate that each of mechanisms A-C is associated with a set of unique kinetic properties, and that the time scale over which one may expect to observe mechanism C is conditioned by the turnover number of the catalytic cycle. In contrast, mechanisms A and B may be relevant to either fast-acting or slow-binding inhibitors. However, slow-binding inhibition according to mechanism A may not be compatible with a fast-acting behavior on the steady-state time scale of a few seconds. We conclude that the recruitment hypothesis (mechanism C) cannot account for slow phlorizin binding to the sodium-coupled glucose transport protein(s), and that mechanism B is the only alternative that may explain the fast-acting slow-binding paradigm.

Reduction of an eight-state mechanism of cotransport to a six-state model using a new computer program.

A computer program was developed to allow easy derivation of steady-state velocity and binding equations for multireactant mechanisms including or without rapid equilibrium segments. Its usefulness is illustrated by deriving the rate equation of the most general sequential iso ordered ter ter mechanism of cotransport in which two Na+ ions bind first to the carrier and mirror symmetry is assumed. It is demonstrated that this mechanism cannot be easily reduced to a previously proposed six-state model of Na+-D-glucose cotransport, which also includes a number of implicit assumptions. In fact, the latter model may only be valid over a restricted range of Na+ concentrations or when assuming very strong positive cooperativity for Na+ binding to the glucose symporter within a rapid equilibrium segment. We thus propose an equivalent eight-state model in which the concept of positive cooperativity is best explained within the framework of a polymeric structure of the transport protein involving a minimum number of two transport-competent and identical subunits. This model also includes an obligatory slow isomerization step between the Na+ and glucose-binding sequences, the nature of which might reflect the presence of functionally asymmetrical subunits.

Caco-2 cell line used as an in vitro model to study cadmium accumulation in intestinal epithelial cells.

109Cd uptake was studied using the highly differentiated TC7 clone of Caco-2 cells as a model of human enterocyte function. Intracellular accumulation of 0.3 microM 109Cd involved a rapid and a slow uptake phase, which resulted in complete equilibration (t(1/2) = 17.3 +/- 1.3 min) with an apparent in-to-out distribution ratio (alphae) of 11.6 +/- 0.8. The amplitude of the rapid phase (U0) and the rate of the slow phase (V) were similarly reduced in the less differentiated PF11 clone, but comparable alphae values were observed at equilibrium. In both clones, the t(1/2) and alphae values increased and decreased, respectively, upon addition of unlabeled Cd to the uptake media. In TC7 cells, 109Cd uptake at 1 min (U1) was unaffected by Ca concentrations four order of magnitude in excess, but both U0 and V demonstrated similar sensitivities to unlabeled Cd, Zn and sulfhydryl-reactive agents. Only U0 disappeared when EDTA was present in the wash solutions. U1 showed saturation kinetics and the data were found compatible with a model assuming rapid initial Cd binding and transport through a unique transport protein (Km = 3.8 +/- 0.7 microM). Cd efflux kinetics demonstrated partial reversibility in EDTA-containing solutions, suggesting that the taken up Cd might be both tightly and loosely bound to intracellular binding sites. However, the displacement of 109Cd measured at 65 min failed to reveal this heterogeneity: the data were found compatible with a model equation assuming the presence of one class of high-capacity high-affinity binding sites. We conclude that a slow-transport fast-intracellular binding mechanism of Cd uptake best accounts for these results and that Cd transport most likely involves a carrier-type of protein unrelated to Ca absorption.

Sugar transport heterogeneity in the kidney: two independent transporters or different transport modes through an oligomeric Protein? 1. Glucose transport studies.

The kinetics of Na+/d-glucose cotransport (SGLT) were reevaluated in rabbit renal brush border membrane vesicles isolated from the whole kidney cortex using a fast-sampling, rapid-filtration apparatus (FSRFA, US patent #5,330,717) for uptake measurements. Our results confirm SGLT heterogeneity in this preparation, and both high (HAG) and low (LAG) affinity glucose transport pathways can be separated over the 15-30 degrees C range of temperatures. It is further shown that: (i) Na+ is an essential activator of both HAG and LAG; (ii) similar energies of activation can be estimated from the linear Arrhenius plots constructed from the Vmax data of HAG and LAG, thus suggesting that the lipid composition and/or the physical state of the membrane do not affect much the functioning of SGLT; (iii) similar Vmax values are observed for glucose and galactose transport through HAG and LAG, thus demonstrating that the two substrates share the same carrier agencies; and (iv) phlorizin inhibits both HAG and LAG competitively and with equal potency (Ki = 15 microM). Individually, these data do not allow us to resolve conclusively whether the kinetic heterogeneity of SGLT results from the expression in the proximal tubule of either two independent transporters (rSGLT1 and rSGLT2) or from a unique transporter (rSGLT1) showing allosteric kinetics. Altogether and compared to the kinetic characteristics of the cloned SGLT1 and SGLT2 systems, they do point to a number of inconsistencies that lead us to conclude the latter possibility, although it is recognized that the two alternatives are not mutually exclusive. It is further suggested, from the differences in the Km values of HAG transport in the kidney as compared to the small intestine and SGLT1 cRNA-injected oocytes, that renal SGLT1 activity is somehow modulated, maybe through heteroassociation with (a) regulatory subunit(s) that might also contribute quite significantly to sugar transport heterogeneity in the kidney proximal tubule.

Kinetic separation and characterization of three sugar transport modes in Caco-2 cells.

The question of sugar transport heterogeneity in the human intestinal Caco-2 cell line was addressed using alpha-methyl-D-glucose (AMG) and 2-deoxy-D-glucose (DG) as substrate analogues for D-glucose, the transport inhibitors phlorizin (PZ) and phloretin (PT), and NaCl or choline chloride uptake media. The data are compatible with the existence of three distinct pathways that can be isolated kinetically according to specific characteristics: 1) an "AMG-strict" system, strictly Na+ dependent and specific for AMG [Michaelis-Menten constant value (K(m)) = 2.0 +/- 0.3 mM] but sensitive to both PZ and PT, with PZ being more potent than PT, 2) a "DG-strict" system, strictly Na+ independent and specific for both DG (K(m) = 5.2 +/- 0.5 mM) and PT; and 3) a "DG/AMG-mixed" system, strictly Na+ dependent, with loose specificities for the glucose analogues DG (K(m) = 0.81 +/- 0.07 mM) and AMG (K(m) = 8.1 +/- 0.8 mM), and the inhibitors PZ and PT, but with PT being more potent than PZ. Since SGLT-1 obtained by polymerase chain reaction from either Caco-2 cells or normal human jejunum demonstrated identical transport properties when expressed in Xenopus laevis oocytes, we conclude that the "AMG-strict" system represents the expression of human SGLT-1 activity in this cell line. Moreover, Western blot analysis revealed that SGLT-1 is located exclusively in the apical membrane. In contrast, neither the nature nor the membrane location of both the DG-strict and DG/AMG-mixed pathways could be resolved unambiguously. Still it has been demonstrated that expression of the latter system is constitutive to all Caco-2 cells and that its Na+ dependence is not the consequence of H(+)-dependent transport activity. Aside from the presence of the DG/AMG-mixed system, a salient feature of Caco-2 cells is that the GLUT-3 protein is located exclusively in the brush-border membrane. Due to these limitations, it is concluded that the Caco-2 cell line cannot be considered as equivalent to either fetal colonic cells or normal enterocytes.

2-Deoxyglucose transport and metabolism in Caco-2 cells.

We investigated the kinetics of 2-deoxy-D-glucose (DG) uptake and metabolism in Caco-2 cells, because this human cell line may represent a valid enterocyte model to assess the dynamics between sugar transport and metabolism and hence to obtain insights into the factors involved during the intracellular phase of glucose absorption. When studied in 14-day-old monolayers, DG uptake is characterized by a lag phase with a time course matching the decrease in intracellular glucose concentrations, and no intracellular glucose 6-phosphate (G-6-P) can be detected at any time during incubation. After 1 h of preincubation of Caco-2 cells in substrate-free transport medium, however, steady-state DG uptake matches 2-deoxy-D-glucose 6-phosphate (DG-6-P) accumulation with undetectable levels of free DG. This complex behavior in DG uptake is linked to high hexokinase activity in Caco-2 cells, and the enzyme has a Michaelis-Menten constant K(m) for glucose that is typical of hexokinase type II (0.120 +/- 0.003 mM). Caco-2 cells also contain low-level glucose-6-phosphatase (G-6-Pase) activity, which may account for the leveling off in DG uptake, and the kinetics of DG transport may be attributed to the existence of a predominant pathway with a K(m) of 1.7 +/- 0.2 mM. Finally, analysis of the growth-related expression of DG transport and hexokinase activity clearly shows that DG uptake is lowest in postconfluent cells when hexokinase is at its highest levels. We thus conclude that 1) transport is the rate-limiting step during DG accumulation, 2) G-6-P is a potent inhibitor of hexokinase activity compared with DG-6-P, so that enzyme inhibition may have physiological relevance in diverting glucose from metabolism during its active reabsorption in the small intestine, and 3) low levels of G-6-Pase activity seem to exclude this enzyme, and hence the endoplasmic reticulum, as important factors during the intracellular phase of glucose transport.

Thermodynamic determination of the Na+: glucose coupling ratio for the human SGLT1 cotransporter.

Phlorizin-sensitive currents mediated by a Na-glucose cotransporter were measured using intact or internally perfused Xenopus laevis oocytes expressing human SGLT1 cDNA. Using a two-microelectrode voltage clamp technique, measured reversal potentials (Vr) at high external alpha-methylglucose (alpha MG) concentrations were linearly related to In[alpha MG]o, and the observed slope of 26.1 +/- 0.8 mV/decade indicated a coupling ratio of 2.25 +/- 0.07 Na ions per alpha MG molecule. As [alpha MG]o decreased below 0.1 mM, Vr was no longer a linear function of In[alpha MG]o, in accordance with the suggested capacity of SGLT1 to carry Na in the absence of sugar (the "Na leak"). A generalized kinetic model for SGLT1 transport introduces a new parameter, Kc, which corresponds to the [alpha MG]o at which the Na leak is equal in magnitude to the coupled Na-alpha MG flux. Using this kinetic model, the curve of Vr as a function of In[alpha MG]o could be fitted over the entire range of [alpha MG]o if Kc is adjusted to 40 +/- 12 microM. Experiments using internally perfused oocytes revealed a number of previously unknown facets of SGLT1 transport. In the bilateral absence of alpha MG, the phlorizin-sensitive Na leak demonstrated a strong inward rectification. The affinity of alpha MG for its internal site was low; the Km was estimated to be between 25 and 50 mM, an order of magnitude higher than that found for the extracellular site. Furthermore, Vr determinations at varying alpha MG concentrations indicate a transport stoichiometry of 2 Na ions per alpha MG molecule: the slope of Vr versus In[alpha MG]o averaged 30.0 +/- 0.7 mV/decade (corresponding to a stoichiometry of 1.96 +/- 0.04 Na ions per alpha MG molecule) whenever [alpha MG]o was higher than 0.1 mM. These direct observations firmly establish that Na ions can utilize the SGLT1 protein to cross the membrane either alone or in a coupled manner with a stoichiometry of 2 Na ions per sugar, molecule.

Glucose transport and glucose 6-phosphate hydrolysis in intact rat liver microsomes.

Glucose transport was investigated in rat liver microsomes in relation to glucose 6-phosphatase (Glu-6-Pase) activity using a fast sampling, rapid filtration apparatus. 1) The rapid phase in tracer uptake and the burst phase in glucose 6-phosphate (Glu-6-P) hydrolysis appear synchronous, while the slow phase of glucose accumulation occurs during the steady-state phase of glucose production. 2) [14C]Glucose efflux from preloaded microsomes can be observed upon addition of either cold Glu-6-P or Glu-6-Pase inhibitors, but not cold glucose. 3) Similar steady-state levels of intramicrosomal glucose are observed under symmetrical conditions of Glu-6-P or vanadate concentrations during influx and efflux experiments, and those levels are directly proportional to Glu-6-Pase activity. 4) The rates of both glucose influx and efflux are characterized by t1/2 values that are independent of Glu-6-P concentrations. 5) Glucose efflux in the presence of saturating concentrations of vanadate was not blocked by 1 mM phloretin, and the initial rates of efflux appear directly proportional to intravesicular glucose concentrations. 6) It is concluded that glucose influx into microsomes is tightly linked to Glu-6-Pase activity, while glucose efflux may occur independent of hydrolysis, so that microsomal glucose transport appears unidirectional even though it can be accounted for by diffusion only over the accessible range of sugar concentrations.

Evidence for a membrane exchangeable glucose pool in the functioning of rat liver glucose-6-phosphatase.

We have investigated the kinetics of tracer uptake into rat liver microsomes in relation to [14C]glucose 6-phosphate (Glu-6-P) hydrolysis by glucose 6-phosphatase (Glu-6-Pase). 1) The steady-state levels of intravesicular tracer accumulated during the rapid (AMP1) and slow (AMP2) phases of uptake both demonstrate Michaelis-Menten kinetics relative to outside Glu-6-P concentrations with Km values similar to those observed for the initial burst (Vi) and steady-state (VSS) rates of Glu-6-P hydrolysis. 2) The AMP1/AMP2 ratio is constant (mean value = 0.105 +/- 0.018) over the whole range of outside Glu-6-P concentrations and is equal to the AMP1max/AMP2max ratio (0.109 +/- 0.032). 3) Linear relationships are observed between the initial rates of glucose transport during the slow uptake phase (V alpha 2) and [AMP1], and between [VSS] and [AMP2]. 4) The value of Vss max exceeds by more than 10-fold that of V alpha 2 max. 5) It is concluded that the substrate transport model is incompatible with those results and that AMP1 represents a membrane exchangeable glucose pool. 6) We propose a new version of the conformational model in which the catalytic site lies deep within a hydrophilic pocket of an intrinsic membrane protein and communicates with the extra- and intravesicular spaces through channels with different glucose permeabilities.

Electrogenic amino acid exchange via the rBAT transporter.

A cDNA clone was isolated from rabbit renal cortex using DNA-mediated expression cloning, which caused alanine-dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na-independent amino acid transporter previously cloned elsewhere. Exposure of cDNA-injected oocytes to neutral amino acids led to voltage-dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut-open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.

Biosynthesis and polarized distribution of neutral endopeptidase in primary cultures of kidney proximal tubule cells.

When cultured in defined medium, kidney proximal convoluted tubule (PCT) cells form a homogeneous population and retain a number of differentiated functions. To characterize this cell system further as a functional model of epithelial polarity, we investigated the biogenic pathway of neutral endopeptidase (NEP), one of the most abundant microvillar membrane proteins in intestinal and kidney cells. We showed that, in contrast with some tumoral cell lines, RNA extracted from PCT cells shows the presence of a single mRNA species encoding NEP. Pulse-chase studies followed by selective immunoprecipitation of NEP molecules present either at the cell surface or in intracellular cell compartments showed that newly synthesized NEP molecules reached the cell surface as early as 30 min after the beginning of the chase with maximum cell surface expression at 60 min. When grown on semipermeable supports, PCT cells were found to target NEP exclusively to the apical plasma membrane. Similar results have been described using MDCK cells to study targeting of recombinant NEP. Thus primary cultures of PCT cells represent a new model with which to investigate the biogenic pathway of endogenous proteins in native epithelial cells.

Heterotropic effects of dipolar amino acids on the activity of the anionic amino acid transport system X-AG in rabbit jejunal brush-border membrane vesicles.

D-Aspartic acid was used as a specific substrate to evaluate the effects of dipolar amino acids on the high affinity anionic amino acid transport system X-AG in rabbit jejunal brush-border membrane vesicles. At pH 6, increasing L-phenylalanine concentrations caused a saturable activation of 0.05 mM D-aspartic acid uptake (Ka = 2.4 mM), and a saturating concentration of effector increased the Vmax of transport (2.6-fold) without any significant effect on the Km. At pH 8, however, a complex activation/inhibition curve was obtained with increasing L-phenylalanine concentrations, and a saturating concentration of effector increased both the Vmax (1.5-fold) and Km (2.1-fold) for transport. Increasing concentrations of L-valine, L-isoleucine, L-methionine, and L-threonine also showed complex activation/inhibition curves of D-aspartic acid uptake at both pH 6 and 8. The maximum level of activation, the plateau reached at saturating concentrations, and the concentration of effector producing either maximum activation or inhibition were, however, different at these two pH values. By using an optimum concentration of 10 mM L-valine at pH 6, the absence of trans-activation and of further activation by a cis-gradient of effector could be demonstrated. These results suggest that two allosteric sites directly accessible from the external medium are responsible for the heterotropic activation of intestinal system X-AG by dipolar amino acids and that, under physiological conditions, such effects might compensate for the lack of specificity of the neutral brush-border system.

Allosterism and Na(+)-D-glucose cotransport kinetics in rabbit jejunal vesicles: compatibility with mixed positive and negative cooperativities in a homo- dimeric or tetrameric structure and experimental evidence for only one transport protein involved.

We first present two simple dimeric models of cotransport that may account for all of the kinetics of Na(+)-D-glucose cotransport published so far in the small intestine. Both the sigmoidicity in the Na+ activation of transport (positive cooperativity) and the upward deviations from linearity in the Eadie-Hofstee plots relative to glucose concentrations (negative cooperativity) can be rationalized within the concept of allosteric kinetic mechanisms corresponding to either of two models involving sequential or mixed concerted and sequential conformational changes. Such models also allow for 2 Na+: 1 S and 1 Na+: 1 S stoichiometries of cotransport at low and high substrate concentrations, respectively, and for partial inhibition by inhibitors or substrate analogues. Moreover, it is shown that the dimeric models may present physiological advantages over the seemingly admitted hypothesis of two different cotransporters in that tissue. We next address the reevaluation of Na(+)-D-glucose cotransport kinetics in rabbit intestinal brush border membrane vesicles using stable membrane preparations, a dynamic approach with the Fast Sampling Rapid Filtration Apparatus (FSRFA), and both nonlinear regression and statistical analyses. Under different conditions of temperatures, Na+ concentrations, and membrane potentials clamped using two different techniques, we demonstrate that our data can be fully accounted for by the presence of only one carrier in rabbit jejunal brush border membranes since transport kinetics relative to glucose concentrations satisfy simple Michaelis-Menten kinetics. Although supporting a monomeric structure of the cotransporter, such a conclusion would conflict with previous kinetic data and more recent studies implying a polymeric structure of the carrier protein. We thus consider a number of alternatives trying to reconcile the observation of Michaelis-Menten kinetics with allosteric mechanisms of cotransport associated with both positive and negative cooperativities for Na+ and glucose binding, respectively. Such models, implying energy storage and release steps through conformational changes associated with ligand binding to an allosteric protein, provide a rational hypothesis to understand the long-time debated question of energy transduction from the Na+ electrochemical gradient to the transporter.