L-Proline Transport Systems of Starfish Pyloric Caeca

1991 ◽  
Vol 158 (1) ◽  
pp. 477-493 ◽  
Author(s):  
GREGORY A. AHEARN ◽  
RACHEL D. BEHNKE
Keyword(s):  
Amino Acid ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Competitive Inhibitor ◽  
Transport Systems ◽  
Proline Transport ◽  
Sigmoidal Functions ◽  
L System ◽  
Carrier Mediated Transport ◽  
Dependent Transport

Purified brush-border membrane vesicles (BBMV) of starfish [Pycnopodia helianthoides (Brandt)] pyloric caecal epithelium were prepared by magnesium precipitation in order to characterize the possible role of this organ in amino acid transport. L-[3H]proline uptake by these vesicles was Na+-dependent and greater at pH7.5 than at pH5.5. L-Pipecolate was a competitive inhibitor of L-proline influx into these BBMV, exhibiting a Ki value of 0.02 mmol l−1. The amino acid inhibitors, L-pipecolate, L-alanine and L-leucine were used as test substrates to block L-prohne influx by the IMINO, NBB and L transport systems, respectively, in order to estimate the contribution of each process to total L-prohne entry into pyloric caecal cells. The carrier-mediated transport constants for L-prohne transfer by these three systems were: Kt=0.18mmoll−1 (IMINO), 0.13mmol l−1 (NBB) and (0.21mmol l−1 (L); Jmax= 1310 pmol mg−1 protein 30 s−1 (IMINO), 360 pmol mg−1 protein 30 s−1 (NBB) and 470pmol mg−1 protein 30s−1 (L). L-Proline influxes through both the IMINO and NBB systems were sigmoidal functions of the external [Na+], while transfer by the L system was Na+-independent. Multiple sodium ions (e.g. 2 or 3 Na+/L-proline) appear to be associated with L-proline transport by both Na+-dependent transport systems, but the nature of this association (i.e. activation or energization) is unclear. Results suggest that starfish pyloric caecal epithelium possesses a similar array of L-proline transport proteins to those found in similar cell types of mammalian intestine or kidney, providing tentative support for an absorptive function for this organ.

Is leucine an allosteric modulator of the lysine transporter in the intestinal basolateral membrane?

1987 ◽  
Vol 253 (5) ◽  
pp. G637-G642 ◽  
Author(s):  
K. Lawless ◽  
D. Maenz ◽  
C. Cheeseman
Keyword(s):  
Amino Acid ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Transport Systems ◽  
Low Concentrations ◽  
Carrier Mediated Transport ◽  
Order Of Magnitude ◽  
Dibasic Amino Acid ◽  
Voltage Clamping ◽  
Stimulation Of

The transport of the dibasic amino acid L-lysine was investigated using basolateral membrane vesicles prepared from rat jejunal mucosal scrapings. The majority of the carrier-mediated transport was unaffected by the presence of sodium in the incubation medium, but voltage clamping of the vesicles did increase lysine uptake, indicating an associated movement of charge. Kinetic analysis of lysine influx and efflux showed the system to be symmetrical, but although the Vmax was comparable to other amino acid transport systems in this membrane, the dissociation constant for the overall reaction (KT) was an order of magnitude larger. This low affinity for lysine would explain the relatively slow rate of transport of this amino acid across the basolateral membrane. Competition experiments indicated that this system has a relatively narrow specificity carrying only lysine, arginine, ornithine, and histidine. In contrast the presence of L-leucine caused a marked stimulation of lysine efflux and influx across the vesicles. This effect was observed with leucine concentrations as low as 0.1 microM. It is concluded that although the lysine transport system in the basolateral membrane is slow in its basal state it can be rapidly turned on by the presence of L-leucine. The remarkably low concentrations required to do this suggest a possible allosteric interaction between the transporter and this neutral amino acid.

Molecular mechanisms involved in the transport of antibiotics into bacteria

Parasitology ◽  
1988 ◽  
Vol 96 (S1) ◽  
pp. S25-S44 ◽  
Author(s):  
I. Chopra
Keyword(s):  
Amino Acid ◽  
Molecular Mechanisms ◽  
Drug Uptake ◽  
Peptide Transport ◽  
Transport Systems ◽  
Facilitated Diffusion ◽  
Phosphotransferase System ◽  
Bacterial Membranes ◽  
Carrier Mediated Transport ◽  
Dependent Transport

SUMMARYMany clinically useful antibacterial drugs have intracellular target sites. Therefore, in order to reach their targets, these compounds must be able to cross bacterial outer and cytoplasmic membranes. Considerable information is available on the mechanisms by which antibiotics cross bacterial membranes and, in many cases, it is now possible to define the molecular basis of their uptake. Passage of drugs across the outer membrane of Gram-negative bacteria can occur by diffusion through porin channels (e.g. β-lactams and tetracyclines), by facilitated diffusion using specific carriers (e.g. albomycin), or by self-promoted uptake (e.g. aminoglycosides and polymyxins). Transfer of antibiotics across the bacterial cytoplasmic membrane is usually mediated by active, carrier-mediated, transport systems normally operating to transport essential solutes into the cell. For example, the antibiotic streptozotocin bears sufficient structural resemblance toN-acetyl-D-glucosamine to be transported by the phosphoenolpyruvate : phosphotransferase system, and D-cycloserine is recognized by the D-alanine, proton motive force dependent transport system. However, in some cases (e.g. tetracycline) although carrier-mediated transport is implied by the observation that drug uptake is energy dependent, the nature of the membrane carrier(s) responsible is unknown. Knowledge acquired from studies on bacterial peptide transport has been successfully used to deliver (or smuggle) amino acid mimetics disguised as peptides into the bacterial cell. These amino acid mimetics, although often poorly transported in their own right, are frequently potent inhibitors of bacterial peptidoglycan or lipopolysaccharide synthesis once they have gained access to the interior of the cell.

Na-dependent L-glutamate transport by eel intestinal BBMV: role of K+ and Cl-

1989 ◽  
Vol 257 (1) ◽  
pp. R180-R188
Author(s):  
P. M. Romano ◽  
G. A. Ahearn ◽  
C. Storelli
Keyword(s):  
Amino Acid ◽  
Glutamate Uptake ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Anguilla Anguilla ◽  
Competitive Inhibitor ◽  
Glutamate Transport ◽  
Electrical Potential Difference ◽  
Intestinal Brush Border Membrane ◽  
Transmembrane Electrical Potential

L-[3H]glutamate uptake into eel (Anguilla anguilla) intestinal brush-border membrane vesicles (BBMV) was a sigmoidal function of extravesicular Na, suggesting that two or more cations accompanied the amino acid during transport. L-[3H]glutamate influx illustrated the following kinetic constants: apparent membrane binding affinity (Kapp) = 0.80 +/- 0.12 mM; influx velocity (Jmax) = 2.61 +/- 0.31 nmol.mg protein-1.min-1; and permeability coefficient (P) = 0.65 +/- 0.10 microliters.mg protein-1. min-1. Results from the imposition of diffusion potentials across vesicle membranes using K-valinomycin or H-carbonyl-cyanide p-chloromethoxyphenylhydrazone suggested that Na-dependent L-glutamate transport was sensitive to transmembrane electrical potential difference. Extravesicular aspartate was a competitive inhibitor of L-[3H]glutamate influx [inhibitory constant (Ki) = 0.28 +/- 0.04 mM]. Intravesicular K and extravesicular Cl ions enhanced maximal amino acid influx and transient L-glutamate accumulation against a concentration gradient (overshoot). Intravesicular K reduced the Kapp of the membrane to L-glutamate, whereas extravesicular Cl increased L-glutamate Jmax. A model for L-[3H]glutamate transport is suggested involving the cotransport of at least two Na and one L-glutamate that is activated by one intravesicular K ion and at least two extravesicular Cl ions.

Expression of Na+-d-glucose cotransporter in brush-border membrane of the chicken intestine

1999 ◽  
Vol 276 (2) ◽  
pp. R627-R631 ◽  
Author(s):  
Carles Garriga ◽  
Nativitat Rovira ◽  
Miquel Moretó ◽  
Joana M. Planas
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Brush Border ◽  
Synthetic Peptide ◽  
Brush Border Membrane ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Dependent Transport

We have studied the expression of Na+-d-glucose cotransporter in brush-border membrane vesicles (BBMVs) of chicken enterocytes to correlate the changes in the apical Na+-dependent transport with the changes in the amounts of transporter determined by Western blot analysis. Two different rabbit polyclonal antibodies were used simultaneously. The antibody raised against amino acids 564–575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 ( antibody 1) specifically detects a single 75-kDa band in the three segments, and this band disappeared when the antibody was preabsorbed with the antigenic peptide. The antibody raised against the synthetic peptide corresponding to amino acids 402–420 of the same protein ( antibody 2) only reacts with jejunal and ileal samples, but no signal is found in BBMVs of rectum. Only when antibody 1 was used was there a linear correlation between the maximal transport rates of hexoses in BBMVs and the relative protein amounts determined by Western blot. These results indicate that the Na+-d-glucose cotransport in the jejunum, the ileum, and the rectum of chickens is due to an SGLT-1 type protein.

Adaptation of phosphate transport in phosphate-deprived LLC-PK1 cells

1985 ◽  
Vol 248 (1) ◽  
pp. F122-F127 ◽  
Author(s):  
J. Caverzasio ◽  
C. D. Brown ◽  
J. Biber ◽  
J. P. Bonjour ◽  
H. Murer
Keyword(s):  
Adaptive Response ◽  
Apical Membrane ◽  
Specific Activity ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Linear Process ◽  
Transport Systems ◽  
Experimental Systems ◽  
Cotransport System ◽  
Dependent Transport

Sodium-dependent transport of phosphate was studied in LLC-PK1 cells that had been deprived of phosphate (Pi). Compared with control cells (fed with 2 mM Pi) a twofold increase in the rate of Na-Pi cotransport was observed in cells incubated for 15 h in a phosphate-free medium, whereas transport of L-alanine and the specific activity of alkaline phosphatase were not changed. The same adaptive response was observed with apical membrane vesicles isolated from Pi-deprived cells. In both experimental systems Pi deprivation caused a change in the Vmax but not in the apparent Km (for Pi) of the cotransport system. Adaptation of the Na-Pi cotransport was triggered by free phosphate concentrations between 0 and 100 microM. Over the first 20 h the adaptive response was found to be a linear process that could be prevented by 70 microM cycloheximide. Adapted cells that were re-treated with phosphate showed a rapid (less than 3 h) decrease in the Na-Pi transport. The data suggest that LLC-PK1 cells adapt to low Pi conditions by increasing the rate of the Na-Pi cotransport, which is located in the apical membrane. Two mechanisms may be involved in the adaptive response: a long-term process involving new protein synthesis, and a short-term response involving activation-inactivation of transport systems already existing.

Harmaline inhibition of Na-dependent transport in renal microvillus membrane vesicles

1980 ◽  
Vol 238 (3) ◽  
pp. F210-F217 ◽  
Author(s):  
P. S. Aronson ◽  
S. E. Bounds
Keyword(s):  
Glucose Transport ◽  
Membrane Vesicles ◽  
Competitive Inhibitor ◽  
Transport Processes ◽  
Coupled Transport ◽  
Na Transport ◽  
Phlorizin Binding ◽  
Drug Concentrations ◽  
Microvillus Membrane ◽  
Dependent Transport

The effects of the hallucinogen harmaline on D-glucose, L-alanine, and Na+ transport were studied in microvillus membrane vesicles isolated from the rabbit renal cortex. Harmaline had no effect on glucose transport in the absence of Na+, but reversibly inhibited sugar flux in the presence of NaCl. Inhibition of Na+-dependent glucose transport was inversely related to the Na+ concentrations. The hallucinogen competitively inhibited the Na+ activation of phlorizin binding to the membranes but did not inhibit phlorizin binding in the absence of Na+. Harmaline inhibited Na+-dependent alanine transport and, at higher drug concentrations, the amino acid flux in the absence of NaCl. Harmaline competitively inhibited the rate of Na+ uptake which, in the absence of glucose and alanine, is known to occur via Na+-H+ exchange. The hallucinogen trans-inhibited the efflux of glucoe and Na+ from membrane vesicles preloaded with the solutes. These findings suggest that harmaline is a direct inhibitor of microvillus membrane transport processes and acts as a competitive inhibitor of Na+ transport sites. Harmaline may therefore be a useful investigative tool for studying mechanisms of Na+-coupled transport in the luminal membrane of the proximal tubular cell.

Myocardial amino acid transport by canine sarcolemma vesicles

1987 ◽  
Vol 252 (6) ◽  
pp. H1070-H1076
Author(s):  
L. H. Young ◽  
B. L. Zaret ◽  
E. J. Barrett
Keyword(s):  
Amino Acid ◽  
Sarcoplasmic Reticulum ◽  
Membrane Vesicles ◽  
Amino Acid Uptake ◽  
Ventricular Myocardium ◽  
Transport Systems ◽  
Acid Uptake ◽  
Leucine Uptake ◽  
Alanine Transport ◽  
Canine Ventricular Myocardium

The transport of L-alanine and L-leucine into membrane vesicles isolated from mature canine ventricular myocardium was studied. Transport was assessed in purified sarcolemma and in vesicles differentially enriched either for sarcolemma or sarcoplasmic reticulum to further localize these transport systems. An imposed inward gradient of a NaNO3 stimulated uptake of L-alanine but not L-leucine by these vesicles. Amino acid uptake by these vesicles occurred into an osmotically active space. The stimulatory effect of Na+ on alanine transport was most striking in the purified sarcolemma vesicles, where Na+-stimulated alanine flux was 45 +/- 14 pmol X mg-1 X min-1. Furthermore, Na+-dependent alanine transport activity appeared to copurify with Na+-K+-ATPase activity, which served as a marker for sarcolemma membrane when these activities were compared in the three different membrane preparations. Leucine transport by sarcolemma was not altered by an imposed Na+ gradient. However, leucine uptake was a saturable function of extravesicular leucine and was inhibited by valine. In contrast, in sarcoplasmic reticulum membrane vesicles leucine uptake increased proportionately with increasing media leucine and was unaffected by valine. Our results demonstrate the feasibility of directly studying the transport of naturally occurring amino acids in membrane vesicles from mammalian heart, and the presence of Na+-dependent alanine transport system and a Na+-independent leucine transporter in the sarcolemma but not in sarcoplasmic reticulum of canine ventricular myocardium.

scholarly journals ATP-dependent transport of glutathione conjugate of 7β,8α-dihydroxy-9α,10α-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles

1998 ◽  
Vol 332 (3) ◽  
pp. 799-805 ◽  
Author(s):  
Sanjay K. SRIVASTAVA ◽  
Xun HU ◽  
Hong XIA ◽  
Richard J. BLEICHER ◽  
Howard A. ZAREN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Competitive Inhibitor ◽  
Dependent Manner ◽  
Plasma Membrane Vesicles ◽  
High Affinity ◽  
Dependent Transport ◽  
Detoxification Pathway ◽  
Stimulation Of

Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 µM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis–Menten kinetics (Km 46 µM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis–Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 µM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 µM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 µM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen.

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