Multiple carriers for dipeptide transport: carrier-mediated transport of glycyl-L-proline in renal BBMV

1991 ◽  
Vol 261 (4) ◽  
pp. F670-F678
Author(s):  
H. A. Skopicki ◽  
K. Fisher ◽  
D. Zikos ◽  
R. Bloch ◽  
G. Flouret ◽  
...  
Keyword(s):  
Brush Border Membrane ◽  
Competitive Inhibition ◽  
Membrane Vesicles ◽  
The Other ◽  
Lower Affinity ◽  
High Affinity ◽  
Carrier Mediated Transport ◽  
Transport Carrier ◽  
Dipeptide Transport ◽  
Lower Capacity

To determine whether multiple carriers are responsible for luminal uptake of glycyl-L-proline (Gly-Pro) in the renal proximal tubule, transport of Gly-[3H]Pro was measured in brush-border membrane vesicles (BBMV). A Line-weaver-Burk analysis of Michaelis-Menten kinetics revealed the presence of two carriers: a lower affinity, higher capacity carrier (Km = 1.3 x 10(-2) M; Vmax = 4.6 x 10(-8) mol.mg-1.min-1) and a higher affinity, lower capacity carrier (Km = 2.7 x 10(-7) M; Vmax = 7.8 x 10(-13) mol.mg-1.min-1). The dipeptides Gly-Sar, beta Ala-His, and pyroGlu-His competitively inhibited the low-affinity carrier. No effect on the Km or Vmax of Gly-Pro transport in this range was seen in the presence of the dipeptides Gly-Gly or cycloHis-Pro. The high-affinity carrier exhibited a different inhibition spectrum. Competitive inhibition of Gly-Pro transport was demonstrated for the dipeptides Gly-Gly and Gly-Sar. However, none of the other peptides tested above altered Gly-Pro transport in the high-affinity range, including pyroGlu-His, which is transported by a high-affinity carrier. At both low (4 x 10(-8) M) and high (4 x 10(-3) M) concentrations, uptake of Gly-Pro was stimulated in the presence of an inwardly directed H+ gradient but was unaffected by the presence of an inward Na+ gradient. In addition, measurements in the presence of valinomycin and an outwardly directed K+ gradient strongly suggest that H(+)-stimulated uptake at both concentrations is electrogenic.(ABSTRACT TRUNCATED AT 250 WORDS)

Dipeptide transport in brush-border membrane vesicles isolated from normal term human placenta

1985 ◽  
Vol 153 (1) ◽  
pp. 83-86 ◽  
Author(s):  
Malliga E. Ganapathy ◽  
Virendra B. Mahesh ◽  
Lawrence D. Devoe ◽  
Frederick H. Leibach ◽  
Vadivel Ganapathy
Keyword(s):  
Human Placenta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Normal Term ◽  
Dipeptide Transport

scholarly journals DapE Can Function as an Aspartyl Peptidase in the Presence of Mn2+

2003 ◽  
Vol 185 (16) ◽  
pp. 4748-4754 ◽  
Author(s):  
Daniel H. Broder ◽  
Charles G. Miller
Keyword(s):  
Divalent Cations ◽  
The Other ◽  
Expression Vectors ◽  
Peptidase Activity ◽  
Native Protein ◽  
Lower Affinity ◽  
High Affinity ◽  
High Affinity Site ◽  
Serovar Typhimurium ◽  
Dipeptidase Activity

ABSTRACT Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn2+. Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn2+-dependent peptidase is DapE (N-succinyl-l,l-diaminopimelate desuccinylase). A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain. Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source. The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-His6). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn2+-dependent aspartyl peptidase activity relative to the MPD dapE+ strain. In addition, purified DapE-His6 exhibited Mn2+-dependent peptidase activity toward aspartyl dipeptides. Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable. Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate. DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids. DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity. Our data indicate that the form of DapE active as a peptidase contains Zn2+ in the high-affinity site and Mn2+ in the low-affinity site.

Folate transport by human intestinal brush-border membrane vesicles

1987 ◽  
Vol 252 (2) ◽  
pp. G229-G236 ◽  
Author(s):  
H. M. Said ◽  
F. K. Ghishan ◽  
R. Redha
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Folate Transport ◽  
Intestinal Brush Border Membrane ◽  
Transport Carrier ◽  
Incubation Buffer

Transport of folic acid (Pte-Glu) across the brush-border membrane of human intestine was studied using brush-border membrane vesicle (BBMV) technique. The transport of Pte-Glu was higher in BBMV prepared from the jejunum than those prepared from the ileum (0.70 +/- 0.05 and 0.14 +/- 0.02 pmol X mg protein-1 X 10 s-1, respectively). The transport of Pte-Glu appeared to be carrier mediated and was pH dependent and increased with decreasing incubation buffer pH; saturable (Kt = 1.69 microM, Vmax = 4.72 pmol X mg protein-1 X 10 s-1); inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate, methotrexate, and 5-formyltetrahydrofolate (Ki = 2.2, 1.4 and 1.4 microM, respectively); not affected by inducing a relatively positive or negative intravesicular compartment; independent of Na+ gradient; and inhibited by 4,4'-diisothiocyanatostlibene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor. The increase in Pte-Glu transport on decreasing incubation buffer pH appeared to be in part mediated through a direct effect of acidic pH on the transport carrier and in part through the pH gradient imposed by activating Pte-Glu-:OH- exchange and/or Pte-Glu-:H+ co-transport mechanisms. The important role of an acidic extravesicular environment in Pte-Glu transport is consistent with a role for the intestinal surface acid microclimate in folate transport. These results demonstrate that Pte-Glu transport in human BBMV occurs by a carrier-mediated system that is similar to that described for rat and rabbit intestinal BBMV.

Uphill transport of beta-alanine in intestinal brush-border membrane vesicles

1990 ◽  
Vol 259 (3) ◽  
pp. G372-G379 ◽  
Author(s):  
Y. Miyamoto ◽  
H. Nakamura ◽  
T. Hoshi ◽  
V. Ganapathy ◽  
F. H. Leibach
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Beta Alanine ◽  
Intestinal Brush Border Membrane ◽  
High Affinity ◽  
Intestinal Brush Border ◽  
Proximal Small Intestine ◽  
Uptake Process

The characteristics of beta-alaline uptake were studied in brush-border membrane vesicles isolated from the proximal small intestine of rabbits and were compared with those of L-alpha-alanine uptake. The uptake of beta-alanine as well as L-alpha-alanine was significantly stimulated by imposing an inwardly directed Na+ gradient. Studies on transstimulation and substrate specificity provide evidence that the transport system serving beta-alanine is distinct from the system serving alpha-alanine. The beta-system also accepts taurine as a substrate. The Na(+)-dependent uptakes of beta-alanine and L-alpha-alanine were differentially influenced by anions. The order in which anions supported uptake was Cl- = SCN- greater than F- greater than NO3- = SO2(-4) for beta-alanine, whereas it was SCN- greater than F- = Cl- = NO3- greater than SO2(-4) for L-alpha-alanine. Cl- appeared to be the preferred anion to support the uptake of beta-alanine. beta-Alanine uptake was greater in the presence of an inwardly directed Cl- gradient than in the presence of Cl- at equal concentrations on both sides of the membrane. The uptake was maximal when a Na+ gradient and a Cl- gradient were present simultaneously. The NaCl gradient-driven beta-alanine uptake was stimulated by an inside-negative K(+)-diffusion potential induced by valinomycin, showing that the uptake process is electrogenic. Stoichiometric analyses suggest that multiple Na+ and one Cl- are associated with the uptake of one beta-alanine molecule. The kinetic study shows that the transporter for beta-alanine is a high-affinity, low-capacity system (Kt = 46 +/- 1 microM; Vmax = 30 +/- 1 pmol.mg protein-1.15 s-1).

G protein-coupled prostaglandin receptor modulates conductive Na+ uptake in lung apical membrane vesicles

1998 ◽  
Vol 274 (4) ◽  
pp. L567-L572
Author(s):  
Somnath Mukhopadhyay ◽  
Asim K. Dutta-Roy ◽  
Gregor K. Fyfe ◽  
Richard E. Olver ◽  
Paul J. Kemp
Keyword(s):  
G Protein ◽  
Protein Function ◽  
Apical Membrane ◽  
Competitive Inhibition ◽  
Rank Order ◽  
Membrane Vesicles ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Uptake Studies ◽  
G Protein Coupled

Because G protein-regulated cation channels in type II pneumocytes constitute the most likely pathway for alveolar Na+ entry, we explored the hypothesis that a G protein-coupled prostaglandin (PG) E2 receptor controls perinatal lung alveolar Na+ transport. [3H]PGE2binding to the alveolar apical membrane was trypsin sensitive and showed a rank order of competitive inhibition: PGE2 = PGE1 > PGD2 > PGF2α. Kinetic analysis demonstrated both high-affinity [dissociation constant ( K D) = 2.1 ± 0.7 nM; maximal binding (Bmax) = 27 ± 7 fmol/mg protein] and low-affinity ( K D = 28 ± 2 nM; Bmax = 265 ± 29 fmol/mg protein) binding sites. Modulation of high-affinity GTPase activity identified a similar potency order (IC50 = 11 mM for PGF2α vs. 10–50 μM for other PGs), suggesting that the receptors are G protein coupled. Finally, 1 μM PGE2(≈IC25) increased conductive22Na+uptake into membrane vesicles only in the presence of 100 μM intravesicular GTP. The K D value for the high-affinity binding site together with the rank order of PG effect on ligand binding and G protein function places this PG receptor in the EP3 subtype, whereas Na+ uptake studies suggest that it helps maintain perinatal lung Na+homeostasis.

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