Regulation of proton transport in urinary epithelia

1983 ◽  
Vol 106 (1) ◽  
pp. 135-141 ◽  
Author(s):  
Q. Al-Awqati ◽  
S. Gluck ◽  
W. Reeves ◽  
C. Cannon
Keyword(s):  
Plasma Membrane ◽  
Substrate Specificity ◽  
Proton Transport ◽  
Transport Activity ◽  
Kidney Medulla ◽  
F1 Atpase ◽  
Yeast Plasma Membrane ◽  
Luminal Membrane ◽  
Turtle Bladder ◽  
Proton Translocating Atpase

In urinary epithelia, like the turtle bladder, protons are transported by a H+ translocating ATPase located in the luminal membrane. We have recently discovered that the H+ pump is stored in small vesicles that lie underneath the luminal membrane. CO2, a major regulator of H+ transport causes these vesicles to fuse with the membrane thereby inserting more H+ pumps. We have now isolated these vesicles from the turtle bladder and from beef kidney medulla. Based on inhibitor sensitivity and substrate specificity this proton translocating ATPase is different from the mitochondrial F0-F1 ATPase, yeast plasma membrane and the gastric H+,K+-ATPase. Solubilization and reconstitution of the enzyme into liposomes shows retention of transport activity and inhibitor sensitivity.

Proton-translocating ATPase from bovine kidney medulla: partial purification and reconstitution

1988 ◽  
Vol 254 (1) ◽  
pp. F71-F79 ◽  
Author(s):  
S. Gluck ◽  
J. Caldwell
Keyword(s):  
Proton Transport ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Intact Protein ◽  
Partial Purification ◽  
Kidney Medulla ◽  
Bovine Kidney ◽  
Vacuolar Acidification ◽  
Proton Translocating Atpase

The proton-translocating ATPase that is responsible both for urinary and vacuolar acidification was partially purified from bovine kidney medulla microsomes. ATPase activity was purified to a maximum specific activity of 1.7 mumol.min-1.mg prot-1 and was inhibited completely by N-ethylmaleimide. The relative molecular weight (Mr) of the intact protein estimated by high-pressure size-exclusion liquid chromatography was 586,000. Nondenaturing gels of the isolated enzyme revealed two protein bands at MrS of 551,000 and 523,000. Sodium dodecyl sulfate-gel electrophoresis of the isolated H+-ATPase revealed component subunits at MrS of 70,000, 56,000, 45,000, 42,000, 38,000, 31,000, 15,000, 14,000, and 12,000. The properties of the isolated H+-ATPase and of microsomal ATP-dependent proton transport correlated closely. The isolated H+-ATPase was reconstituted into phospholipid liposomes and demonstrated N-ethylmaleimide-inhibitable ATP-dependent potential generation, consistent with electrogenic proton transport. In overall structure, the enzyme appears to be a new type of H+-ATPase with several features of the F0F1 class of ion-translocating ATPases but is immunologically and structurally different from the mitochondrial F1-ATPase.

scholarly journals Membrane lipid requirements of the lysine transporter Lyp1 from Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Joury S van‘t Klooster ◽  
Tan-Yun Cheng ◽  
Hendrik R Sikkema ◽  
Aike Jeucken ◽  
D. Branch Moody ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Conformational Changes ◽  
Membrane Lipids ◽  
Lateral Diffusion ◽  
Membrane Lipid ◽  
Transport Activity ◽  
Yeast Plasma Membrane ◽  
Model Protein ◽  
High Fraction

AbstractMembrane lipids act as solvents and functional cofactors for integral membrane proteins. The yeast plasma membrane is unusual in that it may have a high lipid order, which coincides with low passive permeability for small molecules and a slow lateral diffusion of proteins. Yet, membrane proteins whose functions require altered conformation must have flexibility within membranes. We have determined the molecular composition of yeast plasma membrane lipids located within a defined diameter of model proteins, including the APC-superfamily lysine transporter Lyp1. We now use the composition of lipids that naturally surround Lyp1 to guide testing of lipids that support the normal functioning of the transporter, when reconstituted in vesicles of defined lipid composition. We find that phosphatidylserine and ergosterol are essential for Lyp1 function, and the transport activity displays a sigmoidal relationship with the concentration of these lipids. Non-bilayer lipids stimulate transport activity, but different types are interchangeable. Remarkably, Lyp1 requires a relatively high fraction of lipids with one or more unsaturated acyl chains. The transport data and predictions of the periprotein lipidome of Lyp1, support a new model in which a narrow band of lipids immediately surrounding the transmembrane stalk of a model protein allows conformational changes in the protein.

scholarly journals A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport

2003 ◽  
Vol 373 (3) ◽  
pp. 767-774 ◽  
Author(s):  
Hideyuki MITOMO ◽  
Ryo KATO ◽  
Akiko ITO ◽  
Shiho KASAMATSU ◽  
Yoji IKEGAMI ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Atp Binding ◽  
Transport Activity ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Atp Binding Cassette Transporter ◽  
293 Cells ◽  
Atp Binding Cassette

Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg482, Gly482, and Thr482 variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg482]ABCG2 transports [3H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly482 and Thr482). Transport of methotrexate by [Arg482]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S-octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg482]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg482 is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

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