Functional characterization of purified zinc transporter from renal brush border membrane of rat.
Major zinc binding protein purified from renal brush border membrane (BBM) (R. Kumar, R. Prasad, Biochim. Biophys. Acta 1419 (1999) 23) was reconstituted into liposomes and its functional characteristics were investigated. Physical incorporation of the major zinc binding protein into the proteoliposomes was checked by SDS-PAGE, which showed a single band on silver staining. The structural integrity of the proteoliposomes was assessed by phase contrast microscopy, which revealed the proteoliposomes as globular structures and intact boundaries. Further structural integrity/leakiness of the proteoliposomes was checked by monitoring efflux of Zn(2+) from the pre-loaded proteoliposomes in the presence of either 2 mM Ca(2+) or Cd(2+) or Zn(2+). It was observed that even after 2 h of the initiation of efflux, 85-95% of Zn(2+) was retained in the proteoliposomes, thereby indicating that proteoliposomes were not leaky and maintained structural integrity during the uptake study. Zinc uptake into the proteoliposomes followed Michaelis-Menten kinetics with affinity constant (K(m)) of 1.03 mM and maximal velocity (V(max)) of 1333 nmol/mg protein per min. The uptake process followed first-order kinetics with a rate constant (k) of 1. 09x10(-3) s(-1). The specificity of zinc transport system was determined by studying the interaction of divalent cations viz. Ca(2+) and Cd(2+) with the zinc uptake. It was observed that Cd(2+) competitively inhibited the zinc uptake process with inhibitory concentration (K(i)) of 2.9 mM. Kinetic analysis of inhibitory effect of Cd(2+) on zinc uptake revealed an increase in K(m) to 1.74 mM without influencing V(max). Zn(2+) uptake into the proteoliposomes was found to be temperature sensitive and Arrhenius plot showed a breakpoint at 27 degrees C. The apparent energies of activation (E(a)) were found to be 7.09 and 2.74 kcal/mol below and above the breakpoint, respectively. The initial velocity of Zn(2+) uptake increased with the increase in outwardly directed proton gradient ([H](i) greater than [H](o)). The Zn(2+) uptake was inhibited by DCCD, thereby suggesting the involvement of -COOH groups in the translocation of Zn(2+) across the lipid bilayer. The ratio of acidic to basic amino acids (1.26) strongly indicates that it is an acidic protein. The cysteine content in this protein was insignificant, which further corroborates the possibility that the acidic amino acids might be prominent candidates for binding to zinc. The findings of the present study confirms that 40 kDa major zinc binding glycoprotein purified from renal BBM is a zinc transporter involved in the influx of Zn(2+) into the epithelial cells of the renal tubular system.
Kumar
R
R
Department of Biochemistry, Postgraduate Institute of Medical Education and Research, 160 012, Chandigarh, India.
Prasad
R
R
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002104:Cadmium / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D007684:Kidney Tubules / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D008871:Microvilli / Q000378:metabolism*; D010539:Permeability; D011510:Proteolipids / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D015032:Zinc / Q000378:metabolism*
D000596:Amino Acids; D002352:Carrier Proteins; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D007461:Iodoacetates; D008562:Membrane Glycoproteins; D011510:Proteolipids; C020485:proteoliposomes; C017800:zinc-binding protein; D002104:Cadmium; D004024:Dicyclohexylcarbodiimide; D015032:Zinc; D002118:Calcium