Kaler P, Prasad R. Molecular cloning and functional characterization of novel zinc transporter rZip10 (Slc39a10) involved in zinc uptake across rat renal brush-border membrane. Am J Physiol Renal Physiol 292: F217-F229, 2007. First published June 27, 2006 doi:10.1152/ajprenal.00014.2006.-Previously, in our laboratory a 40-kDa zinc transporter protein was purified and functionally reconstituted in proteoliposomes (Kumar R, Prasad R. Biochim Biophys Acta 1419: [23][24][25][26][27][28][29][30][31][32] 1999). Furthermore, we now report the identification of Slc39a10 cDNA encoding the 40-kDa zinc transporter protein by isolating a cloned DNA complementary to zinc transporter mRNA. cDNA was constructed from immunoenriched mRNA encoding the zinc transporter. cDNA was inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli DH5␣ cells were transformed, and colonies were screened for zinc transporter cDNA by insertional inactivation. Plasmid DNA was purified from the ampicillin-sensitive clones, and the cDNA was sequenced from both strands. A basic local alignment research tool (BLAST) search of cDNA revealed that it belongs to the Slc39 gene family of zinc transporters and was designated as Slc39a10. Zinc transporter protein deduced on the basis of cDNA sequence was named rZip10 and consists of 385 amino acids with 9 predicted transmembrane domains. The Slc39a10 gene was abundantly expressed in both rat and human tissues. Increased extracellular zinc concentration resulted in upregulation of Slc39a10 in LLC-PK1 cells expressing rZip10, which was downregulated at higher zinc concentrations. These cells accumulated more zinc than control cells. rZip10-mediated zinc uptake activity was time-, temperature-, and concentration-dependent and saturable which followed Michaelis-Menten kinetics with a Km of 19.2 M and Vmax of 50 pmol⅐min Ϫ1 ⅐mg protein Ϫ1
The present study was conducted to find out any alteration in the expression and activity of alkaline phosphatase in the brush border membrane (BBM) from renal cell carcinoma (RCC) in comparison to normal renal BBM. The specific activity of alkaline phosphatase was drastically reduced in homogenate as well as BBM from RCC kidney when compared to ALP activity in BBM of normal kidney. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (V(max)) and increase in affinity constant (K(m)) of the enzyme. SDS-PAGE studies showed that the BBM proteins having molecular weights ranging from 95 to 170 kDa were poorly expressed in RCC BBM in relative to normal kidney BBM. Incubation of SDS-PAGE gel with BCIP/NBT dye clearly showed that the expression of ALP in tumor renal BBM was markedly reduced as compared to normal kidney. Further, Western blot analysis using anti-alkaline phosphatase antibody also confirmed the reduced expression of ALP in tumor renal BBM. Lipid composition in reference to phospholipids, glycolipids and cholesterol in tumor renal BBM was altered to that of normal renal BBM, indicating alteration in membrane fluidity of tumor renal BBM.
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