Characterization of the rabbit renal na+/dicarboxylate
cotransporter using anti-fusion protein antibodies.
Pajor, Ana M., and Ning Sun.
University of Arizona, Department of Physiology, College of
Medicine, Tucson, AZ 85724
APStracts 3:0204C, 1996.
Polyclonal antibodies were prepared against the rabbit renal
Na+/dicarboxylate cotransporter, NaDC-1. The antibodies were raised
in chickens against a fusion protein consisting of a 60 amino acid
peptide from NaDC-1 and glutathione S-transferase. These antibodies
specifically recognized the fusion protein in Western blots, and
could immunoprecipitate the full-length NaDC-1 after in vitro
translation. The anti-fusion protein antibodies specifically
recognized a protein of 63 kDa in rabbit renal brush border membranes
(BBMV), similar to the predicted mass of 66 kDa. Two proteins of 57
and 115 kDa were recognized in rabbit intestinal brush border
membranes. A protein of 66 kDa was recognized in Xenopus oocytes
injected with NaDC-1 cRNA. Enzymatic deglycosylation of rabbit renal
BBMV resulted in a decrease in mass by 11 kDa, consistent with N
-glycosylation at a single site. Site-directed mutagenesis of the two
consensus sequences for N-glycosylation in the NaDC-1 cDNA showed
that Asn-576, located near the C-terminus, is glycosylated. The non
-glycosylated mutant of NaDC-1 exhibited 50% of wild-type succinate
transport activity when expressed in Xenopus oocytes, suggesting that
glycosylation is not essential for function. The revised secondary
structure model of NaDC-1 contains 11 putative transmembrane domains
and an extracellular glycosylated C-terminus.
Received 19 March 1996; accepted in final form 18 June 1996.
APS Manuscript Number C158-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 4 July 96