Abstract
Mouse kidney, a vitamin D target organ, was investigated for the presence of vitamin D-dependent calcium-binding proteins (CaBP). Mouse kidney cytosol was fractionated by several biochemical methods including gel filtration chromatography, gel permeation high performance liquid chromatography, and chromatofocusing. Mouse kidney was found to possess two CaBPs which completely differed biochemically and exhibited no cross-immunoreactivity. One had a molecular weight of 25,000 and a pI of 5.9. The other, with a molecular weight of 10,000 and a pI of 4.9, was biochemically identical with mouse duodenal 10,000 CaBP. In addition, mouse renal and duodenal 10,000 CaBPs were immunologically identical. Moreover, the 10,000 CaBP was the predominant CaBP in mouse kidney since the latter contained about twice as much 10,000 CaBP as 25,000 CaBP (in mol/mg of renal cytosolic protein). In vitro incorporation of [3H]leucine into renal 10,000 CaBP demonstrated that it is synthesized in situ by mouse kidney. Renal 10,000 CaBP was already present during fetal life, and reached its adult level during the first week after birth. The vitamin D dependency of both mouse renal 10,000 and 25,000 CaBPs was assessed by their decrease in vitamin D-deficient mice and subsequent rise after 1,25-dihydroxyvitamin D3 injection. The concomitant presence of substantial amounts of two vitamin D-dependent CaBPs in mouse kidney is peculiar to this organ, which might consequently provide a unique model for studying the hormonal expressions of 1,25-dihydroxyvitamin D3.
Highlights
Cium-binding proteins (CaBP).Mouse kidney cytosol Purification of duodenal 10,000 CaBP from rat and mouse was fractionated by several biochemical methods in- allowed us todevelop two specific RIAs for each of these two cluding gel filtration chromatography, gel permeation species [14, 16].When studyingthe tissue distribution of high performance liquid chromatography, and chro- 10,000 CaBP in these species, we found a CaBP identical with matofocusing
To investigate the calcium-binding activity, mouse-kidney cytosol was fractionated by several biochemical techniques including gel fitration chromatography, GP-HPLC, and chromatofocusing
T o show that the 10,OOO CaBP we found in mouse kidney present in mouse kidney were compared from an immuno- was an intrinsic proteiwn,e tested mousekidney homogenates chemical point of view using Ouchterlony double immunodif- for their ability to incorporat[e''Hlleucine into CaBP
Summary
Renal 10,000 CaBP was already present For biochemical and immunological studies, normal Swiss mice, during fetal life, and reached its adult level during the 6-8weeks old, were obtained from CERJ, France afned on a normal first week after birth. The vitamin D dependency of diet (UAR, France) Both mouse renal 10,000and 25,000 CaBPswas assessed For vitamin D dependency studies, normal weanling Swiss mice by their decrease in vitamin D-deficient mice and subsequent rise after 1,25-dihydroxyvitamin DS injection. ZOO0 IU of vitamin Ddkg dry diet two vitamin D-dependent CaBPsin mouse kidney is were added to the daily feed (vitamin D-repleted group) In another peculiar to this organ, which might pro- group, each mousereceiveda subcutaneous injection of 1 ng of vide a unique modelfor studying the hormonal expressions of 1,25-dihydroxyvitaminDs.
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