The isolation, characterization and amino terminal sequence of the vitamin D-binding protein (group specific component) from mouse plasma

James L. Borke, Robert D. Litwiller, Michael P. Bell, David N. Fass, David J. McKean, Rajiv Kumar

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Abstract

1. 1. In order to establish a homologous system in which to study the interaction of mouse vitamin D-binding protein (MVDBP) with mouse T-cell lymphocytes, we purified MVDBP from mouse plasma. 2. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that purified MVDBP had an apparent relative molecular weight of 49,000. 3. 3. Previous work in our laboratory has shown that purified rat vitamin D-binding protein (RVDBP) has an apparent relative molecular weight of 52,000. 4. 4. The amino terminal acid sequence of MVDBP is shown below and compared with that of RVDBP. MVDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysAsnGluLeuAlaMetLeuGlyLysGlu RVDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysGlnGluLeuSerThrLeuGlyLysAsp AspPhe AspPhe While 21 out of 24 residues (87.5%) of amino terminus of MVDBP are the same as those in RVDBP, residues 14, 17, 18 and 22 (underlined) are different. 5. 5. The sedimentation coefficient of the protein, determined by sucrose density gradient ultra-centrifugation, is 3.8 for MVDBP and 4.1 for the rat VDBP. 6. 6. The MVDBP purified in this study exhibits only one isoform on isoelectric focusing; the isoelectric point was 4.87 as determined on pH 4.0-6.5 isoelectric focusing gels (IEF). 7. 7. The binding of vitamin D3, 25-hydroxyvitamin D3 and three other analogs was investigated with a charcoal dextran assay. 8. 8. The concentration of 25-hydroxyvitamin D3 at which 50% of protein bound radiolabeled 25-hydroxyvitamin D3 was displaced from MVDBP was found to be 6.8 × 10-9M. A value of 5 × 10-9M was previously reported for the corresponding rat protein. 9. 9. Amino acid composition analysis also showed the mole percent of each residue in MVDBP was very similar to that of RVDBP. The observed properties of mouse VDBP suggest a high degree of homology with the vitamin D-binding protein of rat.

Original languageEnglish (US)
Pages (from-to)1343-1349
Number of pages7
JournalInternational Journal of Biochemistry
Volume20
Issue number12
DOIs
StatePublished - 1988

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ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

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