Partial purification of cytosolic 25 hydroxyvitamin D binding protein from rate enterocytes

Journal Article · Wed Jan 01 04:00:00 EST 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:7034913

Teegarden, D; Meredith, S; Sitrin, M

The authors have characterized and partially purified an enterocyte cytosolic 25 hydroxyvitamin D (250HD) binding protein which may participate in transcellular vitamin D metabolite transport. Triglyceride-rich emulsion particles (1%/wt/vol) containing /sup 3/H-250HD were incubated with rat intestinal cytosol or buffer for 0.5 hr. The reaction mixture was rapidly cooled and the mixture was centrifuged at 65,000g for 1 hr. Aliquots of buffer or cytosol infranatant were taken for scintillation counting. Only 4% of /sup 3/H-250HD transferred from the emulsion particles to buffer whereas 29% transferred to cytosol. Fractionation of cytosol by ammonium sulfate precipitation showed that the 60-80% saturation cut contained the 250HD binding activity. When this ammonium sulfate fraction was labeled with /sup 3/H-250HD and then subjected to gel permeation HPLC using a Toyo Soda G3000SW column, the /sup 3/H-250HD eluted in association with a peak of protein of MW/sup app/=68,000. The protein bound /sup 3/H-250HD eluted as a single peak on DEAE Sephacel anion exchange chromatography. The cytosolic 250HD binding activity was totally inactivated by heating to 60/sup 0/C for 1 hr. Serum vitamin D binding protein (sDBP) is stable under these heating conditions and has a MW/sup app/=58,000. They conclude that rat intestinal cytosol contains a 250HD binding protein whose MW and heat stability characteristics indicate that the cytosolic protein is distinct from sDBP.

• Research Organization: Univ. of Chicago, IL
• OSTI ID: 7034913
• Report Number(s): CONF-8604222-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:4; ISSN FEPRA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIOCHEMICAL REACTION KINETICS
CHROMATOGRAPHY
ENERGY
FRACTIONATION
HEAT
ION EXCHANGE CHROMATOGRAPHY
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MAMMALS
METABOLITES
ORGANIC COMPOUNDS
PROTEINS
PURIFICATION
RATS
REACTION KINETICS
RODENTS
SEPARATION PROCESSES
STABILITY
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES
VITAMIN D
VITAMINS