Biophysical characterization and functional studies on calbindin-D sub 28K : A vitamin D-induced calcium-binding protein

Miscellaneous · Sun Jan 01 00:00:00 EST 1989

OSTI ID:5476081

Leathers, V L

Vitamin D dependent calcium binding protein, or calbindin-D, is the principal protein induced in the intestine in response to the steroid hormone 1,25(OH){sub 2}-vitamin D{sub 3}. A definitive role for calbindin-D in vitamin D{sub 3} mediated biological responses remains unclear. Biophysical and functional studies on chick intestinal calbindin-D{sub 28K} (CaBP) were initiated so that some insight might be gained into its relevance to the process of intestinal calcium transport. Calbindin-D belongs to a class of high affinity calcium binding proteins which includes calmodulin, parvalbumin and troponin C. The Ca {sup 2+} binding stoichiometry and binding constants for calbindin-D{sub 28K} were quantitated by Quin 2 titration analysis. The protein was found to bind 5-6 Ca {sup 2+} ions with a K{sub D} on the order of 10{sup {minus}8}, in agreement with the 6 domains identified from the amino acid sequence. A slow Ca {sup 2+} exchange rate (80 s{sup {minus}1}) as assessed by {sup 43}Ca NMR and extensive calcium dependent conformational changes in {sup 1}H NMR spectra were also observed. Functional studies on chick intestinal CaBP were carried out by two different methods. Interactions between CaBP and intestinal cellular components were assessed via photoaffinity labeling techniques. Specific calcium dependent complexes for CaBP were identified with bovine intestinal alkaline phosphatase and brush border membrane proteins of 60 and 150 kD. CaBP was also found to co-migrate with the alkaline phosphatase activity of chick intestinal brush border membranes as evaluated by gel filtration chromatography. The second procedure for evaluating CaBP functionality has involved the quantitation of CaBP association with vesicular transport components as assessed by ELISA. CaBP, immunoreactivity was observed in purified lysosomes, microsomes and microtubules.

• Research Organization: California Univ., Riverside, CA (United States)
• OSTI ID: 5476081
• Resource Relation: Other Information: Thesis (Ph. D.)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CALCIUM COMPOUNDS
MEMBRANE TRANSPORT
PROTEINS
BIOLOGICAL FUNCTIONS
CHEMICAL COMPOSITION
VITAMIN D
BIOCHEMICAL REACTION KINETICS
CALCIUM 43
CHICKENS
ENZYME IMMUNOASSAY
INTESTINES
NMR SPECTRA
PROTONS
STOICHIOMETRY
ALKALINE EARTH ISOTOPES
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
BARYONS
BIOASSAY
BIRDS
BODY
CALCIUM ISOTOPES
DIGESTIVE SYSTEM
ELEMENTARY PARTICLES
EVEN-ODD NUCLEI
FERMIONS
FOWL
GASTROINTESTINAL TRACT
HADRONS
IMMUNOASSAY
INTERMEDIATE MASS NUCLEI
ISOTOPES
KINETICS
NUCLEI
NUCLEONS
ORGANIC COMPOUNDS
ORGANS
REACTION KINETICS
SPECTRA
STABLE ISOTOPES
VERTEBRATES
VITAMINS
550201* - Biochemistry- Tracer Techniques