Phosphate uptake by absorptive epithelia has been demonstrated to occur by a pH-sensitive, Na-dependent mechanism. The uptake process has been localized to the brush border membrane of intestinal cells where an arsenate-sensitive Na-dependent cotransport mechanism similar to that described for sugars and amino acids has been reported. It is the immediate goal of this proposal to isolate the Na/phosphate cotransporter and determine its structure. The long-term goal is to examine regulation of the cotransporter by vitamin D and dietary phosphate.
The specific aims of this project are to: 1) isolate and purify the functional cotransporter to homogeneity; 2) determine the structure of the cotransporter (sugar content, monomer/dimer, molecular mass, pI, amino acid composition, and amino acid sequence of substrate sites); 3) determine the molecular topography of the protein and active sites (fluorescent quenching of substrate site probes, fluorescence energy transfer, tryptophan and tyrosine fluorescence); 4) examine the cotransporter conformational changes (proteolytic digestion, tryptophan energy transfer and quench reagents, and substrate-site quenching; and 5) examine the effect of vitamin D and dietary phosphate on the number of membrane expressed cotransporters and cytoplasmic cotransporters. These studies will have a general impact on our understanding of the mechanism of sugar, amino acid, and metabolic intermediate transport across plasma membranes of the kidney, intestine, liver, and brain. These studies offer a unique opportunity to examine hormonal regulation of a transport protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK039944-04
Application #
3239985
Study Section
Physiology Study Section (PHY)
Project Start
1987-08-01
Project End
1992-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
4
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555