The main objectives of this proposal are two fold: first, to understand the structural properties of alkaline phosphatase that lead to its anchoring in the membrane and its secretion into the blood and intestinal lumen; second, to determine the regulation of synthesis of the membranous and secreted forms of the enzyme. The knowledge gained from these studies will allow a more rational understanding of the production of serum alkaline phosphatase in man. Moreover, the unique probes that will be developed for the secreted isoenzyme may prove important in understanding and even possibly in diagnosing certain clinical conditions. We will identify cDNA clones encoding both rat membranous and secreted alkaline phosphatase, determine their sequence, and develop nucleotide, and if possible, antibody probes unique to each isoenzyme. These probes will allow us to assay each mRNA in tissues and cultured cell lines under a variety of conditions. The rat intestine is an excellent model tissue for this work, as it contains two mRNAs encoding alkaline phosphatase which are abundant and easily separable (3.0 and 2.7 Kb). Two intestinal cell lines will be studied, one a nonsecreting rat fetal cell, lRD- 98, and a differentiated human cell that secretes the enzyme (CaCo-2). Work with these cells will allow identification of factors that lead to secretion of the enzyme. Northern blotting and in situ hybridization will be used to determine the mRNA production encoding each phosphatase. The cell cultures will be used to define the pathway for phosphatase secretion, using cell fractionation and morphological assessment. The structure of each mRNA and the genomic structure will allow the mechanism of production of the two mRNAs to be determined. The genomic structure will help to identify regulatory regions upstream from the coding region that may regulate tissue specific expression or hormonal responsiveness. The structure of the two isozymes will allow an experimental approach to the problem of phosphatase membrane attachment. The role of covalently bound lipid will be explored by the use of cell-free transcription/translation systems. The possible role of unique COOH-terminal peptide sequences will be assessed by oligonucleotide directed mutagenesis. Knowledge of the secretory and anchoring mechanism in intestine will be useful in understanding release of phosphatase from membrane in other tissues where the enzyme is less abundant (e.g. liver).

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK014038-25
Application #
2136825
Study Section
Special Emphasis Panel (NSS (|))
Project Start
1977-12-01
Project End
1995-11-30
Budget Start
1993-12-01
Budget End
1994-11-30
Support Year
25
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Washington University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Mahmood, Akhtar; Engle, Michael J; Alpers, David H (2002) Secreted intestinal surfactant-like particles interact with cell membranes and extracellular matrix proteins in rats. J Physiol 542:237-44
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Xie, Q; Alpers, D H (2000) The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes. Physiol Genomics 3:8-Jan
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Zhang, Y; Shao, J S; Xie, Q M et al. (1996) Immunolocalization of alkaline phosphatase and surfactant-like particle proteins in rat duodenum during fat absorption. Gastroenterology 110:478-88
Eliakim, R; Alpers, D H; Oren, R et al. (1996) Abundance of surfactant-like particles reflects mucosal integrity in patients with peptic ulcer disease. Gut 39:353-9
Dieckgraefe, B K; Santoro, S A; Alpers, D H (1996) Immunolocalization of alpha-integrin subunits and extracellular matrix components during human colonic organogenesis. Gastroenterology 110:58-71
Alpers, D H; Zhang, Y; Ahnen, D J (1995) Synthesis and parallel secretion of rat intestinal alkaline phosphatase and a surfactant-like particle protein. Am J Physiol 268:E1205-14
Yamagishi, F; Becich, M J; Evans, B A et al. (1994) Clearance of surfactant-like particle proteins from circulation in rats. Am J Physiol 266:G596-605

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