For many years we have been interested in the synthesis and degradation of enterocyte proteins. This is a good system in which to study changes in these parameters, because this cell contains a number of specific proteins which have been purified to homogeneity and against which antibodies have been raised, and because the accumulation of many of these proteins respond to dietary or hormonal stimuli. Although we have successfully studied enterocyte protein degradation, we have been unable to investigate protein synthesis, due to the lack of a suitable cellular or organ culture system. For this reason we have chosen to look at synthesis at a more molecular level, concentrating on the translation of messenger RNA (mRNA) in the cell free system, and making the assumption that alterations in the content of mRNA will reflect synthetic capacity. We will use well characterized methods to determine mRNA content, primary structure of proteins, and processing of the primary translation product. Specific methods to be used will include identification of cell free translation products, microsequencing of primary translation products isolated by immune precipitation from cell free translation systems, protein sequencing of """"""""mature"""""""" proteins, co and post translational cleavage of polypeptides, and cDNA cloning using recombinant DNA techniques. Two classes of proteins will be studied: 1) cytosol proteins, especially fatty acid binding proteins in the enterocyte, and intrinsic factor in the stomach, because of their abundance, relatively small size, and availability of antibodcies. 2) Membrane proteins, especially alkaline phosphatase, because of its dual location on the membrane and in soluble form, and sucrase-isomaltase, because of its relative abundance, ease of isolation, and unusual structural properties. The special problems posed by the study of large proteins in cell free systems are recognized and wil be addressed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK014038-17
Application #
3225192
Study Section
(GCN)
Project Start
1977-12-01
Project End
1987-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
17
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Washington University
Department
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
Mahmood, A; Engle, M J; Hultgren, S J et al. (2000) Role of intestinal surfactant-like particles as a potential reservoir of uropathogenic Escherichia coli. Biochim Biophys Acta 1523:49-55
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
Goetz, G S; Mahmood, A; Hultgren, S J et al. (1999) Binding of pili from uropathogenic Escherichia coli to membranes secreted by human colonocytes and enterocytes. Infect Immun 67:6161-3
Eliakim, R; Goetz, G S; Rubio, S et al. (1997) Isolation and characterization of surfactant-like particles in rat and human colon. Am J Physiol 272:G425-34
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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