Characterization of microvillar membrane proteins of dog small intestine by two-dimensional electrophoresis

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology Vol. 110; no. 3; pp. 483 - 492
• Main Authors: Pemberton, Philip W., Lobley, Robert W., Holmes, Raymond, Sørensen, Susanne H., Simpson, Kenneth W., Batt, Roger M.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.03.1995
• Subjects: Alkaline Phosphatase - isolation & purification; Animals; Cytoskeletal Proteins - chemistry; Cytoskeletal Proteins - isolation & purification; Cytoskeleton; Dipeptidyl Peptidase 4 - isolation & purification; Dog; Dogs; Electrophoresis; Electrophoresis, Gel, Two-Dimensional - methods; Glycoproteins; Humans; Hydrolases; Intestine, Small - chemistry; Intestine, Small - enzymology; Isoelectric Focusing; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - isolation & purification; Membrane Proteins - chemistry; Membrane Proteins - isolation & purification; Microchemistry - methods; Microvillar membrane; Microvilli - chemistry; Microvilli - enzymology; Molecular Weight; Peptide Mapping; Protein mapping; Small intestine; Species Specificity; Microvillar membrane; MOPS; SDS; Protein mapping; Glycoproteins; PAGE; EDTA; Small intestine; DFP; Con-A; PMSF; HEPES; Cytoskeleton; Electrophoresis; Hydrolases; Dog
• ISSN: 1096-4959; 1879-1107
• DOI: 10.1016/0305-0491(94)00209-D

A method for analysing microgram amounts of microvillar membranes by two-dimensional electrophoresis (protein mapping) is described, and has been used to characterize the microvillar proteins of the small intestine of German shepherd, corgi, and beagle dogs. Detergent-solubilized microvillar membranes were radiolabelled with 14C and separated by isoelectric focussing followed by SDS-PAGE. Proteins were detected fluorographically and glycoproteins by lectin-affinity staining. The microvillar hydrolases alkaline phosphatase and dipeptidyl aminopeptidase IV were identified by active-site labelling and aminopeptidase N by immunoprecipitation. Changes following pancreatic duct diversion were consistent with accumulation of pro-sucrase-isomaltase and diminished expression of the sucrase and isomaltase subunits. Cytoskeletal proteins were concentrated in the core fraction remaining after extraction of microvillar membranes with Triton X-100. There were no consistent differences between dogs of different breed, and the canine protein maps were similar to the human.