Robert W. Lobley

An aminopeptidase N deficiency in dog small intestine

This study has identified a naturally occurring, specific deficiency of a brush border aminopeptidase N (ApN) in the small intestines of five clinically healthy dogs. ApN activity in mucosal homogenates of dog small intestine was reduced significantly in deficient animals (13.4 (1.1) nmol min-1 mg-1 protein, n = 5, P < 0.002) compared to healthy control dogs (95.1 (6.7), n = 22). Alkaline phosphatase, gamma-glutamyl transferase, zinc-resistant alpha-glucosidase, maltase, sucrase and lactase in the ApN deficient dogs exhibited comparable activities to those in the control dogs. Microvillar membranes were analysed by one- and two-dimensional electrophoresis. ApN was represented by a single 145kDa band in all control dogs, identified by immunoblotting and immunoprecipitation. Protein maps from deficient dogs were normal apart from the virtual absence of an ApN spot and there were no apparent abnormalities in the glycosylation of microvillar proteins. The findings suggest that intestinal ApN deficiency in these dogs is a primary lesion involving diminished expression of an otherwise normal enzyme protein.

Investigation of the physical properties of dog intestinal microvillar membrane proteins by polyacrylamide gel electrophoresis: a comparison between normal dogs and dogs with exocrine pancreatic insufficiency

Procedures have been validated for the investigation of the physical properties of canine microvillar membrane proteins by SDS-polyacrylamide gel electrophoresis. These have been used to examine mucosal samples from eight control dogs and from five dogs with naturally occurring exocrine pancreatic insufficiency (EPI) in order to evaluate the potential role of the pancreas in the normal turnover of microvillar membrane proteins in the dog. Gel scanning showed that the proportion of total membrane protein in bands corresponding to a molecular mass greater than 200 kDa was up to 20-times higher in dogs with EPI than in control dogs. In particular, a band of apparent molecular mass 218 kDa represented between 8 and 28% of membrane protein in all affected dogs, compared with only 0.5 to 1.8% in controls, and is most likely to contain single chains of both pro-maltase-glucoamylase and pro-sucrase-isomaltase. Incubation of microvillar membranes in vitro with either trypsin or canine pancreatic juice resulted in degradation of this high molecular mass band and a corresponding increase in the amount of protein in three bands representing molecular masses of 150, 133 and 106 kDa. In samples from control dogs aminopeptidase N was identified in the 133 kDa band by Western blotting and incubation with monospecific antiserum. These findings suggest that pancreatic enzymes play a major role in the normal post-translational processing of intestinal microvillar membrane proteins in the dog.

The Kinetics of Monosaccharide Absorption by Human Jejunal Biopsies: Evidence for Active and Passive Processes

The kinetics of initial rates of uptake of glucose, galactose, arabinose and mannitol have been measured in jejunal biopsies from normal subjects in order to investigate the existence of multiple uptake systems. Glucose kinetics fitted best a model of a saturable uptake system (app Kt = 2.06 +/- 0.33 mM, app Jmax = 93.85 +/- 1.19 nmol/10 min/mg dry weight), together with a linear uptake indistinguishable from the passive uptake of arabinose and mannitol (app Kd = 0.80 +/- 0.05 nmol/10 min/mg dry weight/mM for glucose, 0.75 +/- 0.03 for arabinose, and 0.83 +/- 0.03 for mannitol). The saturable uptake, but not the linear uptake, was inhibited by phlorizin and by the absence of sodium. Cytochalasin B and phloretin had no effect on overall uptake. Galactose kinetics in the absence of inhibitors fitted best a model of a single saturable uptake system (app Kt = 11.05 +/- 0.12, app Jmax = 201.9 +/- 1.13) with no evidence of linear uptake. In the presence of phlorizin, or in the absence of sodium, uptake was predominantly linear with app Kds of 0.84 +/- 0.03 and 0.79 +/- 0.01, not significantly different from the linear component of glucose uptake. We conclude that hexose uptake in human jejunum in vitro occurs by both active and passive routes, and that the active uptake of galactose appears to be inhibited at high galactose concentration.

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

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.

GLUTEN-SENSITIVE ENTEROPATHY IN IRISH SETTER DOGS : CHARACTERISATION OF JEJUNAL MICROVILLAR MEMBRANE PROTEINS BY TWO-DIMENSIONAL ELECTROPHORESIS

This study investigated whether gluten-sensitive enteropathy (GSE) in Irish setter dogs was associated with underlying structural abnormalities of microvillar membrane proteins. Jejunal biopsies taken from eight-month-old GSE-affected dogs reared on a normal, gluten-containing diet exhibited partial villous atrophy and contained more intra-epithelial lymphocytes than controls. The morphological abnormalities were reversed by feeding a gluten-free diet for five months and the changes were accompanied by an increase in the mucosal activity of the microvillar hydrolases, particularly aminopeptidase N and dipeptidyl aminopeptidase IV, which reverted to pre-treatment levels after a gluten challenge. Two-dimensional electrophoresis of microvillar membrane proteins isolated from GSE-affected dogs revealed an essentially normal protein map that was comparable to controls. The exception was an intense 85 kDa protein spot that diminished when the affected dogs were fed a gluten-free diet and re-intensified after a gluten challenge.