A quantitative histochemical technique for the characterisation of alpha-glucosidases in the brush-border membrane of rat jejunum.
A quantitative histochemical method to determine the Km and Vmax of alpha-glucosidases in the intestinal epithelium without disruption of the cellular structure is described. 2-Naphthyl-alpha-D-glucoside was used as substrate and hexazonium-p-rosaniline as coupling agent. Using a Leitz MPV2 microdensitometer and a field measuring 4 X 4 micrometers, and reading the test samples against a blank focused on the lamina propria, we observed that the intensity of the colour was a linear function of both the incubation time up to 20 min, and the thickness of the slice up to 20 micrometers. The ratio between the extinction at the absorption maximum and at a second wave-length was constant, whatever the intensity of the colour. By determining the relationship between the extinction and the substrate concentration under standard conditions (slice thickness of of 10 micrometers and incubation time of 10 min), we obtained a saturation curve described by a Km of 0.68 +/- 0.038 mM and a Vmax of 1.41 +/- 0.039 A lambda 480 . 10(-2) . micrometers-1 . min-1. When the hydrolysis of the same substrate by a homogenate of jejunal mucosa was examined biochemically under comparable conditions, a Km of 0.64 +/- 0.012 mM and a Vmax of 57.3 +/- 0.70 mU/mg protein were obtained. When the natural substrate, sucrose, was used in the biochemical study, a Km of 15 +/- 3.5 mM and a Vmax of 149 +/- 24.7 mU/mg protein were obtained. These experiments demonstrate that the kinetic constants of enzyme reactions can be assessed with equal accuracy on histochemical sections as in tissue homogenates.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D003720:Densitometry; D005260:Female; D005959:Glucosidases / Q000378:metabolism*; D006651:Histocytochemistry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000648:ultrastructure; D007583:Jejunum / Q000201:enzymology*; D007700:Kinetics; D008871:Microvilli / Q000201:enzymology*; D051381:Rats; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000378:metabolism*
D005959:Glucosidases; D000520:alpha-Glucosidases