A panel of
monoclonal antibodies was produced against purified microvillus membranes of human small intestinal enterocytes. By means of these probes three
disaccharidases (
sucrase-
isomaltase,
lactase-phlorizin hydrolase, and
maltase-glucoamylase) and four
peptidases (
aminopeptidase N, dipeptidylpeptidase IV, angiotension I-converting
enzyme, and
p-aminobenzoic acid peptide hydrolase) were successfully identified as individual entities by SDS PAGE and localized in the microvillus border of the enterocytes by immunofluorescence microscopy. The
antibodies were used to study the expression of small intestinal
hydrolases in the colonic
adenocarcinoma cell line Caco 2. This cell line was found to express
sucrase-
isomaltase,
lactase-phlorizin hydrolase,
aminopeptidase N, and dipeptidylpeptidase IV, but not the other three
enzymes. Pulse-chase studies with [35S]
methionine and analysis by subunit-specific
monoclonal antibodies revealed that
sucrase-
isomaltase was synthesized and persisted as a single-chain
protein comprising both subunits. Similarly,
lactase-phlorizin hydrolase was synthesized as a large precursor about twice the size of the
lactase subunits found in the human intestine.
Aminopeptidase N and dipeptidylpeptidase IV, known to be dimeric
enzymes in most mammals, were synthesized as monomers. Transport from the rough endoplasmic reticulum to the trans-Golgi apparatus was considerably faster for the
peptidases than for the
disaccharidases, as probed by
endoglycosidase H sensitivity. These results suggest that the major
disaccharidases share a common biosynthetic mechanism that differs from that for
peptidases. Furthermore, the data indicate that the transport of microvillus
membrane proteins to and through the Golgi apparatus is a selective process that may be mediated by transport receptors.