The intestinal aminooligopeptidase (AOP or aminopeptides N) is a 140 kDa glycoprotein with N-linked carbohydrate chains that is initially synthesized in association with endoplasmic reticulum (ER) and transferred to the Golgi where its chains mature. It is then translocated unchanged, presumably by transport vesicles to the brush border membrane surface where it plays an essential role in the surface digestion of oligopeptides of three to seven amino acid residues. An in vivo rat model has been established whereby a segment of jejunal mucosa is isolated for perfusion in an anesthetized animal and precursor 35S-methionine or 3H- fucose are pulsed for five minutes intraintestinally followed by a chase of up to six hours. Maximal incorporation occurs in ER in 50 minutes, in Golgi at 45 minutes, but the arrival at the brush border surface of newly synthesized enzyme occurs many hours later. In the BioBreed Wistar diabetic rat (BBW rat) this enzyme appears to be synthesized normally in the ER where initial glycosylation occurs. Yet a subsequent maturation of the carbohydrate chains is altered; a larger AOP species is seen both in the Golgi and eventually in the brush border. This slightly larger species has also been found in human diabetic intestine. Both N-linked and O-linked carbohydrate chains will be isolated from purified AOP from both rat and human intestine and structurally characterized after purification on high performance liquid chromatography (HPLC) by use of glycosidase probes after radiolabeling metabolically or chemically after their isolation. These would include the endo-beta-glycosidases (Endo H and Endo F), glycosidases and the glycosyl transferases. Isolated chains will also be methylated to determine the types of linkages. Criteria for transport vesicle characterization and specific binding to brush border will also be analyzed. Finally, because AOP can be abruptly regulated to increase its synthesis at least two-fold when a high affinity substrate becomes present in the intestinal lumen, the nature of this regulation will be analyzed by use of a specific cDNA probe. Intestinal RNA and PolyA RNA will be isolated with the new low temperature technique (J.H. Han, et al., Biochemistry 26:1617, 1987) and full length cDNA cloned into the lambda gt18 vector and positive clones identified by monospecific AOP antibody. 32P-cDNA will then be used in a dot blot assay to quantify mRNA for AOP in rat intestine from normal and protein- deficient intestine that have displayed abrupt induction of new AOP synthesis in response to intraintestinal substrate. In this manner, it will be possible to correlate intestinal mRNA levels for AOP with its synthesis rates in vivo and to design further experiments to ascertain the regulation of translation of this important digestive enzyme.