Structural Studies on Microvillus Aminopeptidase from Pig Small Intestine

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Abstract
Two amphiphilic forms of intestinal microvillus aminopeptidase were obtained by purification of the enzyme in the absence and in the presence of aprotinin respectively. By subsequent treatment with trypsin in vitro a hydrophilic form was obtained. These 3 enzyme preparations all contained 3 subunits, A, B and C (MW 168,000, 118,000 and 54,000 respectively). The individual subunits were purified by gel filtration on Ultrogel AcA 22 in the presence of sodium dodecyl sulfate [SDS]. Subunits A and B from aminopeptidase prepared in the presence of aprotinin contained both N-terminal lysine and alanine, whereas N-terminal alanine and glycine were found for both of these subunits from the other amphiphilic form. Conversion of amphiphilic aminopeptidase to the hydrophilic form in vitro led only to a change in the N terminus of the B subunit to a valine. N-terminal serine was found in subunit C from all 3 forms of the aminopeptidase. The amino acid composition and peptide maps of the isolated subunits from one of the amphiphilic forms are reported. Subunit B and C may arise by cleavage of subunit A, whereas it seemed unlikely that C originates from B. Brush border membrane vesicles were cross-linked by dimethyl-3,3''-dithiobispropionimidate, and the aminopeptidase subsequently isolated. Analysis of MW and subunit composition of its constituent polypeptide complexes by 2-dimensional polyacrylamide slab gel electrophoresis in the presence of SDS showed that AB, BBC, BCC, BB, BC and CC complexes had been formed. The subunit structures ABC and BBCC were thus proposed in agreement with the maximum MW of the membrane-bound enzyme previously determined as 330,000.

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