An organ culture employing slices of renal-cortex tissue from piglets of the Yucatan strain was used to study the biogenesis of four microvillar peptidases: endopeptidase-24.11 (EC 3.4.24.11), dipeptidyl peptidase IV (EC 3.4.14.5), aminopeptidase N (EC 3.4.11.2) and aminopeptidase A (EC 3.4.11.7). The viability of the culture system was confirmed by the preservation of ultrastructural integrity and by an unchanged uptake of [3H]alanine into cells during the period of the experiments. After labelling with [35S]methionine, treatment with Mg2+ yielded two fractions, one containing microvilli and another, the Mg2+ pellet, containing intracellular and basolateral membranes. The labelled forms of the peptidases, isolated by immunoprecipitation, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The Mg2+ pellet contained the earliest detectable forms of the enzymes. In each case, a polypeptide of lower Mr than the mature form and sensitive to treatment with endo-beta-N-acetylglucosaminidase H was the first form to be detected. These high-mannose forms were followed, about 30 min after the pulse, by a complex glycosylated form of higher Mr. Only the latter form was observed in microvilli and then only after 90 min of the chase period. A quantitative study of dipeptidyl peptidase IV showed that the forms observed in the Mg2+ pellet were precursors of those in the microvillar fraction. No labelled forms were observed in the cytosol. All four peptidases were thus synthesized within membrane compartments and glycosylated in two steps before assembly in microvilli.

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Endopeptidase-24.11, an integral microvillar membrane enzyme, exists in differently glycosylated forms when purified from pig kidney and intestine [Fulcher, Chaplin & Kenny (1983) Biochem. J. 215, 317-323]. When these glycoproteins, and another form of the kidney enzyme prepared from the Yucatan dwarf strain of piglet, were treated, under controlled conditions, with trifluoromethanesulphonic acid, the proteins were freed of carbohydrate and all had the same apparent subunit Mr (77 000) even though the untreated forms varied from Mr 89 000 to Mr 95 000.

The effects of various inhibitors were studied on the biogenesis of endopeptidase-24.11 (EC 3.4.24.11) and dipeptidyl peptidase IV (EC 3.4.14.5) in slices of renal cortex, from piglets of the Yucatan strain, maintained in organ culture. These microvillar peptidases were synthesized within membrane compartments and underwent glycosylation to yield high-mannose and complex forms [the preceding paper, Stewart & Kenny (1984) Biochem. J. 224, 549-558]. Monensin caused very gross ultrastructural changes in the proximal-tubular cells, resulting from distension of the Golgi sacs. It blocked the processing of the high-mannose to the complex glycosylated forms of the peptidases and prevented their assembly in the microvillar membrane. Swainsonine, an inhibitor of alpha-mannosidase II, generated new 'hybrid' forms of the proteins, intermediate in Mr between the high-mannose and the complex forms, but did not prevent assembly of the hybrid forms in microvilli. Vinblastine, an agent that affects microtubules, delayed, but did not abolish, either the processing or the transport to microvilli. Glucosamine interfered with the initial glycosylation reactions and generated heterogeneous sets of partially glycosylated polypeptides of lower Mr than the high-mannose forms. These results are discussed in relation to the site and mechanism of glycosylation and the involvement of the Golgi complex and microtubules in the biogenesis of these membrane peptidases.