Topology of microvillar membrance hydrolases of kidney and intestine.

Biosynthesis of microvillar proteins

“…The similarity in size (within the resolution of the gel system, 1500Da) of the primary translation product, determined in the absence of microsomal membranes, and a molecular form of the enzyme obtained from tunicamycin-exposed intestinal explants may suggest that the signal does not undergo cleavage during membrane translocation. This finding, albeit provisional until confirmed by stricter experiments such as radiosequencing, bears some topological consequences for aminopeptidase N. Like other microvillar enzymes studied, aminopeptidase N is anchored to the membrane by a small hydrophobic segment near its N-terminus which spans the lipid bilayer (Kenny & Maroux, 1982), possibly giving the enzyme a 'bitopic' membrane configuration in the terminology of Blobel (1980). Aminopeptidase N may therefore be the first example of a bitopic membrane protein with a non-cleaved signal (Fig.…”

“…Early studies devoted to the problem of intracellular transport investigated the movement of glycoproteins, labelled in vivo with various radioactive carbohydrates, through different cellular organelles (for a review see Kenny & Maroux, 1982). Radioautographs from such experiments revealed an initial labelling of the Golgi complex followed by a labelling of other cellular compartments, including the microvillar and basolateral portions of the plasma membrane and lysosomes.…”

“…This has undoubtedly to do with the fact that these enzymes, contrary to viral proteins, each represent only a tiny portion (0.1-1%) of the total mass of protein ofthe cell and that their biosynthesis therefore inevitably must be studied against a high background. By using immunofluorescence labelling, Feracci et al (1982) (Schmitz et al, 1973) and kidney (Booth & Kenny, 1974) tissue and has been used in preparative scale in the purification of several microvillar enzymes (Kenny & Maroux, 1982). Using a modification of the procedure of Kessler et al (1978), it was possible to obtain a Ca2 +-precipitated membrane fraction that contains only 2-4% of the total activity of the microvillar enzymes, indicating a minimal contamination with microvillar membranes (Danielsen et al, 1981a).…”

“…This luminal part of their plasma membrane is demarcated from the basolateral membrane by tight junctions and contains several integral membrane proteins in substantial amounts. Many of these have been purified (Table 1) and their structure and function have been under intensive study during the last decade (for a review, see Kenny & Maroux, 1982). In addition, specific antibodies, polyclonal (for references see as well as monoclonal (Hauri et al, 1980;Gee et al, 1983) have been raised against these enzymes.…”

Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase.

“…The predicted general structure composed of a short intracellular domain, a single transmembrane element, and a large globular extracellular domain is common among membrane hydrolases (45). Multiple potential N-linked glycosylation sites account for the native antigen's high carbohydrate content.…”