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565th MEETING, STIRLING 101 1 Conclusion Although the endoplasmic reticulum, Golgi and plasma membranes contain different enzymes and proteins (Ehrenreich et al., 1973)) the three major regions of the plasma membrane of the hepatocyte differ mainly in the concentration of similar classes of glycoproteins functioning as cell-surface receptors and enzymes. The generation and maintenance of these concentration gradients of glycosylated macromolecules on the hepatocyte plasma membrane appears to be a consequence of a biosynthetic route involving the Golgi apparatus that attaches sugar residues to plasma-membrane precursors and directs their insertion at the sinusoidal region. The final location and orientation of newly inserted molecules is then dictated by their association with other membrane or submembraneous components. I n the hepatocyte a limited loss of plasmamembrane ectocomponents at the canalicular region occurs, owing to detergent catalysed dissolution into the bile fluid (Evans et al., 1976), a process that is intensified in extrahepatic cholestasis. Bennet, G. Leblond, C. P. & Haddad, A. (1974) J. Cell Biol. 60,258-284 Bergeron, J. J. M., Evans, W. H. & Geschwind, I. I. (1973a)J. CelZBiol. 59,771-776 Bergeron, J. J. M., Ehrenreich, J. H., Seikevitz, P. & Palade, G. E. (1973b)J. CeNBiol. 59,73-88 Bergeron, J. J. M., Berridge, M. & Evans, W. H. (1975) Biochim. Biophys. Acta 407,325-337 Cuatrecasas, P. (1974) Annu. Rev. Biochem. 43,169-214 Ehrenreich, J. H., Bergeron, J. J. M., Siekevitz, P. & Palade, G. E. (1973) J. Cell Biol. 59,45-72 Essner, E., Novkoff, A. B. & Masek, B. (1958)J. Biophys. Biochem. Cytol. 4,711-716 Evans, W. H. (1970) Biochem. J. 116,833-842 Evans, W. H. & Gurd, J. W. (1971) Biochem. J. 125,615-624 Evans, W. H. & Gurd, J. W. (1973) Biochem. J. 133,189-199 Evans, W. H., Hood, D. 0. & Gurd, J. W. (1973a) Biochem. J. 135,819-826 Evans, W. H., Bergeron, J. J. M. & Geschwind, I. I. (19736) FEBS Lett. 34,259-262 Evans, W. H., Kremmer, T. & Culvenor, J. C. (1976) Biochem. J. 154,589-595 Farquhar, M. G., Bergeron, J. J. M. & Palade, G. E. (1974) J. Cell Biol. 60,8-25 Fisher, K. A. (1976) Proc. Nafl.Acad. Sci. USA. 73,173-177 Frey, L. D. & Edidin, M. (1970)J. Cell Sci. 7,319-355 Jeejeebhoy, K. N., Ho, J., Greenberg,G. R., Phillips, M. J., Bruce-Robertson,A. & Sodtke, U. (1975) Biochem. J . 146,141-155 Kremmer, T. B., Wisher, M. H. &Evans, W. H. (1976) Biochim. Biophys. Acfain the press Little, J. S. & Widnell, C. C. (1975) Proc. Natl. Acad. ScL U.S.A. 72,4013-4017 Ong, S . H., Whitley, T. H., Stowe, N. W. & Steiner, A. L. (1975) Proc. Nafl.Acad. Sci. U.S.A. n,2022-2026 Paphadjopoulas, D. (1974) J. Theor. Biol. 43,329-337 van Hoeven, R. P., Emmelot, P., Krol, J. H. & Oomen-Muelemans, E. P. M. (1975) Biochim. Biophys. Acfa 380,l-11 Wacker, H. (1974) Biochim. Biophys. Acta 334,417-423 Widnell, C . C. & Unkeless, J. E. (1968) Proc. Natl. Acad. Sci. U.S.A. 61, 1050-1057 Wisher, M. H. &Evans, W. H. (1975) Biochem. J. 146,375-388 Zwaal, R. F. A., Roelefsen, B. & Colley, C. M. (1973) Biochim. Biophys. Acta 300,151-182 Organization of the Kidney Proximal-TubulePIasma Membrane A. JOHN KENNY and ANDREW G. BOOTH Department OfBiochernistry,UniversityofLeeds, 9 Hyde Terrace,Lee& LS2 9LS, U.K. The cells of the kidney proximal tubule display a very obvious polarity. The luminal membrane is specialized to form a brush border comprised of microvilli, and the basal pole shows an infolded plasma membrane enclosing many mitochondria within its folds. The lateral plasma membrane is morphologically unremarkable: adjacent cells appear closely apposed and the intercellular spaces characteristic of intestinal-mucosal cells are not a feature of the proximal tubule. Both poles of the cell are therefore specialized so as to increase their surface areas, especially the luminal surface, which is increased about 100-fold by virtue of its brush border. Although the proximal tubule is capable of some VOl. 4 1012 BIOCHEMICAL SOCIETY TRANSACTIONS secretory activity, the primary physiological function is concerned with absorption. About 80% of the glomerular filtrate is reabsorbed without alteration of its osmolarity. Reabsorption of amino acids and glucose is localized in the proximal tubule and both are known to be carrier-mediated Na+-dependent processes. Histochemical methods have been largely directedtowards the localization of phosphatases,and there is no dispute that the (Na++K+)-activatedATPase* is concentrated in the basal plasma membrane and the non-speck alkaline phosphatase in the microvillus membrane. Serious biochemical contributions to our understanding of these membrane functions have awaited the development of adequate methods for preparing brush border and other proximaltubule plasma membranes. There are now several acceptable procedures for the preparation of brush borders (i.e. clumps of luminal membrane bearing numerous microvilli) and of free microvilli that have been sheared from the remaining membrane during homogenization (see Booth & Kenny, 1974). Marker enzymesfor microvilli are enriched about 15-20-fold in such preparations compared with the cortical homogenate. Electron micrographs of sections of brush-border preparations usually show portions of lateral membranes still attached to the luminal membrane and the cores of these clumps often contain a collection of small vesicles of undefined origin. Preparations of basal-lateral membranes have only recently been investigated. Their resolution from microvilli has been effected by the use of free-flow electrophoresis, the basal-lateral membrane fraction, characterized by (Na++K+)-activated ATPase, migrating more rapidly towards the anode than the microvillus-rich fraction (Heidrich et al., 1972). Such preparations have been reported to contain a Caz+-activatedATPase and a parathyroid-hormonesensitive adenylate cyclase in addition to (Na++K+)-activated ATPase (Kinne, 1975). A speciiic transport system forp-aminohippurate has been attributed to the basal-lateral membranes (Berner & Kinne, 1976). In contrast, the microvillus membrane is much more complex in the variety of enzymes associated with it, although this apparent difference may be due to the paucity of enzymic studies on preparations of basal-lateral membranes. So far, the following have been characterized as microvillus-membrane enzymes: alkaline phosphatase (EC 3.1.3.1)) aminopeptidase M (EC 3.4.1 1.2) (Kinne & Kinne-Saffran, 1969); y-glutamyltransferase (EC 2.3.2.2), maltase (EC 3.2.1.20), 5‘-nucleotidase (EC 3.1.3.5), phosphodiesterase I (EC 3.1.4.1) (Glossmann & Neville, 1972); aminopeptidase A (EC 3.4.11.7), neutral endopeptidase (EC 3.4.24.-), trehalase (EC 3.2.1.28) (George & Kenny, 1973);dipeptidyl peptidase IV (Booth & Kenny, 1974); a cyclic AMP-dependent protein kinase (Kinne, 1975). Structural proteins are also present in microvillus preparations:filamentsresembling actin are readily seen in electron micrographs (Rostgaard & Thuneberg, 1972), and we have purified and characterized an actin from such preparations (Booth & Kenny, 19766). A phlorizin-sensitive glucose-binding protein has been isolated from brush-border preparations (Thomas, 1973), but so far this appears to be the only microvillus protein clearly identified with the transport of small molecules. The proposal that y-glutamyl transferase is involved in amino acid transport in the kidney (Meister, 1973) is now questionable. It is clear that the ‘y-glutamyl cycle’ does not function as an amino acid-transport system. In particular, the activity of one of the enzymes required for the cycle, 5-oxoprolinase, is too low to operate thecycleat aratecommensurate with normal tubular function (Orlowski & Wilk, 1975). Even a more limited role in which y-glutamyl transferase released the amino acid and glutamic acid rather than 5-oxoproline seems unlikely following the report of a patient deficient in y-glutamyl transferase whose biochemical abnormalities included glutathionuria, but who showed no defect in amino acid reabsorption (Schulman et al., 1975). It may well be that the primary role of this microvillus enzyme is that of a glutaminase concerned with NH4+secretion. The luminal pole of the proximal tubule has been long known to be capable of pinocytosis of proteins (see, e.g., Straus, 1964). Proteins substantially larger than 60000 mol.wt. are excluded by the glomerular filter, but the filtrate has been variously estimated to contain between 0.1 and lOmg of protein/100ml (Strober & Waldmann, 1974), and almost * Abbreviation :ATPase, adenosine triphosphatase. 1976 565th MEETING, STIRLING 1013 all of this is reabsorbed. It is generally accepted that the absorbed protein is digested by lysosomes, but the possibility of reabsorption of intact proteins into the blood has been suggested (Maack et al., 1971) and also disputed (Just et al., 1975). It is clear that the initial step in the process involves invagination of the luminal membrane producing apical pits, which pinch off to form the small apical vacuoles that are a prominent feature of the apical cytoplasm. Bode et al. (1974, 1976) have attempted to purify a pinocytic vesicle fraction by free-flow electrophoresis. This fraction was somewhat depleted in microvillus-marker-enzyme activities and the authors concluded that the pinocytic vesicle membrane was different in protein composition from that of the microvillus. However, the homogeneity of the vesicle preparation must remain in doubt in the absence of an authentic marker for pinocytic vesicles. Our interest has focused on the proteins of the microvillus. Two of the peptidases have been purified and characterized in this laboratory. One is a Znz+-metalloenzyme, which we have referred to as neutral endopeptidase (Kerr & Kenny, 197&,6). It has the ability to attack peptides such as insulin B chain and glucagon, but not more highly structured proteins such as insulin, albumin or casein. The specificity shown by the endopeptidase is similar to that of the group of microbial neutral proteinases typified by thermolysin :the peptide bonds attacked are those involving the or-amino group of hydrophobic residues. When purified the enzyme is a monomeric glycoprotein of 93 000mol.W. The other peptidase is dipeptidyl peptidase IV (Kenny et al., 1976), an enzyme that typically releases dipeptides from the N-terminus of peptides with the sequence X-Pro-Y or X-Ala-Y. It also possesses endopeptidase activity in that it attacked certain N-blocked peptides, e.g. Z-Gly-Pro-Leu-Gly-Pro. This enzyme is a serine proteinase that is extremely sensitive to inhibition by di-isopropyl phosphorofluoridate (Dip-F). Titration of the active site with t3*P]Dip-F confirms that the purified enzyme is a dimer comprising two glycoprotein subunits of mol.wt. 130000. Aminopeptidase M (also a Zn2+-protein) has a rather broad specificity in removing N-terminal residues, whereas aminopeptidase A, a protein that is difficult to purify without substantial contamination with aminopeptidase M, is specific for N-terminal glutamic acid and aspartic acid residues. As a group these four enzymes are capable of hydrolysing most peptides of 35 or fewer residues to their constituent amino acids. Indeed an extrapolation from the specific activities of these enzymes in the rabbit kidney (Booth & Kenny, 1974) suggests that they could dispose of about 20mg of such peptides/min in the whole animal, a potential that is several orders of magnitude greater than the expected filtered load of peptide substrates. When purified microvilli are treated with mercaptoethanol and analysed by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis and stained for protein about 20 bands are consistently observed (Booth & Kenny, 19766). Five are major bands, designated 180, 160, 130,95 and 42 (indicating that they have apparent mol.wts. of 180000, 160000, etc.). Apart from two high-molecular-weight peptides (>350000), there are other bands designated 240, 220, 90,75, 70, 65, 55 and 50 and some incompletely resolved bands of mol.wt. less than 40000. Bands 180,160, 130 and 95 also stained for carbohydrate. It is now possible to identify some of the microvillus proteins with four of the five bands. The use of Dip-F as an affinity label has confirmed that band 130 is dipeptidy1 peptidase IV. Aminopeptidase M has been assigned to band 160 and is the major glycoprotein of the membrane. Neutral endopeptidase (a glycoprotein), together with a structural protein (lacking carbohydrate residues), a-actinin, have been assigned to band 95, and actin, the major protein of the microvillus, is identified with band 42. The contribution of each of these proteins (except a-actinin) to the total protein of the microvillus has been determined, in the case of the three enzymes by comparison of the specific activities of the purified enzyme with that of the microvillus preparation, and in the case of actin by a computer analysis that is capable of resolving overlapping peaks of the stained gel pattern. Actin comprises 9 % of the microvillus protein and the three peptidases together are another 12%. Alkaline phosphatase can be labelled with 32P,and this approach has shown that the monomeric enzyme has a rno1.W. of 80000 and contributes only 0.04%of the microvillus protein and is not detectable as a stained band. Band VOl. 4 Extrinsic glycoprotein Aminopeptidase M Dipeptidyl peptidase IV Neutral endopeptidase a-Actinin Alkaline phosphatase Actin y-Glutamyl transferase AminopeptidaseA PhosphodiesteraseI 5’-Nucleot idase Trehalase Maltase - - 60oOo(?) - @oOo( ?) 180000 160000 130000 95 000 95 000 80000 42 000 Subunit mo1.w. Location Membrane Membrane Membrane Membrane Core Membrane Core Membrane Membrane Membrane Membrane Membrane Membrane Class Extrinsic Intrinsic Intrinsic Intrinsic Extrinsic Intrinsic Extrinsic Intrinsic Intrinsic Intrinsic Intrinsic Intrinsic Intrinsic - 0 0 + + + + + +- Glycosylation + 0 0 0 + + 0 0 0 0 + + + Solubilization by papain - - 4.1 3.9 4.1 2.5(?) 0.04 9.0 - Proportion of microvillus protein (%) - - 450 530 760 470( ?) 10 3800 - No. of molecules per microvillus Subunit mol.wts. are for the native rather than the purified proteins, where these are known to be significantly different. The term extrinsic indicates a protein extractable by simple salt media. -, Not known; +, detectable; 0 , not detectable. Table 1. Kidney microvillusproteins 4 ?I Ez F 8 a 8 8 565th MEETING, STIRLING 1015 fa) Fig. 1. Proposed model of a kidney microvillus showing the organization of the core and membraneproteins (a)Projection of the view along the axis of a microvillus. (b) Projection of a longitudinal section of the central region between arrows in (a). The designation of the membrane proteins is as follows: (A) extrinsic proteins; (B) intrinsic proteins released by papain; (C) intrinsic proteins not released by papain. The drawing is approximately to scale, the diameter of the microvillus is 100nm, the lipid bilayer is 7nm in thickness and the longitudinal spacing of the cross-bridges is 36nm, a value dictated by geometry of the actin double helix. 180, also a glycoprotein, may be classified as an ‘extrinsic’ protein, since it is extractable by simple salt solutions. Extraction of microvilli with NaCl or EDTA releases about 15-20 % of the protein. None of the known enzymes are detectable in the extract, which appears to contain proteins corresponding to bands 180,95 and 42 and attributable to the extrinsic glycoprotein, a-actinin and actin respectively. The other known components of the microvillus membrane are all intrinsic membrane proteins that resist release by simple salt extraction and require treatment with either proteinases or detergents for their solubilization. Papain is effective in releasing aminopeptidases M and A, as well as dipeptidyl peptidase IV and y-glutamyl transferase. Neutral endopeptidase, alkaline phosphatase, trehalase and phosphodiesterase I are wholly resistant to papain treatment and are arguably more deeply inserted into the VOl. 4 1016 BIOCHEMICAL SOCIETY TRANSACTIONS lipid bilayer of the membrane. Dipeptidyl peptidase IV has been purified from Triton X-100-extracted kidney microsomal fraction (R.D. C. Macnair & A. J. Kenny, unpublished work). The various strands of information derived from electron microscopy, enzymology and protein chemistry now permit a somewhat speculative model of the microvillus organization to be suggested. The core is composed of actin filaments arranged in a 1+6 array (Rostgaard & Thuneberg, 1972; Booth & Kenny, 19766). Knowing the length of a microvillus (l.5,um) and the half-pitch of the actin double helix (36nm) containing 13-14 subunits, we can calculate that there are some 3800 molecules of actin/microvillus, amounting to 9.0% of the microvillus protein. We can therefore estimate the number of monomers of some of the other microvillus proteins (Table 1). The role of a-actinin (Mooseker & Tilney, 1975; Booth & Kenny, 1976~)may well be to provide cross-bridges between actin filaments, and between actin and the membrane. In intestinal microvilli the ratio of actin: a-actinin is 8: 1 (Mooseker &Tihey, 1975), and if the same is true of kidney microvilli one would predict a-actinin to constitute 2.5% of the microvillus protein. One way in which the a-actinin cross-bridging might be accommodated is shown in Fig. 1. The dimensions of the a-actinin molecule are 30nmx2nm (Podlubnaya et al., 1975) and in this model one-third are involved in actin-actin links and the remainder in actin-membrane bridges. The functional role of the microvillus peptidases is a question now demanding attention. We have already indicated that their potential as peptide hydrolases for substrates in the glomerular filtrate seems greatly in excess of requirements. This degree of ‘overkill’, coupled with the fact that three of the peptidases are major intrinsic proteins in the membrane, makes an alternative role a strong possibility. Two questions require attention. (1) D o these outward-facing enzymes of the microvillus membrane become inwardfacing peptidases in a pinocytic vacuole? Although unable to initiate attack on proteins this group of enzymes could complement the subsequent attack by the lysosomal cathepsins. (2) Are these proteins concerned primarily with amino acid transport? There are a number of attractive features in this suggestion: they are large intrinsic proteins(93000-160000mol.wt.);twoofthem aredimeric, at least when released from the membrane (though not necessarily dimeric in situ); there is some similarity between their specificities as peptidases and the specificities of the group-specific transport systems (for review, see Young & Freedman, 1971) such that aminopeptidase A resembles the acidic system, aminopeptidase M or neutral endopeptidase the neutral system, dipeptidyl peptidase IV the iminoglycine system. The scheme fails to account for the basic system that transports diamino acids, and clearly the correlation of the specificities is far from perfect. Nevertheless we believe that the hypothesis is one that can be tested in isolated tubules or microvillus vesicles and in the perfused kidney. We thank the Medical Research Council for support. Berner, W. & Kinne, R. (1976) Pfltigers Arch. 361,269-277 Bode, F., Pockrandt-Hemstedt,H., Baumann, K. & Kinne, R. (1974) J. Cell Biol. 63,998-1008 Bode, F., Baumann, K. & Kinne, R. (1976) Biochim. Biophys. Acta 433,294-310 Booth, A. G. & Kenny, A. J. (1974) Biochem. J. 142,575-581 Booth, A. G. & Kenny, A. J. (1976~)J. Cell. Sci. 21,449-464 Booth, A. G. & Kenny, A. J. (19766) Biochem. J. 159,395407 George, S. G. & Kenny, A. J. (1973) Biochem. J. 134,43-57 Glossmann, H. &Neville, D. M., Jr. (1972) J. Biol. Chem. 247,7779-7789 Heidrich, H.-G., Kinne, R., Kinne-Saffran, E. & Hannig, K. (1972) J. Cell Biol. 54,232-245 Just, M., Rockel, A., Stanjek, A. & Bode, F. (1975) Naunyn-Schmiedeberg’sArch. Pharmacol. 289,229-236 Kenny, A. J., Booth, A. G., George, S. G., Ingram, J., Kershaw, D., Wood, E. J. & Young, A. R. (1976) Biochem. J. 157,169-182 Kerr, M. A. & Kenny, A. J. (1974~)Biochem. J. 137,477-488 Kerr, M. A. & Kenny, A. J. (19746) Biochem. J. 137,489-495 Kinne, R. K. H. (1975) Med. Clins. N . Am. 59,615-627 U m e , R. K. H. & KinneSaffran, E. (1969) Pftigers Arch. 308,l-15 1976 565th MEETING, STIRLING 1017 Maack, T., MacKensie, D. D. S. & Kinter, W. B. (1971) Am. J. Physiol. 221,1609-1616 Meister, A. (1973) Science 180, 33-39 Mooseker, M.S. & Tilney, L. G. (1975) J. Cell Biol. 67,725-743 Orlowski, M. & Wilk, S. (1975) Em. J. Biochem. 53,581-590 Podlubnaya, Z . A. Tskhovrebova, L. A., Zaalishvili, M. M. & Stefanenko, G. A. (1975) J. Mol. Biol. 92,357-359 Rostgaard, J. & Thuneberg, L. (1972) Z . Zellforsch.Mikrosk. Anat. 132,473-496 Schulman, J. D., Goodman, S. I., Mace, J. W., Patrick, J. D., Tietze, F. &Butler, E. J. (1975) Biochem. Biophys. Res. Commun. 65,68-74 Straw, W. (1964) J. Cell Biol. 21,295-308 Strober, W. & Waldmann, T. A. (1974) Nephron 13,35-66 Thomas, L. (1973) Biochim. Biophys. Acra 291,454-464 Young, J. A. & Freedman, B. S. (1971) Clin. Chern. 17,245-266 Biochemical Differentiation of the Plasma Membrane of the Intestinal Epithelial Cell ROBERT H. MICHELL, ROGER COLEMAN and BARBARA A. LEWIS* Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K. The brush border and basolateral regions of the surface of the intestinal epithelial cell (enterocyte) are exposed to environments which differ even more than those which bathe the different functional regions of the surface of the hepatocyte or the renal tubular cell, and therefore the biochemical differentiation of the enterocyte plasma membrane is also more striking. In addition, it has been easier to study, since the highly adapted brush-border membrane of the enterocyte is one of the easiest of plasma membranes to isolate pure and in quantity (see Isselbacher, 1974). From the study of brush-border preparations a picture has emerged of a membrane with a variety of terminal digestive hydrolases (especially aminopeptidase, disaccharidases and alkaline phosphatase) attached to its luminal surface (Louvard et al., 1975). These are large proteins (Maestracci et al., 1975), which are heavily glycosylated (Kelly & Alpers, 1973; Weiser, 1973; Quaroni et al., 1974) and are moored to the membrane by relatively small hydrophobic polypeptide segments, probably at their N-terminal ends (Maroux & Louvard, 1976). Some of the end-products of digestion that these enzymes generate are then passed across the membrane by transport systems which are closely coupled to the hydrolases and are Na+-independent (Ramaswamy et al., 1976). Some enter the cell via one of several electrogenically driven Na+/solute symport systems (Crane, 1965,1974; Schultz & Curran, 1970; Berger et a[., 1972; Murer & Hopfer, 1974; Hopfer et al., 1973, 1976; Sigrist-Nelson et al., 1976; Alvarado, 1976) and others are taken into the cell by specific transport systems that are independent of Na+ (e.g. fructose; Sigrist-Nelson & Hopfer, 1974). At the cytoplasmic surface of the brush-border membrane, and attached to it by aactinin bridges, is an actin array which forms the microvillus core that also interacts with myosin-like proteins in the terminal web (Mooseker & Tilney, 1975). This raises the exciting, but still largely unexplored, possibility that the brush border is motile (Thuneberg & Rostegaard, 1969; Sandstrom, 1971; Mooseker, 1974) and that this somehow facilitates its digestive and absorptive function, perhaps by preventing the formation of unstirred layers of fluids at either surface of the brush-border membrane. The remainder of the enterocyte plasma membrane (the basolateral region) is separated from the brush border by the terminal bar. Thus, in contrast with the brush border, it interacts with the protective and controlled internal environment of the body and might be expected to be much more similar to the plasma membranes of other cells. It is this region of the plasma membrane which plays the key role in such processes as generating * Present address: Department of Biochemistry, University of Bristol, Medical School, Bristol BS8 1TD.U.K. VOl. 4