The use of a monoclonal antibody for the rapid purification of kidney neutral endopeptidase ("enkephalinase") solubilized in octyl glucoside

The neutral endopeptidase ("enkephalinase") of the rabbit brush border membrane has been purified to homogeneity by a rapid immunoaffinity method using a monoclonal antibody. In contrast with other methods used so far, a complete extraction of enkephalinase from the brush border membrane can be achieved with octyl glucoside, without loss of activity. The solubilized enzyme can be selectively separated from the other proteins in a single step using an immunoaffinity column consisting of the monoclonal antibody covalently linked to Sepharose CL-4B. It is demonstrated that enkephalinase can then be recovered in an active form by elution at low pH. The purified enzyme obtained by this method is completely inhibited by thiorphan and appears as a single 94,000 dalton protein after polyacrylamide gel electrophoresis under denaturing and reducing conditions.     

Identification of P-glycoprotein in renal brush border membranes

A monoclonal antibody (C219) that recognizes the P-glycoprotein (Mr = 170,000) in plasma membranes of multidrug-resistant Chinese hamster ovary (CHO) cell lines was used to assay renal brush border membrane (BBM) and basolateral membrane (BLM) fractions for the presence of a cross-reactive polypeptide. The C219 antibody bound to a 155,000 dalton protein in immunoblots of rat BBM but not BLM proteins resolved by sodium dodecyl sulfate gel electrophoresis. The corresponding human kidney BBM and dog kidney BBM proteins had molecular weights of 170,000 and 160,000 respectively. The glycoprotein nature of the renal protein was shown by its sensitivity to N-glycanase treatment which reduced the apparent molecular weight of the dog protein to 120,000. In addition, dog P-glycoprotein could be bound to and eluted from immobilized wheat germ agglutinin. The molecular weight, antibody crossreactivity, glycosidase sensitivity and lectin binding show that this protein is a normal kidney analogue of the P-glycoprotein induced in multidrug resistant cell lines.     

Functional analysis of the purified glucocorticoid receptor

Glucocorticoid-receptor complex (GR) has been purified from rat liver by differential affinity for DNA before and after activation, followed by ion-exchange chromatography. The purified GR has mol. wt 94,000 dalton. The protein contains three functional domains: (A) a steroid-binding domain; (B) a DNA-binding domain; and (C) a domain necessary for normal biological function. A second protein, with mol. wt 72,000 dalton, copurifies with the GR. This protein does not bind steroid, does not interact with antibodies raised against the GR and does not show the same susceptibility to limited proteolytic cleavage as the 94,000 dalton protein. Analysis of the specific interaction of the purified GR with the mouse mammary tumour virus gene, assayed by glycerol-gradient centrifugation, shows that one molecule of 94,000 dalton protein binds to each of the specific binding sites in the long terminal repeat region. Analysis of the fractions from the glycerol gradients show that the 72,000 dalton protein is associated to the binding species (94,000 dalton receptor protein) in about equimolar amounts. Analysis of the molybdate-stabilized non-activated receptor complex using monoclonal antibodies raised against the 94,000 dalton receptor protein indicates that the molybdate-stabilized complex is a hetero-oligomer. The hetero-oligomer consists of only one molecule of the 94,000 dalton receptor protein, in association with other non-steroid-binding proteins.     

Defect of a fiber cell-specific 94-kDa protein in the lens of inherited microphthalmic mutant mouse Elo

Deficiency in a 94,000-dalton protein in the non-crystallin fraction from the Elo mouse lens was shown. To perform further investigations, we raised an antibody against the 94,000-dalton protein isolated from normal mouse lens. Western blot analysis with the antibody indicated that the protein was only present in the lens and not in the brain, lung, heart, liver, and kidney. In the lens, it was unique to the cortex and nucleus fractions, not being present in the epithelial cells. Furthermore, it was observed in the water-soluble fraction as well as in the urea-soluble fraction. The antibody weakly but clearly reacted with the chick CP97 lens peptide, a fiber cell-specific protein, and anti-CP97 antibody also reacted with the 94,000-dalton protein. From these results, we concluded that the protein corresponds to CP97 cytoskeletal protein in the mouse lens. The protein was deficient in the lenses from Elo mice, but microphthalmic lenses from CTA mice contained a normal level.     

Biosynthesis and degradation of altered immature forms of intestinal dipeptidyl peptidase IV in a rat strain lacking the enzyme.

We have used a strain of rat (Fischer 344) lacking brush border membrane dipeptidyl peptidase IV activity to examine its effect on the intestinal assimilation of prolyl peptides. In addition, we have examined the biochemical basis for the enzyme deficiency. An analysis of several brush border membrane hydrolases in different regions of the small intestine demonstrates that these rats lack only dipeptidyl peptidase IV. They also have a greatly reduced ability to hydrolyze and absorb in vivo peptides of the NH2-X-Pro-Y type which are known substrates for the enzyme. Immunoblot analysis with polyclonal and monoclonal antibody indicates that the animals lack an identifiable dipeptidyl peptidase IV protein in intestinal epithelial cells. Levels and types of dipeptidyl peptidase IV mRNA were analyzed in several tissues and found to be similar to that of control animals. Biosynthetic labeling of intestinal explants revealed that two distinct forms (102 and 108 kDa) of dipeptidyl peptidase IV are initially synthesized by deficient rats, in contrast to the single protein (106 kDa) observed in normal animals. Pulse-chase labeling experiments (+/- endoglycosidase H) show that these two altered forms of dipeptidyl peptidase IV, although initially glycosylated with N-linked high mannose carbohydrate, fail to be processed to the mature complex glycosylated form and undergo intracellular degradation.     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.     

Purification of a putative Na+/D-glucose cotransporter from pig kidney brush border membranes on a phlorizin affinity column

Phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, was derivatised to 3-aminophlorizin and subsequently coupled to Affi-Gel 15. Affinity chromatography of pig kidney brush border membranes solubilised in Triton X-100 allowed the purification of a 60 kDa protein on this resin. We consider this protein to be the Na+/D-glucose cotransporter, or part of it, for the following reasons: (i) binding of this protein to Affi-Gel 15 specifically requires phlorizin covalently attached to the resin and is lowered when phlorizin is replaced by phloretin; (ii) binding of the 60 kDa protein to a phlorizin affinity column requires the presence of Na+; (iii) polyclonal as well as monoclonal antibodies against the 60 kDa protein inhibit binding of phlorizin to brush border membranes from rabbit and pig kidney.     

Biochemical studies of the human thymocyte cell-surface antigens T6, T9 and T10

Three human thymic cell-surface antigens T6, T9 and T10, previously defined by monoclonal antibodies, were analyzed using immunoprecipitation techniques. The antigen T6 was found to be a 49,000 dalton glycoprotein, which is associated with β2-microglobulin, the small subunit (12,000 daltons) of the HLA-A, -B, and -C antigens. The target antigen for the monoclonal reagent anti-T9 was found to be a glycoprotein of 94,000 daltons, which appears as a disulfide-linked dimer of 190,000 daltons on the cell surface. The antigen precipitated by the anti-T10 antibody is a 45,000 dalton glycoprotein. We present preliminary evidence that all three cell-surface proteins may be integral membrane proteins. These findings, in addition to the distribution patterns, suggest that the T6 antigen is the human homolog of the murine thymus leukemia (TL) antigen.     

Characterization of aminopeptidase N from Torpedo marmorata kidney

Summary— A major antigen of the brush border membrane of Torpedo marmorata kidney was identified and purified by immunoprecipitation. The sequence of its 18 N terminal amino acids was determined and found to be very similar to that of mammalian aminopeptidase N (EC 3.4.11.2). Indeed aminopeptidase N activity was efficiently immunoprecipitated by monoclonal antibody 180K1. The purified antigen gives a broad band at 180 kDa after SDS-gel electrophoresis, which, after treatment by endoglycosidase F, is converted to a thinner band at 140 kDa. This antigen is therefore heavily glycosylated. Depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of 18S (2% CHAPS) or as a single peak of 7.8S (1% CHAPS plus 0.2 % C₁₂E₉), showing that Torpedo aminopeptidase N behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a 160 kDa monomer. The antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells (600 gold particles/μm² of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolatéral membranes, vacuoles or vesicles). Monoclonal antibodies prepared here will be useful tools for further functional and structural studies of Torpedo kidney aminopeptidase N.     

Association of Na(+)-H(+) exchanger isoform NHE3 and dipeptidyl peptidase IV in the renal proximal tubule

In an attempt to identify proteins that assemble with the apical membrane Na(+)-H(+) exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC ), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na(+)-H(+) exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity.     

Comparison of two pig intestinal brush border peptidases with the corresponding renal enzymes.

Intestinal dipeptidyl peptidase IV and gamma-glutamyltransferase were compared to the corresponding kidney enzymes with respect to immunological and electrophoretic properties. The influences of selected effectors on the two enzymes were also studied. The two kidney peptidases exhibited the reaction of total identity with the corresponding intestinal enzymes in immunodiffusion. Furthermore, the intestinal dipeptidyl peptidase IV and gamma-glutamyl transferase showed the same inhibition patterns as the corresponding kidney enzymes and the acceptor specificity of the intestinal gamma-glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic acid. It is suggested that the intestinal brush border peptidases, dipeptidyl peptidase IV and gamma-glutamyltransferase, are closely related to the corresponding enzymes obtained from the kidney.     

Is there a tripeptidyl peptidase in the renal brush-border membrane?

A recent claim that the renal brush border contains a tripeptidyl peptidase [Andersen & McDonald (1987) Am. J. Physiol. 253, F649-F655] was examined. In a fluorescent assay, the hydrolysis of Gly-Pro-Met-2-naphthylamide (-NH-Nap) and Gly-Pro-Leu-NH-Nap by pig kidney microvilli was strongly inhibited by amastatin or di-isopropyl phosphorofluoridate (inhibitors of aminopeptidases and dipeptidyl peptidase IV). The products formed were shown to be Gly-Pro and Met-NH-Nap (or Leu-NH-Nap) and free 2-naphthylamine. Specific antibodies to pig and rat aminopeptidase N abolished the apparent tripeptidyl peptidase activity. We conclude that these substrates are hydrolysed by the sequential attack of dipeptidyl peptidase IV and aminopeptidase N and that pig and rat brush borders lack a detectable tripeptidyl peptidase.     

A monoclonal antibody against human kidney gamma-glutamyl transpeptidase: preparation, immunochemical, and immunohistochemical characterization

To perform immunohistochemical study of the distribution of gamma-glutamyl transpeptidase in human organs, a highly specific antibody against the human enzyme is required. We prepared monoclonal antibody against gamma-glutamyl transpeptidase from human kidney, using the hybridoma technique. The antibody was of the IgG1 type and the light chain belonged to the kappa class. The antibody reacted specifically with the 63 KD heavy subunit of the enzyme. Examination of the specificity of the antibody performed by immunohistochemical staining of human kidney sections revealed that the antigen was localized on the brush border and along the basolateral membrane of the epithelial cells of both the convoluted and the straight portions of the proximal tubule. This antibody was also reactive in several human organs other than kidney, including epididymis, prostate, seminal vesicle, pancreas, and normal liver, and in human hepatoma. These findings indicate the existence of an antigenic determinant common to human kidney and other organs. The monoclonal antibody did not crossreact with mouse, rat, guinea pig, rabbit, or pig kidney.     

Distinction of three types of D-glucose transport systems in animal cells

Immunoblotting of plasma membrane fractions from rat kidney cortex with antibody to human erythrocyte glucose transporter showed a single major cross-reacting material of 48K in basolateral membrane fractions possessing a facilitated diffusion system for D-glucose, but not in brush border membrane fractions which have a Na-dependent active transport system. Cytochalasin B inhibited D-glucose uptake in basolateral membrane vesicles but not in brush border vesicles. Cross-reacting materials of 44-55K were detected in several animal cells exhibiting facilitated diffusion systems, including a hormone dependent system. These results indicate molecular difference between glucose transporters of facilitated diffusion systems and active transport systems.     

Monoclonal antibodies against rat kidney antigens.

The proximal tubule of the nephron is subdivided into three structurally and functionally distinct segments, which can be differentiated with the help of special methods. With the aim of producing selective markers for these three portions of the proximal tubule, we raised monoclonal antibodies against the brush border membranes of the rat kidney. Immunohistochemistry was carried out with eleven different monoclonal antibodies to sections of rat kidney and other tissues at the light- and electron-microscopical level. These monoclonal antibodies mainly detect antigens located on the brush border of the proximal tubule, and they allow a distinction between its three segments. However, some antibodies also recognize other portions of the nephron, or even the glomerulus or stromal elements. Sites recognized by the antibodies are not limited to the kidney, but staining is observed on the intestinal brush border, the intralobular ducts of the pancreas, the bile canaliculi of the liver and on the macrophage clusters of the spleen. These antibodies are interesting reagents which can be applied to study biochemical differences between brush border membranes. In addition, they recognize antigenically related sites in other organs with reabsorptive or secretory tasks.     

Brush border membrane hydrolysis of S-benzyl-cysteine-p-nitroanilide, and activity of aminopeptidase M

A peptidase activity which can hydrolyze cysteinylglycine and S-benzyl-cysteine-p-nitroanilide was purified from rat renal brush border membranes. The purified peptidase exhibits an even greater specific activity when assayed with substrates for aminopeptidase M, leucine-p-nitroanilide and alanine-p-nitroanilide. All three activities copurify and coelectrophorese. In addition titration of the three activities in isolated brush border membrane vesicles with Fab antibodies prepared against the highly purified peptidase produced similar inactivation profiles. Therefore, the activity within brush border membrane towards S-benzyl-cysteine-p-nitroanilide is due to the action of aminopeptidase M (EC 3.4.11.2.)     

Brush border motility. Microvillar contraction in triton-treated brush borders isolated from intestinal epithelium

The brush border of intestinal epithelial cells consists of an array of tightly packed microvilli. Within each microvillus is a bundle of 20-30 actin filaments. The basal ends of the filament bundles are embedded in and interconected by a filamentous meshwork, the terminal web, which lies directly beneath the microvilli. When calcium and ATP are added to isolated brush borders that have been treated with the detergent, Triton X-100, the microvillar filament bundles rapidly retract into and through the terminal web region. Biochemical studies of brush border contractile proteins suggest that the observed microvillar contraction is actomyosin mediated. We have shown previously that the major protein of the brush border's actin (Tilney, L. G., and M. S. Mooseker. 1971. Proc. Natl. Acad. Sci. U. S. A. 68:2611-2615). The brush border also contains a protein with the same molecular weight as the heavy chain subunit of myosin (200, 000 daltons). In addition, preparations of demembranated brush borders exhibit potassium-EDTA ATPase activity of 0.02 mumol phosphate/mg-min (22 degrees C); this assay is diagnostic for myosin-like ATPase isolated from vertebrate sources. Other proteins of the brush border include a 30,000 dalton protein with properties similar to those of tropomyosin, and a protein with the same molecular weight as the Z band protein, alpha-actinin (95,000 daltons). How these observations bear on the basis for microvillar movements in vivo is discussed within the framework of our recent model for the organization of actin and myosin in the brush border (Mooseker, M. S., and L. G. Tilney. 1975. J. Cell Biol. 67:725-743).     

Presence of an extensive clathrin coat on the apical plasmalemma of the rat kidney proximal tubule cell

The nature of the cytoplasmic coat present on the apical invaginations of the kidney proximal tubule cell was investigated by immuneoverlay and immunocytochemistry of renal brush borders with anticlathrin antibodies. When kidney cortex was prepared for electron microscopy using methods that enhance visualization of clathrin coats, the apical invaginations at the base of the brush border microvilli were seen to be backed by a nearly continuous coating which resembles but is more extensive than the lattice-like clathrin coats found around brain coated vesicles. When isolated brush border fractions were prepared under conditions that preserve the coats, separated by SDS PAGE, and transferred to nitrocellulose, the presence of clathrin heavy and light chains was detected by immuneoverlay using two different affinity-purified anticlathrin IgGs--one that we prepared, which detects only the clathrin light chains, and the other, prepared by Louvard et al. ( Louvard , D., C. Morris, G. Warren, K. Stanley, F. Winkler , and H. Reggio , 1983, EMBO [Eur. Mol. Biol. Organ.] J., 2:1655-1664), which detects both the heavy and light chains. As viewed by light microscopy (immunofluorescence or immunoperoxidase), staining with both anticlathrins was concentrated at the base of the proximal tubule microvilli. Immunoelectron microscopic localizations carried out on brush border fractions (using peroxidase and gold conjugates) demonstrated specific binding of anticlathrin IgGs to the lattice-like cytoplasmic coat. When brush border fractions were reacted with monoclonal antibodies prepared against gp330 and maltase, proteins that serve as markers for the membrane of the apical invaginations and microvilli, respectively ( Kerjaschki , D., L. Noronha - Blob , B. Sacktor , and M. G. Farquhar , 1984, J. Cell Biol., 98:1505-1513), the two proteins retained their restrictive distribution in the brush border. The findings demonstrate (a) that the cytoplasmic coat of the proximal tubule intermicrovillar apical invaginations is composed of clathrin heavy and light chains, and (b) that the differential distribution of proteins in these two brush border microdomains is maintained in appropriately prepared brush border fractions.     

Protein kinase C in mouse kidney: subcellular distribution and endogenous substrates

The subcellular distribution, kinetic properties, and endogenous substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) were examined in mouse kidney cortex. Protein kinase C associated with the particulate, mitochondrial, and brush border membrane fractions was assayed after solubilization in 0.2% Triton X-100 under conditions shown to be noninhibitory to catalytic activity. Of recovered activity, 52% was associated with the cytosolic fraction; mitochondrial and brush border membrane associated protein kinase C constituted 12 and 3%, respectively, of the activity recovered in the particulate fraction. Protein kinase C associated with brush border membranes exhibited a high affinity for ATP (apparent Km = 62 +/- 10 microM) and the highest apparent maximal velocity (1146 +/- 116 pmol P/(mg protein.min] of the renal fractions examined. Maximal stimulation of protein kinase C by diacylglycerol (in the presence of phosphatidylserine) was achieved at both 25 and 300 microM calcium in all renal fractions. These results are consistent with previous reports demonstrating that diacylglycerol increases the apparent affinity of protein kinase C for calcium. Phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol, was able to substitute for diacylglycerol and stimulate cytosolic and particulate renal protein kinase C. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a specific inhibitor of protein kinase C, led to significant inhibition of catalytic activity in all renal subcellular fractions. Endogenous substrates for protein kinase C were demonstrated in renal cytosolic (26, 45, 63, and 105 kilodaltons (kDa], particulate (26, 33, 68, and 105 kDa), mitochondrial (43 kDa), and brush border membrane (26, 41, 52, 88, and 105 kDa) fractions. The possible physiological significance of protein kinase C in mammalian kidney is discussed.     

Immunolocalization of ecto-5'-nucleotidase in the kidney by a monoclonal antibody

A monoclonal antibody IgG, has been raised against ecto-5'-nucleotidase purified from rat kidney homogenate. The specificity of the antibody was verified by immunoprecipitation. The distribution of the corresponding antigen in the rat kidney was studied by immunocytochemistry (FITC and PAP technique) in 1 micron thick cryostat sections. The antibody reacted with the brush border of proximal tubules, the apical cell membrane and the apical cytoplasm of intercalated cells in connecting tubules and collecting ducts and with interstitial cells of the cortex. Among the interstitial cells exclusively stellate shaped fibroblasts were reactive whereas rounded interstitial cells (type II interstitial cells) as well as pericytes and endothelial cells of peritubular capillaries were unreactive. Compared to the staining intensity of the fibroblasts in the cortical labyrinth the reactivity of the fibroblasts in the medullary rays of the cortex was weak or absent. Interstitial cells of the entire medulla were unreactive. Concerning the fibroblasts in the periarterial connective tissue, those surrounding the larger arteries (arcuate arteries, cortical radial arteries) were negative, those alongside afferent and efferent arterioles were positive. Endothelia of lymphatic capillaries travelling within the periarterial connective tissue were also positive. All components of the juxtaglomerular apparatus were negative. The findings are consistent with an interstitial production of adenosine, available extracellularly and thus being able to reach the major target sites of adenosine, the smooth muscles of glomerular arterioles, including the granular cells at the glomerular vascular pole.