Rapid purification of dipeptidyl peptidase IV from rat liver plasma membrane

Dipeptidyl peptidase IV (EC 3.4.14.5) was solubilized from rat liver plasma membranes with sulphobetaine 14 and purified by successive affinity chromatography on Con A-Sepharose, wheat germ lectin-Sepharose and arginine-Sepharose columns. The specific activity of the final preparation was 49.4 mumol Gly-Pro p-nitroanilide/min per mg protein, representing a 1098-fold purification of the homogenate. SDS-polyacrylamide gel electrophoresis of the arginine-Sepharose eluate showed a single protein band with a molecular weight of 105,000. The isoelectric point was determined to be 3.9 under non-denaturing conditions with sulphobetaine 14. The preparation was free of post-proline cleaving enzyme. The content of aminopeptidase M was 0.2% of the total protein.     

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.     

Preparative purification of dipeptidyl peptidase IV.

Dipeptidyl-Peptidase IV was purified from pig kidney by ammonium sulfate fractionation, gel filtration, QAE-cellulose chromatography and affinity columns with Gly-Pro- and Concanavalin A-Sepharose. The specific activity of the purified enzyme is 41.8 units/mg. Polyacrylamide gel electrophoresis and silver staining show a single band. The enzyme preparation is free of aminopeptidase and dipeptidase activity, proved fluorimetrically and by gas chromatography/mass spectrometry. The most important procedure for removal of contaminating enzyme activities is a stepwise NaCl-gradient on a QAE-ZetaPrep ion exchange disk.     

Identification of dipeptidyl peptidase IV as a protein shared by the plasma membrane of hepatocytes and liver biomatrix

The histotypic organization of liver parenchyma involves specific intercellular contacts and interaction of hepatocytes with supporting biomatrix. Evidence from this laboratory identified a peptide (Hep105, apparent Mr 105 000) that is shared by the plasma membrane of rat hepatocytes and rat liver biomatrix. This report identifies Hep105 as a peptide component of dipeptidyl peptidase IV (DPPIV; EC 3.4.14.-). A monoclonal antibody (MAb 236.3) was shown to immunoprecipitate DPPIV from non-ionic detergent extracts of surface-labeled 125I hepatocytes. The immunoprecipitate contained two 125I-labeled peptides: Hep105 and a peptide of apparent Mr 150000 (Hep150). Proteolysis of 125I-labeled Hep105 and Hep150 by Staphylococcus aureus V8 protease yielded essentially identical patterns of 125I-labeled peptide degradation products, indicating that Hep105 and Hep150 are structurally related. Only Hep150 exhibited DPPIV activity on transblot analysis, an observation that is consistent with the interpretation that it is the monomeric form of the enzyme. Heating (100 degrees C, 5 min) of purified Hep150 in the presence of sodium dodecylsulfate (SDS) resulted in its conversion to Hep105 and the disappearance of any demonstrable enzymatic activity. 3H-labeled diisopropyl fluorophosphate was incorporated into Hep105, indicating that Hep105 contains the active site for DPPIV. Purified rat liver biomatrix was shown to possess significant DPPIV activity. Taken together, these data indicate that Hep105 s a peptide component of DPPIV.     

Purification and characterization of dipeptidyl peptidase IV in rat liver lysosomal membranes.

Dipeptidyl peptidase IV (m-DPP IV) in rat liver lysosomal membranes was purified about 50-fold over the lysosomal membranes with 38% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The enzyme amounts to about 3% of lysosomal membrane protein constituents. The purification procedures included: extraction of lysosomal membranes by Triton X-100, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme (M(r) 240,000) is composed of two identical subunits with an apparent molecular weight of 110,000. The enzyme contains about 12.4% carbohydrate and the carbohydrate moiety was composed of mannose, galactose, fucose, N-acetylglucosamine, and neuraminic acid in a molar ratio of 14:17:2:24:11. Susceptibility to neuraminidase and immunoreactivity of the enzyme in intact tritosomes were examined to study the topology of the enzyme in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the enzyme were not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that both the oligosaccharide chains and the main protein portion of the enzyme are on the inside surface of the tritosomal membranes. Subcellular localization of DPP IV was determined by means of enzyme immunoassay, which indicated that bile canalicular membranes and lysosomal membranes are the major sites of localization, and DPP IV activity in lysosomes was separated into a membrane bound form (60%) and a soluble form (40%). Immunoelectron microscopy clearly confirmed that DPP IV occurs not only in the bile canalicular domain but also in the lysosomes of rat liver.     

Selective fluorescence probes for dipeptidyl peptidase activity-fibroblast activation protein and dipeptidyl peptidase IV

Development of suitable tools to assess enzyme activity directly from their complex cellular environment has a dramatic impact on understanding the functional roles of proteins as well as on the discovery of new drugs. In this study, a novel fluorescence-based chemosensor strategy for the direct readout of dipeptidase activities within intact living cells is described. Selective activity-based probes were designed to sense two important type II transmembrane serine proteases, fibroblast activation protein (FAP) and dipeptidyl peptidase IV (DPP-IV). These serine proteases have been implicated in diverse cellular activities, including blood coagulation, digestion, immune responses, wound healing, tumor growth, tumor invasion, and metastasis. Here, we validated that Ac-GPGP-2SBPO and GPGP-2SBPO probes are excellent reporters of both proteolytic activities. Furthermore, the novel probes can differentiate between FAP and DPP-IV proteolytic activities in cellular assay. Potentially, this assay platform is immediately useful for novel drug discovery.     

Activities of dipeptidyl peptidase II, dipeptidyl peptidase IV, prolyl endopeptidase, and collagenase-like peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis.

We examined the activities of peptidases in the synovial membrane from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Dipeptidyl peptidase II (DPP II), prolyl endopeptidase (PEP), and collagenase-like peptidase (CLP) activities were higher in knee joint synovial membrane from patients with RA than in that from patients with OA. DPP II and PEP activities in knee joint synovial membrane of patients with RA increased in parallel with the increase in joint fluid volume, whereas DPP IV activity decreased in parallel with the increase in joint fluid volume. These results suggest that these peptidases in the synovial membrane may play some role in immunological disturbances in the joints of patients with RA. Measurement of these peptidases in synovial membrane may be useful in the diagnosis of the severity of local joint inflammation.     

Proteins of the kidney microvillar membrane. The amphipathic form of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV was solubilized from the microvillar membrane of pig kidney by Triton X-100. The purified enzyme was homogeneous on polyacrylamide-gel electrophoresis and ultracentrifugation, although immunoelectrophoresis indicated that amino-peptidase M was a minor contaminant. A comparison of the detergent-solubilized and proteinase (autolysis)-solubilized forms of the enzyme was undertaken to elucidate the structure and function of the hydrophobic domain that serves to anchor the protein to the membrane. No differences in catalytic properties, nor in sensitivity to inhibition by di-isopropyl phosphorofluoridate were found. On the other hand, several structural differences could be demonstrated. Both forms were about 130,000 subunit mol.wt., but the detergent form appeared to be larger by no more than about 4,000. Electron microscopy showed both forms to be dimers, and gel filtration revealed a difference in the dimeric mol.wt. of about 38 000, mainly attributable to detergent molecules bound to the hydrophobic domain. Papain converted the detergent form into a hydrophilic form that could not be distinguished in properties from the autolysis form. A hydrophobic peptide of about 3500 mol.wt. was identified as a product of papain treatment. The detergent and proteinase forms differed in primary structure. Partial N-terminal amino acid sequences were shown to be different, and the pattern of release of amino acids from the C-terminus by carboxypeptidase Y was essentially similar. The results are consistent with a model in which the protein is anchored to the microvillar membrane by a small hydrophobic domain located within the N-terminal amino acid sequence of the polypeptide chain. The significance of these results in relation to biosynthesis of the enzyme and assembly in the membrane is discussed.     

A collagen-binding glycoprotein on the surface of mouse fibroblasts is identified as dipeptidyl peptidase IV.

A dipeptidyl aminopeptidase (DPP) was detected in plasma membranes from normal (3T3) and transformed (3T12) mouse fibroblasts. This enzyme was active in cleaving the prolyl bond in the synthetic dipeptide nitroanilide Gly-Pro-NH-Np, which is a specific substrate for DPP IV (Km 0.63 mM and Vmax 6.1 nmol/min per mg at pH 6.0 and 37 degrees C). However, it did not degrade Pro-NH-Np or other dipeptide nitroanilides such as Gly-Arg-NH-Np or Val-Ala-NH-Np. The enzyme was totally inhibited by di-isopropyl phosphorofluoridate (Pri2-P-F) and by phenylmethanesulphonyl fluoride, indicating a serine catalytic site for the proteinase. DPP IV is a glycoprotein that specifically recognized immobilized gelatin and type I collagen. Upon molecular exclusion chromatography, the proteinase exhibited an apparent Mr of 100,000. SDS/polyacrylamide-gel electrophoresis under non-reducing and reducing conditions revealed that the [3H]Pri2-P-protein was exclusively represented by a polypeptide of Mr 55,000. This suggested that DPP IV consists of two non-covalently linked 55,000-Mr subunits. Fibroblast adhesion to native or denatured collagen was significantly inhibited by the two dipeptide inhibitors of DPP IV, Gly-Pro-Ala and Ala-Pro-Gly, but not by the peptides Gly-Pro and Gly-Gly-Gly, which are not inhibitors of the proteinase. Moreover, preliminary fractionation of DPP IV by molecular exclusion chromatography and affinity chromatography indicated that this material was active in disrupting cell adhesion to collagens. Taken together, the above data suggest that a fibroblast membrane-associated collagen-binding glycoprotein, DPP IV, may play a role in cell attachment to collagen.     

Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes.

Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase expressed at high levels in rat kidney, liver and lung. We established eight monoclonal antibodies against partially purified DPP IV from rat liver plasma membranes. By means of a competitive dot blot assay with purified DPP IV, these antibodies were shown to recognize four different epitopes of the glycoprotein, designated A - D. The epitopes are located on the extracellular domain of DPP IV, as shown by papain digestion of liver plasma membranes. Treatment of DPP IV with neuraminidase and glycopeptide N-glycosidase F, as well as incubation of hepatocytes with the alpha-mannosidase I inhibitor deoxymannojirimycin, revealed that epitope A may be formed by a mannose-rich sugar chain and epitope D might represent a complex carbohydrate structure in the mature glycoprotein, while the epitopes B and C are formed by the protein moiety. Concanavalin A reduced the binding of monoclonal antibody to epitope A by 78%. Binding to epitope D was blocked by 73% with wheat germ lectin, and by more than 99% with sialic acid; epitopes B and C were unaffected by any of the lectins or sugars tested. The immunological cross-reactivity with DPP IV from Morris hepatoma 7777 was demonstrated with monoclonal antibodies against epitopes A-C. Epitope D was not recognized on hepatoma DPP IV. However, in addition to DPP IV, four hepatoma plasma membrane glycoproteins were precipitated by the monoclonal antibody against the epitope D, indicating that this epitope is not uniquely restricted to DPP IV.     

Purification and characterization of barley dipeptidyl peptidase IV

Barley (Hordeum vulgare L.) storage proteins, which have a high content of proline (Pro) and glutamine, are cleaved by cysteine endoproteases to yield peptides with a Pro next to the N-terminal and/or C-terminal amino acid residues. A peptidase cleaving after Xaa-Pro- at the N terminus of peptides was purified from green barley malt. It was identified as a serine-type dipeptidyl peptidase (DPP), based on inhibitor studies, and the nature of the cleavage product. It is a monomeric glycoprotein with an apparent molecular mass of 105 kD (85 kD after deglycosylation), with a pI of 3.55 and a pH optimum at 7.2. Substrate specificity was determined with a series of fluorogenic peptide substrates with the general formula Xaa-Pro-AMC, where Xaa is an unspecified amino acid and AMC is 7-amino-4-methylcoumarin. The best substrates were Xaa = lysine and arginine, while the poorest were Xaa = aspartic acid, phenylalanine, and glutamic acid. The K(m) values ranged from 0.071 to 8.9 microM, compared with values of 9 to 130 microM reported for mammalian DPP IVs. We discuss the possible role of DPP IV in the degradation of small Pro-containing peptides transported from the endosperm to the embryo of the germinating barley grain.     

Hydrolysis and transport of proline-containing peptides in renal brush-border membrane vesicles from dipeptidyl peptidase IV-positive and dipeptidyl peptidase IV-negative rat strains

In this investigation, we have demonstrated that the renal brush-border membrane of Fischer 344 rats from the Japanese Charles River Inc. specifically lacks dipeptidyl peptidase IV (DPP IV) activity, whereas the renal brush-border membrane of Fischer 344 rats from three different sources within the United States possesses normal levels of DPP IV activity. Comparison of the brush-border proteins between Charles River (U.S.A.) Fischer 344 rats (DPP IV positive) and Japanese Charles River Fischer 344 rats (DPP IV negative) revealed that a protein band (Mr = 100,000), apparently identical with DPP IV, was absent in the membranes from Japanese Charles River Fischer 344 rats. We examined the handling of radiolabeled beta-casomorphin fragment 1-5 (Tyr-Pro-[3H]Phe-Pro-Gly), a specific substrate for DPP IV, in renal brush-border membrane vesicles isolated from DPP IV-positive and DPP IV-negative rats. Although the membrane vesicles from DPP IV-positive rats were able to hydrolyze the pentapeptide to di- and tripeptides with the subsequent active transport of these products via the H+ gradient-dependent peptide transport system, the membrane vesicles from DPP IV-negative rats failed to hydrolyze the pentapeptide and hence lacked the ability to transport the radiolabel actively from the parent peptide. The H+ gradient-dependent glycyl-sarcosine uptake and the Na+ gradient-dependent proline uptake, however, were normal in DPP IV-negative rats. Urine analysis revealed that the DPP IV-negative rats excreted proline- and hydroxyproline-containing peptides in significantly increased amounts in their urine compared with control rats. Furthermore, following intravenous administration of Tyr-Pro-Phe-Pro-NH2, a peptide that is exclusively hydrolyzed by DPP IV, urinary excretion of the peptide in the intact form was many-fold greater in DPP IV-negative rats than in control rats. These data provide conclusive evidence for the obligatory role of DPP IV in the renal handling of proline (and hydroxyproline)-containing peptides.     

Comparative immunohistochemistry and histochemistry of dipeptidyl peptidase IV in rat organs during development

Summary The occurrence of dipeptidyl peptidase (DPP) IV during development in Wistar rat organs was studied on day 10, 16 and 21 of gestation and on day 1, 4, 8, 13, 21, 26 and 60 after birth comparing immunohistochemistry and activity histochemistry. A polyclonal antibody, as well as monoclonal antibodies recognizing four different epitopes (A-D) of the DPP IV molecule, were employed for the immunohistochemical studies. In all investigated tissues, immunoreactivity with the polyclonal antibody appeared earlier than DPP IV activity and was already present on day 10 of gestation in the plasma membranes of embryonic and extraembryonic (decidual) cells. At these and other sites, e.g. brain capillary endothelium and tracheal or bronchial epithelium, immunoreactivity with the polyclonal antibody decreased or disappeared after birth and enzyme activity never developed. Immunoreactivity with the monoclonal antibodies appeared later than that with the polyclonal antibody, and mostly in those structures where DPP IV activity was subsequently found. The monoclonal antibody against epitope D showed a high reactivity in the epididymal duct, renal collecting ducts and in all domains of the hepatocyte plasma membrane, where neither DPP IV activity nor immunoreactivity with the other antibodies were observed. Our results also suggest that DPP IV might be present as a molecule before it becomes catalytically active and that immunoreactivity occurs at more sites than DPP IV activity. However, it cannot be excluded that the polyclonal antibody and the monoclonal antibody against the epitope D cross-react with as yet uncharacterized proteins, which express common epitopes during embryonic development, but are not present in the tissues of adult Wistar rats.     

Interaction of purified brush-border membrane aminopeptidase N and dipeptidyl peptidase IV with lectin-sepharose derivatives

The glycoprotein nature of two peptidases purified from the rat intestinal brush-border membrane was examined by their interaction with several lectin-Sepharose derivatives. Aminopeptidase N (EC 3.4.11.2), which contains 20% carbohydrate by weight, was bound minimally (less than 30%) by columns of Con A-, RCAI- and WGA-Sepharose. Alternatively, a greater proportion of dipeptidyl peptidase IV (EC 3.4.14.-) was bound by these immobilized lectins with 50% of the enzyme binding to Con A-Sepharose. Treatment of both enzymes with neuraminidase enhanced the binding of aminopeptidase to RCAI-Sepharose by 4-fold but did not alter the binding patterns of dipeptidyl peptidase IV. A sequential fractionation of the two peptidases with columns of Con A- and RCAI-Sepharose gave four fractions of each enzyme with differing lectin-binding specificities. Approximately 60% of the dipeptidyl peptidase IV interacted with either one or both of the lectins while only 30% of the aminopeptidase N did so. Kinetic analysis of the four isolated fractions revealed some differences, possibly related to variations in the carbohydrate moiety. The findings confirm that these two purified rat intestinal brush-border membrane peptidases are glycoproteins and, while they share a common physiologic function and source, they apparently have very different and possibly unique asparagine-linked oligosaccharide side-chains. In addition, a considerable degree of microheterogeneity exists in the carbohydrate structure of these two enzymes.     

Ultrastructural localization of dipeptidyl peptidase IV in rat salivary glands by immunocytochemistry

Summary The ultrastructural localization of dipeptidyl peptidase IV (DPP IV) (EC 3.4.14.5) in rat submandibular and parotid glands was studied immunocytochemically by the peroxidase-antiperoxidase (PAP) method, using a monospecific antiserum against rat kidney DPP IV. There were no differences in the immunocytochemical localization of DPP IV between submandibular and parotid glands. In these glands, DPP IV was primarily found to be associated with the luminal and intercellular canalicular plasma membranes of acinar cells and with the luminal plasma membranes of intercalated and striated duct cells. Occasionally, immunoreaction of DPP IV was detected in cytoplasmic vesicles (vacuoles), lysosomes, and multivesicular bodies in some acinar cells as well as in ductal epithelial cells. Furthermore, the reaction product was also found within the lumina of peri-acinar and peri-ductal capillaries and in the cytoplasm of some fibroblasts in the interstitial connective tissue. These data suggest that DPP IV in the submandibular and parotid glands may play some role in the secretion or reabsorption processes of secretory proteins and peptides in these glands.     

Quantitative immunogold localization of dipeptidyl peptidase IV (DPP IV) in rat liver cells

We investigated ultrastructural localization of dipeptidyl peptidase IV (DPP IV) [EC3.4.14 5] in rat liver cells quantitatively by post embedding protein A-gold technique. In the hepatocyte, DPP IV was mainly localized on the bile canalicular surface and the lysosomal membranes, but were scarcely detectable on the sinusoid-lateral surface. A small number of DPP IV was also detected in the trans region of the Golgi apparatus, suggesting that this part may play important roles in intracellular transport or recycling of this enzyme. In the endothelial cell, DPP IV existed on the whole surface of the plasma membrane and the lysosomes. In the Kupffer cell DPP IV was mainly localized in lysosomes and a few were detected on the plasma membrane.     

Development of a dual fluorogenic and chromogenic dipeptidyl peptidase IV substrate

A new far-red dual fluorogenic and chromogenic substrate, 5-glycylprolylglycylprolyl-9-di-3-sulfonyl-propylaminobenza[a]phenoxazonium perchlorate (GPGP-2SBPO), was developed for dipeptidyl peptidase IV (DPP-IV) sensing. The glycylprolylglycylprolyl tetrapeptide was chosen as the recognition sequence due to its stability under physiological conditions. In contrast, the truncated substrate, GP-2SBPO, containing only a glycylprolyl peptide, is unstable. Proteolysis of GPGP-2SBPO was assayed by monitoring the absorbance and fluorescence signals from the released fluorochrome, 2SBPO, at 625 and 670nm, respectively.     

Expression of the rat CD26 antigen (dipeptidyl peptidase IV) on subpopulations of rat lymphocytes.

The T cell activation antigen CD26 has been recently identified as the cell surface ectopeptidase dipeptidyl peptidase IV (DPP-IV). DPP-IV is found on many cell types, including lymphocytes, epithelial cells, and certain endothelial cells. The MRC OX61 monoclonal antibody (MAb) which specifically recognises rat DPP-IV was used to examine the expression of CD26/DPP-IV on rat lymphocytes. The molecular nature of the antigen was examined by immunoprecipitation from thymocytes, splenocytes, and hepatocytes. Analysis by one- and two-dimensional gel electrophoresis indicated that the native form of CD26 includes a 220-kDa homodimer. On tissue sections MRC OX61 MAb stained nearly all thymocytes and in the spleen and lymph nodes predominantly stained the T cell areas. However, in immunofluorescence experiments OX61 stained 80 to 87% of lymph node cells and 78 to 85% of spleen cells. Furthermore, two-colour immunofluorescence analysis of the CD4+, CD8+, and Ig+ lymphocyte subsets indicated that only 2 to 5% of each of these subsets lacked OX61 staining. Spleen cells and thymocytes of both CD4+ and CD8+ subsets stained much more intensely with OX61 after these cells were stimulated with phytohemagglutinin. These findings indicate that rat CD26 antigen expression is not confined to the T cell population as has been suggested, but also occurs on B cells, and is increased on T cells following their activation.     

Evidence for tripeptide-proton symport in renal brush border membrane vesicles. Studies in a novel rat strain with a genetic absence of dipeptidyl peptidase IV

We have investigated the transport characteristics of L-phenylalanyl-L-prolyl-L-alanine in renal brush-border membrane vesicles isolated from Japan Fisher 344 rats. This particular rat strain genetically lacks dipeptidyl peptidase IV. Owing to the absence of this enzyme, the tripeptide was found to be completely resistant to hydrolysis by the renal brush-border membrane vesicles. Uptake of the tripeptide into these membrane vesicles in the presence of an inwardly directed Na+ gradient was slightly greater than in the presence of a K+ gradient, but there was no evidence for active transport. On the contrary, uptake was very rapid in the presence of an inside-alkaline transmembrane pH gradient, and accumulation of the tripeptide inside the vesicles against a concentration gradient could be demonstrated under these conditions. The uptake was drastically reduced by dissipation of the pH gradient. The uptake was stimulated by an inside-negative membrane potential and inhibited by an inside-positive membrane potential. Moreover, the uptake was greater in voltage-clamped membrane vesicles than in control vesicles. Many di- and tripeptides inhibited this pH gradient-stimulated uptake of Phe-Pro-Ala. The apparent dissociation constant for the tripeptide was 48 microM. High performance liquid chromatography analysis of the intravesicular content at the peak of the overshoot revealed that the tripeptide was transported across the membrane almost entirely in the intact form. These data provide the first direct evidence for the presence of an electrogenic tripeptide-proton symport in renal brush-border membranes.