Regulation of ENaCs by proteases: An increasingly complex story.

Proteolytic processing of epithelial Na+ channel (ENaC) subunits has an important role in the regulation of ENaC gating. This Commentary addresses the potential roles of specific proteases and protease inhibitors in the control of Na+ channel gating.

gamma -glutamyltransferase and its isoform mediate an endoplasmic reticulum stress response.

Although use of multiple alternative first exons generates unique noncoding 5'-ends for gamma-glutamyltransferase (GGT) cDNAs in several species, we show here that alternative splicing events also alter coding exons in mouse GGT to produce at least four protein isoforms. GGTDelta1 introduces CAG four bases upstream of the primary ATG codon and encodes an active GGT heterodimeric ectoenzyme identical to constitutive GGT cDNA but translational efficiency is reduced 2-fold. GGTDelta2-5 deletes the last eight nucleotides of exon 2 through most of exon 5 in-frame, selectively eliminating residues 96-231 from the amphipathic N-terminal subunit, including four N-glycan consensus sites, while leaving the C-terminal hydrophilic subunit intact. GGTDelta7 introduces 22 bases from intron 7 causing a frameshift and a premature stop codon so a truncated polypeptide is encoded terminating with 14 novel residues but retaining the first 339 residues of the native GGT protein. GGTDelta8-9 deletes the terminal four nucleotides of exon 8 plus all of exon 9 and inserts 24 bases from intron 9 in-frame so the C-terminal subunit of the encoded polypeptide loses residues 401-444 but gains eight internal hydrophobic residues. In contrast to the product of GGTDelta1, those derived from GGTDelta2-5, Delta7, Delta8-9 all lack transferase activity and persist as single-chain glycoproteins retained largely in the endoplasmic reticulum as determined by immunofluorescence microscopy and constitutive endoglycosidase H sensitivity in metabolically labeled cells. The developmental-stage plus tissue-specific regulation of the alternative splicing events at GGTDelta7 and GGTDelta8-9 implies unique roles for these GGT protein isoforms. The ability of the GGTDelta1 and GGTDelta7 to mediate the induction of C/EBP homologous protein-10, CHOP-10, and immunoglobulin heavy chain binding protein, BiP, implicates a specific role for these two GGT protein isoforms in the endoplasmic reticulum stress response.

Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state.

MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.

Influenza M2 proton channel activity selectively inhibits trans-Golgi network release of apical membrane and secreted proteins in polarized Madin-Darby canine kidney cells.

The function of acidification in protein sorting along the biosynthetic pathway has been difficult to elucidate, in part because reagents used to alter organelle pH affect all acidified compartments and are poorly reversible. We have used a novel approach to examine the role of acidification in protein sorting in polarized Madin-Darby canine kidney (MDCK) cells. We expressed the influenza virus M2 protein, an acid-activated ion channel that equilibrates lumenal and cytosolic pH, in polarized MDCK cells and examined the consequences on the targeting and delivery of apical and basolateral proteins. M2 activity affects the pH of only a subset of acidified organelles, and its activity can be rapidly reversed using ion channel blockers (Henkel, J.R., G. Apodaca, Y. Altschuler, S. Hardy, and O.A. Weisz. 1998. Mol. Biol. Cell. 8:2477-2490; Henkel, J.R., J.L. Popovich, G.A. Gibson, S.C. Watkins, and O.A. Weisz. 1999. J. Biol. Chem. 274:9854-9860). M2 expression significantly decreased the kinetics of cell surface delivery of the apical membrane protein influenza hemagglutinin, but not of the basolaterally delivered polymeric immunoglobulin receptor. Similarly, the kinetics of apical secretion of a soluble form of gamma-glutamyltranspeptidase were reduced with no effect on the basolaterally secreted fraction. Interestingly, M2 activity had no effect on the rate of secretion of a nonglycosylated protein (human growth hormone [hGH]) that was secreted equally from both surfaces. However, M2 slowed apical secretion of a glycosylated mutant of hGH that was secreted predominantly apically. Our results suggest a role for acidic trans-Golgi network pH in signal-mediated loading of apical cargo into forming vesicles.

Low permeabilities of MDCK cell monolayers: a model barrier epithelium.

Barrier epithelia such as the renal collecting duct (in the absence of antidiuretic hormone) and thick ascending limb, as well as the stomach and mammalian bladder, exhibit extremely low permeabilities to water and small nonelectrolytes. A cell culture model of such epithelia is needed to determine how the structure of barrier apical membranes reduce permeability and how such membranes may be generated and maintained. In the present studies, the transepithelial electrical resistance and isotopic water and urea fluxes were measured for Madin-Darby canine kidney (MDCK) type I and type II cells, as well as type I cells expressing the mucin protein, MUC1, in their apical membranes. Although earlier studies had found the unstirred layer effects too great to permit measurement of transepithelial permeabilities, use of ultrathin semipermeable supports in this study overcame this difficulty. Apical membrane diffusive water permeabilities were 1.8 +/- 0.4 x 10(-4) cm/s and 3.5 +/- 0.5 x 10(-4) cm/s in MDCK type I and type II cells, respectively, at 20 degrees C. Urea permeability in type I cells at the same temperature was 6.0 +/- 0.9 x 10(-6) cm/s. These values resemble those of other barrier epithelial apical membranes, either isolated or in intact epithelia, and the water permeability values are far below those of other epithelial cells in culture. Transfection of MDCK type I cells with the major human urinary epithelial mucin, MUC1, led to abundant expression of the fully glycosylated form of the protein on immunoblots, and flow cytometry revealed that virtually all the cells expressed the protein. However, MUC1 had no effect on water or urea permeabilities. In conclusion, MDCK cells grown on semipermeable supports form a model barrier epithelium. Abundant expression of mucins does not alter the permeability properties of these cells.

Differential glycosylation of MUC1 in tumors and transfected epithelial and lymphoblastoid cell lines.

The membrane-bound mucin-like protein MUC1 with a specified number of tandem repeats has been expressed by transfection of the cDNAs in both the epithelial cell lines MDCK and LLC-PK1, and human lymphoblastoid cell lines T2 and C1R. The structure and glycosylation states of the MUC1 in these four lines were compared with that of the endogenous MUC1 found in the human pancreatic (HPAF) and breast (BT-20) tumor cell lines using flow cytometry and Western blot analysis with anti-MUC1 antibodies, which are either sensitive or insensitive to the glycosylation state of the tandem repeat, and pretreatment of cells with phenyl-alpha-galactosaminide, an inhibitor of mucin sialylation. A similar analysis of MUC1 expression in transfected normal and O-glycosylation defective CHO cells reveals that the addition of galactose to the core oligosaccharide structure is apparently responsible for the anomalous difference in M(r), between the mature and propeptide forms of the MUC1. Both the tumor cells and the transfected lymphoblastoid cells consistently express significant steady state levels of both the heavily glycosylated mature forms and the poorly glycosylated propeptide forms of the MUC1, whereas MUC1 is found predominantly as the mature extensively glycosylated species in the transfected epithelial cells. Immunofluoresence microscopy of cross sections of the polarized epithelial cells grown on culture filter inserts reveals that the MUC1 is clearly present at the apical surface of the cells, consistent with its expression in normal tissues. Thus, the successful expression of the MUC1 by transfection of either lymphoblastoid cells or epithelial cells yields model systems both for studying the natural structure/function relationships of the protein domains within the MUC1 molecule and for further elucidating the previously reported MHC-independent T-cell recognition of the MUC1.

Differential expression of MUC1 on transfected cell lines influences its recognition by MUC1 specific T cells.

In adenocarcinomas of the breast and pancreas, underglycosylation of the glycoprotein MUC1, also expressed by normal breast and pancreatic ductal epithelial cells, results in new protein epitopes to which the immune system mounts a cytotoxic T cell response. This cytotoxic immune response is directed primarily against epitopes on the tandem repeat domain of MUC1, and is unconventional in that it is major histocompatibility complex (MHC)-unrestricted. It is therefore necessary to investigate the molecular basis of this immune response in order to enhance and optimize it for immune therapy purposes. In the present study, we characterize new MUC1 transfected human lymphoblastoid cell lines C1R and T2, and a pig kidney epithelial line LLC-PKI, that express MUC1 with either two repeats (MUC1-2R) or 22 repeats (MUC1-22R), and use them as stimulators and targets for cytotoxic T cells (CTL) in vitro. We show that MUC1-2R is processed and glycosylated similarly to MUC1-22R. In contrast to MUC1-22R, MUC1-2R is not recognized by CTL on T2 and C1R cells known for no or low MHC class I expression. It is however recognized when expressed at high density on xenogeneic LLC-PKI cells. We propose that in MHC-unrestricted recognition, a large number of MUC1 epitopes is necessary to effectively engage the T cell receptor, and that in the presence of a low number of epitopes, engagement of the CD8 co-receptor by MHC class I molecules may be required for completing the signal through the T cell receptor.

Synthesis and release of amphipathic gamma-glutamyl transferase by the pulmonary alveolar type 2 cell. Its redistribution throughout the gas exchange portion of the lung indicates a new role for surfactant.

gamma-Glutamyl transferase (gamma-GT) catalyzes a transpeptidation reaction which is involved in the metabolism of glutathione. Glutathione is abundant within the epithelial lining fluid of the lung. However, little is known about gamma-GT expression in the epithelial cells of the lung alveolus. Herein we show that the pulmonary alveolar epithelial type 2 cell expresses the gene for gamma-GT. We were unable to detect expression in the pulmonary alveolar epithelial type 1 cell or in the pulmonary alveolar macrophage. gamma-GT expression in the pulmonary alveolar epithelial type 2 cell is via mRNA III, a transcript that was initially cloned from the liver. This cell synthesizes gamma-GT protein and releases enzyme activity into a surfactant-associated pool within the lung alveolus. The specific activity of this surfactant-associated enzyme is almost 10-fold higher than that of whole lung. This activity results from amphipathic gamma-GT since it partitions with lung surfactant phospholipid and with the detergent phase of Triton X-114. Activity can be dissociated from each by papain proteolysis. These results demonstrate that gamma-GT is expressed in the differentiated pulmonary alveolar epithelial type 2 cell and that amphipathic gamma-GT protein is released by this cell along with lung surfactant. These results suggest that surfactant may serve an expanded role in lung cell biology as the vehicle for the redistribution of amphipathic signal anchored proteins throughout the gas exchange surface of the lung.

Gamma-glutamyltranspeptidase expression regulates the growth-inhibitory activity of the anti-tumor prodrug gamma-L-glutaminyl-4-hydroxy-3-iodobenzene.

gamma-L-glutaminyl-4-hydroxy-3-iodobenzene (I-GHB), a novel iodinated analog of gamma-L-glutaminyl-4-hydroxybenzene (GHB), demonstrates greater anti-tumor activity in human and in murine melanoma cell lines. These phenolic amides are substrates for gamma-glutamyltranspeptidase (GGTP; E.C. 2.3.2.2), a cell-membrane-associated ecto-enzyme which is elevated in a number of tumor systems. We now present data to show that the growth-inhibitory activity of I-GHB and GHB may be mediated via GGTP-catalyzed reactions. The growth-inhibitory activity of I-GHB and GHB in pigmented B16-BL6 melanoma cells was blocked significantly by rabbit anti-rat GGTP polyclonal antibodies. The combination of L-serine and sodium borate, a specific transition-state inhibitor of GGTP, as well as acivicin, a glutamine antagonist and irreversible GGTP inhibitor, inhibited the killing of BL6 cells by GHB and I-GHB. To further define the role of GGTP expression in the regulation of phenolic amide cytotoxicity, GGTP-negative Chinese hamster ovary cells (CHO-K1) were transfected with a functional rat renal cDNA representing the full-length GGTP transcript. I-GHB and GHB were significantly more cytotoxic in GGTP cDNA transfected Chinese hamster ovary (CHO-K1-GGTP) cells than in non-transfected CHO-K1 cells. The combination of L-serine and sodium borate blocked the cytotoxic activity of these pro-drugs and also inhibited GGTP-catalyzed formation of polymerized products from these phenolic amides in intact BL6 melanoma and CHO-K1-GGTP cells. Furthermore, melanin formation from GHB was not observed in non-transfected CHO-K1 cells lacking GGTP expression. The combined data strongly suggest that GGTP-catalyzed hydrolysis of the anti-tumor pro-drugs I-GHB and GHB to 4-aminophenols mediates the expression of antitumor activity.

Role of apical and basolateral secretion in turnover of glutathione in LLC-PK1 cells.

Previous clearance measurements have established that the rapid turnover of renal proximal tubular glutathione is in part due to apical secretion and degradation by gamma-glutamyltranspeptidase, an ectoenzyme that is primarily associated with the brush-border membrane. The relationship between glutathione turnover and secretion was further characterized using confluent cultures of LLC-PK1 cells grown on nitrocellulose supports. The resulting cell layer was impermeable to [3H]inulin and exhibited a polarized expression of gamma-glutamyltranspeptidase. Incubating cells with 5 mM buthionine sulfoximine, an inhibitor of glutathione synthesis, produced an 86% inhibition of [35S]cystine incorporation into intracellular glutathione. Under these conditions, the prominent intracellular pool of glutathione turns over with an apparent half-life of 4 h and a first-order rate constant of 0.17 h-1. This turnover is unaffected by pretreatment with AT-125, an inhibitor of gamma-glutamyltranspeptidase. The rate of accumulation of glutathione in the apical and basolateral medium of cells pretreated with AT-125 was 22 and 34 nmol.mg protein-1.h-1, respectively. The combined secretion was equivalent to the calculated turnover rate of intracellular glutathione (57 nmol.mg protein-1.h-1). Therefore, the combined processes of apical and basolateral secretion can account for the turnover of intracellular glutathione in LLC-PK1 cells.

Expression of rat renal gamma-glutamyltranspeptidase in LLC-PK1 cells as a model for apical targeting.

In the rat, gamma-glutamyltranspeptidase (gamma GT) is transcribed into four unique mRNAs from a single gene by use of at least three different promoters and alternative splicing. For the first time, two distinct full-length cDNAs encoding the protein for rat renal gamma-glutamyltranspeptidase have now been isolated. Characterization by restriction enzyme mapping and nucleotide sequencing indicates that the two cDNAs, corresponding to transcripts I and II, differ only in the 5' noncoding region. However, transcription from promoter I, most proximal to the coding sequence, apparently began 20 bases upstream from the major transcription start previously reported. Since in vitro transcription and translation of these two new gamma GT cDNAs were found to produce a full-length peptide (M(r) approximately 62,000), both cDNAs were used to transfect LLC-PK1 (porcine) cells, a polarized cell line most representative of the renal proximal tubule. Rat gamma GT was expressed in transfected cells as judged by immunofluorescence analysis, direct immunoprecipitation after metabolic labeling with [35S]methionine, and an increase in gamma GT specific enzymatic activity (up to 5-fold). When clonal cell lines (I or II) were grown on Falcon filter inserts, the increased gamma GT activity was found only at the apical surface, consistent with polarized expression of the rat gamma GT. In contrast, transfection of the same cells with cDNA of human growth hormone resulted in both apical (70%) and basal lateral (30%) secretion of the expressed hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of multiple proteins structurally related to gamma-glutamyl transpeptidase in non-neoplastic adult rat hepatocytes in vivo and in culture.

Freshly isolated hepatocytes from normal adult rat liver do not express measurable gamma-glutamyl transpeptidase (GGT) mRNA in contrast to the significant GGT mRNA levels expressed by normal adult rat kidney and hyperplastic bile ductular tissue from bile duct-ligated rats. However, the induction of GGT activity in rat hepatocytes by two-thirds hepatectomy was accompanied by the appearance of a high level of GGT mRNA. We are now able to demonstrate that normal adult rat hepatocytes express 5 protein bands which cross-react with 2 different anti-rat kidney GGT antisera. The apparent molecular weights were 26.9, 58.0, 63.9, 73.5, and 83.4 kDa, respectively. Expression of the 26.9- and 58.0-kDa proteins strikingly parallels the pattern of induction of GGT enzymatic activity. This suggests that these 2 proteins correspond to the active dimeric enzyme previously described in kidney and neoplastic hepatocellular tissue. In normal hepatocytes, the 73.5-kDa protein represents 50% of the total GGT-immunoreactive protein, in contrast to kidney, where this band contains less than 4% of the GGT protein. The kinetics of expression of the 73.5-kDa protein upon induction of GGT activity in hepatocytes, as well as in culture turnover studies, suggests that this protein is a precursor form of the active enzyme, such as the described 78/79-kDa single-chain glycoprotein propeptide of GGT. It appears that in normal hepatocytes, this precursor is not processed to the same extent as in kidney or in hyperplastic bile ductular tissue.

O-linked glycosylation of rat renal gamma-glutamyltranspeptidase adjacent to its membrane anchor domain.

Large domains rich in serine and threonine, that are likely to exhibit clusters of O-linked oligosaccharides, have been reported adjacent to the anchor of several cell surface proteins. No such domain is evident in the primary sequence of rat renal gamma-glutamyltranspeptidase. However, papain treatment of the amphipathic enzyme (Triton-purified gamma-glutamyltranspeptidase, T gamma GT), pretreated with galactose oxidase and NaB3H4 (Frielle, T., and Curthoys, N. P. (1983) Biochemistry 22, 5709-5714), yields the hydrophilic enzyme (papain-treated Triton-purified gamma-glutamyltranspeptidase, PT gamma GT) and a labeled peptide which contains both the amino-terminal membrane anchor and the sequence Pro27-Thr28-Thr29-Ser30. Since [3H]galactose was identified in this peptide, the presence of O-linked oligosaccharides was investigated. Carbohydrate analysis is consistent with the presence of two simple O-linked oligosaccharides on T gamma GT and one on PT gamma GT. Lectin blot analysis of T gamma GT and PT gamma GT was carried out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The small subunits of both T gamma GT and PT gamma GT and the large amphipathic subunit of T gamma GT all react with the peanut agglutinin lectin, but the large subunit of PT gamma GT exhibits no such reactivity. The reactivity with PNA is consistent with the presence of one oligosaccharide with the structure galactose beta 1-3N-acetylgalactosamine alpha 1-Ser/Thr attached to each subunit of T gamma GT. The papain-sensitivity of the oligosaccharide from the larger subunit is consistent with O-glycosylation at the Thr28-Thr29-Ser30 sequence. The results of lectin blot analysis with wheat germ agglutinin imply that the content of N-linked oligosaccharides is unaffected by papain treatment of the transpeptidase. These data represent the first direct evidence for O-glycosylation of a microvillar hydrolase at a site immediately adjacent to the membrane anchor and indicates that even small clusters of Thr and Ser can be O-glycosylated. Isolated O-linked oligosaccharides may have functional significance since single Ser and Thr residues are consistently found near the membrane anchor of many cell surface proteins.

The identification of two subcellular sites for cleavage of gamma-glutamyltranspeptidase propeptide.

In order to determine the subcellular site(s) of rat renal gamma-glutamyltranspeptidase propeptide cleavage labeled immunoprecipitates were obtained from preparations of either intracellular membranes or brush border membrane vesicles. Heterodimer accounts for 25% of the label associated with transpeptidase in intracellular membranes from 5 to 40 min postinjection of [35S]methionine, consistent with a cotranslational cleavage of propeptide in the endoplasmic reticulum. Labeled propeptide and heterodimer appear in the brush border membrane fraction between 20-30 min postinjection and accumulate for 1 h and 4h, respectively. Subsequently, the propeptide disappears with a half-life of 1 h while the heterodimer is relatively stable. These results confirm our previous proposal for two distinct subcellular sites for transpeptidase propeptide cleavage (Capraro, M.A. and Hughey, R.P. (1983) FEBS Lett. 157, 139-143).

Evidence for stable homodimers and heterodimers of gamma-glutamyltranspeptidase subunits under protein-denaturing conditions.

gamma-Glutamyltranspeptidase is synthesized as a core glycosylated propeptide (Mr 75,000) which is subsequently cleaved to yield a stable heterodimeric structure (subunit Mr 50,000 and 30,000). The propeptide represents an insignificant mass of the transpeptidase but higher molecular weight bands designated H1 (Mr 85,000) and H2 (Mr 100,000) are readily observed by protein staining or immunoblot analysis of the enzyme or crude membranes after SDS-polyacrylamide gel electrophoresis. Although H1 and H2 represent the predominant antigenic forms of transpeptidase in tissues which exhibit relatively low specific enzyme activity, neither their structure nor their physiological function is known. In order to determine the relationship between H1 and H2, and the large (L) and small (S) subunits of the transpeptidase, individual bands (H1, H2, L and S) of the purified renal enzyme were cut from a Coomassie-stained SDS gel, eluted and re-electrophoresed. Isolated S produced S and dimers of S (Mr 60,000), while isolated L produced L and dimers of L corresponding to H2. Equivalent mixtures of L and S also produced H1. Utilizing IgG affinity-purified against either L or S, immunoblot analysis confirmed that H2 is a dimer of L, and H1 is a heterodimer of L and S. However, monoclonal IgG which recognizes both transpeptidase propeptide and native heterodimer did not react with H1. Thus, it is clear that isolated L and S can form and maintain unique dimeric structures during SDS-polyacrylamide gel electrophoresis. With this information it should now be possible to ascertain the basis for the apparent predominance of H1 and H2 in non-renal tissues.

Use of acivicin in the determination of rate constants for turnover of rat renal gamma-glutamyltranspeptidase.

The specific enzymatic activity of renal gamma-glutamyltranspeptidase is decreased from control levels (0.86 unit-1 mg-1) to minimal values within 2 h postinjection of 100-g rats with acivicin, an irreversible inhibitor of the enzyme. The recovery of transpeptidase specific activity was followed from 2 to 24 h postinjection and the data were used to calculate the absolute rate constants for degradation (kd = 0.47 +/- 0.03 day-1) and synthesis (ks = 0.41 +/- 0.04 unit-1 mg-1 day-1). This corresponds to a half-life for the renal transpeptidase of 1.46 +/- 0.09 days and 99% recovery of the specific activity by 10 days postinjection. Recovery was followed for 14 days and closely approximates this theoretical curve. The data from control experiments designed to test for secondary effects of the drug, acivicin, show that neither the relative rate of synthesis nor apparent rate of degradation for either total protein or gamma-glutamyltranspeptidase is significantly altered by acivicin treatment of rats. The results also show that the acivicin-inhibited transpeptidase is not degraded differently than enzymatically active enzyme. The individual heterodimer subunits also exhibit similar apparent half-lives in both control and treated animals. Thus, recovery of renal gamma-glutamyltranspeptidase specific activity after acivicin treatment can be used in vivo to determine absolute values of ks and kd for this enzyme. These values have not been reported for any other constituent of the renal brush-border membrane.

Processing of the propeptide form of rat renal gamma-glutamyltranspeptidase.

The biosynthesis of rat renal gamma-glutamyltranspeptidase (EC 2.3.2.2) was studied by sodium dodecyl sulfate gel electrophoresis and fluorography of specific immunoprecipitates obtained at varying times' postinjection with [35S]methionine. At 20 min postinjection 3 endo-beta-N-acetylglucosaminidase H-sensitive bands were observed representing the propeptide (Mr 75 000) large subunit (Mr 49 500) and small subunit (Mr 29 000) of transpeptidase. The alterations in Mr are consistent with removal of 6 N-linked coreoligosaccharides from the propeptide; 4 from the large subunit and 2 from the small subunit. All 3 bands became more diffuse and less endoglycosidase H-sensitive by 40 min and completely resistant by 60 min postinjection. At 20 h postinjection no propeptide remained. Thus, the primary propeptide cleavage reaction occurs prior to the loss of endoglycosidase H sensitivity while about 30% of the propeptide is processed along with the heterodimer and cleaved at a later time.

Rat renal gamma-glutamyltransferase activity: a reaction of glutamine synthetase.

The experimental observations of Welbourne (Am. J. Physiol. 226, 544-548 (1974)) led him to propose that a gamma-glutamyltransferase activity contributes significantly to basal renal ammonia production. He measured the transferase activity in vitro as gamma-glutamylhydroxamate or ammonia production from glutamine and hydroxylamine. We report that in crude homogenates of rat kidney, gamma-glutamyltransferase activity requires the presence of a divalent cation, arsenate, and ADP. These conditions are also required for the maximal expression of the gamma-glutamyltransferase reaction catalyzed by glutamine synthetase. Glutamine synthetase and gamma-glutamyltransferase activities exhibit an identical distribution during differential centrifugation. In addition, the two activities comigrate sucrose gradient velocity centrifugation, and exhibit identical heat inactivation and L-methionine sulfoximine inhibition profiles. Furthermore, both activities are found to be localized primarily in the outer stripe region of the renal medulla. Based on these observations, it is concluded that the primary gamma-glutamyltransferase activity as assayed in rat renal tissue is a partial reaction of glutamine synthetase, an enzyme which does not catalyze the production of ammonia and gamma-glutamyl peptides from glutamine.

Comparison of the association and orientation of gamma-glutamyltranspeptidase in lecithin vesicles and in native membranes.

gamma-Glutamyltranspeptidase purified following solubilization with Triton X-100 can associate with single-layered [14C]lecithin vesicles. Enzyme activity and radiolabeled vesicles were shown to co-migrate during Sepharose 4B chromatography and isopycnic sucrose gradient centrifugation. The enzyme-vesicle complex exhibits a density corresponding to that of a single enzyme molecule bound to a single vesicle, gamma-Glutamyltranspeptidase purified following a solubilization with papain does not bind to vesicles. In addition, papain treatment of vesicles containing the Triton-purified transpeptidase results in the release of 95% of the transpeptidase activity without release of internally trapped [3H]sucrose. The released transpeptidase is chromatographically identical to the papain-purified transpeptidase. gamma-Glutamyltranspeptidase activity associated with both native membranes and with lecithin vesicles exhibits a temperature-induced transition in its energy of activation. In contrast, the proteolytic- and detergent-solubilized forms of the enzyme exhibit a single energy of activation over the entire temperature range. These results suggest that gamma-glutamyltranspeptidase binding to vesicles is due to a papain sensitive sequence of amino acids and that the enzyme.vesicle complex closely approximates the interaction and orientation of gamma-glutamyltranspeptidase with brush border membranes.