Cellular settings mediating Src Substrate switching between focal adhesion kinase tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) tyrosine 734

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.     

Degradation of atrial natriuretic factor in the rat.

The biologically active circulating form of atrial natriuretic factor (ANF) in the rat is the 28-amino-acid peptide ANF-(Ser-99-Tyr-126). Degradation of this peptide in vivo as well as in vitro, in whole blood, in plasma and by the isolated mesenteric artery was investigated. Studies in vivo in the rat demonstrated that the elimination and degradation of ANF was extremely fast: within 3 min more than 95% of the injected immunoreactive material was eliminated from circulation. The production of a short C-terminal peptide was detected on injection of 125I-ANF-(Ser-99-Tyr-126) into the rat. This peptide increased proportionately with incubation time. Experiments in vitro in the presence of whole blood or plasma did not cause any major destruction of ANF even after incubation for 60 min. After this prolonged incubation in plasma, ANF-(Ser-99-Tyr-126) was partially converted into ANF-(Ser-103-Tyr-126), a less potent peptide. Isolated mesenteric-artery preparation appeared to degrade ANF in a manner very similar to the system in vivo. These results suggest that degradation of ANF may occur either after internalization in the vascular cells or by a membrane-bound enzyme in the vasculature.     

The binding of calcium to human fibronectin

The binding of calcium to human plasma fibronectin has been measured by equilibrium dialysis at 25° in 0.1 M NaCl 50mM Tris HCL, pH 7.4. Curve fitting of the binding data indicates that fibronectin has two strong calcium binding sites per chain (Mr 220,000), KD = 1.3 mM and approximately 12 weak sites, KD = 2.3 mM. No significant displacement of bound calcium by magnesium was observed at magnesium concentrations up to 1 mM. Calcium binding to a pair of tryptic fragments of fibronectin (Mr ? 160,000 and 180,000) that bind to gelatin has also been investigated. These fragments have a single class of calcium binding sites, with 2.2 sites per chain, KD = 1.1 mM. Negligible calcium binding to tryptic fragments derived from other regions of the fibronectin molecule was observed.     

Tyrosine phosphorylation of beta3 integrin provides a binding site for Pyk2

Integrins expressed on leukocytes possess the ability to maintain themselves in a non-adhesive state, thus preventing unwarranted adhesion and uncontrolled inflammation. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(V)beta(3). Firm adhesion to the extracellular matrix and directed cellular motility requires the reorganization of the actin cytoskeleton. The ability of beta(3) to recruit signaling and scaffolding molecules to propagate alpha(V)beta(3) -mediated signals is regulated in part by the phosphorylation of the beta(3) cytoplasmic tail. The identities of integrin-associated signaling molecules within alpha(V)beta(3) podosomes and in particular the proximal binding partners of the beta(3) cytoplasmic tail are not completely known. Here we show that alpha(V)beta(3) ligation induces Pyk2-Tyr-402 phosphorylation and its association with the beta(3) cytoplasmic tail in a beta(3)-Tyr-747 phosphorylation-dependent manner. Pyk2 binding to the beta(3) cytoplasmic tail is direct and dependent upon Pyk2-Tyr-402 and beta(3) -Tyr-747 phosphorylations. These data identify Pyk2 as a phosphorylated beta(3) binding partner, providing a potential structural and signaling platform to achieve alpha(V)beta(3) -mediated remodeling of the actin cytoskeleton.     

Separation and characterisation of tryptic fragments from the adenosine triphosphatase of sarcoplasmic reticulum.

The two halves of the ATPase, M, 115,000, from sarcoplasmic reticulum produ-ed by limited trypsin treatment have been purified in sodium dodecylsulphate. The fragment of Mr60,000 has been purified by electrophoresis on cellulose acetate slabs and that of Mr 55,000 by gel filtration. The two halves of the 60,000 Mr fragment (Mr33,000 and 24,000) produced by more extensive trypsin treatment have also been purified by gel filtration in sodium dodecylsulphate. The sum of the amino acid analyses of the constituent tryptic fragments is in good agreement with that for the whole ATPase. The amino acid compositions of the two halves of the ATPase were strikingly similar. N-terminal analysis shows that the ATPase and its constituent tryptic polypeptides all possess a single N-terminal alanine implying no further cleavage of the polypeptide by trypsin. Attempts to solubilize selectively the tryptic fragments from the membrane by a variety of denaturing and solubilising agents under a variety of conditions have proved unsuccessful, suggesting that the interaction between the tryptic polypeptides is stronger than between the lipid and the protein. The possibility that the interaction between the tryptic polypeptides includes disulphide bonding has been eliminated.     

Thrombin-like enzyme from the venom of Bitis gabonica. Purification, properties, and coagulant actions

Gabonase, an enzyme which acts on fibrinogen and factor XIII in uniquely thrombin-like ways, was purified to electrophoretic homogeneity from the venom of Bitis gabonica. On sodium dodecyl sulfate-polyacrylamide electrophoresis, the reduced protein behaved as a single chain with Mr = 30,600. The enzyme contains 20.6% carbohydrate, no free sulfhydryl groups and hence, from amino acid analysis, five disulfide bonds. Its extinction coefficient (E1%1cm) at 280 nm is 9.6. Its pI is 5.3. Gabonase has an active serine residue, is inactivated by phenylmethanesulfonyl fluoride, and has an active histidine which reacts with the chloromethyl ketone of tosyl-L-lysine. Its NH2-terminal amino acid sequence (Val-Val-Gly-Gly-Ala-Glu-Cys-Lys-Ile-Asp-Gly-His-Arg-Cys-Leu-Ala-Leu-Leu -Tyr-) is homologous to the B chain of thrombin. The activity of the enzyme is stabilized by calcium ion. It exhibits strong N alpha-p-tosyl-L-arginine methyl esterase activity, hydrolyzes tripeptide nitroanilide derivatives weakly or not at all, and cleaves no peptide bonds in insulin, glucagon, or the S peptide of ribonuclease. Gabonase clots fibrinogen with a specific activity of 45 NIH thrombin-equivalent units/mg, releasing both fibrinopeptides A and B and showing substrate inhibition at fibrinogen concentrations of 3 mg/ml or greater. The enzyme also activates factor XIII. It is not inactivated by either heparin or hirudin.     

Action of human cathepsin G on the oxidized B chain of insulin.

The specificity of cathepsin G, a serine neutral proteinase from human neutrophil leucotyes, was determine dby its action on the insulin B chain. The most susceptible bonds were Phe-24-Phe-25, Leu-15-Tyr-16 and Tyr-16-Leu-17. Other bonds hydrolysed were Leu-6-Cys(O3H)-7, Leu-11-Val-12, Leu-17-Val-18 and Phe-25-Tyr-26. These results suggest that the specificity of cathespin G is closer to that of pig chymotrypsin C than ox Chymotrypsin A. Tables listing amino acid composition, N-terminal residue, and yields of isolated peptides have been deposited as Supplementary Publication SUP 50 075 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7B2, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977) 161,1.     

Immunochemical analysis of subunit structures of 1,4-dihydropyridine receptors associated with voltage-dependent Ca2+ channels in skeletal, cardiac, and smooth muscles

Previous purification studies of the 1,4-dihydropyridine receptor associated with the calcium channel of rabbit skeletal muscle had shown that it is composed of a large glycoprotein of Mr 140,000-145,000 associated with a smaller component of Mr 32,000-34,000. Specific antisera have now been prepared against the larger component (anti-140 serum) and the smaller one (anti-32 serum). The specificity of these two antisera has been analyzed by immunoblot assays with microsomal preparations of rabbit skeletal muscle. Under disulfide-reducing conditions the anti-140 serum specifically labeled a polypeptide of Mr 140,000 while the anti-32 serum labeled three polypeptides of Mr 32,000, 29,000, and 26,000. Under nonreducing conditions both the anti-140 and the anti-32 sera specifically recognized a single large polypeptide of Mr 170,000. The same type of approach showed that the dihydropyridine receptor in cardiac and smooth muscles had a polypeptide composition similar to that found in skeletal muscle with a large polypeptide of Mr 170,000-176,000 made of two different chains of about Mr 140,000 and 34,000-32,000 associated by disulfide bridges.     

Calmodulin binding proteins in rat liver mitochondria

Calmodulin binding proteins have been found in submitochondrial fractions obtained from highly purified rat liver mitochondria. The matrix fraction contains two major calmodulin binding proteins: one, having Mr of 145,000, apparently is carbamoyl-phosphate synthetase. Another has a Mr of 58,000 and has not been associated with enzyme activities. A major calmodulin binding protein of unknown function and having Mr of 32,000 has been found in the Triton X-100 solubilizate of the inner membrane. Minor amounts of two calmodulin binding proteins having Mr of about 37,000 and 56,000 have been found in the outer membrane.     

Succinate thiokinase in pigeon breast muscle mitochondria

Succinate thiokinase has been purified from pigeon breast muscle. It has been confirmed that the enzyme is entirely specific for ATP, and Km is very high (approximately 0.8 mM). Activity in mitochondrial sonicates is low enough for it to be doubtful whether the enzyme can support citric acid cycle flux in the tissue. The enzyme appears to have an Mr of 80 000-100 000, and to have two unequal subunits. As determined by SDS gel electrophoresis one subunit certainly has an Mr of 40 000.     

The Interaction of Protein-tyrosine Phosphatase α (PTPα) and RACK1 Protein Enables Insulin-like Growth Factor 1 (IGF-1)-stimulated Abl-dependent and -independent Tyrosine Phosphorylation of PTPα

Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration.     

The influence of adrenergic and cholinergic blocking drugs on the glycogen content of the brain in rats deprived of paradoxical sleep

Recent studies have pointed out biochemical and pharmacological phenomena associated with the mechanism or mechanisms of sleep, especially in its paradoxical phase (Jouvet, 1964; Mandel, 1964). Our previous experiments have shown that paradoxical sleep (PS) deprivation leads to the fall of total glycogen content in certain regions of the brains of cats (Mr?ulja, Raki? and Radulova?ki, 1967; Mr?ulja and Raki?, 1968) and rats (Karad?i? and Mr?ulja, 1969). It was shown that changes of glycogen content correspond to PS deprivation and that PS deprivation is a specific stress to which the CNS responds selectively. Alterations in the glycogen concentration in a number of different brain structures lead us to conclude that neural areas affected by PS deprivation are widely distributed. Jouvet (1962) was one of the first to suggest that a neurohumoral mechanism may be concerned in the control of and characteristics of sleep. Experiments have shown that both cholinergic and adrenergic mechanisms may be involved in the initiation, maintenance and control of sleep. It has also been pointed out that paradoxical sleep can be started and maintained by cholinergic drugs (Matsuzaki, Okada and Shuto, 1967, 1968), blocked or reduced by anticholinergic compounds (Matsuzaki et al., 1968), and stimulated by noradrenaline or by its precursor, DOPA (Matsumoto and Jouvet, 1964). Bowers, Hartmann and Freedman (1966) showed that the ACh level of the rat telencephalon decreases with PS deprivation while the levels of norpinephrine and serotonin remain the same (Barchas and Freedman, 1963). More recently, Pujol, Mouret, Jouvet and Glowinski (1968) found the increased turnover of cerebral norepinephrine during rebound of PS in the rat. It is also of interest to point out that probably both adrenergic and cholinergic processes participate in the glycogenolytic effect of physostigmine (Mr?ulja, Terzi? and Varagi?, 1968). It was suggested that physostigmine initiates the cholinergic processes which then trigger off adrenergic processes. The aim in the present work was to determine the glycogen content in certain brain regions of rats which were subjected to PS deprivation lasting 72 hr and treated with some cholinergic or beta-adrenergic blocking agents, as well as with a catecholamine depleting drug.     

Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of protein phosphatase 1I activating kinase from bovine brain cytosolic and particulate fractions

The activating kinase of protein phosphatase 1I is distributed in approximately equal amounts between the cytosolic and particulate fractions of bovine brain homogenates. Both species of this protein kinase have been purified to near homogeneity. The cytosolic form, purified about 7,000-fold, has an apparent Mr = approximately 75,000, as estimated by gel filtration chromatography on Sephacryl S-300. The enzyme contains two subunits, with apparent Mr = 52,000 and 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both subunits undergo phosphorylation when the enzyme is incubated with Mg2+ and [gamma-32P]ATP. Peptide maps of the two subunits are different, and rabbit antibodies to the 52-kDa subunit show only very minor cross-reactivity to the 46-kDa subunit. These observations indicate that the two subunits are different. The species of protein phosphatase 1I activating kinase that is associated with the membrane fraction has an apparent Mr = approximately 105,000 as estimated by gel filtration. This species also contains two subunits, with apparent Mr = 52,000 and 46,000, the properties of which are very similar, if not identical, to those of the two subunits comprising the cytosolic form of the protein kinase.     

Acetyl-coenzyme-A carboxylase from rat liver. Subunit structure and proteolytic modification.

The subunit structure of rat liver acetyl-coenzyme-A carboxylase has been studied by polyacrylamide gel electrophoresis in the presence of dodecylsulfate. A number of individual preparations of the enzyme purified by the same procedures exhibited three different types of electrophoretic patterns as follows: first, a single slow-moving protein bands (Mr 230000); secondly, two adjacent fast-moving protein band (M4 124000 and 118 000); finally, all three protein bands. With the use of the [14C]biotin-labelled enzyme, the biotinyl prosthetic group was shown to be associated with the polypeptide of 230000 Mr as well as with that of 124000 Mr, but not with the polypeptide of 118000 Mr. Studies were next made with the labelled enzyme to examine the possibility that the two light polypeptides might have been formed by proteolytic modification of the heavy polypeptide during the procedures used for the purification of the enzyme. Treatment of the enzyme with trypsin or chymotrypsin resulted in cleavage of the heavy polypeptide into two nonidentical polypeptides with molecular weights of approximately 120000. Incubation of the enzyme with proteases derived from rat liver converted the heavy polypeptide into lighter polypeptides of 80000-130000 Mr. Acetyl-CoA carboxylase isolated from crude rat liver extracts by means of immunoprecipitation with specific antibody invariably showed only the heavy polypeptide. The biotin content of the enzyme was found to be 1 mol per 237000 g protein. These results indicate that rat liver acetyl-CoA carboxylase, unlike bacterial and plant biotin enzymes, has only one kind of subunit, which has a molecular weight of 230000 and contains one molecular of biotin. Thus, the mammalian enzyme exhibits a highly integrated subunit structure.     

Extracellular proteins of Vibrio cholerae: nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic El Tor strain 017 and characterization of the hlyA mutation in the non-haemolytic classical strain 569B

The EI T or haemolysin, product of hlyA, is exported from Vibrio cholerae as a Mr 80,000 protein which can be subsequently cleaved to give two proteins of Mr 65,000 and 15,000. Nucleotide sequence analysis has demonstrated that hlyA encodes a protein of Mr 82,250 with a potential 18-amino-acid signal sequence. The non-haemolytic classical strain 569B has been shown to have a structural gene defect rather than a defect in secretion. By non-reciprocal recombination it was possible to transfer this defect onto a plasmid and show that a truncated hlyA product of Mr 27,000 is made in Escherichia coli K-12 minicells. Nucleotide sequence analysis demonstrates an 11-base-pair deletion which would result in a Mr 26,940 protein probably loosely associated with the membrane.     

Human liver alkaline phosphatase, purification and partial sequencing: homology with the placental isozyme

Human liver alkaline phosphatase (AP) has been purified to homogeneity. The enzyme has a molecular weight of 150,000 in its native state and consists of two identical subunits of Mr 75,000. After treatment with endoglycosidase F the molecular weight is reduced to 50,000 indicating a high degree of glycosylation. The amino-terminal sequence up to 22 residues was found to be Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-Lys-Tyr-(Ala)-Arg-Asp-Gln-Ala-Gln-?- Thr-Leu-Lys-Tyr. The amino-terminal portions of human and bovine liver AP are identical. The amino termini of the human liver and human placental AP isozymes have appreciable homology. Conformationally the amino termini are very similar.     

Extracellular proteins secreted by the basidiomycete Schizophyllum commune in response to carbon source

The secretion of 1,4-beta-D-glucanases by the basidiomycete Schizophyllum commune in response to cellulose or cellobiose has been studied. The proteins were labeled with 35S, and the secretion of enzymes was measured by beta-glucosidase and carboxymethyl cellulase activities and by immunoprecipitation with specific antibodies. The antigen proteins used were a beta-glucosidase (Mr, 93,000), an avicelase (avicelase II; Mr, 64,000), and a carboxymethyl cellulose (carboxymethyl cellulase I; Mr 41,000). The beta-glucosidase was initially secreted as an Mr 110,000 form, which was followed later by lower-molecular-weight (88,000 to 93,000) forms. The avicelase II, which accounted for about 50% of the secreted labeled protein, had an Mr of 64,000. Secretion of the related avicelase I (Mr 61,000) followed later. The carboxymethyl cellulose I was secreted in two molecular weight forms, Mr 44,000 and 41,000. The evidence is consistent with the idea that three genes account for the secreted glucanase activities. Other species result from different glycosylation or proteolytic cleavage processing, which may occur during or after secretion. The beta-glucosidase secretion appears to be regulated differently than that of avicelase II or carboxymethyl cellulase I; the latter two were regulated coordinately under the conditions used in this work. No common immune determinants between the three antigens were observed.     

In vitro translation and processing of rat kidney gamma-glutamyl transpeptidase

Rat kidney gamma-glutamyl transpeptidase is composed of two nonidentical glycosylated subunits. The enzyme is localized on the lumenal surface of the brush-border membranes of proximal tubule epithelial cells; it is attached to the membranes via an NH2-terminal segment of the larger of the two subunits. Tissue-labeling experiments followed by immunoprecipitation with antibodies directed against the enzyme and its two subunits demonstrate that a glycosylated single chain precursor (Mr = 78,000), containing the elements of both the subunits, is initially synthesized. Pulse-chase studies in the presence of pactamycin, and inhibitor of protein synthesis initiation, indicate that the larger of the two subunits is located at the NH2 terminus of the Mr = 78,000 precursor. The initial events in the biosynthesis and processing of gamma-glutamyl transpeptidase were investigated by in vitro translation of rat kidney mRNA. Such translation results in the synthesis of a Mr = 63,000 unglycosylated polypeptide which has been shown immunologically to contain the domains for both subunits. The Mr = 63,000 species is processed to a Mr = 78,000 core-glycosylated polypeptide when translation of mRNA is carried out in the presence of dog pancreas microsomes. This processing does not appear to be associated with cleavage of an NH2-terminal leader sequence. The Mr = 78,000 polypeptide is integrated into the microsomal membranes with an orientation that is analogous to that found on the brush-border membranes. Glycosylation and membrane integration of transpeptidase are cotranslational events. Upon longer incubation, the Mr = 78,000 species sequestered within the microsomal vesicles is cleaved to species corresponding in size to the two subunits of the kidney enzyme.