Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To analyze the influence of cell differentiation and the effects of hormones on the subcellular distribution of apical antigens in polarized epithelial cells, we have compared the localization of three brush border (BB) hydrolases [neutral endopeptidase (ENDO), aminopeptidase N (APN), and dipeptidylpeptidase IV (DPPIV)] in primary cultures of renal proximal tubule cells grown in various culture media. The degree of cell differentiation modulated by medium composition was estimated by measuring proximal functions, including glucose transport, specific enzymatic activities, and PTH responsiveness. In the dedifferentiated state observed in cells grown in 1% fetal calf serum (FCS)-supplemented medium, the three hydrolases are abnormally concentrated in a cytoplasmic vesicle compartment with weak expression on both membrane domains. By contrast, in serum-free hormonally defined medium (DM: insulin, 5 microgram/ml; dexamethasone, 5 x 10(-8) M), which markedly enhances morphological and functional cell differentiation, the distribution of hydrolases parallels that observed in the normal tubule. When added to the DM devoid of hormones, insulin has little polarizing effect, whereas dexamethasone dramatically increases the apical expression of the hydrolases, which then almost disappear from the basolateral membrane and cytoplasmic vesicular compartments. This glucocorticoid hormone augments the amount of immunoreactive antigen detectable on the apical domain in paraformaldehyde-fixed cells but does not change the total enzymatic activity. This suggests the presence in tubular cells of a dexamethasone-dependent polarizing machinery that requires de novo RNA and protein synthesis, and probably acts mainly by targeting a storage cytoplasmic pool of enzyme to the apical domain.
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PMID:Polarized membrane expression of brush-border hydrolases in primary cultures of kidney proximal tubular cells depends on cell differentiation and is induced by dexamethasone. 197 36

Endopeptidase-24.11 is a 90-kDa surface glycoprotein with the ability to hydrolyze a variety of biologically active peptides. Interest in this enzyme is based on the consensus that it may play a role in the termination of peptide signals in the central nervous system. In the present study, we have investigated the distribution of endopeptidase-24.11 in two nerves of the peripheral nervous system of newborn pigs: the sciatic, composed of a mixture of myelinated and nonmyelinated axons, and cervical sympathetic trunk in which greater than 99% of the axons are nonmyelinated. The endopeptidase was monitored enzymatically, as well as by immunoblotting and immunocytochemistry using mono- and polyclonal anti-endopeptidase antibodies. Endopeptidase-24.11 was detected in both the sciatic nerve and the cervical sympathetic trunk. Membrane preparations from sciatic nerve hydrolyzed 125I-insulin B-chain, and more than 50% of the activity was inhibited by phosphoramidon with an IC50 concentration of 3.2 nM. Moreover, a 90-kDa polypeptide was detected by immunoblotting of sciatic nerve membranes. The type of cells expressing the endopeptidase was determined by immunohistochemistry. In teased nerve preparations, these cells were identified morphologically as myelin- and non-myelin-forming Schwann cells. Endopeptidase-24.11 was also expressed by cultured Schwann cells from sciatic nerve and cervical sympathetic trunk maintained for 3 h in vitro. The presence of endopeptidase-24.11 on the Schwann cell surface raises the possibility of a potential role for the enzyme in nerve development and/or regeneration.
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PMID:Endopeptidase-24.11, a cell-surface peptidase of central nervous system neurons, is expressed by Schwann cells in the pig peripheral nervous system. 207 94

The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg31-Arg32) and a type II activity that cleaves the C-peptide/A-chain junction (Lys64-Arg65). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, we have undertaken comparative 1H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models of prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the 1H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic 1H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. Unexpectedly, nonlocal perturbations are observed in the insulin moiety of proinsulin, as monitored by the resonances of internal aromatic groups. Remarkably, these perturbations are reverted by site-specific cleavage of the connecting peptide at the CA junction but not the BC junction. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. We propose that this structure (designated the "CA knuckle") provides a recognition element for type II proinsulin endopeptidase.
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PMID:NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition. 225 1

Microvillar membranes derived from the brush border of the renal proximal tubule are very rich in peptidases. Pig kidney microvilli contain endopeptidase-24.11 associated with a battery of exopeptidases. The manner by which some neuropeptides are degraded by the combined attack of the peptidases of this membrane has been investigated. The contribution of individual peptidases was assessed by including inhibitors (phosphoramidon, captopril, amastatin and di-isopropyl fluorophosphate) with the membrane fraction when incubated with the peptides. Substance P, bradykinin and angiotensins I, II and III and insulin B-chain were rapidly hydrolysed by kidney microvilli. Oxytocin was hydrolysed much more slowly, but no products were detected from [Arg8]vasopressin or insulin under the conditions used for other peptides. The peptide bonds hydrolysed were identified and the contributions of the different peptidases were quantified. For each of the susceptible peptides, the main contribution came from endopeptidase-24.11 (inhibited by phosphoramidon). Peptidyl dipeptidase A (angiotensin-I-converting enzyme) was of less importance, even in respect of angiotensin I and bradykinin. When [2,3-Pro3,4-3H]bradykinin was also investigated at a lower concentration (20 nM), the conclusions in regard to the contributions of the two peptidases were unchanged. The possibility that endopeptidase-24.11 might attack within the six-residue disulphide-bridged rings of oxytocin and vasopressin was examined by dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ation and by reduction and carboxymethylation of the products after incubation. Additional peptides were only observed after prolonged incubation, consistent with hydrolysis at the Tyr2-Ile3 and Tyr2-Phe3 bonds respectively. These results show that a range of neuropeptides are efficiently degraded by microvillar membranes and that endopeptidase-24.11 plays a key role in this process.
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PMID:Metabolism of neuropeptides. Hydrolysis of the angiotensins, bradykinin, substance P and oxytocin by pig kidney microvillar membranes. 243 10

Endopeptidase-2, the second endopeptidase in rat kidney brush border [Kenny & Ingram (1987) Biochem. J. 245, 515-524] has been further characterized in regard to its specificity and its contribution to the hydrolysis of peptides by microvillar membrane preparations. The peptide products were identified, after incubating luliberin, substance P, bradykinin and angiotensins I, II and III with the purified enzyme. The bonds hydrolysed were those involving a hydrophobic amino acid residue, but this residue could be located at either the P1 or P1' site. Luliberin was hydrolysed faster than other peptides tested, followed by substance P and bradykinin. Human alpha-atrial natriuretic peptide and the angiotensins were only slowly attacked. Oxytocin and [Arg8]vasopressin were not hydrolysed. No peptide fragments were detected on prolonged incubation with insulin, cytochrome c, ovalbumin and serum albumin. In comparison with pig endopeptidase-24.11 the rates for the susceptible peptides were, with the exception of luliberin, much lower for endopeptidase-2. Indeed, for bradykinin and substance P the ratio kcat./Km was two orders of magnitude lower. Since both endopeptidases are present in rat kidney microvilli, an assessment was made of the relative contributions to the hydrolysis of luliberin, bradykinin and substance P. Only for the first named was endopeptidase-2 the dominant enzyme; for bradykinin it made an equal, and for substance P a minor, contribution.
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PMID:The metabolism of neuropeptides. Hydrolysis of peptides by the phosphoramidon-insensitive rat kidney enzyme 'endopeptidase-2' and by rat microvillar membranes. 246 6

The relationship between growth and cytodifferentiation was studied in cultured human mammary myoepithelial cells under serum-free culture conditions. Myoepithelial-cell differentiation was monitored by quantifying cells showing immunoreactivity to the muscle isoform of actin; to the membrane glycoprotein common acute lymphoblastic leukemia antigen (CALLA); and to type IV collagen. Growth was quantified either by measuring the actual increase in cell number, or in a more-sensitive assay using immunoreactivity to the cell-proliferation-associated nuclear antigen Ki-67 as a measurement of the number of cells leaving the G0-phase of the cell cycle. The results showed that: (a) Primary cultures of myoepithelial cells on DME-F12 supplemented with cholera toxin (CT) alone resulted in the formation of quiescent cell islets (in the G0-phase of the cell cycle) showing phenotypic traits preserved from the in vivo situation (actin- and CALLA-positive cells with little or no type-IV-collagen immunoreactivity). (b) After addition of epidermal growth factor (EGF), with an ED50 of 1-10 ng/ml, in the presence of CT, the cells entered the G1-phase of the cell cycle, without further increase in cell number. At the same ED50 of EGF, the frequency of CALLA-positive cells decreased, while the number of cells immunoreactive for type IV collagen increased with a maximal effect of EGF seen after 7-11 days. During the same period, the cells remained fully differentiated with respect to actin immunoreactivity. (c) Further addition of insulin (I) to the medium in the presence of EGF and CT resulted in the cells entering an exponential growth phase associated with simultaneous decrease in actin immunoreactivity with a maximal effect of I after 11 days of exposure. The dose-response curve to I was virtually identical for stimulating cell proliferation and for reducing the frequency of actin-immunoreactive cells (ED50 in the range of 30 ng/ml), suggesting that the two processes were controlled by the same initial I-receptor interaction. (d) Some reduction in the number of actin-positive cells was exerted by I-EGF-CT independently of the mitogenic response, but this reduction was further augmented if the cells were allowed to proliferate. (e) Time-course studies of quiescent (G0-phase) cells stimulated to exponential growth revealed that entrance of cells into the G1-phase of the cell cycle preceded the loss of muscle actin filaments. (f) Exponentially growing actin-negative epithelial cells did not resume a myoepithelial phenotype in density-arrested postconfluent cultures.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Growth factor control of myoepithelial-cell differentiation in cultures of human mammary gland. 246 50

A lysate of purified insulin secretory granules, which contains two types of proinsulin processing activity (type 1, Arg-Arg-directed and type II, Lys-Arg-directed (Davidson, H.W., Rhodes, C.J., and Hutton, J. C. (1988) Nature 333, 93-96), was found to process proalbumin by specific proteolytic cleavage of the COOH-terminal side of the Arg-2-Arg-1 sequence. The subcellular distribution of proalbumin processing activity in insulinoma tissue paralleled that for proinsulin conversion and occurred principally in a secretory granule fraction. Cleavage appeared to result from the Arg-Arg-directed type 1 proinsulin processing endo-peptidase. It was Ca2+-dependent (K0.5 activation = 1.0-1.5 mM Ca2+), unaffected by group-specific inhibitors of serine, cysteinyl, or aspartyl proteinases, and had an acidic pH optimum (5.5). Active-site inhibitor studies showed this activity had a preference for dibasic over monobasic amino acid sequences and indicated that the sequence of the dibasic site was an important determinant of the susceptibility of the substrate to cleavage. The activity did not process the proalbumin Christchurch mutant (Arg-2-Arg-1 to Arg-2-Gln-1). It was inhibited by the variant alpha 1-antitrypsin Pittsburgh (Met358 to Arg358; K0.5 = 100 nM) but not by other related proteins normally co-secreted with albumin from hepatocytes, namely alpha 1-antitrypsin M, alpha 2-macroglobulin, or antithrombin III. The insulin secretory granule proalbumin processing activity was indistinguishable from a proalbumin endopeptidase reported in rat liver membranes and similar to the yeast KEX-2 protease. These findings suggest that a highly conserved set of proprotein endopeptidases exists, which are specific for a dibasic sequence but broadly specific for proprotein substrates. Such enzymic activities appear to be active within both the constitutive and regulated pathways of secretion. Intraorganellar Ca2+ and pH appear to play a key role in regulating their activities.
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PMID:Proalbumin to albumin conversion by a proinsulin processing endopeptidase of insulin secretory granules. 250 14

An extracellular Zn-endopeptidase was purified to homogeneity from the culture filtrates of Streptococcus faecalis (human oral strain 0G1-10) by a procedure that comprised concentration in an Amicon Hollow Fiber System, ammonium sulfate precipitation, gel permeation chromatography, hydrophobic interaction chromatography (batch operation on phenyl-sepharose Cl-4B), followed by fast protein liquid chromatography (FPLC) on a phenyl-Superose HR 5/5 column, and finally FPLC on a Superose 12 HR 10/30 column. The enzyme is a 31.5-kDa strongly hydrophobic protein with an isoelectric point of 4.6 and a broad pH optimum of 6 to 8. The substrate specificity of the enzyme is similar to that of the mammalian membrane endopeptidase-24.11 and Streptococcus thermophilus thermolysin (EC 3.4.24.4) in hydrolyzing preferentially the Phe24-Phe25 bond in insulin B-chain, followed by cleavage of the His5-Leu6 bond. The enzyme was especially active on Azocoll and gelatin; soluble and insoluble collagens were hydrolyzed at a lower rate. S. faecalis sex pheromone-related peptides and several mammalian bioactive peptides were cleaved at sites involving pronounced hydrophobicity. The enzyme did not hydrolyze small synthetic peptide derivatives (phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg and 2-furylacryloyl-L-Leu-Gly-L-Ala) that are typically attacked by "true" bacterial collagenases. Chemical modification indicated the importance of histidyl, carboxyl, and tyrosyl groups in enzyme activity, suggesting that this enzyme may thus be classified as a metalloprotease II (EC 3.4.24.4). The enzyme is strongly inhibited by a 720-kDa factor present in rat inflammatory exudate. The pronounced ability of the enzyme to attack collagenous materials and certain bioactive peptides suggests its participation in inflammatory processes involving the presence of S. faecalis.
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PMID:Purification and substrate specificity of a strongly hydrophobic extracellular metalloendopeptidase ("gelatinase") from Streptococcus faecalis (strain 0G1-10). 253 44

Inhibitor studies were performed on the two endopeptidase activities involved in proinsulin conversion in isolated insulin secretory granules [Davidson, Rhodes & Hutton (1988) Nature (London) 333, 93-96]. The active-site-directed peptides L-alanyl-L-arginyl-L-arginylmethyldimethylsulphonium and L-alanyl-L-lysyl-L-arginylmethyldimethylsulphonium inhibited these activities in accordance with the observed cleavage pattern, suggesting that the primary amino acid sequence of the dibasic site was an important determinant of the endopeptidase substrate specificities.
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PMID:The inhibition of proinsulin-processing endopeptidase activities by active-site-directed peptides. 264 90

Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae. Two molecular forms of proteinase A (PRA), Mr 45,000 and 54,000, (estimated on SDS-PAGE) were obtained. Both forms were inhibited by pepstatin and other acid proteinase inhibitors. The enzyme digested hemoglobin most rapidly at pH 2.7-3.2 and casein at pH 2.4-2.8 and 5.5-6.0. The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4-6 M urea. Urea also stimulated the enzyme activity by 30-50%. As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X-Tyr and X-Phe type. A proteinase B (PRB) preparation of approximately Mr 33,000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases. The purified enzyme could be stabilized with 40% glycerol and stored at -20 degrees C without significant loss of activity for several months. The third endopeptidase, designated PRY, of Mr 72,000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB. Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15-Tyr16 peptide bond of the oxidized beta-chain of insulin. On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at -20 degrees C without glycerol. The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and properties of three endopeptidases from baker's yeast. 266 27


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