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

The proteolytic processing of frog (Rana esculenta) proopiomelanocortin in melanotropic cells of the intermediate pituitary gland has been examined through purification of the mature fragments by reverse-phase high-pressure liquid chromatography and microsequencing of isolated peptides. alpha-Melanotropin, beta-melanotropin, Lys-gamma-melanotropin, corticotropin-like intermediate lobe peptide, and hinge peptide have been isolated and chemically characterized. The results show a high preservation in the processing sites of frog proopiomelanotropin when compared to bovine counterparts. They reveal also a great conservation of the processing enzyme equipment of melanotropic cells in tetrapods species. Identification of Lys-gamma-melanotropin suggests the occurrence of an endopeptidase able to cleave between two basic residues. On the other hand alpha-melanotropin does not appear to be N-acetylated, as previously found in the clawed-toad Xenopus laevis, and this feature might distinguish amphibian from mammalian proopiomelanocortin processing.
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PMID:Study of frog (Rana esculenta) proopiomelanocortin processing in the intermediate pituitary. Identification of alpha-melanotropin, beta-melanotropin, Lys-gamma-melanotropin, and corticotropin-like intermediate lobe peptide. 165 Dec 91

Iron oxide particles of average size 0.5-1.5 microns, covered by a silane coat carrying amino groups (Bio-Mag, Advanced Magnetics, Boston), were derivatized by reaction with N-[(gamma-maleimidobutyryl)oxy]-succinimide (GMBS), N-hydroxysuccinimidyl iodoacetate (NHIA), 2-iminothiolane (2-It), or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The derivatized particles were suitable for the reaction with sulfhydryl groups and subsequently coated with monoclonal antibodies (MoAbs) of different classes and isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) as well as polyclonal rabbit anti-mouse IgG (RAM). The antibodies were reduced by dithiothreitol (DTT) and covalently conjugated to the BioMag derivatives via liberated sulfhydryls of the hinge region. The observed conjugation ratios, expressed as protein/iron (micrograms/mg), could be reproducibly varied for optimization. These ratios were dependent on the type and amount of antibody offered for coupling to the derivatized particles, decreasing as follows: polyclonal = IgM greater than IgG2b greater than IgG2a = IgG3 greater IgG1. The conjugation ratios were also dependent on the type and amount of the spacer used to derivatize the BioMag particles, decreasing as follows: GMBS greater than NHIA greater than 2-It greater than SPDP. The magnetically responsive magnetite-antibody conjugates ("magneto-beads"), carrying MoAb BMA 081 (anti-CD8; IgG2a), MoAb BB10 (anti-CD10/CALLA; IgG2b), MoAb VIL-A1 (anti-CD10; IgM), and polyclonal RAM, coupled similarly via 3.6 mumol of GMBS spacer per mg of Fe, were further investigated with respect to a depletion effect on specific cell subsets. The rates of cell depletion were found to be strongly dependent on the individual characteristics of the antibody used.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hinge-thiol coupling of monoclonal antibody to silanized iron oxide particles and evaluation of magnetic cell depletion. 215 62

Normal human pituitaries were extracted in boiling water and acetic acid, and the alpha-amidated peptide products of pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N), as well as their glycine-extended precursors, were characterized by sequence-specific radioimmunoassays, gel-chromatography, h.p.l.c. and amino acid sequencing. alpha MSH and gamma 1MSH constituted 0.27-1.32% and 0.10-5.10%, respectively, of the POMC-derived products [calculated as the sum of adrenocorticotropic hormone (ACTH)-(1-39), ACTH-(1-14) and alpha MSH immunoreactivity]. alpha MSH and ACTH-(1-14) were only present in non- or mono-acetylated forms. Only large forms of gamma 1MSH and gamma 2MSH were present in partly glycosylated states. The hinge peptides were amidated to an extent two to three orders of magnitude greater than alpha MSH and gamma 1MSH. Most (99%) of the HP-N was of low molecular mass and consisted mainly of HP-N-30. The remaining part was high-molecular-mass HP-N, probably HP-N-108, although the presence of HP-N-44 could not be completely excluded. These results show that all the possible amidated POMC-related peptides are present in normal human pituitary. It also shows that cleavage in vivo at all dibasic amino acids but one, takes place at the N-terminal POMC region; the exception is at the POMC-(49-50) N-terminal of the gamma MSH sequence. The pattern of peptides produced suggests that the generation of amidated peptides is mainly regulated at the endopeptidase level.
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PMID:Alpha-amidated peptides derived from pro-opiomelanocortin in normal human pituitary. 283 46

Streptococcus sanguis, an initiator of human dental plaque, produces an endopeptidase which cleaves immunoglobulin A1 (IgA1) at the hinge region. A rapid nephelometric assay was developed for the quantitation of IgA1-specific protease activity. The protease was produced in dialysis cultures which yielded cell-free fluids having 14 times the specific activity of conventional cultures. Assay was based on the difference in detectable IgA1 concentrations at the start and termination of the reaction; IgA1 concentrations were determined by rate of complex formation with IgA-specific antibody. The rates of IgA1 cleavage were linear during incubations up to 3 h if enzyme preparations were sufficiently diluted. The assay resolution was less than that achieved with sodium dodecyl sulphate-polyacrylamide gel electrophoresis, but 1 h incubation of protease-IgA1 reaction mixtures was adequate for measurement. The pH optimum for the reaction was 7.0 and the calculated Km was 5.6 X 10(-5)M IgA1. The optimal incubation temperature was in the range of 37-40 degrees C; the enzyme lost all activity at 55 degrees C.
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PMID:Nephelometric assay for the immunoglobulin A1-protease produced by the oral bacterium Streptococcus sanguis. 676 Aug 37

Raw-starch-digesting amylase (RSDA) is a key extracellular enzyme of mesophilic Bacillus circulans F-2 which uses raw starch granules as a carbon source. Previous work has demonstrated that there are two domains of the enzyme during digestion with subtilisin, and that RSDA activity is selectively inactivated by limited proteolysis with subtilisin, which cleaves the enzyme between these hydrolytic and adsorption domains (Kim, C.-H., Kwon, S.-T., Taniguchi, H. and Lee, D.-S. (1992) Biochim. Biophys. Acta 1122, 243-250). In this work we show that a similar phenomenon is observed during limited proteinase K, thermolysin and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63 and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the proteinase-sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive towards anti-RSDA were detected through whole bacterial cultivation. 93, 75, 63, 55, 38 and 31-kDa proteins were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results suggest that enzyme heterogeneity of the raw-starch hydrolysis system might arise from the endogenous proteolytic activity of the bacterium.
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PMID:Domain structure and multiplicity of raw-starch-digesting amylase from Bacillus circulans: extensive proteolysis with proteinase K, endopeptidase Glu-C and thermolysin. 769 Nov 84

The complete amino-acid sequence of the hinge region in the subfragment-2 (S-2) derived from adult chicken cardiac ventricular muscle myosin has been determined by direct protein sequencing. The entire amino-acid sequence of this hinge composed of 143 residues was established by structural analysis of CNBr peptides, lysyl and arginyl endopeptidase peptides of carboxymethylated S-2. By sequence comparison with the corresponding region of the same chicken cardiac myosin which was recently deduced from its cDNA eight amino-acid differences were recognized. Comparing the sequence of this hinge with those of other cardiac myosins such as rat alpha- and beta-myosin heavy chains (MHC), rabbit alpha-MHC and human alpha- and beta-MHCs relatively lower degrees of sequence identities, namely 74.8%, 77.6%, 76.1% 75.5% and 75.5%, are observed. On the other hand, more than 89.5% sequence identities are shown among these mammalian cardiac myosins. These results indicate that avian cardiac MHC has diverged earlier than mammalian cardiac myosin has diverged to alpha- and beta-MHC. Amino-acid substitutions in this hinge region form a cluster on the C-terminal sequence region. On the contrary, in the N-terminal portion, completely conserved segments are observed, suggesting that these regions may contribute to the myosin ATPase activity and muscle contraction.
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PMID:Primary structure of the hinge region in adult chicken cardiac myosin subfragment-2. 843 95

Complete and rapid peptide and glycopeptide mapping of a mouse monoclonal immunoglobulin (IgG2b) were carried out by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry (LC/ ESI IT-MS/MS). It was possible to obtain spectra of a minor glycopeptide at a quantity as low as 1.8 pmol. Reduced and carboxymethylated mouse antidansyl monoclonal IgG2b (RCM-IgG2b) was digested with lysyl-endopeptidase. Proteolytic peptides were subjected to capillary HPLC separation followed by analysis with an ion trap mass spectrometer. The complete amino acid sequence of the IgG2b was characterized by using LC/ ESI IT-MS/MS. The structures of 12 different types of O-linked oligosaccharides attached to Thr-221AH in the hinge region and those of three major types of N-linked oligosaccharides attached to Asn-297H have been characterized.
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PMID:Complete and rapid peptide and glycopeptide mapping of mouse monoclonal antibody by LC/MS/MS using ion trap mass spectrometry. 966 36

Group A Streptococcus has evolved numerous mechanisms to evade the host immune system to survive, disseminate, and cause disease. Recently a secreted protein named Mac-1 was identified and shown to enhance survival of the pathogen. A new variant of Mac-1 (designated Mac-2) also was recently described and shown to differ from Mac-1 by approximately 50% amino acid sequence divergence in the middle one-third of the molecule. To gain new information about the role of Mac-1 and Mac-2 in host-pathogen interactions, solution binding experiments were performed using surface plasmon resonance and purified Mac proteins. Mac-1 bound the same lower hinge region of human IgG as Fc receptors with 2.5 microM affinity, which lead to proteolytic cleavage of the antibody. Similar Km (6.8-18.9 microM) and kcat (0.02-0.13 s(-1)) values of the Mac-1 endopeptidase activity were obtained for IgG1, IgG2, IgG3, and IgG4. Mac-2 variant, in contrast, bound human IgG poorly (KD = 16 mM) and had weak endopeptidase activity against IgG. Instead, Mac-2 bound FcgammaRII and FcgammaRIII with 5 and 75 microM affinity, respectively. This binding competitively blocked IgG from recognition by Fc receptors. Taken together, Mac proteins block immunoglobulin recognition by Fc receptors and degrade immunoglobulins, thereby enhancing survival of the pathogen through the inhibition of phagocytosis, endocytosis of IgG-opsonized particles, and antibody-dependent cell-mediated cytotoxicity. Consequently, these proteins may be potential therapeutic targets.
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PMID:Insight of host immune evasion mediated by two variants of group a Streptococcus Mac protein. 1546 62

Streptococcus pyogenes, an important pathogen in humans, secretes an IgG specific endopeptidase named IdeS. To elucidate the mechanism that is responsible for this specificity, we have here characterized the activity of IdeS in detail. Both gamma chains of human IgG or its Fc fragment were cleaved in the hinge region after Gly236 by IdeS, but other proteins or synthetic peptides containing sequences such as the P(4)-P(1) segment in the IgG cleavage site, or long peptides resembling the IgG hinge, were not hydrolyzed at all. This is likely due to a second binding site interacting with the Fc part of IgG. The lack of IdeS activity on peptide substrates necessitated the development of an assay with IgG as the substrate for kinetic studies. IdeS showed a sigmoidal velocity curve at physiological IgG concentrations, and a declining enzyme rate at higher IgG concentrations. This atypical velocity curve suggests product inhibition and/or allosteric control, which again indicates the presence of an exosite involved in substrate binding. The pseudoequilibrium constant for IdeS hydrolysis of IgG was 90 microM. The enzyme exhibited activity in the pH range of 5.1-7.6, with an optimum at pH 6.6. IdeS was stable above pH 10 but not at acidic pH. It exhibited an activity maximum around 37 degrees C and a decreased thermal stability at 42 degrees C. Iodoacetate and iodoacetamide inhibited IdeS, as expected for a cysteine protease, and biochemical evidence verified this classification. E-64 and chicken cystatin, specific inhibitors of family C1 and C13 cysteine proteases, were without effect on enzyme activity, as were class specific serine, aspartic, and metallo protease inhibitors. No significant similarities were found in protein sequence comparisons with known enzyme families, suggesting that IdeS represents a novel family of cysteine proteases.
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PMID:Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding. 1558 66

Thimet oligopeptidase (EC 3.4.24.15) is a zinc(II) endopeptidase implicated in the processing of numerous physiological peptides. Although its role in selecting and processing peptides is not fully understood, it is believed that flexible loop regions lining the substrate-binding site allow the enzyme to conform to substrates of varying structure. This study describes mutant forms of thimet oligopeptidase in which Gly or Tyr residues in the 599-611 loop region were replaced, individually and in combination, to elucidate the mechanism of substrate selection by this enzyme. Decreases in k(cat) observed on mutation of Tyr605 and Tyr612 demonstrate that these residues contribute to the efficient cleavage of most substrates. Modeling studies showing that a hinge-bend movement brings both Tyr612 and Tyr605 within hydrogen bond distance of the cleaved peptide bond supports this role. Thus, molecular modeling studies support a key role in transition state stabilization of this enzyme by Tyr605. Interestingly, kinetic parameters show that a bradykinin derivative is processed distinctly from the other substrates tested, suggesting that an alternative catalytic mechanism may be employed for this particular substrate. The data demonstrate that neither Tyr605 nor Tyr612 is necessary for the hydrolysis of this substrate. Relative to other substrates, the bradykinin derivative is also unaffected by Gly mutations in the loop. This distinction suggests that the role of Gly residues in the loop is to properly orientate these Tyr residues in order to accommodate varying substrate structures. This also opens up the possibility that certain substrates may be cleaved by an open form of the enzyme.
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PMID:Hydrogen bond residue positioning in the 599-611 loop of thimet oligopeptidase is required for substrate selection. 1895 47


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