Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A human liver cDNA library was screened by colony hybridization with two mixtures of synthetic oligodeoxyribonucleotides as probes. These oligonucleotides encoded regions of beta-factor XIIa as predicted from the amino acid sequence. Four positive clones were isolated that contained DNA coding for most of factor XII mRNA. DNA sequence analysis of these overlapping clones showed that they contained DNA coding for part of an amino-terminal extension, the complete amino acid sequence of plasma factor XII, a TGA stop codon, a 3' untranslated region of 150 nucleotides, and a poly(A)+ tail. The cDNA sequence predicts that plasma factor XII consists of 596 amino acid residues. Within the predicted amino acid sequence of factor XII, we have identified three peptide bonds that are cleaved by kallikrein during the formation of beta-factor XIIa. Comparison of the structure of factor XII with other proteins revealed extensive sequence identity with regions of tissue-type plasminogen activator (the epidermal growth factor-like region and the kringle region) and fibronectin (type I and type II homologies). As the type II region of fibronectin contains a collagen-binding site, the homologous region in factor XII may be responsible for the binding of factor XII to collagen. The carboxyl-terminal region of factor XII shares considerable amino acid sequence homology with other serine proteases including trypsin and many clotting factors. A preliminary structural model of beta-factor XIIa is proposed based on the known high resolution x-ray diffraction structures of trypsin, chymotrypsin, and elastase.
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PMID:Characterization of human blood coagulation factor XII cDNA. Prediction of the primary structure of factor XII and the tertiary structure of beta-factor XIIa. 387 53

The binding of mouse epidermal growth factor-urogastrone (EGF-URO) to membranes from term human placenta is peptide-specific, saturable (about 20 pmol of EGF-URO bound maximally/mg of protein), reversible, and of high affinity (KD about 400 pM). Optimal binding is observed at pH 7.6. At low pH (3.5 to 5.0). EGF-URO can be reversibly dissociated from the receptor; however, exposure to pH < 3 irreversibly inactivates the receptor. The binding, which does not exhibit ligand cooperativity, exhibits an association rate constant of 6.1 x 10(-4) s-1 and a dissociation rate constant of 6.1 x 10(-4) s-1. The dissociation constant determined from the rate constants, 240 pM, is in reasonable agreement with the constant estimated by equilibrium methods. Both monovalent and divalent cations augment EGF-URO binding 2- to 3-fold. Although in general, divalent cations enhance binding at lower concentrations (optimum, 5 mM) than do monovalent cations (optimum, approximately 80 mM), there is no cation-specific effect. Neither guanine nor adenine nucleotides affect EGF-URO binding. Whereas the proteolytic enzymes (trypsin, chymotrypsin, papain, and pepsin) inactivate the receptor, neuraminidase and phospholipases A2, C, and D augment EGF-URO binding. Neuraminidase increases the number of available sites without affecting ligand affinity. Wheat germ agglutinin, concanavalin A, and phytohemagglutinin all compete for the binding of EGF-URO. The data complement previous observations of EGF-URO binding obtained in intact cells and provide a basis for the solubilization, characterization, and isolation of this receptor from a rich tissue source.
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PMID:Characterization of the receptor for epidermal growth factor-urogastrone in human placenta membranes. 625 85

Microsomal membranes form human placenta, which bind 5-20 pmol of 125I-epidermal growth factor (EGF) per mg protein, have been affinity-labeled with 125I-EGF either spontaneously or with dimethylsuberimidate. Coomassie blue staining patterns on SDS polyacrylamide gels are minimally altered, and the EGF-receptor complex appears as a specifically labeled band of 180,000 daltons which is not removed by urea, neutral buffers, or chaotropic salts but is partially extracted by mild detergents. Limited proteolysis by alpha chymotrypsin and several other serine proteases yields labeled fragments of 170,000, 130,000, 85,000, and 48,000 daltons. More facile cleavage by papain or bromelain rapidly degrades the hormone-receptor complex to smaller labeled fragments of about 35,000 and 25,000 daltons. These fragments retain the binding site for EGF, are capable of binding EGF, and remain associated with the membrane. Alpha chymotryptic digestion of receptor solubilized by detergents yields the same fragments obtained with intact vesicles, suggesting that the fragments may represent intrinsic proteolytic domains of the receptor.
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PMID:Proteolytic domains of the epidermal growth factor receptor of human placenta. 626 4

The cell membrane receptor for epidermal growth factor (EGF) appears to be a glycoprotein of Mr 170,000 and mediates the mitogenic and metabolic responses of cells with EGF receptors (EGF-R). Normal rat kidney (NRK) have about 3 X 10(5) EGF-R per cell. Upon transformation of NRK cells by Kirsten sarcoma virus, the transformed derivative (KNRK) loses the ability to bind 125I-EGF. Membranes from NRK and KNRK cells were included in EGF-dependent phosphorylation reactions to search for evidence of the EGF-R. A phosphorylated protein of Mr 170,000 was detected in both NRK and KNRK membranes. The Mr 170,000 protein was identified to be EGF-R by immunoprecipitation with monoclonal antibody to the receptor. Furthermore, two-dimensional peptide mapping using trypsin and chymotrypsin digestions of the iodinated receptors from both NRK and KNRK cells showed essentially identical patterns. These data indicate that the EGF-R is present in KNRK cells with apparently the same protein structure as the NRK counterpart.
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PMID:Nonfunctional epidermal growth factor receptor in cells transformed by Kirsten sarcoma virus. 632 50

One of the esteroproteinases present in the submandibular glands of female mice was purified and characterized. The enzyme, designated proteinase F in this report, had a pI value of 4.6 and a molecular weight of 27600, being comprised of two subunits of 10000 and 18000 daltons. The amino acid composition of proteinase F resembled that of the epidermal growth factor-binding protein, but antiserum against proteinase F only reacted weakly against the binding protein. Proteinase F had an optimum pH at around 9.0 and was strongly inhibited by Cu2+ and Hg2+ (42 and 76% inhibition, respectively, at a concentration of 4 x 10(-6) M). It was also inhibited by aprotinin, phenylmethylsulfonylfluoride, iodoacetamide, leupeptin, antipain, and benzamidine but neither by trypsin inhibitors from pancrease, soybean, or ovomucoid, nor by TLCK, TPCK, and epsilon-amino-n-caproic acid. Although its actual physiological function has yet to be determined, these properties indicate that proteinase F is a new enzyme, being distinguished from known proteinases, kallikrein, plasmin, trypsin, chymotrypsin, tonin, angiotensin-converting enzyme, proteinase A (beta-nerve growth factor endopeptidase), proteinase D (epidermal growth factor-binding protein), P-esterase, renin A, and renin C. Proteinase F was present in the submandibular glands of female mice more abundantly than in those of males, but it increased in males following castration. Thus, proteinase F appears to be affected by male hormones in vivo.
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PMID:A new esteroproteinase (proteinase F) from the submandibular glands of female mice. 633 33

The objective of this work was to devise methods for the isolation and culture of duct epithelium from rhesus monkey pancreas with the expectation that such methods would be applicable to the human pancreas. This objective is important because of the role duct epithelium appears to play in human diseases such as pancreatic cancer and cystic fibrosis. Pieces of freshly procured pancreas were minced and enzymatically dissociated, resulting in a digest that contained a few isolated ductules (intralobular ducts) as well as numerous small tissue fragments consisting of roughly equal proportions of ductular and acinar cells. These fragments were suspended in a rat tail collagen gel and cultured for up to 2 weeks in a medium supplemented with cholera toxin, epidermal growth factor, and other additives. A few cystic ductular fragments were initially observed among a large number of predominantly solid fragments. Later, most of the solid fragments also became cystic and eventually resembled the ductules except for being spherical. Autoradiographic analysis of DNA synthesis showed that the cysts possessed a proliferative potential. The cysts consisted almost entirely of ductule-like epithelium with no recognizable acinar cells, and exhibited greatly reduced concentrations of the acinar marker enzymes amylase, chymotrypsin, and gamma-glutamyl transferase. In contrast, the specific activity of the duct marker enzyme carbonic anhydrase was elevated in freshly isolated digests compared with the whole pancreas and this elevated activity was maintained for 4-5 days of culture, after which it declined. Other evidence for the ductular nature of the cysts was their low density relative to freshly isolated acinar tissue, their ability to distend (suggestive of fluid/electrolyte secretion), and the accumulation of mucins at the apical borders of the cells. The results show that fragments of rhesus monkey pancreas that are enriched in ductular epithelium assume some of the properties of ductular cells when cultured in a collagen gel. These epithelial preparations should facilitate biochemical and physiological studies of this important pancreatic cell type.
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PMID:Isolation and culture of rhesus monkey pancreatic ductules and ductule-like epithelium. 750 63

Mannose-binding protein (MBP) plays an important role in host defense by recognizing sugar residues on certain pathogens and activating the complement cascade. Recently, we described a new protease, designated MBP-associated serine protease (MASP) which is required for complement activation by MBP. We have cloned the cDNA that encodes this protease and found that the deduced amino acid sequence contains an epidermal growth factor-like domain, two short consensus repeats and a serine protease domain. The overall structure of MASP is similar to serine proteases of the first complement component complex, C1r-C1s. Unlike C1r-C1s, however, MASP has a histidine loop structure common to many serine proteases such as trypsin and chymotrypsin. The MASP gene was mapped on the long arm of chromosome 3 which is different from C1r-C1s as well as from trypsin and chymotrypsin. These findings suggest that MASP may have emerged prior to C1r-C1s from a common ancestor. This implies that MBP-MASP, a complex of lectin and serine protease, presumably evolved prior to adaptive immune recognition involving antibody and the classical complement pathway.
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PMID:Molecular characterization of a novel serine protease involved in activation of the complement system by mannose-binding protein. 801 3

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored membrane protein thought to play a primary role in the generation of pericellular proteolytic activity, and to be involved in cancer cell invasion and metastasis. This protein is composed of three homologous domains, the NH2-terminal of which is involved in the high-affinity binding (Kd approximately 0.1-1.0 nM) to the epidermal growth factor-like module of urokinase-type plasminogen activator (uPA). Here we report that intact uPAR binds the low molecular weight fluorophore 8-anilino-1-naphthalenesulfonate (ANS) to form a 1:1 stoichiometric complex and that the resulting enhancement of the ANS fluorescence probes the functional state of uPAR as judged by several independent criteria. First, the uPAR-mediated increase in ANS fluorescence can be titrated by uPA as well as by its receptor binding derivatives (the amino-terminal fragment and the growth factor-like module). Second, an anti-uPAR monoclonal antibody, capable of preventing uPA binding, can also titrate the uPAR-dependent ANS fluorescence whereas other antibodies not interfering with uPA binding are unable to exert this effect. Third, the dissociation profile of uPA-uPAR complexes as a function of increasing concentrations of guanidine hydrochloride closely parallels the loss of the ANS binding site in uPAR. Finally, liberation of the NH2-terminal domain from uPAR by limited chymotrypsin cleavage after Tyr87 leads to a loss of both enhanced ANS fluorescence and high-affinity uPA binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor. 804 85

To study the role of a putative high affinity Ca2+ binding site in the protease domain of factor X, we prepared a deletion mutant (E2FX) lacking the Gla and first epidermal growth factor-like domains. E2FX possesses a single high affinity Ca2+ binding site (Kd = 154 microM). Asp-70 or Glu-80 (chymotrypsin numbering system) was then converted to Lys. These mutants, E2FXD70K and E2FXE80K, allow examination of the Ca(2+)-dependent assembly of activation complexes involving substrate and enzyme forms of factor X that do not bind Ca2+. E2FXD70K and E2FXE80K share similar properties. They retain functional activity, but the D70K mutant no longer binds Ca2+, and neither mutant exhibits a Ca(2+)-dependent increase in peptide chromogenic substrate activity. Activation of E2FXD70K by the soluble tissue factor-factor VIIa complex was still Ca(2+)-dependent (Kd(app) = 133 +/- 38 microM), but the Kd(app) for Ca2+ was decreased relative to E2FX (Kd(app) = 205 +/- 53 microM). Prothrombin or prethrombin 1 activation by the E2FXaD70K in the presence of factor Va was also Ca(2+)-dependent (Kd(app) = 124 +/- 47 microM and 102 +/- 38 microM, respectively), and the Kd(app) was again lower than that of E2FXa (272 +/- 79 microM and 179 +/- 54 microM, respectively). In the absence of factor Va, activation of prethrombin 1 by E2FXaD70K was not influenced by Ca2+, but activation by E2FXa was enhanced (Kd(app) = 161 +/- 32 microM). The results suggest the presence of functionally important Ca2+ binding sites in components of the factor X and prothrombin activation complexes that do not involve factor X. Furthermore, the Ca2+ binding sites in factor X and trypsin appear to be structurally similar. The Asp or Glu-->Lys mutations within this site may create an internal salt bridge that conserves factor X (Xa) function even in the absence of Ca2+.
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PMID:Asp-70-->Lys mutant of factor X lacks high affinity Ca2+ binding site yet retains function. 806 84

The receptor for human urokinase-type plasminogen activator (uPAR) is synthesized as a 313-residue-long polypeptide containing 28 cysteine residues, the pattern of which defines three homologous repeats within the protein. These entities are believed to represent a novel protein domain structure, of which the NH2-terminal domain of uPAR can be covalently cross-linked to the epidermal growth factor-like module of urokinase after receptor-ligand interaction. The NH2-terminal domain of a recombinant, soluble uPAR derivative, labeled with [35S]cysteine, was isolated after limited proteolysis with chymotrypsin. The four disulfide bonds present within this domain were assigned by a combination of plasma desorption mass spectrometry, amino acid composition, and sequence analyses of peptides generated by trypsin, endoproteinase Asp-N, and thermolysin. The following disulfide bond structure was determined: Cys3-Cys24, Cys6-Cys12, Cys17-Cys45, and Cys71-Cys76. Similar cysteine pairing is likely to be found within other members of this protein superfamily, i.e. the membrane inhibitor of reactive lysis, Ly-6, and the remaining two domains of uPAR. However, an additional pair of cysteines present within these domains probably forms a fifth disulfide bond.
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PMID:Localization of the disulfide bonds in the NH2-terminal domain of the cellular receptor for human urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins. 839 46


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