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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII by thrombin must be accompanied by a limited proteolysis of the arginyl-glycyl bond located in the N-terminal region of the a chain, liberating the "Activation peptide." The possibility of activating Factor XII with other porteinases was examined using Factor Xa [EC 3.4.21.6], Factor XIIa, kallikreins [EC 3.4.21.8], urokinase [EC 3.4.99.26], trypsin [EC 3.4.21.4], ficin [EC 3.4.22.3], papain [EC 3.4.22.2], and bromelain [EC 3.4.22.4]. Among these enzymes, only bromelain and trypsin showed clear activating effects.
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PMID:On the activation of bovine plasma factor XIII. Amino acid sequence of the peptide released by thrombin and the terminal residues of the subunit polypeptides. 122 22

Modified (Arg63-Ile64 reactive-site peptide bond hydrolyzed) soybean trypsin inhibitor (Kunitz) with all reactive amino groups, except that of Ile64, protected was described in the preceding paper (Kowalski, D., and Laskowski, M., Jr. (1976), Biochemistry, preceding paper in this issue). Treatment of this inhibitor with tert-butyloxycarbonyl-Ala- and tert-butyloxycarbonyl-Ile-N-hydroxy-succinimide esters yields inactive endo-tert-butyloxycarbonyl-Ala63A-and endo-tert-butyloxycarbonyl-Ile63A-modified inhibitors. The tert-butyloxycarbonyl groups were removed by treatment of the proteins with trifluoroacetic acid. After renaturation and purification, the resultant endo-Ala63A- and endo-Ile63A-modified inhibitors co-electrophorese with modified inhibitor both on disc gels (pH 9.4) and sodium dodecyl sulfate gels (after reduction of disulfide bonds) and show end groups corresponding to the 63A residue. These derivatives fail to form stable complexes with trypsin, extending the previous observation (Kowalski, D., and Laskowski, M., Jr. (1972), Biochemistry 11, 3451) that acylation of the P1' residue in modified inhibitors leads to inactivation. However, the incubation of endo-Ala63A- and endo-Ile63A-modified inhibitors with trypsin at pH 6.5 leads to the synthesis of the Arg63-Ala63A and Arg63-Ile63A peptide bonds in 4% yield. This is very close to the yield anticipated from a semiquantitative theory for the value of the equilibrium constant for reactive-site peptide bond. An alternative chemical method of insertion is also described. Controlled treatment of modified inhibitor with the N-carboxyanhydride of Glu produced inactive endo-Glu63A-modified inhibitor. Incubation of this inactive derivative with trypsin at pH 6.5 leads to 16% synthesis of the Arg63-Glu63A peptide bond. The higher yield of single chain protein in this case is attributed to the influence of the negative charge of the Glu63A side chain. Thus, the insertion of an amino acid residue between the P1 and P1' residues in soybean trypsin inhibitor (Kunitz) converts a trypsin inhibitor into a trypsin substrate.
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PMID:Chemical-enzymatic insertion of an amino acid residue in the reactive site of soybean trypsin inhibitor (Kunitz). 125 50

The complete amino acid sequence of rat thyrocalcitonin has been determined by automated Edman degradations of the intact molecule, a cyanogen bromide fragment, and by degradations of mixtures of peptides produced by hydrolysis of the hormone with trypsin and chymotrypsin. The sequence determined was H2N-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Leu-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ser-Ile-Gly-Val-Gly-Ala-Pro-NH2. This sequence differs in only two positions from that found in the human hormone, i.e. leucine-16 in the rat vs phenylalanine-16 in the human, and serine-26 in the rat vs alanine-26 in the human. These similarities and differences are consistent with the previously reported immunological properties of the hormones isolated from these two species.
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PMID:The complete amino-acid sequence of rat thyrocalcitonin. 127 75

The surface topography of a 190-residue COOH-terminal colicin E1 channel peptide (NH2-Met 333-Ile 522-COOH) bound to uniformly sized 0.2-micron liposomes was probed by accessibility of the peptide to proteases in order (1) to determine whether the channel structure contains trans-membrane segments in addition to the four alpha-helices previously identified and (2) to discriminate between different topographical possibilities for the surface-bound state. An unfolded surface-bound state is indicated by increased trypsin susceptibility of the bound peptide relative to that of the peptide in aqueous solution. The peptide is bound tightly to the membrane surface with Kd < 10(-7) M. The NH2-terminal 50 residues of the membrane-bound peptide are unbound or loosely bound as indicated by their accessibility to proteases, in contrast with the COOH-terminal 140 residues, which are almost protease inaccessible. The general protease accessibility of the NH2-terminal segment Ala 336-Lys 382 excludes any model for the closed channel state that would include trans-membrane helices on the NH2-terminal side of Lys 382. Lys 381-Lys 382 is a major site for protease cleavage of the surface-bound channel peptide. A site for proteinase K cleavage just upstream of the amphiphilic gating hairpin (K420-K461) implies the presence of a surface-exposed segment in this region. These protease accessibility data indicate that it is unlikely that there are any alpha-helices on the NH2-terminal side of the gating hairpin K420-K461 that are inserted into the membrane in the absence of a membrane potential. A model for the topography of an unfolded monomeric surface-bound intermediate of the colicin channel domain, including a trans-membrane hydrophobic helical hairpin and two or three long surface-bound helices, is proposed.
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PMID:Constraints imposed by protease accessibility on the trans-membrane and surface topography of the colicin E1 ion channel. 128 5

Penetration of a cell membrane as an early event in infection of cells by mammalian reoviruses appears to require a particular type of viral particle, the infectious subvirion particle (ISVP), which is generated from an intact virion by proteolytic cleavage of the outer capsid proteins sigma 3 and mu 1/mu 1C. Characterizations of the structural components and properties of ISVPs are thus relevant to attempts to understand the mechanism of penetration by reoviruses. In this study, a novel, approximately 13-kDa carboxy-terminal fragment (given the name phi) was found to be generated from protein mu 1/mu 1C during in vitro treatments of virions with trypsin or chymotrypsin to yield ISVPs. With trypsin treatment, both the carboxy-terminal fragment phi and the amino-terminal fragment mu 1 delta/delta were shown to be generated and to remain attached to ISVPs in stoichiometric quantities. Sites of protease cleavage were identified in the deduced amino acid sequence of mu 1 by determining the amino-terminal sequences of phi proteins: trypsin cleaves between arginine 584 and isoleucine 585, and chymotrypsin cleaves between tyrosine 581 and glycine 582. Findings in this study indicate that sequences in the phi portion of mu 1/mu 1C may participate in the unique functions attributed to ISVPs. Notably, the delta-phi cleavage junction was predicted to be flanked by a pair of long amphipathic alpha-helices. These amphipathic alpha-helices, together with the myristoyl group at the extreme amino terminus of mu 1/mu 1N, are proposed to interact directly with the lipid bilayer of a cell membrane during penetration by mammalian reoviruses.
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PMID:A carboxy-terminal fragment of protein mu 1/mu 1C is present in infectious subvirion particles of mammalian reoviruses and is proposed to have a role in penetration. 132 74

Sendai virus mutants, KDe-21 and KDe-62, which had undergone multiple cycles of replication in Madin Darby canine kidney (MDCK) cells in the absence of exogenous proteases were isolated. The fusion (F) protein of the mutants regained proteolytic cleavability in MDCK cells and chick embryos, but the F protein remained non-cleavable in other cell lines. Unlike the F protein of wild-type (wt) virus, the mutant F was resistant to trypsin but was sensitive to elastase and, to a lesser extent, to chymotrypsin. Sequence analyses of the F gene and the F protein revealed an amino acid substitution at the cleavage site, Arg(116) to Ile, which conferred trypsin resistance and enhanced cleavability at Ile(116) by elastase and host proteases present in MDCK cells and in chicken embryos. In contrast to the pneumopathogenicity in mice of wt Sendai virus, the KDe mutants were non-pathogenic; cleavage activation of the F protein did not occur in the lungs and thereby infection was terminated after an initial cycle of replication.
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PMID:Changes in specific cleavability of the Sendai virus fusion protein: implications for pathogenicity in mice. 133 65

Gourd seed inhibitors were purified in the following manner: gourd seeds were ground and extracted with 10 mM ammonium carbonate, pH 7.8. The extract was precipitated with 65-90% acetone and the acetone precipitates were gel filtered in a Cellulofine GCL-90-m column. Fractions of 3000 Da showing trypsin inhibitory activity were combined and purified further by ion exchange and reversed phase chromatographies. Three inhibitors, LLTI-I, II, and III were thus purified to homogeneity and the amino acid sequences of these inhibitors were: [sequence: see text] The exact sequences are unique but very similar to proteinase inhibitors belonging to the squash family. Based on the sequence, it is assumed that the peptide bond (Arg-Ile) found in the three inhibitors is the reactive site for trypsin. The Ki values estimated for complexes of LLTI-I, II, and III with bovine trypsin were 3.6 x 10(-10) M, 6.5 x 10(-11) M, and 3.0 x 10(-11) M, respectively.
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PMID:Purification and characterization of serine proteinase inhibitors form gourd (Lagenaria leucantha Rusby var. Gourda Makino) seeds. 136 3

Three inhibitors (CMCTI-I, II, and III) were isolated from oriental pickling melon (Cucumis melo L. var. Conomon Makino) seeds by acetone precipitation, gel filtration, and reversed phase chromatography. The amino acid sequences of these inhibitors were: [table; see text] The reactive sites (P1 and P1' sites) of these inhibitors are presumed to be the Lys-Ile indicated by an arrow, comparing them with other squash family inhibitors. All three inhibitors can inhibit lysyl endopeptidase and trypsin at the enzyme-inhibitor ratio of 1:1.
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PMID:Amino acid sequences of trypsin inhibitors from oriental pickling melon (Cucumis melo L. var. Conomon Makino) seeds. 136 38

Monoclonal antibodies were raised that specifically recognize the NH2-terminal neoepitope sequence present in link protein cleavage products derived from stromelysin-degraded proteoglycan aggregate. Competitive enzyme-linked immunosorbent assay, using synthetic peptides as inhibitors, showed that one of these antibodies (CH-3) required, for antibody recognition, the free NH2-terminal amino acid isoleucine (residue 17 of the intact protein) in the sequence NH2-IQAENG at the stromelysin cleavage site of link protein 3. Human proteoglycan aggregate was digested with recombinant human stromelysin, bovine chymotrypsin, bovine trypsin, and porcine elastase, and their respective link protein degradation products were tested for immunoreactivity with antibody CH-3. Only stromelysin- and chymotrypsin-generated link protein 3 were recognized by antibody CH-3. Both of these enzymes generate link protein NH2 termini with the sequence 17IQAENG. . .; hence these studies indicated that monoclonal antibody CH-3 recognized this neoepitope sequence in only specific proteolytically modified link protein molecules. Since the occurrence of link protein 3 increases with aging, the incidence of CH-3 epitope in proteoglycans isolated from human knee articular cartilage of individuals of different ages was investigated. The prevalence of CH-3 epitope was found to be highest in newborn and adolescent articular cartilage samples. However, little CH-3 epitope was detected in older adult cartilage, although considerably more link protein 3 was present in these samples. These results suggest that additional proteolytic agents are responsible for the increased occurrence of link protein degradation products with aging.
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PMID:Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation. Application to studies of human link protein cleavage by stromelysin. 137 86

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine. 138 24


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