Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have explored the heterogeneity in the proteolytic processing of the N-terminus of human tissue plasminogen activator. We demonstrate that normal propeptide processing occurs following Arg-4, preceding the sequence Gly-Ala-Arg-Ser+1. Generation of the previously designated Ser+1 occurs via secondary proteolysis following secretion. By site-directed mutagenesis, we have eliminated this cleavage site resulting in a derivative containing the propeptide sequence. N-terminal sequence analysis of this form indicated that signal peptide cleavage occurs following Ser-13. The pro-tPA derivative had near normal serine protease and plasminogen activating activities, and could be stimulated by fibrin. An additional derivative, containing the tribasic sequence from the human protein C propeptide preceding Ser+1, was secreted with full processing of the propeptide. Our data have defined the cleavages for the signal peptide and propeptide and demonstrate that a tribasic sequence can be used to eliminate N-terminal heterogeneity in this molecule. In addition, we demonstrate that, unlike several other serine proteases, a propeptide sequence does not alter the activity of this enzyme.
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PMID:Signal and propeptide processing of human tissue plasminogen activator: activity of a pro-tPA derivative. 193 Jan 75

Activated protein C has been observed to bind to the light chains of factor Va and factor VIII. Fragments of the factor VIII light chain were produced by recombinant DNA techniques and expressed in Escherichia coli. Three fragments of the light chain were studied; L4 (residues 1974-2332), L3.2 (residues 1560-1829 and 2046-2332), and L3.3 (residues 1560-2052). Two fragments, L4 and L3.3, which overlapped sequences between residues 1974-2052, inhibited the anticoagulant activity of activated protein C. Comparison of the sequences of factors V and VIII in this region revealed that residues 2005-2018 in the factor VIII sequence were homologous with residues 1861-1874 in the factor V sequence. The peptides Arg-Ala-Gly-Met-Gln-Thr-Phe-Leu-Ile (RAGMQTPFLI; residues 1865-1874) from the factor V sequence and His-Ala-Gly-Met-Ser-Thr-Leu-Phe-Ile-Val (HAGMSTLFIV; residues 2009-2018) from the factor VIII sequence were synthesized. Both peptides were observed to inhibit the anticoagulant activity of activated protein C and its inactivation of factors Va and VIII. Furthermore RAGMQTPFLI quenched the fluorescence of the dansyl-Glu-Gly-Arg-modified protease. Polyclonal antibodies against RAGMQTPFLI bound to factor Va and inhibited the anticoagulant activity of activated protein C and the inactivation of factor Va. These results indicate that a portion of the binding sites for activated protein C on the light chains of factors V and VIII are contained in the sequences RAGMQTPFLI or HAGMSTLFIV, respectively.
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PMID:Identification of the binding site for activated protein C on the light chain of factors V and VIII. 213 54

Thrombomodulin is an endothelial cell membrane protein which plays a central regulatory role in the protein C anticoagulant pathway. The human thrombomodulin intronless gene was isolated from a genomic DNA library and used to isolate the coding region. A mammalian expression vector, phd-TMD1, encoding all the extracellular domains of human thrombomodulin but lacking the transmembrane and cytoplasmic domains was constructed. Stable phd-TMD 1 transformants, in both hamster AV12-644 and human 293 cells, expressed functionally active recombinant thrombomodulin as a secreted, soluble product. Soluble thrombomodulin was secreted as two major proteins of 105 kDa and 75 kDa, both of which were purified to homogeneity. The kinetic properties for protein C activation of the two proteins were very different: the Kd for thrombin, Km for protein C, and Ca2+ optima were 3.0 nM, 1.5 microM, and 1-3 mM for the 105-kDa protein and 16 nM, 2.3 microM, and 0.2-0.5 mM for the 75-kDa protein. In clotting and platelet activation assays, the 105-kDa protein was a much more potent anticoagulant than the 75-kDa protein. Both forms of the protein had the amino-terminal sequence Ala19-Pro-Ala-Glu-Pro-Gln. Amino acid composition analysis indicated that both forms of the protein had the same amino acid content which was consistent with the predicted protein comprising residues Ala19 to Ser515. The difference in size appeared to be due to glycosylation as both forms were of similar size following chemical deglycosylation. These studies suggest that (1) secretable thrombomodulin derivatives can be used to study structure-function relationships of the extracellular domains of this important regulatory protein, (2) the extent of glycosylation has profound effects on the kinetic and anticoagulant properties of human thrombomodulin, and (3) soluble recombinant human thrombomodulins may be developed as clinically significant therapeutic anticoagulants.
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PMID:Stable expression of a secretable deletion mutant of recombinant human thrombomodulin in mammalian cells. 216 69

A binding site for thrombomodulin on human thrombin (alpha-thrombin) was elucidated by identifying an epitope for a monoclonal antibody for thrombin (MT-6) which inhibited the activation of protein C by the thrombin-thrombomodulin complex by directly inhibiting the binding of thrombin to thrombomodulin. An 8.5-kDa fragment isolated by digestion of thrombin with Staphylococcus aureus V8 protease followed by reversed-phase high performance liquid chromatography (HPLC) and a peptide isolated by reversed-phase HPLC after reduction of the 8.5-kDa fragment, which was composed of three peptides linked by disulfide-bonds, bound directly to MT-6 and thrombomodulin. The amino acid sequence of the peptide coincided with the sequence of residues Thr-147 to Asp-175 of the B-chain of thrombin. A synthetic peptide corresponding to Thr-147 to Ser-158 of the B-chain inhibited the binding of thrombin to thrombomodulin. Elastase-digested thrombin, which was cleaved between Ala-150 and Asn-151, lost its binding affinity for both MT-6 and thrombomodulin. These findings indicate that the binding site for thrombomodulin is located within the sequence between Thr-147 and Ser-158 of the B-chain.
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PMID:Localization of thrombomodulin-binding site within human thrombin. 216 98

Apamin is a single-chain, disulfide-bonded, 18-amino acid peptide that elicits mouse T cell responses when presented by cells expressing syngeneic Ad or Ab class II MHC molecules. We previously showed that both the unfolding of this peptide by APC and the integrity of its N terminus segment were required for efficient apamin T cell recognition. To seek further information on the sites through which this peptide interacts with Ia and/or TCR, we used a panel of Ad- or Ab-restricted, apamin-specific THC to probe the antigenicity of a series of synthetic apamin analogs. These included peptides either truncated at the N terminus, or substituted by Ala at position 2, 4, 6, 7, 8, or 10. Analysis of THC responses to apamin analogs and use of the latter in competition assays for peptide presentation revealed the following: 1) optimal apamin T cell recognition critically involved Lys4, Ala5, Pro6, Glu7, and Leu10. The role of these residues in either "Ia or TCR binding regions" was found to depend upon the restricting Ia molecules at play. Thus, Lys4, Glu7, and Leu10 were TCR-binding residues in both Ad- and Ab-apamin complexes, whereas Lys4 participated in apamin/Ab but not, or to a marginal extent, in apamin/Ad interaction. Furthermore, Pro6 was associated either with an Ia contact region or a TCR interaction site when apamin was presented by Ab or Ad molecules, respectively. Unfolded apamin and the unrelated chicken OVA323-339 peptide were found to bind to the same, or closely related site(s) of Ad, as shown by their ability to compete reciprocally for recognition by appropriate Ad-restricted THC. Four distinct TCR V beta genes (V beta 2, V beta 4, V beta 6, and V beta 8) were found to be used in our panel of 16 apamin-specific THC. These data indicate that apamin interacts with Ad or TCR through a motif resembling other beta-sheeted, Ad-binding sequences; however, based on the spacing of the critical residues (i.e., 4, 7, and 10), the possibility exists that apamin processing permits the folding of this sequence into an alpha-helix.
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PMID:Structural analysis of the interaction of apamin with Ia and its recognition by Ad- or Ab-restricted mouse T cells. 247 20

The vitellaria are an extensive network of glandular cells and ducts distributed throughout the peripheral tissues of the liver fluke Fasciola hepatica. Eggshell precursor proteins are produced and stockpiled in the vitelline cells of mature flukes. Vitelline protein C has an extraordinary composition: the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) and histidine each comprise about 20% of the residues, while glycine represents 41-42% in all variants of what appears to be a microheterogeneous protein family. Protein C has an apparent molecular weight of 16,000-17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the protein appears homogeneous following polyacrylamide gel electrophoresis in Tris-glycine with SDS and a acetic acid-urea, electrophoresis in borate, however, suggests that the vitelline protein consists of four or more closely related proteins weighing from 16,000 to 18,500. Isoelectric focusing of the protein family in the presence of 8 M urea resolved only two species having pI values of 6.89 and 6.99. A single N-terminus having the sequence H-H-W-D-G-DOPA-G-DOPA-G was detected. The primary structure of vitelline protein C is characterized by a repeated motif consisting of (G-X)n, where X is Ser, DOPA, or His. Most of the His occurs as G-H repeats in a pepsin-resistant fragment of the protein. Previously, a 31-kDa protein, representing up to 6% of the total protein in the fluke, was reported [Waite, J. H., & Rice-Ficht, A (1987) Biochemistry 26, 7819-7825] to contain significant levels of DOPA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A histidine-rich protein from the vitellaria of the liver fluke Fasciola hepatica. 277 56

The three major capsid proteins of adeno-associated virus type 2 (AAV2) virions are designated A, B, and C and have molecular sizes of 90, 72, and 60 kDa, respectively. These proteins are related, and genetic studies have shown they are encoded by a long open reading frame located in the right half of the genome. The coding capacity distal to the first ATG in this reading frame is only 503 amino acids (i.e., a protein about the size of protein C), but an open frame sequence devoid of ATG codons extends upstream for an additional 184 codons. Although the amino terminus of the C capsid protein is blocked, partial amino acid sequence analyses of peptides from C have confirmed that it is encoded within the portion of the reading frame distal to the first ATG at nucleotide (nt) location 2810. The amino terminus of the B capsid protein is not blocked, and its sequence begins with alanine. The triplet encoding this alanine lies 64 codons upstream from the initiation site for protein C and is immediately preceded by the threonine codon, ACG, at nt 2615. This ACG codon lies in the most favorable sequence context for protein synthesis initiation. All three AAV2 capsid proteins are labeled in vitro with formyl[35S]methionyl-tRNAf, indicating that synthesis of each protein is initiated independently. Our data suggest that the nt 2615 ACG codon directs the methionyl-tRNA-dependent initiation of the AAV2 B capsid protein. Proteins B and C may be synthesized from the same mRNA species and their relative abundance could be determined by the efficiencies of their respective initiation codons.
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PMID:Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon. 299 84

Seventy-four peptide amides of 7-amino-4-methylcoumarin (Mec) of the type Boc-Xaa-Yaa-Arg-NH-Mec were newly synthesized and tested to find specific substrates for blood-clotting proteases and trypsin. The Xaa and Yaa residues of these substrates have been replaced by 12 and 15 different amino acids, respectively. Among these peptides, the followings were found to be most sensitive substrates for individual enzymes: Boc-Asp(OBzl)-Pro-Arg-NH-Mec (kcat = 160 s-1, Km = 11 microM, kcat/Km = 15,000,000 M-1 s-1) for human alpha-thrombin, Z-less than Glu-Gly-Arg-NH-Mec (kcat = 19 s-1, Km = 59 microM, kcat/Km = 320,000 M-1 s-1) for bovine factor Xa, Boc-Gln-Gly-Arg-NH-Mec (kcat = 5.8 s-1, Km = 140 microM, kcat/Km = 42,000) for bovine factor XIIa, Boc-Asp(OBzl)-Ala-Arg-NH-Mec (kcat = 9.2 s-1, Km = 120 microM, kcat/Km = 77,000 M-1 s-1) for bovine activated protein C, and Boc-Gly-Phe-Arg-NH-Mec (kcat = 29 s-1, Km = 230 microM, kcat/Km = 130,000 M-1 s-1) for bovine plasma kallikrein. Moreover, Boc-Glu(OBzl)-Ala-Arg-NH-Mec (kcat = 46 s-1, Km = 370 microM, kcat/Km = 120,000 M-1 s-1) was newly found as a good substrate for human factor XIa. Bovine trypsin effectively hydrolyzed peptide-NH-Mec substrates containing Ala and Pro at the P2 site. The most reactive substrate was Boc-Gln-Ala-Arg-NH-Mec (kcat = 120 s-1, Km = 6.0 microM, kcat/Km = 20,000,000 M-1 s-1).
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PMID:Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin. 327 5

A series of 14 tripeptide 4-nitroanilide substrates of the type Z-AA-Gly-Arg-NA and Z-AA-Phe-Arg-NA where AA = Ala, Asn, Glu, Lys, Phe, Pro, or Ser were used to map the S3 subsite of several serine proteases involved in blood coagulation. The enzymes studied included bovine thrombin, factor IXa, factor Xa, factor XIa, human beta-factor XIIa (factor XIIa fragment), and activated bovine and human protein C. Kinetic constants (kcat, KM, and kcat/KM) for the enzymatic hydrolysis of the substrates by each enzyme were determined and used to compare the relative reactivities of the individual enzymes. Most of the enzymes reacted with all the substrates, although a few showed considerable specificity. Human beta-factor XIIa showed the highest reactivity of all the coagulation proteases studied and was also very substrate specific (kcat/KM ranged over 470-fold). The best substrate was Z-Lys-Phe-Arg-NA with kcat/KM = 140 000 M-1 s-1. Activated bovine protein C (best substrate = Z-Ser-Phe-Arg-NA), factor Xa (best substrate = Z-Glu-Gly-Arg-NA), and thrombin (best substrate = Z-Lys-Gly-Arg-NA) were the group of enzymes that showed next highest reactivity toward the substrates. Activated bovine protein C, thrombin, and factor Xa displayed relatively little substrate specificity. Activated human protein C (best substrate = Z-Ser-Phe-Arg-NA) and factor XIa (best substrate = Z-Glu-Gly-Arg-NA) are moderately reactive enzymes. Activated human protein C is an extremely specific enzyme since it has such a large range of kcat/KM values.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates. 637 Mar 1

More than 100 chromogenic and fluorogenic peptide substrates are now available for the evaluation of coagulation and related parameters. Many of these substrates exhibit undesirable physical properties, such as insolubility, surface adsorption, and interaction with endogenous plasma proteins. Some of these substrates are capable of inhibiting serine protease generation during activation in the global assay. In order to develop synthetic chromogenic substrates with desirable physical and biochemical characteristics, modified amino acids, such as CHG, CHT, and Nleu, have been utilized. Similarly, to provide a favorable molecular environment to facilitate enzyme and synthetic substrate interactions, various molecular manipulations, such as the introduction of bulky groups, is helpful in developing substrates for protein Ca and C1-esterase. Substrates for Factor Xa, CH3-O-CO-CHG-Arg-pNA (bovine Xa, Km 2.5 X 10(-4) M; human, Km 3.5 X 10(-4) M); thrombin, H-D-CHT-Ala-Arg-pNA (bovine thrombin, Km 3 X 10(-6) M; human thrombin, Km 6 X 10(-6) M); plasmin, H-D-Nleu-CHT-Lys-pNA (human plasmin, Km 2.2 X 10(-5) M) were found to have identical or superior biochemical characteristics to the earlier substrates. These newer substrates were found to be more soluble (greater than 5 X 10(-4) M) in physiologic buffer, less susceptible to autoamidolysis at reaction conditions, and did not produce opacity of the test solution in final concentrations of 5 X 10(-4) M. Comparable results on normal and pathologic plasma samples were obtained in various laboratory assays that utilize currently available substrates for Factors Xa and IIa, kallikrein, and plasmin (R = greater than 0.9). We propose that prior to the application of a new synthetic substrate in a given assay, a careful biochemical and physical screening of the substrate, the assay conditions, and the interaction of substrates with plasma proteins is highly desirable.
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PMID:Newer synthetic peptide substrates in coagulation testing: some practical considerations for automated methods. 665 59


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