A new sensitive and highly specific chromogenic peptide substrate for Factor Xa

Aurell, L.; Friberger, P.; Karlsson, Göran; Claeson, Göran

doi:10.1016/0049-3848(77)90018-4

Cited by 141 publications

(49 citation statements)

References 13 publications

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“…2a). The P2-Gly was most effective as previously reported [7,24,26,35,361, resulting in the increase of the k,,, value. In addition, the Gly and Ala residues contributed to increase the l/Km value.…”

Section: Bovine Factor Xasupporting

confidence: 63%

Highly sensitive peptide‐4‐methylcoumaryl‐7‐amide substrates for blood‐clotting proteases and trypsin

Kawabata

Miura

Morita

et al. 1988

European Journal of Biochemistry

240

137

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Seventy-four peptide amides of 7-amino-4-methylcoumarine (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 The Xaa and Yaa residues of these substrates have been replaced by 12 and 15 different amino acids, respectively.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(OBz1)-Pro-Arg-NH-Mec (k,,, = 160 s-', K, = 11 pM, k,,,/K, = 15000000 M-' s-') for human a-thrombin, Z-< Glu-Gly-Arg-NH-Mec (k,,, = 19 s-', K, = 59 pM, k,,,/K, = 320000 M-' s-') for bovine factor Xa, BocGln-Gly-Arg-NH-Mec (k,,, = 5.8 s-', K, = 140 pM, k,,,/K, = 42000) for bovine factor XIIa, Boc-Asp(OBz1)-Ala-Arg-NH-Mec (k,,, = 9.2 s-', K,,, = 120 pM, k,,,/K, = 77000 M-' s -l ) for bovine activated protein C, and Boc-Gly-Phe-Arg-NH-Mec (k,,, = 29 s-', K, = 230 pM, kcat/Km = 130000 M-' s-') for bovine plasma kallikrein. Moreover, Boc-Glu(OBz1)-Ala-Arg-NH-Mec (k,,, = 46 s-', K, = 370 pM, kcat/Km = 120000 M-' s-'> 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 (k,,, = 120 sK1, K, = 6.0 pM, k,,,/K, = 20000000 M-' s-').Peptide amides of 7-amino-4-methylcoumarine (Mec) originally developed for the sensitive assay for a-chymotrypsin [l] [7], and horseshoe crab clotting factors [12]. Most of these substrates were designed based on the information from the COOH-terminal sequences of the 'activation peptides', which are liberated during the conversions of plasma serine protease zymogens to their active enzymes. The utility of these fluorogenic peptide substrates has been established for the assay of trace amounts of enzymes because of their high sensitivities. Moreover, the specific substrates for a-thrombin, factor Xa, plasma kallikrein and urokinase so far developed by our group [7] are now commercially available.

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Section: Bovine Factor Xasupporting

confidence: 63%

Highly sensitive peptide‐4‐methylcoumaryl‐7‐amide substrates for blood‐clotting proteases and trypsin

Kawabata

Miura

Morita

et al. 1988

European Journal of Biochemistry

240

137

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“…This construct allows for the rapid purification of the expressed protein by Ni 2ϩ affinity chromatography. The intact RII␣ domain is released from the N-terminal His-tag by factor Xa cleavage (21,22 15 N/ 13 C-enriched RII ␣ -(1-44)) containing 150 mg/ml ampicillin is inoculated with a 5-ml starting culture of E. coli. The 1-liter culture is grown at 37°C to an A 600 of 0.5 prior to induction with 1 mM isopropyl ␤-D-thiogalactoside.…”

Section: Methodsmentioning

confidence: 99%

The A-kinase Anchoring Domain of Type IIα cAMP-dependent Protein Kinase Is Highly Helical

Newlon¹,

Roy²,

Hausken³

et al. 1997

Journal of Biological Chemistry

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Subcellular localization of the type II cAMP-dependent protein kinase is controlled by interaction of the regulatory subunit with A-Kinase Anchoring Proteins (AKAPs). This contribution examines the solution structure of a 44-residue region that is sufficient for high affinity binding to AKAPs. The N-terminal dimerization domain of the type II␣ regulatory subunit of cAMP-dependent protein kinase was expressed to high levels on minimal media and uniformly isotopically enriched with 15 N and 13 C nuclei. Sequence-specific backbone and side chain resonance assignments have been made for greater than 95% of the amino acids in the free dimerization domain using high resolution multidimensional heteronuclear NMR techniques. Contrary to the results from secondary structure prediction algorithms, our analysis indicates that the domain is highly helical with a single 3-5-residue sequence involved in a ␤-strand. The assignments and secondary structure analysis provide the basis for analyzing the structure and dynamics of the dimerization domain both free and complexed with specific anchoring proteins.The protein kinases constitute a diverse family of enzymes with well over one thousand members discovered to date. They differ in substrate specificity (serine/threonine-versus tyrosine-specific) as well as in their role in specific signal transduction pathways. Despite this considerable diversity, genetic and structural analyses of the catalytic portions of these enzymes have shown that they evolved from a common ancestor and share a conserved overall fold (1-3). This catalytic core is often flanked by well defined structural domains that confer functional properties upon the enzyme. For example, SH2, SH3, pleckstrin, C1, C2, and Lim domains have been implicated in mediating specific protein-protein or protein-lipid interactions that control specificity and/or alter activity in particular protein kinases (4 -6). With this added structural complexity, in many cases it is still unclear how signals from outside the cell reach a specific target in the dense reaches of the cytoplasm and nucleus in a timely fashion. An attractive proposal has recently been raised to address this particular problem. The "targeting hypothesis" proposes that phosphorylation events are controlled, in part, by subcellular localization of protein kinases and phosphatases in the cell via the interaction of targeting domains on the enzymes with specific anchoring proteins (7-10). The principle behind localization is that the protein kinase will be held in close proximity to its target and released when the activation signal is discharged. A pressing issue, then, is to define the molecular basis for these targeting interactions.The correct intracellular targeting of protein kinases and phosphatases confers specificity to the enzymes, in part, by placing them in close proximity to their preferred substrates. Targeting of these enzymes occurs via association with specific proteins that are found in different locations in the cell (7, 8, 10 -12). The classic ...

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“…Factor X was activated by incubating 50 Mug with 100 Ml RW-X-Sepharose in the presence of 10 mM CaCl2 at 370C for 3-5 min. Activation was at least 95% complete, as assessed by both the hydrolysis of S-2222 (34) and by polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol. a2M, ATIII, and a1PI were purified to homogeneity from human plasma as previously described (25)(26)(27).…”

Section: Introductionmentioning

confidence: 99%

Regulation of factor Xa in vitro in human and mouse plasma and in vivo in mouse. Role of the endothelium and plasma proteinase inhibitors.

Fuchs¹,

Pizzo²

1983

J. Clin. Invest.

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A B S T R A C T The regulation of human Factor Xa was studied in vitro in human and mouse plasma, and in vivo in mouse. In human plasma, '251-Factor Xa bound to a1-proteinase inhibitor, antithrombin III, and a2-macroglobulin in a ratio of 4.9:1.9:1 as determined by gel electrophoresis and by adsorption to IgG-(antiproteinase inhibitor)-Sepharose beads. The distribution of Factor Xa in mouse plasma was similar. The clearance of Factor Xa in mice was rapid (50% clearance in 3 min) and biphasic. a1-Proteinase inhibitor-trypsin, even at a 2,000-fold molar excess, failed to inhibit the clearance of Factor Xa, while a2-macroglobulin-trypsin inhibited only the later phase of clearance. The plasma clearance of diisopropylphosphoryl-Factor Xa was more rapid than native Factor X. (50% clearance in 2.5 min), and the clearance was blocked by diisopropylphosphoryl-thrombin. Electrophoresis experiments confirmed that by 2 min after injection into the murine circulation, 90% of the bound Factor Xa was on a2-macroglobulin, in marked contrast to the in vitro results. Organ distribution studies at 3 and 15 min with 125I_ Factor X. demonstrated that the majority of radioactivity was in the liver, with significant radioactivity also present in lung and kidney. Autopsies performed 30 s after injection of 125I-Factor Xa also demonstrated significant binding to the aorta and vena cava. These studies indicate that Factor Xa binds to specific thrombin-binding sites on endothelial cells, and that this binding alters its proteinase inhibitor specificity. Factor Xa binds to a2-macroglobulin in vivo, whereas the predominant in vitro inhibitor of Factor X. is a,-proteinase inhibitor.

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A new sensitive and highly specific chromogenic peptide substrate for Factor Xa

Cited by 141 publications

References 13 publications

Highly sensitive peptide‐4‐methylcoumaryl‐7‐amide substrates for blood‐clotting proteases and trypsin

Highly sensitive peptide‐4‐methylcoumaryl‐7‐amide substrates for blood‐clotting proteases and trypsin

The A-kinase Anchoring Domain of Type IIα cAMP-dependent Protein Kinase Is Highly Helical

Regulation of factor Xa in vitro in human and mouse plasma and in vivo in mouse. Role of the endothelium and plasma proteinase inhibitors.

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