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M C Owen

Publications and source records attributed to M C Owen.

18 recordsLinked to original sources
PubMed1992

A competitive ELISA for lipoprotein(a).

A competitive ELISA for lipoprotein(a) (Lp(a)) is described. The method uses a commercially available polyclonal anti-Lp(a) antibody and an IgG biotinstreptavidin-horseradish peroxidase detection system. The method is simple and robust with an assay sensitivity of 0.7 ng/well (1.4 micrograms/l). The antibody cross-reactivity was 0.14% against LDL and 0.70% against plasminogen. The coefficients of variation obtained with control sera of 266 and 552 mg/l were: 5.0% and 4.6% (n = 6), respectively for the intraassay; and 10.8% and 9.5% (n = 16), respectively for the interassay. The method showed an excellent correlation with a commercial immunoradiometric assay (IRMA), y (ELISA) = 0.94x (IRMA) - 8, (r = 0.98). A recovery study in which a 200 mg/L standard and four plasma samples were diluted with different proportions of a low plasma sample, gave linear relationships and also confirmed the specificity of the antibody.

Binding, Competitive↗
PubMed1991

P1 variant antithrombins Glasgow (393 Arg to His) and Pescara (393 Arg to Pro) have increased heparin affinity and are resistant to catalytic cleavage by elastase. Implications for the heparin activation mechanism.

The heparin affinity of normal and two P1 variants of antithrombin-III (AT) was studied by gradient elution with NaCl in Tris buffer on heparin-Sepharose. At pH 7.4 normal AT eluted at [Na+] 0.78 mol/l and the variants both showed increased affinity with AT Pescara eluting at [Na+] 0.86 mol/l and AT Glasgow at [Na+] 0.92 mol/l. We have earlier proposed a model for heparin activation in which the native state of AT maintains a salt bridge involving the P1 Arg-393 residue. Binding of heparin induces a higher heparin affinity conformation in which the salt bridge is disrupted to reveal the reactive centre for inhibition of thrombin. The Glasgow and Pescara variants, lacking a reactive centre P1 basic residue, would be unable to form this salt bridge, and we suggested that the high affinity conformation which they adopt as their native state would resemble the heparin induced conformation. To examine this model, we measured the heparin induced fluorescence of two P1 variants and tested the susceptibility of their reactive loops to catalytic cleavage. Both variants had fluorescence spectra indistinguishable from normal AT. In the absence of heparin, neither variant was more susceptible than normal to catalytic cleavage by human neutrophil elastase. These findings suggest that the conformation of these P1 variants is different to that of fully heparinized normal AT.

Antithrombin III↗
PubMed1990

Antithrombin III.

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Amino Acid Sequence↗
PubMed1989

Conformational lability of vitronectin: induction of an antigenic change by alpha-thrombin-serpin complexes and by proteolytically modified thrombin.

We previously showed that the alpha-thrombin-antithrombin III complex causes antigenic change in vitronectin as monitored by the monoclonal anti-vitronectin antibody 8E6 (Tomasini & Mosher, 1988). We have extended these studies to other protease-serpin complexes and to gamma-thrombin, a proteolytic derivative of alpha-thrombin. In the presence of heparin, recognition of vitronectin by 8E6 was increased 64- or 52-fold by interaction with the complex of alpha-thrombin and heparin cofactor II or the Pittsburgh mutant (Met358----Arg) of alpha 1-protease inhibitor, respectively. This was comparable to the value obtained with the alpha-thrombin-antithrombin III complex. Factor Xa-serpin complexes were approximately 4-fold less effective than the corresponding thrombin complexes. alpha-Thrombin-serpin complexes but not Xa-serpin complexes formed disulfide-bonded complexes with vitronectin. Antigenic changes and disulfide-bonded complexes were not detected when trypsin- or chymotrypsin-serpin complexes were incubated with vitronectin. gamma-Thrombin caused 7- and 34-fold increases in recognition of vitronectin by MaVN 8E6 in the absence and presence of heparin, respectively. In contrast, alpha-thrombin by itself had no effect. The antigenic change induced by gamma-thrombin was maximal when gamma-thrombin and vitronectin were equimolar, was not dependent on cleavage of vitronectin, and was abolished by inhibition of gamma-thrombin with Phe-Pro-Arg-chloromethyl ketone but not with diisopropyl fluorophosphate. These data indicate that alpha-thrombin is the component in alpha-thrombin-serpin complexes that induces the antigenic change in vitronectin, probably via a region that is preferentially exposed in gamma-thrombin.

Animals↗
PubMed1989

Molecular characterization of antithrombin Barcelona-2: 47 arginine to cysteine.

The molecular characterization of antithrombin Barcelona-2 is reported. The abnormal antithrombin was isolated from plasma by chromatography on heparin-Sepharose at pH 6.0, and ion exchange on DEAE-Sephadex at pH 8.6 and 6.0. The tryptic peptides were mapped by reverse-phase HPLC and amino acid sequencing and mass spectrometry showed arginine-47 to be replaced by cysteine. The affinity of Barcelona-2 for heparin is dramatically decreased. The new cysteine does not form a mixed disulphide with DTNB, implying it is present as a disulphide with some other available thiol molecule such as cysteine. This extra bulk at position 47 accounts for the low heparin affinity compared with two other mutations (Rouen-1 47 His; Rouen-2 47 Ser) at this residue. These results confirm the view that Arg-47 is an important residue in heparin binding. No dimers of Barcelona-2 were observed suggesting that steric hindrance of the new cysteine at residue 47 prevents dimerisation.

Amino Acid Sequence↗
PubMed1988

Antithrombin Glasgow, 393 Arg to His: a P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity.

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41-year-old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin-Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCl. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C-terminal small fragment resulting from catalytic cleavage with elastase between P3 and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.

Adult↗
PubMed1988

Proposed heparin binding site in antithrombin based on arginine 47. A new variant Rouen-II, 47 Arg to Ser.

Antithrombin Rouen-II, a new inherited variant of antithrombin-III, was found in two members of a family with no definite history of thrombosis. The subjects had normal antigenic concentrations of antithrombin and normal progressive inhibitory activity. However, the variant had defective heparin and heparan sulfate cofactor activities, and was not activated by a synthetic pentasaccharide representing the minimum heparin sequence. The abnormal antithrombin was isolated using heparin-Sepharose chromatography, and on electrophoresis at pH 8.6 migrated more anodally than normal. Two-dimensional peptide mapping of tryptic and Staphylococcus aureus V8 protease digests was performed and the abnormal peptide was located by tryptophan staining. Amino acid sequence studies demonstrated a substitution of arginine at residue 47 by a serine. Evidence strongly suggests that arginine 47 is a prime heparin binding site in antithrombin and that it forms part of a proposed positively charged linear site (to which heparin binds) that stretches across the surface of the molecule from the A to the D helix.

Adult↗
PubMed1987

Heparin binding defect in a new antithrombin III variant: Rouen, 47 Arg to His.

Antithrombin III (AT-III) Rouen is a hereditary abnormal antithrombin with normal progressive inhibitory activity and reduced heparin cofactor activity. It was isolated from the plasma of a woman who suffered a sudden idiopathic sensorineural hearing loss and balance impairment. There was no familial history of thrombosis. By heparin-Sepharose chromatography, AT-III Rouen was separated from the normal antithrombin on elution with increasing concentrations of NaCl. AT-III Rouen eluted earlier than is normal at both pH 7.4 and pH 6.0. At the lower pH, the antithrombins bound more avidly to the column, with the abnormal AT-III eluting closer to the normal than at the higher pH. Two-dimensional peptide mapping of tryptic and Staphylococcus aureus V8 protease digests of carboxymethylated antithrombins was performed on thin-layer silica plates. The abnormal peptide was located by tryptophan staining, and amino acid analysis and sequence studies demonstrated a substitution of an arginine at residue 47 for a histidine. Results from this study suggest that replacement of arginine 47 by a partially positively charged histidine has less effect on the heparin binding affinity than dose replacing it with a neutral cysteine side chain as in AT-III Toyama, in which no heparin binding was observed. In addition, heparin binding per se is not a sufficient condition to activate AT-III.

Adult↗
PubMed1975

The isolation and identification of haemoglobin Lephore Boston (Washington) in an Australian family.

Haemoglobin Lepore is a haemoglobin variant associated with a thalassaemia-like disorder. It has been only rarely detected in Anglo-Saxons and its occurrrence in an Australian family of British and stock is reported for the first time. The appearances of the blood film resemble those seen in various hypochromic anaemias, inclusing thalassaemia traits, and on this account it is of clinical importance to recognize it in order to avoid unnecessary investigation and treatment. The chemical structure of nine examples of haemoglobin Lepore has been confirmed by peptide mapping and amino acid analysis, and the genetic mechanisms postulated for the production of haemoglobin Lepore are discussed.

Amino Acids↗
PubMed1975

A sensitive technique by differential scanning for detecting haemoglobins of fetal origin.

A simple modification is made to the ultraviolet scanning technique for the detection of the tryptophan notch which is characteristic of haemoglobins of fetal origin. This involves a differential scan using Hb A solution in the reference compartment. This technique has all the advantages of the standard scan method but extends the scope of it by being much more sensitive and decisive.

Buffers↗
PubMedDate not supplied

Plakalbumin, alpha 1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis.

An old puzzle in protein biochemistry concerns the ready conversion of ovalbumin, by proteolysis, to the much more stable derivative, plakalbumin. Ovalbumin is now known to belong to the serpin superfamily, most of which are serine proteinase inhibitors. We report here studies of two such members of the family, the human plasma proteins alpha 1-antitrypsin and antithrombin, and show that they undergo a similar change in stability on selective proteolysis. This change, which is accompanied by a loss of inhibitory activity, can best be considered as an irreversible molecular transition from a native stressed (S) conformation, to a more ordered relaxed (R) form. The maintenance of the native S conformation, and hence the maintenance of inhibitory activity, is critically dependent on the integrity of an exposed loop of polypeptide. We propose that the susceptibility of this peptide loop to proteolytic cleavage gives it an incidental role as a physiological switch which allows the inactivation of individual inhibitors by specific proteolysis. The vulnerability of this exposed loop in each inhibitor also explains the pathological action of a number of venoms and toxins. In particular, the demonstration here of the cleavage of antithrombin, by leukocyte elastase, explains an observed change in blood coagulation that accompanies severe inflammation and which can result in fatal thrombosis.

Animals↗