PubMed Health⌕ Search

Biomedical subjects

R L Lundblad

Publications and source records attributed to R L Lundblad.

At least 55 records · Page 3Linked to original sources
PubMed1985

Evidence for a prevalent dimorphism in the activation peptide of human coagulation factor IX.

We have independently isolated and characterized cDNA and genomic clones for the human coagulation factor IX. Sequence analysis in both cases indicates that threonine is encoded by the triplet ACT as the third residue of the activation peptide. This is in agreement with some earlier reports but in disagreement with others that show the alanine triplet GCT at this position. The discrepancy can thus be accounted for by natural variation of a single nucleotide in the normal population. Amino acid sequence analyses of activated factor IX from plasma samples of four individuals yielded two cases of alanine and two cases of threonine at the third position of the activation peptide. In factor IX from pooled plasma and in factor IX from a heterozygous individual, however, both alanine and threonine were found. Taken together, the findings show that a prevalent nondeleterious dimorphism exists in the activation peptide of human coagulation factor IX.

Alleles↗
PubMed1985

Characterization of the clotting activities of structurally different forms of activated factor IX. Enzymatic properties of normal human factor IXa alpha, factor IXa beta, and activated factor IX Chapel Hill.

Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha.

Blood Coagulation↗
PubMed1985

Structure and function of factor IX: defects in haemophilia B.

The genetics of haemophilia B and the structure-function relationships of factor IX interactions with cofactors and substrates have been reviewed. Emphasis has been placed on contributions to our understanding made by analysis of variants. Amino acid substitutions at or near the site of activation lead to inactive factor IX or to factor IX species with decreased clotting activity. Release of the activation peptide is necessary for optimal interaction of factor IX with its cofactors and substrates. Abnormalities in the calcium binding region, whether Gla independent or dependent, also decrease clotting activity. The defects in haemophilia Bm variants somehow affect factor VII-tissue factor interactions with factor X. Other mutations may affect the factor IX heavy chain, probably at or near the active site. Amino acid substitutions may cause conformational changes in factor IX that interfere with other interactions such as with antithrombin III and factor VIII. Recombinant DNA techniques have been employed to analyse normal and abnormal factor IX genes. DNA sequence analysis of factor IX cDNA clones revealed the primary structure of the mature protein and a predicted leader peptide. Knowledge of the primary sequence of factor IX allowed identification of the specific defect in the factor IX Chapel Hill variant. Analysis of normal factor IX genomic clones has determined that the 35 kb gene is composed of eight coding exons and seven intervening sequences. Sequence analysis of the CRM+ variants will identify mutations disrupting the normal interactions of factor IX. Southern analysis of CRM- variants has revealed gross factor IX gene deletions in some cases. Such deletions have been employed for carrier deletion in some families. Restriction fragment length polymorphisms in the factor IX gene have also proven useful for carrier identification. Manipulations of the cloned factor IX gene to make specific mutations in vitro and improvements in the technology for expression of deliberately modified genes will further elucidate the relationships between factor IX structure and function.

Amino Acid Sequence↗
PubMed1984

Interaction of substrates with glutamine synthetase after limited proteolysis.

Previous studies [Dautry-Varsat, A., Cohen, G. N., & Stadtman, E.R. (1979) J. Biol. Chem. 254, 3124-3128; Lei, M., Aebi, U., Heidner, E. G., & Eisenberg, D. (1979) J. Biol. Chem. 254, 3129-3134] have shown that Escherichia coli glutamine synthetase (GS) can be cleaved by proteases to form a limited digestion species called nicked glutamine synthetase (GS). The present study gives the amino acid sequence of the protease-sensitive region of glutamine synthetase. The present study also shows that GS is enzymatically active, but this activity is low compared to the activity of GS. The apparent Michaelis constant value for glutamate was 90 mM for GS as compared to 3 mM for GS, while the Michaelis constant values for ATP were similar for GS and GS*. The dissociation constant values for ATP, as determined by intrinsic fluorescence measurements, were similar for GS and GS*. Glutamate decreased the dissociation constant value of ATP for GS because of synergism between the two binding sites; glutamate did not decrease the dissociation constant value of ATP for GS*. The glutamate analogue methionine sulfoximine bound very tightly to GS and inactivated the enzyme in the presence of ATP. Methionine sulfoximine did not appear to bind to GS* and did not inactivate GS* in the presence of ATP. The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine bound to GS and inactivated the enzyme by forming a covalent bond with it. Glutamate accelerated this inactivation because of the synergism between the ATP and glutamate binding sites of GS.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1984

Bovine alpha- and beta-thrombin. Reduced fibrinogen-clotting activity of beta-thrombin is not a consequence of reduced affinity for fibrinogen.

beta-Thrombin, a product of the limited proteolysis of alpha-thrombin, is characterized by greatly reduced fibrinogen-clotting activity as compared to alpha-thrombin but with unchanged activity toward ester substrates. The present study was designed to elucidate the basis for the changes in the catalytic activity resulting from the conversion of bovine alpha-thrombin to bovine beta-thrombin. Fibrinogen was utilized as a competitive inhibitor in the hydrolysis of a peptide nitroanilide substrate by bovine alpha- and beta-thrombin. The Ki values obtained for fibrinogen in these experiments were similar for alpha- and beta-thrombin (about 10 microM). Similar values for Ki were obtained when fibrinogen was used to inhibit the inactivation of bovine alpha- and beta-thrombin by diisopropylphosphorofluoridate. These experiments suggested that the conversion of bovine alpha- to beta-thrombin does not affect the fibrinogen-binding site on thrombin. Differences in the reactivity of functional groups at the active site were then explored. beta-Thrombin was observed to undergo modification at the active site histidine at a slower rate than that of alpha-thrombin when reacted with either tosyllysyl chloromethyl ketone or diethyl pyrocarbonate. It is suggested that the difference in the fibrinogen-clotting activity of these two forms of thrombin can result from changes in the reactivity of the active site histidine residue.

Affinity Labels↗
PubMed1984

Self-association of bovine prothrombin fragment 1 in the presence of metal ions. Use of a covalent cross-linking reagent to study the reaction.

The interaction of metal ions with prothrombin fragment 1 has been shown by other investigators to result in conformational change(s). Previous studies based on hydrodynamic measurements from other laboratories have suggested that prothrombin fragment 1 will, in addition to undergoing conformational change, self-associate in the presence of metal ions but there is a question regarding the specificity and role of metal ions in this process. The present study has made use of a covalent cross-linking agent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin fragment 1 and has focused on factors influencing the covalent dimerization of bovine prothrombin fragment 1 during reaction with this reagent. Optimal dimerization of bovine prothrombin fragment 1 required the involvement of cations in two separable processes. The "first role" is probably a reflection of a slow conformational change in the bovine prothrombin fragment 1 representing isomerization of a proline-containing peptide bond (Marsh, H. C., Scott, M. E., Hiskey, R. G., and Koehler, K. A. (1979) Biochem. J. 183, 513-517). The "second role" most likely reflects ion bridging with another prothrombin fragment 1 molecule. The ion specificities of these two roles are clearly different. Ions stabilizing the proper conformation (first role) are Ca2+, Sr2+, Mg2+, Mn2+, with Ba2+ having less capability in filling this role. Ions which have activity in the second role (ion bridge formation) are Ca2+, Sr2+, and Ba2+. Mn2+ and Mg2+ cannot fulfill the requirements of this second role and actually inhibit the function of Ca2+.

Animals↗
PubMed1984

Observations on the reaction of 2-hydroxy-5-nitrobenzyl bromide with a peptide-bound tryptophanyl residue.

Previous studies on the isolation of peptides containing tryptophanyl residues modified with 2-hydroxy-5-nitrobenzyl bromide demonstrated multiple products of reaction at the same residue as well as technical difficulties in the primary structure analysis of peptides containing the modified tryptophanyl residue. The present study was undertaken to explore the reaction of 2-hydroxy-5-nitrobenzyl bromide with the single tryptophanyl residue in a synthetic peptide, experimental allergenic encephalitogenic peptide. The modification of this peptide was accomplished in sodium acetate, pH 4.75, and reagent removed by gel filtration. Amino acid analysis of the modified peptide suggested that only the tryptophanyl residue had been modified under these experimental conditions. The modified peptide could be separated into multiple derivatives by high-performance liquid chromatography. Although it is clear that some of the observed heterogeneity reflects a difference in the degree of substitution at the single tryptophanyl residue, several of the derivatives appear to have the same extent of substitution. It is suggested that the heterogeneity observed is a reflection of the establishment of a new diastereoisomeric center in the peptide. These results are consistent with previous observations from other laboratories and provide a basis for the explanation of apparent heterogeneity of peptides obtained from modified proteins.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1983

Identification of the molecular defect in factor IX Chapel Hill: substitution of histidine for arginine at position 145.

Hemophilia B Chapel Hill is a mild hereditary hemorrhagic disorder in which the factor IX antigen is present in normal amounts but factor IX biological activity is markedly reduced. Previous studies have demonstrated that purified factor IX Chapel Hill has 8% of the activity of normal human factor IX and that the activation of factor IX Chapel Hill is defective in that only one of the two peptide bonds hydrolyzed during activation of normal factor IX is cleaved. The tryptic peptides from normal human factor IX and factor IX Chapel Hill were subjected to analysis by high-performance liquid chromatography. Comparison of the elution profile of the peptides obtained from factor IX Chapel Hill and normal factor IX demonstrated that the tripeptide Leu-Thr-Arg, which is derived from the normal molecule (positions 143-145) immediately amino-terminal from the Arg-Ala peptide bond at 145-146 that is cleaved during the activation of factor IX with factor XIa, was absent in the digest obtained from factor factor IX Chapel Hill. The elongated "activation peptide" from factor factor IX Chapel Hill was obtained by further high-performance liquid chromatographic fractionation and subjected to primary structure analysis. The following sequence, corresponding to positions 143-147, was obtained: Leu-Thr-His-Ala-Glu. Thus, the primary molecular defect in factor factor IX Chapel Hill is the substitution of histidine for arginine at position 145. This substitution precludes cleavage by factor XIa at this peptide bond, and the activation peptide region remains associated with the light chain of factor IXa Chapel Hill.

Amino Acid Sequence↗
PubMed1982

Influence of metal ions on prothrombin self-association. Demonstration of dimer formation by intermolecular cross-linking with dithiobis(succinimidylpropionate).

Interaction of certain metal ions with prothrombin and prothrombin fragment 1 has been shown to result in conformational change(s). Self-association of prothrombin and prothrombin fragment 1 in the presence of divalent cations has been reported. The present study has made use of a covalent cross-linking reagent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin in the presence of divalent cations. In the presence of certain divalent cations, prothrombin dimer is the product of such cross-linking. Optimal dimerization of bovine prothrombin requires preincubation with calcium ions prior to the addition of cross-linking reagent. Calcium ions are also required for dimerization after this preincubation period. A similar time dependence is not seen with human prothrombin. The dimerization of bovine prothrombin is also supported by strontium and gadolinium to an extent comparable to that of calcium and to a lesser extent by barium or manganese. Magnesium ions do not support dimerization. The results suggest that certain divalent cations either induce or stabilize a conformation of prothrombin which can self-associate to form dimers. The results further suggest that divalent cations are also necessary for the actual cross-linking process subsequent to this conformational change.

Amino Acid Sequence↗
PubMed1982

Measurement of human factor IXa activity in an isolated factor X activation system.

To determine the functional properties of factor IX isolated from the plasma of CRM+ hemophilia B patients, an assay system using proteins isolated from human plasma had to be developed which would be amenable to kinetic studies under a variety of experimental conditions. The present study describes the activation of factor X by factor IXa, isolated from normal human plasma, in an assay system which allows manipulation of calcium, phospholipid and factor VIIIa concentrations. Initial rate measurements with factor VIIIa present in the system were made by incubating factor VIII with factor Xa immediately before factor IXa assay. With this approach, the initial rate of factor X activation was constant, with little evidence for a lag period. Within the framework of the assay system described in the present study, it should be possible to examine not only genetic variants of factor IX, but also variants of factor VIII, as well as providing a means of routine factor IXa and factor VIII(a) assays.

Blood Coagulation Tests↗
PubMed1982

The acceleration by polylysine of the activation of factor X by factor IXa.

The present study reports that polylysine can function as a cofactor in the conversion of factor X to factor Xa by factor IXa. In the presence of polylysine, factor X is converted to factor Xa by factor IXa as demonstrated by both clotting and amidolytic assays. The activation of factor X by factor IXa requires the prior activation of factor IX to IXa by factor XIa. Conversion of factor X to factor Xa by factor IXa is not observed in the absence of polylysine. The activation reaction proceeds optimally at pH 8.0 with an equal weight ratio of polylysine to factor X. The effect of polylysine is readily reversed by low concentrations of NaCl or elevated temperature suggesting that electrostatic interactions are of primary importance in the polylysine facilitation of the activation of factor X by factor IXa.

Animals↗
PubMed1981

Structure-function relations in platelet-thrombin reactions. Inhibition of platelet-thrombin interactions by lysine modification.

The chemical modification of lysine residues in human alpha-thrombin has been used to study the interaction of thrombin with human platelets. Phosphopyridoxylation of thrombin using pyridoxal 5'-phosphate (pyridoxal-P) has been shown to inhibit the fibrinogen clotting activity of thrombin but not the catalytic activity (Griffith, M. J. J. Biol. Chem. 254, 3401-3406). Phosphopyridoxylation resulted in marked inhibition of the platelet-activating activity of thrombin. The concentration of pyridoxal-P-thrombin required to induce half-maximal platelet aggregation and release was 60 times greater than that of unmodified thrombin. Binding studies using pyridoxal-P-125I-thrombin showed a loss of both high and low affinity binding of thrombin to the surface of intact gel filtered platelets. In contrast, thrombin modified with pyridoxal-P in the presence of heparin incorporated up to 1 mol of pyridoxal-P per mol of thrombin. The heparin-protected pyridoxal-P-thrombin was only slightly inhibited in its interaction with platelets, and binding studies with the heparin-protected pyridoxal-P-125I-thrombin showed selective loss of low affinity binding but preservation of high affinity binding. These results provide further support for the hypothesis that residues at the macromolecular binding site of thrombin are involved in the binding of thrombin to platelets and further separate this functional region of thrombin into two lysine-containing subregions, one which is protected from modification by heparin which is involved in high affinity binding, and another which is not protected by heparin which is involved in low affinity binding.

Blood Platelets↗
PubMed1981

Dissociation of antithrombin III--thrombin complex. Formation of active and inactive antithrombin III.

Dissociation of the complexes formed during the reaction of thrombin with antithrombin III was studied by determining the kinetics of release of thrombin and active antithrombin III from the complexes. The rate of thrombin release increased gradually between pH 7.0 and 8.5 and then sharply increased at pH 9.0. The rate of active antithrombin III release did not change significantly over the same pH range. The results suggested that thrombin is released after reaction with antithrombin III via two pathways which are differentially affected by the pH of the solution. At pH 7.0 thrombin is released predominantly by dissociation of a non-acylated enzyme--inhibitor complex since active antithrombin III was released in relatively low amounts. The results suggested that thrombin inhibition by antithrombin III does not require the formation of an acyl bond between the active-site serine of thrombin and a carbonyl carbon of antithrombin III. The release of thrombin by acylation--deacylation appeared to be limited by the rate of enzyme acylation which was favored by more alkaline pH. This further suggests that acyl bond formation is a secondary reaction that can occur during thrombin inhibition by antithrombin III but is not necessary for thrombin inhibition.

Antithrombin III↗
PubMed1981

Characterization of the defect in activation of factor IX Chapel Hill by human factor XIa.

Factor IXChapel Hill (Factor IXCH), an abnormal Factor IX molecule isolated from the plasma of a patient with mild hemophilia B, has previously been shown to exhibit delayed activation by Factor XIa and calcium. In this study, we have found that Factor IXCH is cleaved upon incubation with human Factor XIa and calcium; however, cleavage of this protein is not observed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, the rate of disappearance of the zymogen parallels both the appearance of the heavy chain and the generation of clotting activity. In addition, a protein band that migrates with an apparent molecular weight of 45,000 also increases in parallel with clotting activity. Factor IXCH and normal Factor IX (Factor IXN), after incubation with Factor XIa and calcium, were subjected to amino terminal sequence analysis. Activated Factor IXN is cleaved at an arginine-alanine (Arg-Ala) bond and an arginine-valine (Arg-Val) bond as demonstrated by formation of the three amino terminal sequences corresponding to the amino terminal of the light chain, heavy chain, and activation peptide. However, activated Factor IXCH has only two amino terminal sequences, corresponding to the original amino terminal sequence and the heavy chain (formed by cleavage at the Arg-Val bond). It is concluded that the major defect in Factor IXCH is the inability of Factor XIa to cleave the Arg-Ala bond at a significant rate. The rate of formation of clotting activity of Factor IXCH is approximately 60% of the rate of formation of clotting activity of Factor IXN. The specific clotting activity of activated Factor IXCH is between 20 and 33% of activated Factor IXN.

Amino Acid Sequence↗
PubMed1979

The covalent differences between bovine alpha- and beta-thrombin. A structural explanation for the changes in catalytic activity.

The partial covalent structure of bovine beta-thrombin has been determined by the use of automated Edman degradation and carboxypeptidase digestion of the component polypeptide chains separated by gel filtration following either reduction and carboxymethylation or performic acid oxidation. beta-Thrombin has been found to contain three peptide chains derived by proteolysis of the parent alpha-thrombin molecule. The A chain of alpha-thrombin has been cleaved at two points yielding a peptide (A1 chain) which contains 17 amino acids, beginning with threonine 14 and ending with lysine 30. The B chain of alpha-thrombin has been cleaved at two positions to yield a B1 chain which begins with the NH2-terminal isoleucine and terminates with lysine 65 and a B2 chain which begins with lysine 74 and continues through COOH-terminal serine 259. The A1 chain and B2 chain are linked by a disulfide bridge. Although there is no evidence for a covalent bond between the B1 chain and the B2-A1 chains, the B1 chain is tightly bound to the remainder of the molecule, for separation is achieved only under denaturing conditions.

Amino Acid Sequence↗