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Biomedical subjects

E W Davie

Publications and source records attributed to E W Davie.

At least 19 recordsLinked to original sources
PubMed1992

Role of fibrinogen alpha and gamma chain sites in platelet aggregation.

Fibrinogen (Fbg) mediates platelet aggregation by its interaction with the platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3). Peptides containing the amino acid sequence RGD derived from the alpha chain (residues alpha 95-97 and residues alpha 572-574) and the sequence HHLGGAKQAGDV derived from the carboxyl terminus of the gamma chain of Fbg (residues gamma 400-411) inhibit these interactions. To determine the role of these sequences in intact Fbg, recombinant human Fbg (rFbg), mutant rFbgs with an RGD-->RGE substitution at either position alpha 97 or alpha 574, and a rFbg gamma'-containing variant that has a carboxyl-terminal interruption in the HHLGGAKQAGDV sequence have been expressed in transfected BHK cells. Purified rFbg and the two RGE mutant Fbgs were similar to plasma Fbg in platelet aggregation assays. In contrast, the gamma' variant Fbg was markedly defective in platelet aggregation. These data support the proposals that the carboxyl-terminal region of the gamma chain of Fbg is essential for optimal platelet aggregation and that the alpha-chain RGD sequences are neither necessary nor sufficient for platelet aggregation.

Amino Acid Sequence↗
PubMed1992

A microsomal endopeptidase from liver with substrate specificity for processing proproteins such as the vitamin K-dependent proteins of plasma.

The microsomal fraction of rabbit liver contains an endopeptidase that cleaves synthetic peptides that mimic the amino acid sequences of the processing sites of many proproteins, including the vitamin K-dependent proteins. The endopeptidase (M(r) 69,000) was extracted from liver microsomes with 1% Lubrol and purified about 2,700-fold. The substrate employed for isolation and characterization of the enzyme was the decapeptide acetyl-Ala-Arg-Val-Arg-Arg-Ala-Asn-Ser-Phe-Leu (prothrombin peptide), in which hydrolysis occurred on the carboxyl side of the paired Arg-Arg residues. The purified enzyme, whose activity was enhanced 1.8-fold by 0.1 mM CoCl2, has a Km = 80 microM and Vmax = 21,000 nmol.min-1.mg-1 and a pH optimum of 8.7. Proteolytic cleavage of decapeptide substrates was dependent on an arginine residue at positions P1 and P4. The enzyme was completely inhibited by EDTA and 1,10-phenanthroline as well as by p-chloromercuriphenylsulfonic acid and Hg2+. Inhibitors of serine proteases and cysteine proteases had no effect. Based on the substrate preference, the endopeptidase appears to be a good candidate for the enzyme responsible for the precursor processing of the vitamin K-dependent proteins and a number of other proproteins that are synthesized via the secretory pathway in liver and other tissues.

4-Chloromercuribenzenesulfonate↗
PubMed1992

Apple four in human blood coagulation factor XI mediates dimer formation.

Human blood coagulation factor XI is a dimer composed of two identical subunits. Each subunit contains four apple domains as tandem repeats followed by a serine protease region. A disulfide bridge between Cys321 of each fourth apple domain links the subunits together. The role of Cys321 in the dimerization of factor XI was examined by mutagenesis followed by expression of its cDNA in baby hamster kidney cells. The recombinant proteins were then purified from the tissue culture medium and shown to have full biological activity. Normal recombinant factor XI was secreted as a dimer as determined by SDS-PAGE, while recombinant factor XI-Cys321 Ser migrated as a monomer under these conditions. Gel filtration studies, however, revealed that each protein existed as a dimer under native conditions, indicating that the disulfide bond between Cys321 of each factor XI monomer was not necessary for dimer formation. The fourth apple domain (apple4) of factor XI was then introduced into tissue plasminogen activator (tPA) to investigate its role in the dimerization of other polypeptide chains. The fusion protein, containing apple4 (apple4-tPA), formed dimers as detected by SDS-PAGE and gel filtration. Furthermore, dimerization was specific to apple4, while apple3 had no effect on dimerization. These data further indicated that the apple4 domain of factor XI mediates dimerization of the two subunits and the interchain disulfide bond involving Cys321 was not essential for dimer formation.

Amino Acid Sequence↗
PubMed1992

Liver-specific expression of the gene coding for human factor X, a blood coagulation factor.

Factor X is a vitamin K-dependent glycoprotein that plays an essential role in both the intrinsic and extrinsic pathways of blood coagulation. Studies on a recombinant lambda phage containing the 5'-flanking region of the human factor X gene showed that the factor X gene was linked to and was located at the 3' end of the factor VII gene: the initiation codon of the factor X gene was 2823 base pairs (bp) downstream from the polyadenylation site of the factor VII gene. This 2.8-kilobase intergenic region, and progressively deleted fragments of it, was fused to the chloramphenicol acetyltransferase gene, and transient expressions in HepG2 cells, human fibroblasts, and Chinese hamster ovary cells were measured. A liver-specific promoter element, FXP1-binding site, essential for hepatocyte-specific transcription was identified. This promoter sequence, further localized to -63 to -42 bp in DNase I footprint studies, was homologous to LF-A1 or hepatic nuclear factor-4 recognition sequence and was equally functional in the normal and inverse orientations. FXP1 site bound to nuclear protein(s) from HepG2 cells and complex formation was partially abolished by the presence of duplex oligonucleotides containing liver factor-A1 or hepatic nuclear factor-4-binding sequences. Two additional positive elements located upstream of the promoter region, spanning from -215 to -149 bp (FXP2 site), and -457 to -351 bp (FXP3 site), were also established by reporter gene assays.

Amino Acid Sequence↗
PubMed1992

Expression of human blood coagulation factor XI: characterization of the defect in factor XI type III deficiency.

Human factor XI (FXI) is a blood coagulation factor participating in the early phase of the intrinsic pathway of blood coagulation. It circulates in blood as a glycoprotein composed of two identical chains held together by a single disulfide bond between the fourth apple domains. FXI has been expressed in baby hamster kidney (BHK) cells, where it was synthesized as a single-chain molecule that was converted to the dimer before secretion. The recombinant protein was fully active in a clotting assay, indicating that it interacted readily with other components of the coagulation cascade. A mutant FXI in which Phe283 was converted to Leu (Phe283Leu) was also expressed in BHK cells. This amino acid change occurs in the fourth apple domain of FXI and corresponds to the type III deficiency in Ashkenazi Jews. The mutant protein was secreted at reduced levels (about 8%) compared with normal FXI. This was due to a defect in the dimerization of the molecule rather than a decrease in the transcription of type III messenger RNA. Once secreted, however, the mutant protein consisted of a dimer with full biologic activity. The in vitro expression of FXI indicated that the impaired dimerization and secretion of the Phe283Leu mutant can account for the defect found in patients who are homozygous for the type III FXI deficiency.

Amino Acid Sequence↗
PubMed1991

Primary structure of the catalytic subunit of calf thymus DNA polymerase delta: sequence similarities with other DNA polymerases.

The 125- and 48-kDa subunits of bovine DNA polymerase delta have been isolated by SDS-polyacrylamide gel electrophoresis and demonstrated to be unrelated by partial peptide mapping with N-chlorosuccinimide. A 116-kDa polypeptide, usually present in DNA polymerase delta preparations, was shown to be a degraded form of the 125-kDa catalytic subunit. Amino acid sequence data from Staphylococcus aureus V8 protease, cyanogen bromide, and trypsin digestion of the 125- and 116-kDa polypeptides were used to design primers for the polymerase chain reaction to determine the nucleotide sequence of a full-length cDNA encoding the catalytic subunit of bovine DNA polymerase delta. The predicted polypeptide is 1106 amino acids in length with a calculated molecular weight of 123,707. This is in agreement with the molecular weight of 125,000 estimated from SDS-polyacrylamide gel electrophoresis. Comparison of the deduced amino acid sequence of the catalytic subunit of bovine DNA polymerase delta with that of its counterpart from Saccharomyces cerevisiae showed that the proteins are 44% identical. The catalytic subunit of bovine DNA polymerase delta contains the seven conserved regions found in a number of bacterial, viral, and eukaryotic DNA polymerases. It also contains five additional regions that are highly conserved between bovine and yeast DNA polymerase delta, but these regions share little or no homology with the alpha polymerases. Four of these additional regions are also highly homologous to the herpes virus family of DNA polymerases, whereas one region is not homologous to any other DNA polymerase that has been sequenced thus far.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Primary structure of the catalytic subunit of human DNA polymerase delta and chromosomal location of the gene.

The catalytic subunit (Mr approximately 124,000) of human DNA polymerase delta has been cloned by PCR using poly(A)+ RNA from HepG2 cells and primers designed from the amino acid sequence of regions highly conserved between bovine and yeast DNA polymerase delta. The human cDNA was 3443 nucleotides in length and coded for a polypeptide of 1107 amino acids. The enzyme was 94% identical to bovine DNA polymerase delta and contained the numerous highly conserved regions previously observed in the bovine and yeast enzymes. The human enzyme also contained two putative zinc-finger domains in the carboxyl end of the molecule, as well as a putative nuclear localization signal at the amino-terminal end. The gene coding for human DNA polymerase delta was localized to chromosome 19.

Amino Acid Sequence↗
PubMed1991

Recombinant human fibrinogen and sulfation of the gamma' chain.

Human fibrinogen and the homodimeric gamma'-chain-containing variant have been expressed in BHK cells using cDNAs coding for the alpha, beta, and gamma (or gamma') chains. The fibrinogens were secreted at levels greater than 4 micrograms (mg of total cell protein)-1 day-1 and were biologically active in clotting assays. Recombinant fibrinogen containing the gamma' chain incorporated 35SO4 into its chains during biosynthesis, while no incorporation occurred in the protein containing the gamma chain. The identity of the sulfated gamma' chain was verified by its ability to form dimers during clotting. In addition, carboxypeptidase Y digestion of the recombinant fibrinogen containing the gamma' chain released 96% of the 35S label from the sulfated chain, and the radioactive material was identified as tyrosine O-sulfate. These results clarify previous findings of the sulfation of tyrosine in human fibrinogen.

Amino Acid Sequence↗
PubMed1991

Factor XI deficiency in Ashkenazi Jews in Israel.

BACKGROUND AND METHODS: Severe factor XI deficiency, which is relatively common among Ashkenazi Jews, is associated with injury-related bleeding of considerable severity. Three point mutations--a splice-junction abnormality (Type I), Glu117----Stop (Type II), and Phe283----Leu (Type III)--have been described in six patients with factor XI deficiency. Clinical correlations with these mutations have not been carried out. We determined the relative frequency of the mutations and their association with plasma levels of factor XI clotting activity and bleeding, analyzing the mutations with the polymerase chain reaction and restriction-enzyme digestion. RESULTS: The Type II and Type III mutations had similar frequencies among 43 Ashkenazi Jewish probands with severe factor XI deficiency; these two mutations accounted for 49 percent and 47 percent, respectively, of a total of 86 analyzed alleles. Among 40 of the probands and 12 of their relatives with severe factor XI deficiency, patients homozygous for Type III mutation had a significantly higher level of factor XI clotting activity (mean [+/- SD] percentage of normal values, 9.7 +/- 3.8 percent; n = 13) than those homozygous for Type II mutation (1.2 +/- 0.5 percent, n = 16) or compound heterozygotes with Type II/III mutation (3.3 +/- 1.6 percent, n = 23), as well as significantly fewer episodes of injury-related bleeding. Each of these three groups had a similarly increased proportion of episodes of bleeding complications after surgery at sites with enhanced local fibrinolysis, such as the urinary tract, or during tooth extraction. CONCLUSIONS: Type II and Type III mutations are the predominant causes of factor XI deficiency among Ashkenazi Jews. Genotypic analysis, assay for factor XI, and consideration of the type and location of surgery can be helpful in planning operations in patients with this disorder.

Alleles↗
PubMed1991

Location of the disulfide bonds in human plasma prekallikrein: the presence of four novel apple domains in the amino-terminal portion of the molecule.

The location of 16 of the 18 disulfide bonds in human plasma prekallikrein was determined by amino acid sequence analysis of cystinyl peptides produced by chemical and enzymatic digestions. A unique structure, named the apple domain, was established for each of the four tandem repeats in the amino-terminal portion of the molecule. The apple domains (90 or 91 amino acids) contain 3 highly conserved disulfide bonds linking the first and sixth, second and fifth, and third and fourth half-cystine residues present in each repeat. The fourth tandem repeat contains an extra disulfide bond that forms a second small loop within the apple domain. The carboxyl-terminal portion of plasma prekallikrein containing the catalytic region of the molecule was found to have disulfide bonds located in positions similar to those of other serine proteases.

Amino Acid Sequence↗
PubMed1991

Location of the disulfide bonds in human coagulation factor XI: the presence of tandem apple domains.

Factor XI is a plasma glycoprotein that participates in the blood coagulation cascade. Of the 19 disulfide bonds present in each of the subunits of the human protein, 16 were determined by amino acid sequence analysis of peptide fragments produced by chemical and enzymatic digestion. Four apple domains of 90 or 91 amino acids were identified in the tandem repeats present in the amino-terminal portion of each subunit of factor XI. The disulfide bonds in the carboxyl-terminal portion of the molecule were similar to those in the catalytic region of other serine proteases. The two identical subunits of factor XI were connected by a single disulfide bond at Cys321 linking each of the fourth apple domains while each of the Cys residues at position 11 in the first apple domains forms a disulfide bond with another Cys residue.

Amino Acid Sequence↗
PubMed1991

Two types of abnormal genes for plasminogen in families with a predisposition for thrombosis.

The gene coding for plasminogen has been compared with several abnormal genes from Japanese patients by the polymerase chain reaction and DNA sequence analysis. Two types of abnormal genes coding for plasminogen were identified in these patients. In the type I mutation, a guanosine in GCT coding for Ala-601 near the active-site histidine was replaced by an adenosine resulting in ACT coding for threonine. This mutation was also shown by the loss of a cleavage site for Fnu4HI endonuclease, a restriction enzyme that recognizes GCTGC but not ACTGC. In the type II mutation, a guanosine in GTC coding for Val-355 was replaced by a thymidine resulting in TTC coding for phenylalanine. This change was readily shown by digestion with Ava II endonuclease, a restriction enzyme that recognizes GGTCC and not GTTCC. The type I mutation has been found to be identical to a plasminogen variant identified in Japanese patients by amino acid sequence analysis and also detected by isoelectric focusing, whereas the type II mutation is a unique amino acid substitution in the connecting region between the third and fourth kringles in plasminogen. DNA sequence analysis also revealed that the abnormal genes carry several silent nucleotide substitutions located primarily within introns and 5' and 3' flanking regions.

Base Sequence↗
PubMed1990

Nucleotide sequence of the gene for the b subunit of human factor XIII.

Factor XIII (Mr 320,000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (Mr 75,000 each) and two b subunits (Mr 80,000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in lambda phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92% of the gene. The introns contained four Alu repetitive sequences, one each in introns A, E, I, and J. A fifth Alu repeat was present in the flanking 3' end of the gene. Two partial KpnI repeats were also found in the introns, including one in intron I and one in intron J. The KpnI repeat in intron J was 89% homologous to a sequence of approximately 2200 nucleotides flanking the gene coding for human beta globin and approximately 3800 nucleotides from the L1 insertion present in the gene for human factor VIII. Intron H also contained an "O" family repeat, while two potential regions for Z-DNA were identified within introns G and J. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.

Amino Acid Sequence↗
PubMed1990

Amino acid sequence of human protein Z, a vitamin K-dependent plasma glycoprotein.

Protein Z is a vitamin K-dependent glycoprotein isolated and characterized from human and bovine plasma. A cDNA coding for human protein Z has been obtained by the isolation of phage clones from a liver cDNA library and in vitro amplification of two other liver libraries. Protein Z is synthesized with a prepro-leader sequence of 40 amino acids. The mature protein is composed of 360 residues including a Gla domain of 13 carboxyglutamic acid residues, two epidermal growth factor domains, and a carboxyl terminal region which is highly homologous to the catalytic domain of serine proteases. Human protein Z, however, contains an Asp instead of Ser and a Lys instead of His in the catalytic triad of the active site.

Amino Acid Sequence↗
PubMed1990

Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system.

The organization and structure of the gene coding for plasminogen has been determined by a combination of in vitro amplification of leukocyte DNA from normal individuals and isolation of unique clones from three different human genomic libraries. These clones were characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene for human plasminogen spanned about 52.5 kilobases of DNA and consisted of 19 exons separated by 18 introns. DNA sequence analysis revealed that the five kringle structures in plasminogen were coded by two exons. The nucleotides in the introns at the intron-exon boundaries were GT-AG analogous to those found in other eukaryotic genes. Three polyadenylation sites for plasminogen mRNA were also identified. When the amino acid sequences deduced from the genomic DNA and cDNAs of plasminogen were compared with that of the plasma protein determined by amino acid sequence analysis, an apparent amino acid polymorphism was observed in several positions of the polypeptide chain. Nucleotide sequence analysis of the amplified genomic DNAs and genomic clones also revealed that the plasminogen gene was very closely related to several other proteins, including apolipoprotein(a). This protein may have evolved via duplication and exon shuffling of the plasminogen gene. The presence of another plasminogen-related gene(s) in the human genomic library was also observed.

Amino Acid Sequence↗