scholarly journals An apparently higher molecular weight gamma-chain variant in a new congenital abnormal fibrinogen Tochigi characterized by the replacement of gamma arginine-275 by cysteine

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 480-487 ◽  
Author(s):  
N Yoshida ◽  
K Ota ◽  
M Moroi ◽  
M Matsuda
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Positive Staining ◽  
Gamma Chain ◽  
Peptide Peak ◽  
Sds Page ◽  
Fibrin Monomers ◽  
Chain Variant

Abstract A gamma-chain variant with an apparently higher molecular weight than the normal gamma-chain was detected in a new congenital abnormal fibrinogen with impaired polymerization of the fibrin monomer and with normal release of fibrinopeptides A and B in a 51-year-old male. Purified fibrinogen analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the reduced condition in the system of Laemmli contained two protein bands in the gamma-chain region (molecular weight, 50,500 as compared with 50,000 for the normal), both with normal crosslinking ability. The presence of two types of gamma- chains was more clearly detected when reduced and carboxymethylated fibrinogen was analyzed by SDS-PAGE or when reduced fragment D2 was analyzed on SDS-PAGE followed by Western blotting, and identified by positive staining for anti gamma-chain monoclonal antibody. Cyanogen bromide- or lysylendopeptidase-cleavage of purified gamma-chains analyzed on reverse-phase high performance liquid chromatography showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. Amino acid sequence analysis demonstrated that the gamma arginine-275 of gamma-chain variant was replaced by a cysteine. These data suggest that some regions or conformations containing gamma 275 will affect the polymerization of fibrin monomers. The propositus' two daughters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Tochigi, and the gamma-chain variant as gamma Tochigi.

scholarly journals An apparently higher molecular weight gamma-chain variant in a new congenital abnormal fibrinogen Tochigi characterized by the replacement of gamma arginine-275 by cysteine

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 480-487
Author(s):  
N Yoshida ◽  
K Ota ◽  
M Moroi ◽  
M Matsuda
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Positive Staining ◽  
Gamma Chain ◽  
Peptide Peak ◽  
Sds Page ◽  
Fibrin Monomers ◽  
Chain Variant

A gamma-chain variant with an apparently higher molecular weight than the normal gamma-chain was detected in a new congenital abnormal fibrinogen with impaired polymerization of the fibrin monomer and with normal release of fibrinopeptides A and B in a 51-year-old male. Purified fibrinogen analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the reduced condition in the system of Laemmli contained two protein bands in the gamma-chain region (molecular weight, 50,500 as compared with 50,000 for the normal), both with normal crosslinking ability. The presence of two types of gamma- chains was more clearly detected when reduced and carboxymethylated fibrinogen was analyzed by SDS-PAGE or when reduced fragment D2 was analyzed on SDS-PAGE followed by Western blotting, and identified by positive staining for anti gamma-chain monoclonal antibody. Cyanogen bromide- or lysylendopeptidase-cleavage of purified gamma-chains analyzed on reverse-phase high performance liquid chromatography showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. Amino acid sequence analysis demonstrated that the gamma arginine-275 of gamma-chain variant was replaced by a cysteine. These data suggest that some regions or conformations containing gamma 275 will affect the polymerization of fibrin monomers. The propositus' two daughters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Tochigi, and the gamma-chain variant as gamma Tochigi.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

scholarly journals Fibrinogen Kyoto II, a new congenitally abnormal molecule, characterized by the replacement of A alpha proline-18 by leucine

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 149-153
Author(s):  
N Yoshida ◽  
M Okuma ◽  
H Hirata ◽  
M Matsuda ◽  
K Yamazumi ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Replacement ◽  
Amino Acid Sequence Analysis ◽  
Alpha Chain ◽  
Molecular Weights ◽  
Peptide Peak ◽  
Fibrin Monomers

A new case of heterozygous dysfibrinogenemia characterized by an amino acid replacement in the NH2-terminal region of the fibrin alpha-chain was found in a 27-year-old woman with a bleeding problem. Her one-stage prothrombin time and activated partial thromboplastin time were slightly prolonged, and the purified fibrinogen from this patient had a markedly prolonged thrombin or reptilase time. Release of fibrinopeptides A and B was normal, but the polymerization of fibrin monomers was impaired. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fibrinogen under the reduced condition showed no abnormalities in the apparent molecular weights of its three chains. Reverse-phase high performance liquid chromatography (HPLC) of the lysylendopeptidase-cleaved purified A alpha-chains showed a decrease in one peptide compared with the normal amount and the appearance of an abnormal peptide peak. These peptides were treated with thrombin and further separated on HPLC. Amino acid sequence analysis of the abnormal peptide indicated that A alpha proline-18, the second residue from the NH2-terminus of the fibrin alpha-chain, was replaced by leucine. The synthetic peptide Gly-Pro-Arg-Pro inhibited both thrombin- and reptilase-induced fibrin aggregation, but Gly-Leu- Arg-Pro showed little or no inhibition under the same conditions. The discovery of this abnormal fibrinogen supports the findings that A alpha proline-18 is important as part of the polymerization site in the NH2-terminus of the fibrin alpha-chain. The propositus' mother had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Kyoto II.

scholarly journals Polymerization defect of fibrinogen Baltimore III due to a gamma Asn308- ---Ile mutation

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1659-1663 ◽  
Author(s):  
S Bantia ◽  
WR Bell ◽  
CV Dang
Keyword(s):  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequence Analysis ◽  
Gamma Chain ◽  
Reverse Phase ◽  
Lysyl Endopeptidase ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Relative Molecular Weight

Fibrinogen Baltimore III, a congenital abnormal fibrinogen with impaired fibrin monomer polymerization, displays a normal gamma-chain and a gamma-variant that has an apparently lower relative molecular weight (mol wt) than normal on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Reverse phase high-performance liquid chromatography (HPLC) analysis of the lysyl endopeptidase digest of the purified gamma-chains of fibrinogen Baltimore III revealed the presence of a peptide that is not found in the digest of the normal fibrinogen gamma-chain. Amino acid sequence analysis of this peptide indicated that the gamma-chain residue 308, asparagine, is replaced by isoleucine. Concanavalin A bound both normal and variant gamma-chains of fibrinogen Baltimore III, indicating that the carbohydrate moiety is not altered and is not responsible for the increase in electrophoretic mobility of the Baltimore III gamma-chain. This study suggests that the integrity of gamma Asn308 is critical for fibrin monomer polymerization, since alteration to either a basic (fibrinogen Kyoto I, Asn----Lys) or hydrophobic (Asn----Ile) residue results in significantly delayed polymerization of fibrinogen to fibrin.

scholarly journals Polymerization defect of fibrinogen Baltimore III due to a gamma Asn308- ---Ile mutation

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1659-1663 ◽  
Author(s):  
S Bantia ◽  
WR Bell ◽  
CV Dang
Keyword(s):  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequence Analysis ◽  
Gamma Chain ◽  
Reverse Phase ◽  
Lysyl Endopeptidase ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Relative Molecular Weight

Abstract Fibrinogen Baltimore III, a congenital abnormal fibrinogen with impaired fibrin monomer polymerization, displays a normal gamma-chain and a gamma-variant that has an apparently lower relative molecular weight (mol wt) than normal on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Reverse phase high-performance liquid chromatography (HPLC) analysis of the lysyl endopeptidase digest of the purified gamma-chains of fibrinogen Baltimore III revealed the presence of a peptide that is not found in the digest of the normal fibrinogen gamma-chain. Amino acid sequence analysis of this peptide indicated that the gamma-chain residue 308, asparagine, is replaced by isoleucine. Concanavalin A bound both normal and variant gamma-chains of fibrinogen Baltimore III, indicating that the carbohydrate moiety is not altered and is not responsible for the increase in electrophoretic mobility of the Baltimore III gamma-chain. This study suggests that the integrity of gamma Asn308 is critical for fibrin monomer polymerization, since alteration to either a basic (fibrinogen Kyoto I, Asn----Lys) or hydrophobic (Asn----Ile) residue results in significantly delayed polymerization of fibrinogen to fibrin.

scholarly journals Fibrinogen Kyoto II, a new congenitally abnormal molecule, characterized by the replacement of A alpha proline-18 by leucine

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 149-153 ◽  
Author(s):  
N Yoshida ◽  
M Okuma ◽  
H Hirata ◽  
M Matsuda ◽  
K Yamazumi ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Replacement ◽  
Amino Acid Sequence Analysis ◽  
Alpha Chain ◽  
Molecular Weights ◽  
Peptide Peak ◽  
Fibrin Monomers

Abstract A new case of heterozygous dysfibrinogenemia characterized by an amino acid replacement in the NH2-terminal region of the fibrin alpha-chain was found in a 27-year-old woman with a bleeding problem. Her one-stage prothrombin time and activated partial thromboplastin time were slightly prolonged, and the purified fibrinogen from this patient had a markedly prolonged thrombin or reptilase time. Release of fibrinopeptides A and B was normal, but the polymerization of fibrin monomers was impaired. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fibrinogen under the reduced condition showed no abnormalities in the apparent molecular weights of its three chains. Reverse-phase high performance liquid chromatography (HPLC) of the lysylendopeptidase-cleaved purified A alpha-chains showed a decrease in one peptide compared with the normal amount and the appearance of an abnormal peptide peak. These peptides were treated with thrombin and further separated on HPLC. Amino acid sequence analysis of the abnormal peptide indicated that A alpha proline-18, the second residue from the NH2-terminus of the fibrin alpha-chain, was replaced by leucine. The synthetic peptide Gly-Pro-Arg-Pro inhibited both thrombin- and reptilase-induced fibrin aggregation, but Gly-Leu- Arg-Pro showed little or no inhibition under the same conditions. The discovery of this abnormal fibrinogen supports the findings that A alpha proline-18 is important as part of the polymerization site in the NH2-terminus of the fibrin alpha-chain. The propositus' mother had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Kyoto II.

scholarly journals A lower molecular weight gamma-chain variant in a congenital abnormal fibrinogen (Kyoto)

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 703-707 ◽  
Author(s):  
N Yoshida ◽  
M Okuma ◽  
M Moroi ◽  
M Matsuda
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Protein Band ◽  
Lower Molecular Weight ◽  
Molar Ratio ◽  
Positive Staining ◽  
Gamma Chain ◽  
Abnormal Protein ◽  
Chain Variant

A gamma-chain variant with a lower molecular weight than the normal gamma chain was detected in a new congenital abnormal fibrinogen with impaired polymerization of the fibrin monomer and with normal release of fibrinopeptides A and B in a 45-year-old male. Purified fibrinogen analyzed on SDS-polyacrylamide gel electrophoresis under the reduced condition contained an abnormal protein band with an apparent molecular weight of 48,000 compared with the gamma chain with a molecular weight of 50,000. This abnormal protein band was found to be a gamma-chain variant from the molar ratio of A alpha chain:B beta chain:gamma chain:abnormal protein (about 2:2:1:1), with positive staining for carbohydrate and crosslinking ability. Crosslinked fibrin contained three types of gamma-gamma dimers with apparent molecular weights of 94,000 (the same as normal major gamma-gamma dimer), 92,000 and 90,000, and the plasmic digests of crosslinked fibrin in the presence of calcium retained three types of gamma-gamma dimer remnants. This suggests that the abnormal gamma-chain variant has a shorter polypeptide chain not in the NH2-terminal but in the COOH-terminal portion, probably at or near the polymerization site. This patient's two daughters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Kyoto, and the gamma- chain variant as gamma Kyoto.

scholarly journals A lower molecular weight gamma-chain variant in a congenital abnormal fibrinogen (Kyoto)

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 703-707 ◽  
Author(s):  
N Yoshida ◽  
M Okuma ◽  
M Moroi ◽  
M Matsuda
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Protein Band ◽  
Lower Molecular Weight ◽  
Molar Ratio ◽  
Positive Staining ◽  
Gamma Chain ◽  
Abnormal Protein ◽  
Chain Variant

Abstract A gamma-chain variant with a lower molecular weight than the normal gamma chain was detected in a new congenital abnormal fibrinogen with impaired polymerization of the fibrin monomer and with normal release of fibrinopeptides A and B in a 45-year-old male. Purified fibrinogen analyzed on SDS-polyacrylamide gel electrophoresis under the reduced condition contained an abnormal protein band with an apparent molecular weight of 48,000 compared with the gamma chain with a molecular weight of 50,000. This abnormal protein band was found to be a gamma-chain variant from the molar ratio of A alpha chain:B beta chain:gamma chain:abnormal protein (about 2:2:1:1), with positive staining for carbohydrate and crosslinking ability. Crosslinked fibrin contained three types of gamma-gamma dimers with apparent molecular weights of 94,000 (the same as normal major gamma-gamma dimer), 92,000 and 90,000, and the plasmic digests of crosslinked fibrin in the presence of calcium retained three types of gamma-gamma dimer remnants. This suggests that the abnormal gamma-chain variant has a shorter polypeptide chain not in the NH2-terminal but in the COOH-terminal portion, probably at or near the polymerization site. This patient's two daughters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Kyoto, and the gamma- chain variant as gamma Kyoto.

Study on Degradation of Radix Isatidis Polypeptide in Artificial Gastrointestinal Environment

2014 ◽  
Vol 989-994 ◽  
pp. 1020-1024
Author(s):  
Nan Nan ◽  
Xi Jing Liu
Keyword(s):  
Gel Electrophoresis ◽  
Free Amino ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Radix Isatidis ◽  
Sds Page ◽  
Electrophoresis Method ◽  
Amino Acid Levels ◽  
Different Molecular Weight

Radix Isatidis is a traditional Chinese medicine for treatment of influenza and inflammation in China. In this paper, in order to study the degradation situation of Radix Isatidis polypeptide in artificial gastrointestinal environment, the SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) method was used to detect the degradation of Radix Isatidis polypeptide in artificial intestinal juice and gastric juice, and it showed that Radix Isatidis peptides could be degradated to different degrees. HPLC (High Performance Liquid Chromatography) was used to determine the change of peptides degradation, and it indicated that free amino acid levels did not change significantly. The result after degradation was also detected by BCA method, and it showed that there were still a large number of polypeptides in the liquid. From this experiment we can come to this conclusion that Radix Isatidis polypeptides in artificial gastrointestinal juice mostly degraded into a series of different molecular weight peptides.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

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