scholarly journals The localization of a vitamin K-induced modification in an N-terminal fragment of human prothrombin

1974 ◽  
Vol 143 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Tore Skotland ◽  
Turid Holm ◽  
Bjarne Østerud ◽  
Ragnar Flengsrud ◽  
Hans Prydz
Keyword(s):  
Amino Acid ◽  
Vitamin K ◽  
Vitamin K Antagonists ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Molecular Weights ◽  
A Value ◽  
Terminal Fragment ◽  
Cnbr Cleavage

1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to homogeneity from human serum. 2. The apparent molecular weight is 27000±2000 in sodium dodecyl sulphate–polyacrylamide-gel electrophoresis, whereas a value of about 19600 is obtained by calculation based on amino acid and carbohydrate analyses. The N-terminal sequence is an Ala-Asx bond. The fragment contains about 16% carbohydrate, binds phospholipids in the presence of Ca2+and is adsorbed to BaSO4. The pKa of its BaSO4-binding group(s) is 3.1–3.5. 3. By CNBr cleavage of fragment PF-I two peptides (C-1 and C-2) were obtained with molecular weights of about 5900 (C-2) and 12400 (C-1) on the basis of amino acid and carbohydrate analyses. Only the smaller (N-terminal) peptide is adsorbed to BaSO4 and, since the ability of the whole protein to bind to BaSO4 is known to be absent in samples obtained from patients treated with vitamin K antagonists, this peptide probably contains the site of a modification to the structure of the protein which occurs during biosynthesis and depends on vitamin K. This peptide does not contain hexosamine or sialic acid.

Subunit studies of coupling factor 1 of bean chloroplasts

1976 ◽  
Vol 54 (5) ◽  
pp. 481-487 ◽  
Author(s):  
M. P. Silvanovich ◽  
R. D. Hill
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coupling Factor ◽  
Leaf Extracts ◽  
Molecular Weights ◽  
Major Species ◽  
Chloroplast Coupling Factor ◽  
Coupling Factors

A bean chloroplast coupling factor (CF1) with latent Ca2+-dependent ATPase activity was studied. Immunodiffusion of bean (Phaseolus vulgaris) chloroplast and etioplast coupling factors and spinach coupling factor against antiserum to spinach coupling factor showed partial identity of the bean coupling factor with that of spinach. An immunoelectrophoretic comparison, under dissociating conditions, of bean leaf extracts and spinach extracts containing CF1 subunits (as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) gave identical results for both extracts. At least six distinct polypeptide species were found. The major species had molecular weights of 42 000, 59 000 and 63 000 daltons. Amino acid analysis of electrophoretically purified bean CF1 gave results similar to those published for spinach CF1.

scholarly journals Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement

1976 ◽  
Vol 155 (1) ◽  
pp. 5-17 ◽  
Author(s):  
K B M Reid
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Sequence Data ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Disulphide Bond ◽  
Small Peptides ◽  
Molecular Weights ◽  
A Chain

1. Digestion of human subcomponent C1q with pepsin at pH4.45 for 20h at 37 degrees C fragmented most of the non-collagen-like amino acid sequences in the molecule to small peptides, whereas the entire regions of collagen-like sequence that comprised 38% by weight of the subcomponent C1q were left intact. 2. The collagen-like fraction of the digest was eluted in the void volume of a Sephadex G-200 column, was was showm to be composed of two major fragments when examined by electrophoresis on polyacrylamide gels run in buffers containing sodium dodecyl sulphate. These fragments were separated on CM-cellulose at pH4.9 in buffers containing 7.5M-urea. 3. Human subcomponent C1q on reduction and alkylation yields equimolar amounnts of three chains, which have been designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. One of the pepsin fragments was shown to be composed of the N-terminal 95 residues of the A chain linked, via residue A4, by a single disulphide bond to a residue in the sequence B2-B6 in the N-terminal 91 residues of the B chain. The second pepsin fragment was shown to be composed of a disulphide-linked dimer of the N-terminal 94 residues of the C chain, the only disulphide bond being located at residue C4.4. The mol. wts. of the unoxidized and oxidized pepsin fragments were estimated from their amino acid compositions to be 20 000 and 18 200 for the A-B and C-C dimers and 11 400, 8800 and 9600 for the collagen-like fragments of the A, B and C chains respectively. Estimation of the molecular weights of the peptic fragments by polyacrylamide-gel electrophoresis run in the presence of sodium dodecyl sulphate gave values that were approx. 50% higher than expected from the amino acid sequence data. This is probably due to the high collagen-like sequence content of these fragments.

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.

Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum

1982 ◽  
Vol 60 (3) ◽  
pp. 263-271 ◽  
Author(s):  
Serge Côté ◽  
Paul Nadeau ◽  
James M. Neelin ◽  
Dominick Pallotta
Keyword(s):  
Amino Acid ◽  
Physarum Polycephalum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Mobility ◽  
Apparent Molecular Weight ◽  
Chromosomal Protein ◽  
Basic Proteins ◽  
Isolation And Characterization ◽  
Core Histones

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid–urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, andH4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid–urea or SDS–polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid–urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.

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.

Production and Characterization of Ovalbumin and Ovotransferrin from Chicken Egg White

2012 ◽  
Vol 8 (2) ◽  
Author(s):  
Jianguo Liu ◽  
Jing Liu ◽  
Xuefang Zhang
Keyword(s):  
Amino Acid ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Egg White ◽  
Molecular Weights ◽  
Chicken Egg ◽  
Isoelectric Points ◽  
Iron Binding Capacity

An efficient and easily scaled up method to separate ovalbumin and ovotransferrin simultaneously from chicken egg white using ultrafiltration is proposed. The purities of ovalbumin and ovotransferrin obtained were 94% and 89%, with the yields of 82% and 76%, respectively. The resulting ovalbumin and ovotransferrin products was then characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, dynamic light scattering, circular dichroism, and amino acid analysis to confirm their molecular weights, isoelectric points, aggregate sizes, molecular secondary structures, and amino acid compositions. The iron-binding capacity of the purified ovotransferrin was also evaluated.

scholarly journals Structure and properties of human interferon-α from Namalwa lymphoblastoid cells

1982 ◽  
Vol 207 (3) ◽  
pp. 397-408 ◽  
Author(s):  
G Allen
Keyword(s):  
Amino Acid ◽  
Interferon Alpha ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Human Interferon ◽  
Interferon Α ◽  
British Library ◽  
Lymphoblastoid Cells ◽  
Molecular Weights

The chromatographic properties of human interferon-alpha from Namalwa lymphoblastoid cells on Sephadex G-75 are described. The interferons are separated into two groups of four, with apparent molecular weights 19050 and 22000. Some of the latter form dimers at high concentrations. Fractions containing interferon were studied by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Seven of the components had apparent molecular weights in this system, after reduction, of between 18400 and 20900: one component is probably glycosylated and has an apparent molecular weight of 27500. Amino acid sequences of peptides derived from interferon mixtures were determined and are related to published sequences deduced from the nucleotide sequences of cloned complementary DNA coding for interferon-alpha. The results show that the major interferon-alpha species isolated from Namalwa cells do not undergo C-terminal processing. Amino acid analyses of peptides are presented in Supplementary Publication SUP 50117 (28 pages), which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1981) 193, 5.

scholarly journals Accelerated breakdown of reticulocyte protein formed under conditions of amino acid depletion

1978 ◽  
Vol 176 (1) ◽  
pp. 151-158 ◽  
Author(s):  
C S Chandler ◽  
F J Ballard
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Stringent Response ◽  
Molecular Weights ◽  
Protein Inhibition ◽  
Deficient Medium ◽  
Amino Acid Depletion ◽  
Rabbit Reticulocytes

1. Labile protein is formed when rat or rabbit reticulocytes are incubated in medium deficient in individual amino acids, especially histidine, valine or alanine. The fraction of unstable protein is increased to about 35% of the total protein synthesized when the histidinyl-tRNA-charging inhibitor, histidinol, is added to histidine-deficient media. 2. The molecular weights of the labile proteins measured by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of urea are less than haemoglobin and probably represent prematurely terminated haemoglobin chains. 3. Although protein synthesis is always lower under conditions that produce labile protein, inhibition of protein synthesis by fluoride or cycloheximide does not give an effect similar to amino acid depletion. 4. The synthesis of protein in deficient medium does not alter the degradation rate of pre-existing protein in reticulocytes and is thus unrelated to the stringent response in bacteria. 5. We propose that amino acid-deficient medium leads to a decreased charging of the appropriate tRNA, a concomitant decrease in protein synthesis and the degradation of nascent peptides.

scholarly journals Intraspecies heterogeneity of epidermal keratins isolated from bovine hoof and snout

1979 ◽  
Vol 177 (1) ◽  
pp. 187-196 ◽  
Author(s):  
L D Lee ◽  
J Kubilus ◽  
H P Baden
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polypeptide Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Helical Structure ◽  
Gene Products ◽  
Post Translational Modifications ◽  
Molecular Weights ◽  
Bovine Hoof

The alpha-keratins, the principal components of the tonafilaments, were extracted, characterized and compared in bovine hoof and snout epidermis. The alpha-fibrous proteins of these tissues are similar with respect to their molecular weights, amino acid composition and percentage of helical structure. However, distinct differences in the polypeptides comprising these proteins were observed. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these proteins consistently showed that the polypeptide chain in snout, designated as band B (mol.wt. 67,000), was completely absent from hoof preparations. This was confirmed with several alternative preparative procedures. The peptides produced by digestion of the intact keratins from hoof and snout with CNBr were distinctly different. Finally, digestion of keratins from hoof and snout with trypsin yielded products that differed in size and resistance to further digestion. Thus, in addition to the interspecies polypeptide heterogeneity documented in the literature, this report establishes the intraspecies heterogeneity of keratins and suggests that these differences are due to either the expression of different gene products or differences in post-translational modifications in these two tissues.

scholarly journals Properties of salt-resistant lipase and lipoprotein lipase purified from human post-heparin plasma

1979 ◽  
Vol 179 (3) ◽  
pp. 555-559 ◽  
Author(s):  
A M Ostlund-Lindqvist
Keyword(s):  
Amino Acid ◽  
Amino Acid Composition ◽  
Lipoprotein Lipase ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Molecular Weights ◽  
Heparin Plasma

Lipoprotein lipase and salt-resistant lipase were isolated from human post-heparin plasma. The proteins of human post-plasma lipoprotein lipase and salt-resistant lipase were identified and demonstrated to be immunologically different. Significant differences between the two enzymes in their relative amino acid composition were demonstrated, which indicates that the two enzymes are different proteins. When analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the enzymes seemed to have monomer molecular weights similar to that of lipoprotein lipase purified from bovine milk.

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