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.

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.

Electrophoretic determination of sulfhydryl groups and its application to complex protein samples, in vitro protein synthesis mixtures, and cross-linked proteins

1986 ◽  
Vol 64 (2) ◽  
pp. 154-160 ◽  
Author(s):  
Helga Stan-Lotter ◽  
Philip D. Bragg
Keyword(s):  
Protein Synthesis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sulfhydryl Groups ◽  
Additional Parameter ◽  
In Vitro Protein Synthesis ◽  
Molecular Weights ◽  
Complex Protein ◽  
Vitro Protein

Without prior fractionation, the number of sulfhydryl groups of individual polypeptides in a protein mixture can be determined, provided their molecular weights and approximate isoelectric points are known. Urea-denatured protein samples are reacted with iodoacetamide and iodoacetate in a modified version of Creighton's procedure. After separation by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and isoelectric focusing, the number of sulfhydryl groups is determined by counting the protein bands which have additional negative charges. This method requires little material and provides an additional parameter, besides the molecular weight and isoelectric point, for the identification and characterization of a protein. The sensitivity may be enhanced for nonradioactive proteins by using 14C-labeled iodoacetamide and iodoacetate. The procedure has been applied to prokaryotic in vitro protein synthesis mixtures, bacterial membrane protein, and trypsin-cleaved or chemically cross-linked subunits of the F1 ATPase from Escherichia coli.

scholarly journals Synthesis of proteins from [35S]methionine by guinea pig megakaryocytes in vivo and time course of appearance of newly synthesized proteins in platelets

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 887-891 ◽  
Author(s):  
BP Schick
Keyword(s):  
Protein Synthesis ◽  
Guinea Pigs ◽  
Time Course ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Sds Page ◽  
Relationship Of

The relationship of protein synthesis to megakaryocyte maturation has been studied in guinea pigs in vivo. Guinea pigs were injected with a single dose of [35S]methionine. Megakaryocytes and platelets were isolated daily for 4 days, and proteins from both cells were isolated by DEAE-Sephacel chromatography and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. All proteins in megakaryocytes corresponding to stained bands on the SDS- PAGE gels were radiolabeled at 3 hours after injection. The greatest loss of radioactivity from the megakaryocytes occurred between 1 and 3 days after injection. Only trace labeling of platelet proteins was seen at 3 hours, representing almost entirely three bands at molecular weights 47,000, 52,000, and 66,000. At 24 hours only about 13% of the maximal labeling was present, but not all proteins were labeled. The maximal labeling was at 3 days. The pattern of labeling of platelets at 3 days was identical to that of megakaryocytes at 3 hours. The protein pattern of nonmegakaryocytic marrow cells was different from that of the platelets and megakaryocytes. Data presented here suggest that most protein synthesis in megakaryocytes is completed at least 24 hours before release of the platelets to the circulation, and suggest some specificity in the proteins that are synthesized at the terminal stages of maturation.

scholarly journals Synthesis of proteins from [35S]methionine by guinea pig megakaryocytes in vivo and time course of appearance of newly synthesized proteins in platelets

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 887-891 ◽  
Author(s):  
BP Schick
Keyword(s):  
Protein Synthesis ◽  
Guinea Pigs ◽  
Time Course ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Sds Page ◽  
Relationship Of

Abstract The relationship of protein synthesis to megakaryocyte maturation has been studied in guinea pigs in vivo. Guinea pigs were injected with a single dose of [35S]methionine. Megakaryocytes and platelets were isolated daily for 4 days, and proteins from both cells were isolated by DEAE-Sephacel chromatography and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. All proteins in megakaryocytes corresponding to stained bands on the SDS- PAGE gels were radiolabeled at 3 hours after injection. The greatest loss of radioactivity from the megakaryocytes occurred between 1 and 3 days after injection. Only trace labeling of platelet proteins was seen at 3 hours, representing almost entirely three bands at molecular weights 47,000, 52,000, and 66,000. At 24 hours only about 13% of the maximal labeling was present, but not all proteins were labeled. The maximal labeling was at 3 days. The pattern of labeling of platelets at 3 days was identical to that of megakaryocytes at 3 hours. The protein pattern of nonmegakaryocytic marrow cells was different from that of the platelets and megakaryocytes. Data presented here suggest that most protein synthesis in megakaryocytes is completed at least 24 hours before release of the platelets to the circulation, and suggest some specificity in the proteins that are synthesized at the terminal stages of maturation.

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.

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 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.

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.

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