scholarly journals Yeast phosphoglycerate mutate. Cyanogen bromide cleavage and amino acid sequence of an active-site peptide

1976 ◽  
Vol 153 (2) ◽  
pp. 145-149 ◽  
Author(s):  
L A Fothergill ◽  
G I Hodgson
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Cyanogen Bromide ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Phosphoglycerate Mutase ◽  
Large Fragment ◽  
X Ray ◽  
Cnbr Cleavage

The molecular weight and amino acid composition of phosphoglycerate mutase from yeast were determined. CNBr cleavage produced a large (190-residue) fragment and a small (60-residue) fragment. Tryptic and chymotryptic peptides derived from the large fragment were fractionated by ion-exchange chromatography. Peptides from two histidine-containing regions were isolated and the amino acid sequences were determined. Correlation of these data with X-ray-crystallographic evidence shows that the histidine residue in the sequence Arg-Leu Asn-Glu-Arg-His-Tyr-Gly-Asp-Leu-Glu-Gly-Lys is located at the active site.

Isolation of a Crosslinked Peptide from Hitman Fibrin and Development of a Radioimmunoassay

1979 ◽  
Author(s):  
R. Canfield ◽  
B. Lahiri ◽  
R. D’Alisa ◽  
V. Butler ◽  
H. Nossel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Edman Degradation ◽  
Cnbr Cleavage ◽  
Normal Hemostasis

Factor XIIIa introduces up to six crossllnklng bonds per molecule of fibrin; the bonds between the γ chains on adjacent fibrin molecules form most rapidly. Since cross linking is essential for normal hemostasis and is likely to be important in tests to detect thrombosis, we have attempted to develop a radioimmunoassay that exhibits specificity for the γ chain crosslinks. The immunogen consisted of a 54 amino acid, crosslinked peptide, isolated from purified human γ-γ chains following CNBr cleavage, gel filtration on Sephadex G-50 and ion-exchange chromatography on SP-Sephadex. Amino acid analysis and Edman degradation through step 24 confirmed the sequence of Chen and Doolittle (Biochemistry 10: i486, 1971), and the two degradation steps that failed to liberate the expected PTH-amino acids matched the reported location of the Gin-Lys crosslinks. Antisera were obtained against this immunogen coupled either to bovine thyroglobulin or bovine serum albumin. All antisera elicited bound immunogen that was covalently coupled to ribonuclease radiolabeled with 125I as a tracer. The unlabeled γ-γ, crosslinked peptide effectively inhibited binding (0.03-0.08 picomoles for 50% inhibition), while with some antisera up to 500 times more of the 27 amino acid γ monomer peptide was required for the same degree of inhibition. Fibrinogen and fragment D also were poor Inhibitors. The results Indicate that it is possible by radioimmunoassay to distinguish the COOH-termlnal region of the γ-γ dlmer from that of uncrosslinked molecules.

Chemical and serological properties of a cyanogen bromide peptide of southern bean mosaic virus protein

1980 ◽  
Vol 26 (12) ◽  
pp. 1450-1459 ◽  
Author(s):  
J. H. Tremaine ◽  
W. P. Ronald ◽  
E. M. Kelly
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Mosaic Virus ◽  
Cyanogen Bromide ◽  
Tomato Bushy Stunt Virus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Southern Bean Mosaic Virus ◽  
Gel Diffusion

Southern bean mosaic virus (SBMV) protein was cleaved with cyanogen bromide and a highly basic peptide, CB-1, was isolated by ion exclusion and ion-exchange chromatography. Twelve peptides were separated from a tryptic digest of CB-1 by ion-exchange chromatography and the composition of these peptides was similar to that of peptides released from EDTA-swollen virus particles by limited tryptic digestion. The composition and N-termini of the tryptic peptides indicated CB-1 was from the N-terminus of SBMV protein and contained 48 amino acid residues. The CB-1 peptide moved rapidly to the cathode in polyacrylamide gel electrophoresis at pH 3.9 and contained nine arginine residues, three lysine residues, and no acidic amino acid residues. It was shown to interact with purified viral RNA, sodium dextran sulfate, and calf thymus DNA.Antiserum to sodium dodecyl sulfate (SDS)-dissociated virus gave a reaction of partial identity between the CB-1 peptide and the SDS-dissociated virus in SDS gel diffusion tests. The CB-1 peptide did not react with antiserum to SDS-dissociated, trypsin-treated virus. Gel diffusion tests conducted in saline agar gels between trypsin-treated virus and SBMV, with SBMV antiserum, did not show differences in their serological properties. Antiserum to the CB-1 peptide conjugated to tomato bushy stunt virus reacted with SBMV but SBMV antiserum did not react with CB-1 or the CB-1-tomato bushy stunt virus conjugate.

scholarly journals Studies on Marsupial Protein. II. Amino Acid Sequence of the -Chain of Haemoglobin from the Grey Kangaroo, Macropus Giganteus

1969 ◽  
Vol 22 (6) ◽  
pp. 1437 ◽  
Author(s):  
GM Air ◽  
EOP Thompson
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Tryptic Digestion ◽  
Grey Kangaroo

The amino acid sequence of the jS-chain of haemoglobin from M. giganteus has been determined. The soluble peptides formed by tryptic digestion were isolated by gel filtration, ion-exchange chromatography, and paper ionophoresis, and amino acid sequences determined by the "dansyl"-Edman procedure. Special procedures were necessary for three peptides which were insoluble.

Carnivora: The Amino Acid Sequence of the Adult European Mink (Mustela lutreola, Mustelidae) Hemoglobins

1990 ◽  
Vol 45 (3-4) ◽  
pp. 223-228 ◽  
Author(s):  
Aftab Ahmed ◽  
Meeno Jahan ◽  
Gerhard Braumtzer
Keyword(s):  
Amino Acid ◽  
Gas Phase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Mustela Lutreola ◽  
European Mink ◽  
Globin Chains ◽  
The Family ◽  
Insight Into

Abstract The complete amino acid sequences of the hemoglobins from the adult European mink (Mustela lutreola) are presented. The erythrocytes contain two hemoglobin components and three globin chains. The isolation of globin chains achieved by ion-exchange chromatography on a column of CM -cellulose in 8 M urea buffer. The primary structure of globin chains and of the tryptic peptides determined in liquid-and gas-phase sequenators. The alignment of the a-and β-chains with those of reported sequences from other carnivora species belonging to the family Mustelidae may give an insight into the evolution of this molecule.

scholarly journals Two globin strains in the giant annelid extracellular haemoglobins

1987 ◽  
Vol 241 (2) ◽  
pp. 441-445 ◽  
Author(s):  
T Gotoh ◽  
F Shishikura ◽  
J W Snow ◽  
K I Ereifej ◽  
S N Vinogradov ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lumbricus Terrestris ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
Reverse Phase ◽  
Terminal Amino ◽  
Polypeptide Chains

The constituent polypeptide chains I, II, III and IV of the giant extracellular haemoglobin of the oligochaete Lumbricus terrestris were isolated by mono Q ion-exchange chromatography and C8 reverse-phase chromatography. The N-terminal amino acid sequences of Lumbricus chains I, III and IV were determined and aligned with those of Lumbricus chain II and the four chains of the extracellular haemoglobin of the polychaete Tylorrhynchus heterochaetus. Three invariant amino acid residues, Cys-7, Val-15 and Trp-19, were found to occur in the N-terminal segments (17-22 residues) of the eight chains of Lumbricus and Tylorrhynchus haemoglobins. In addition, it was found that the eight sequences could be separated into two groups: ‘A’, consisting of Lumbricus chains I and II and Tylorrhynchus chains I and IIA, having invariant Lys-14 and Lys-16, and ‘B’, consisting of Lumbricus chains III and IV and Tylorrhynchus IIB and IIC, having invariant Cys-6, Ser-8 and Asp-11. This result suggests that there are two strains of globin chain in the annelid extracellular haemoglobins.

The Amino Acid Sequence of Streptomyces griseus Trypsin. I. The Peptic Peptides

1973 ◽  
Vol 51 (7) ◽  
pp. 1077-1088 ◽  
Author(s):  
L. Jurášek ◽  
L. B. Smillie
Keyword(s):  
Amino Acid ◽  
Serine Proteases ◽  
System Analysis ◽  
Streptomyces Griseus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Terminal Sequence ◽  
Unique Amino Acid ◽  
High Voltage Electrophoresis

Streptomyces griseus trypsin (S.G.T.) isolated from pronase was digested with pepsin. The peptic peptides were isolated by high-voltage electrophoresis on paper and ion-exchange chromatography on Chromobead P resin using the Technicon autoanalyzer system. Analysis of the purified peptides provides 28 unique amino acid sequences accounting for approximately 95% of the S.G.T. molecule. A portion of the residues not accounted for can be ascribed to free leucine, phenylalanine, tyrosine, and tryptophan present in the peptic digest. The NH2-terminal sequence of S.G.T. was shown to be Val–Val–Gly–Gly–Thr–Arg–Ala–Ala–Gln–Gly–Glu–Phe and is highly homologous with NH2-terminal sequences of other Asp–Ser–Gly serine proteases.

Cyanogen Bromide Fragments of Rabbit Skeletal Tropomyosin

1973 ◽  
Vol 51 (1) ◽  
pp. 56-70 ◽  
Author(s):  
R. S. Hodges ◽  
L. B. Smillie
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Sequence ◽  
Cyanogen Bromide ◽  
Coiled Coil ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Coil Structure ◽  
Polypeptide Chains

Previous studies have demonstrated that rabbit skeletal tropomyosin consists of two or more chemically non-identical but highly homologous polypeptide chains. Attempts by a variety of techniques to prepare pure tropomyosin chains in amounts adequate for chemical characterization have been unsuccessful to date. To provide more extensive information for the purpose of elucidating the relationship between amino acid sequence and the coiled-coil structure of tropomyosin, a cyanogen bromide treatment of the S-carboxymethylated protein was carried out. The fragments were separated into small and large components by gel filtration on Sephadex G-50. The small fragments were fractionated by ion-exchange chromatography and electrophoresis on paper and their sequences elucidated by conventional methods. Coupled with previous data, these results indicate a minimum of seven unique methionine sequences and are consistent with a high degree of homology in the tropomyosin polypeptide chains. From the mixture of the larger cyanogen bromide polypeptides, a fragment was isolated by ion-exchange chromatography on QAE-Sephadex. In aqueous buffer it had a molecular weight of 35 000 and an α-helical content of about 60% as estimated by circular dichroism. In 8 M urea its molecular weight was reduced to 15 000, a value in reasonable agreement with a minimal molecular weight of 17 000 calculated from its amino acid composition. From its histidine content (two residues) and the known COOH-terminal amino acid sequence of the protein, the fragment was concluded to be derived from the COOH-terminal half of the molecule. These results are consistent with a degree of 'coiled-coil' structure in a fragment representing about one-half of the tropomyosin molecule.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

Bovine Factors X1And X2: Activation Peptide Based Chromatographic Differences

1979 ◽  
Author(s):  
Takashi Morita ◽  
Craig Jackson
Keyword(s):  
Amino Acid ◽  
Anion Exchange ◽  
Gel Filtration ◽  
Heart Association ◽  
Personal Communication ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Factor X ◽  
Activation Peptide ◽  
Activation Peptides

Bovine Factor X is eluted in two forms (X1and X2) from anion exchange chromatographic columns. These two forms have indistinguishable amino acid compositions, molecular weights and specific activities. The amino acid sequences containing the γ-carboxyglutamic acid residues have been shown to be identical in X1 and X2(H. Morris, personal communication). An activation peptide is released from the N-terminal region of the heavy chain of Factor X by an activator from Russell’s viper venom. This peptide can be isolated after activation by gel filtration on Sephadex G-100 under nondenaturing conditions. The activation peptides from a mixture of Factors X1 and X2 were separated into two forms by anion-exchange chromatography. The activation peptide (AP1) which eluted first was shown to be derived from Factor X1. while the activation peptiae (AP2) which eluted second was shown to be derived from X2 on the basis of chromatographic separations carried out on Factors X1 and X2 separately. Factor Xa was eluted as a symmetrical single peak. On the basis of these and other data characterizing these products, we conclude that the difference between X1 and X2 are properties of the structures of the activation peptides. (Supported by a grant HL 12820 from the National Heart, Lung and Blood Institute. C.M.J. is an Established Investigator of the American Heart Association).

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