scholarly journals Isolation, properties and amino acid sequences of three neurotoxins from the venom of a sea snake, Aipysurus laevis

1976 ◽  
Vol 153 (1) ◽  
pp. 79-87 ◽  
Author(s):  
N Maeda ◽  
N Tamiya
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Amino Acid Sequences ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Toxin B ◽  
Almost All

Aipysurus laevis venom was chromatographed on CM-cellulose and Bio-Rex 70 columns. Three neurotoxic components, toxins Aipysurus laevis a, b and c, were isolated. The toxins a, b and c corresponded to 22, 33 and 21% respectively of the proteins in the original venom, and accounted for almost all the lethal activity of the venom. The three toxins a, b and c were monodisperse on disc electrophoresis at pH4; toxins a and b moved at the same velocity and c a little faster. They were monodisperse also on sodium dodecyl sulphate-polyacrylamide-disc-gel electrophoresis, giving a molecular weight of 7600. The molecular weight of toxin b estimated by gel filtration was 7000. The amino acid sequence analyses of these toxins revealed that they consisted of 60 amino acid residues and that Aipysurus laevis b was [25-methionine, 28-arginine] Aipysurus laevis a. Aipysurus laevis c was [28-lysine] Aipysurus laevis a, the tryptic peptide sequence relying on homology. The LD50 values of these toxins for 20g mice were 0.076 μg/g body wt. They inhibited the acetylcholine-induced contracture but did not affect the CKl-induced contracture of the isolated muscle.

Studies on Chymotrypsin-P. I. Characterization

1971 ◽  
Vol 49 (9) ◽  
pp. 999-1004 ◽  
Author(s):  
M. C. Shaw ◽  
T. Viswanatha
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Physicochemical Properties ◽  
Gel Filtration ◽  
Amino Acid Residues ◽  
The Difference ◽  
Filtration Technique

The physicochemical properties of chymotrypsin-P obtained by the papain activation of chymotrypsinogen have been investigated. The molecular weight of this enzyme as determined by gel filtration technique has been found to be 24 000 ± 1000. The amino acid residues occupying the N-terminal positions and the composition of the B- and C-chains of chymotrypsin-P are identical with those found in α-chymotrypsin. Thus the difference between the two enzymes is restricted to the composition of their A-chains.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

Isolation of Two Bovine Amelogenin Peptides and Their Amino Acid Sequences

1987 ◽  
Vol 1 (2) ◽  
pp. 276-281 ◽  
Author(s):  
J.-H. Yeh ◽  
T. Takagi ◽  
S. Sasaki
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Sequences ◽  
Polyacrylamide Gel ◽  
The Other ◽  
Edman Degradation ◽  
Amino Acid Residues ◽  
Peptide Fractions

Two peptide fractions of bovine amelogenin having a highly aggregative property to form polymers were purified by chromatography, SDS-polyacrylamide gel electrophoresis, and HPLC. Amino acid sequences of purified peptides were determined by automated Edman degradation. One peptide was found to be composed of 63 amino acid residues having a molecular weight of 7105, and the other of 86 residues having that of 9683. The sequence of the smaller peptide was identical to the C-terminal 63 residues of the amelogenin molecule of 170 residues previously reported, but the larger contained eight residues which are absent in the amelogenin sequence. There is a possibility that the latter peptide might be synthesized independently from mRNA spliced at different positions.

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa

1978 ◽  
Vol 56 (10) ◽  
pp. 927-933 ◽  
Author(s):  
W. S. Lin ◽  
M. Kapoor
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamine Synthetase ◽  
Neurospora Crassa ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Affinity Column ◽  
Subunit Structure ◽  
Α Helix

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate–Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate α-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360 000 and as having a sedimentation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate α-helix content of 23–24%. The subunit generated by treatment with urea was found to be 45 000 daltons by gel filtration methods and a molecular weight of 46 000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45 000 daltons.

scholarly journals Cloning and Comparison of fliC Genes and Identification of Glycosylation in the Flagellin ofPseudomonas aeruginosa a-Type Strains

1998 ◽  
Vol 180 (12) ◽  
pp. 3209-3217 ◽  
Author(s):  
Cynthia D. Brimer ◽  
T. C. Montie
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Type Strains

ABSTRACT Pseudomonas aeruginosa a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of severalPseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The flagellin amino acid sequences of several a-type strains (170018, 5933, 5939, and PAK) were compared, and that of 170018 was compared with that of PAO1, a b-type strain. The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results. A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939. These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody. Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.

Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

1999 ◽  
Vol 65 (7) ◽  
pp. 3001-3007 ◽  
Author(s):  
Frederic Chavagnat ◽  
Michael G. Casey ◽  
Jacques Meyer
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Streptococcus Thermophilus ◽  
Maximal Activity ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
T7 Promoter ◽  
Reading Frame ◽  
Endopeptidase Activity

ABSTRACT The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of thepepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7

1992 ◽  
Vol 38 (9) ◽  
pp. 891-897 ◽  
Author(s):  
Hiroshi Tsujibo ◽  
Yukio Yoshida ◽  
Katsushiro Miyamoto ◽  
Chiaki Imada ◽  
Yoshiro Okami ◽  
...  
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Amino Terminal

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 °C, respectively. Chi-A was stable in the range of pH 5–10 up to 40 °C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulons WL-12. Key words: marine bacterium, Alteromonas sp., chitinase.

scholarly journals Polymeric C-terminal cross-linked material from type-I collagen. A modified method for purification, anomalous behaviour on gel filtration, molecular weight estimation, carbohydrate content and lipid content

1980 ◽  
Vol 189 (1) ◽  
pp. 111-124 ◽  
Author(s):  
N D Light ◽  
A J Bailey
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Type I Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Type I ◽  
Molecular Weight Range ◽  
Carbohydrate Analysis ◽  
Weight Range

Polymeric cross-linked C-terminal peptide material (poly-alpha 1CB6) from mature bovine tendon type-I collagen was prepared and purified by a modification of the method previously described [Light & Bailey (1980) Biochem. J. 185, 373-381]. Poly-alpha 1CB6 was shown to exhibit concentration-dependent aggregation effects on gel filtration due to interaction with a filtration medium. The material had an amino acid content that was very similar to a mixture of alpha 1CB6 and alpha 1CB5. The material was shown to be polydisperse with a mol.wt. range of 50 000-350 000, but chromatographic fractions were relatively homogeneous over this molecular weight range with respect to amino-acid composition. The heterogeneity of the material was not due to incomplete CNBr peptide cleavage, as poly-alpha 1CB6 did not contain detectable quantities of methionine. The material showed no discrete bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but gave a constant blue stain throughout the molecular weight range described above. Lipid analysis showed that the partially purified material contained elevated levels of stearate when compared to the crude CNBr-digested starting material. This may indicate the specific association of a stearic-acid-rich lipid with the peptide material. On carbohydrate analysis poly-alpha 1CB6 was shown to contain only galactose and glucose at levels of 0.72 and 0.28% respectively. The carbohydrate and amino acid analyses indicated that (alpha 1CB6)2-(alpha 1CB5)1 may be the basic cross-linked structural unit of poly-alpha 1CB6)2-(alpha 1CB5)1 units, although the carbohydrate analysis indicated that the higher molecular weight oligomers may be enriched in alpha 1CB6.

scholarly journals Thermostable NADP+-Dependent Medium-Chain Alcohol Dehydrogenase from Acinetobacter sp. Strain M-1: Purification and Characterization and Gene Expression inEscherichia coli

2000 ◽  
Vol 66 (12) ◽  
pp. 5231-5235 ◽  
Author(s):  
Akio Tani ◽  
Yasuyoshi Sakai ◽  
Takeru Ishige ◽  
Nobuo Kato
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Purification And Characterization ◽  
Gene Coding ◽  
Molecular Masses ◽  
Expression Inescherichia Coli

ABSTRACT NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C2 to C14 and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed inEscherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes.

scholarly journals The isolation, properties and amino acid sequence of erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata

1972 ◽  
Vol 130 (2) ◽  
pp. 547-555 ◽  
Author(s):  
N. Tamiya ◽  
H. Abe
Keyword(s):  
Amino Acid ◽  
Aspartic Acid ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Amino Acid Residues ◽  
Sea Snake ◽  
Acetylcholine Contracture ◽  
Laticauda Semifasciata ◽  
A Minor ◽  
Erabutoxin B

Erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata, was isolated in pure form by repeated column chromatography on CM-cellulose columns. The toxin was crystallizable and monodisperse in rechromatography, disc electrophoresis and isoelectric focusing (isoelectric point, pH9.23–9.25). The molecular weight of the toxin, as estimated by gel filtration, was 7000. The toxin showed the same lethal activity to mice (0.13μg/g body wt., intramuscular injection) and the same effect on isolated frog muscle as erabutoxins a and b, the main toxic components of the venom. The toxin inhibited the acetylcholine contracture but not the potassium chloride contracture of muscle. Erabutoxin c consisted of 62 amino acid residues, containing one fewer lysine and one more histidine than erabutoxin a and one fewer lysine and one more aspartic acid (or asparagine) than erabutoxin b. Erabutoxin c was reduced, S-carboxymethylated and hydrolysed with trypsin. The only fragment different from the corresponding fragments from erabutoxin b was hydrolysed further with pepsin. One of the peptic fragments, which was assumed to have the aspartic acid (or asparagine) residue in question at the C-terminal end, was treated with carboxypeptidase A. The C-terminal residue was found to be an asparagine. It was therefore concluded that erabutoxin c was [51-asparagine]-erabutoxin b.

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