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 Characterization of gastric mucoproteins isolated by equilibrium density-gradient centrifugation in caesium chloride

1974 ◽  
Vol 141 (3) ◽  
pp. 633-639 ◽  
Author(s):  
Bryan J. Starkey ◽  
David Snary ◽  
Adrian Allen
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Equilibrium Density ◽  
Amino Acid Residues ◽  
Gastric Mucus ◽  
Terminal Amino Acid ◽  
Terminal Amino

1. The mucoprotein from pig gastric mucus has been purified by equilibrium centrifugation in a CsCl gradient. 2. This procedure removes the non-covalently bound protein, which is closely associated with the mucoprotein and not easily removed from it by gel filtration. 3. The purified mucoprotein is separable by gel filtration into a high-molecular-weight mucoprotein A (mol.wt. 2.3×106) and a low-molecular-weight mucoprotein B/C (mol.wt. 1.15×106). 4. These two mucoproteins have the same chemical analysis namely fucose 11.3%, galactose 26%, glucosamine 19.5%, galactosamine 8.3% and protein 13.6%. 5. Mucoprotein A contains 3.1% ester sulphate. 6. These mucoproteins are isolated without enzymic digestion and have a higher protein content than the blood-group-substance mucoproteins from proteolytic digestion of gastric mucus. Detailed amino acid analysis shows that the extra protein in the non-enzymically digested material is composed of amino acids other than serine and threonine. 7. Mucoproteins A and B/C contain respectively 130 and 9 half-cystine residues per molecule of which about 78 and 6 residues are involved in disulphide linkages. 8. Cleavage of these disulphide linkages by mercaptoethanol splits both mucoproteins into four equally sized subunits of mol.wt. 5.2×105for mucoprotein A and 2.8×104for mucoprotein B/C. 9. The sole N-terminal amino acid of mucoprotein A is aspartic acid, whereas mucoprotein B/C has several different N-terminal amino acid residues.

scholarly journals Purification of a high-molecular-weight somatoliberin (growth-hormone-releasing factor) from pig hypothalami

1983 ◽  
Vol 209 (3) ◽  
pp. 643-651 ◽  
Author(s):  
J E C Sykes ◽  
P J Lowry
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Amino Acid ◽  
Response Curve ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Amino Acid Residues ◽  
Growth Hormone Releasing Factor

Preliminary observations [Sykes & Lowry (1980) J. Endocrinol. 85, 42P-43P] had suggested that the major hypothalamic somatoliberin (growth-hormone-releasing factor) was a larger peptide than the other characterized hypothalamic factors, with an elution position on Sephadex G-50 between those of neurophysin and corticotropin. The present paper reports the isolation and preliminary characterization of pig hypothalamic somatoliberin. Acid extracts of pig stalk median eminence were purified by gel filtration and preparative and analytical high-pressure liquid chromatography to yield a preparation that was specific in the release of somatotropin (growth hormone) in vitro, giving a steep dose-response curve at doses in the range 0.20-3.0 ng. Amino acid analysis revealed a non-cysteine-containing peptide with a high number of glutamate (or glutamine) and aspartate (or asparagine) residues. The peptide had about 56-57 amino acid residues and an apparent molecular weight of 6400, in keeping with its elution position on a column of Sephadex G-50.

α-Glutamic acid-β-naphthylamidase from Neisseria catarrhalis

1969 ◽  
Vol 15 (11) ◽  
pp. 1293-1300 ◽  
Author(s):  
Sidney T. Cox ◽  
Francis J. Behal
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Metal Ion ◽  
Gel Filtration ◽  
Aminopeptidase Activity ◽  
Amino Acid Residues ◽  
Ph Optimum ◽  
Hydrolysis Of ◽  
Derivatives Of

A second bacterial peptidase-like enzyme, arylamidase-II, has been isolated from cell free extracts of Neisseria catarrhalis. Arylamidase-II action is limited primarily to the hydrolysis of α-glutamic acid and α-aspartic acid derivatives of β-naphthylamine and short peptides of glutamic acid. The enzyme was purified 450-fold by gel filtration, ion exchange, and calcium phosphate chromatography. Its pH optimum and molecular weight were 7.7 and 170 000, respectively. Aside from its restricted substrate specificity, arylamidase-II has been found to be closely related in its mechanism of action, molecular weight, pH optimum, and metal ion dependence to arylamidase-I, which catalyzes the hydrolysis of neutral amino acid derivatives of β-naphthylamine. Arylamidase-II exhibits aminopeptidase activity, requiring the amino acid residues in the N-terminal and penultimate position to be of the L-configuration in order for hydrolysis to occur.

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.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

Effect of extractant and molecular size on the optical and chemical properties of soil humic acids

Soil Research ◽  
1969 ◽  
Vol 7 (3) ◽  
pp. 229 ◽  
Author(s):  
JHA Butler ◽  
JN Ladd
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Humic Acids ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Molecular Size ◽  
Carboxyl Content ◽  
Peptide Bonds ◽  
Molecular Weights ◽  
Amino Acid Contents

Humic acids extracted from soil with sodium pyrophosphate have greater proportions of lower molecular weight material, less acid-hydrolysable amino acid nitrogen contents, but greater carboxyl contents and extinction values (260 and 450 nm) than humic acids extracted subsequently from the same sample with alkali. Humic acids extracted with alkali from fresh soil samples have intermediate values. Extinction values at 260 nm are directly correlated with carboxyl contents for a given soil. Different crop histories have no significant effect on the measured properties of the extracted humic acids. An alkali-extracted humic acid has been fractionated by gel filtration into seven fractions of different nominal molecular weight ranges. As the molecular weights of the fractions increase, both aliphatic C-H (based on infrared absorption at 2900 cm-1) and acid-hydrolysable amino acid contents increase, whereas extinction values at 260 nm and carboxyl contents decrease. The infrared spectra of the high molecular weight fractions have peaks at 1650 and 1510 cm-1 which correlate with acid-hydrolysable amino acid contents and which correspond to amide I and II bands of peptide bonds. Alkaline hydrolysis to split peptide bonds eliminates both these peaks. The spectra also have peaks at 1720 and 1210 cm-1 which correlate with the carboxyl content.

scholarly journals α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

Influence of Blood Group and Secretor Status on Carbohydrate Structures in Human Gastric Mucins: Implications for Peptic Ulcer

1995 ◽  
Vol 89 (4) ◽  
pp. 405-415 ◽  
Author(s):  
R. L. Sidebotham ◽  
J. H. Baron ◽  
J. Schrager ◽  
J. Spencer ◽  
J. R. Clamp ◽  
...  
Keyword(s):  
Peptic Ulcer ◽  
Amino Acid ◽  
Blood Group ◽  
Average Length ◽  
Amino Acid Residues ◽  
Secretor Status ◽  
Increased Risk ◽  
The Mean ◽  
The Difference ◽  
Carbohydrate Chains

1. The content and distribution of carbohydrate was examined in mucus glycopolypeptides from human antral mucosae. 2. The mean amount of carbohydrate per 1000 amino acid residues was found to be similar in glycopolypeptides with A, B or H activity. It was slightly, though significantly, less in glycopolypeptides lacking these determinants, because carbohydrate chains were of a shorter average length than in the A-, B- or H-active preparations. This difference was reflected in the sizes of oligosaccharide—alcohols released from representative glycopolypeptides with alkaline borohydride. 3. Differences between A-, B- or H-active and non-secretor glycopolypeptides in terms of the mean number of carbohydrate chains per 1000 amino acid residues were found to be small, and without significance. 4. The average number of peripheral monosaccharide units per 1000 amino acid residues was greater in A-active than in H-active, and least in non-secretor, glycopolypeptides. This order was reversed for monosaccharide units incorporated into skeletal (core plus backbone) structures. The difference in each case was statistically significant. 5. These findings suggest that the increased risk of peptic ulcer associated with blood group O and non-secretor status is unlikely to be attributable to an inherent deficiency in the protective mucus layer, linked to differences between mucins that are associated with A, B or H activity. Other hypotheses linked to infection with Helicobacter pylori are examined.

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