Isolation and characterization of a zinc-containing metalloprotease expressed by Vibrio tubiashii

2003 ◽  
Vol 49 (8) ◽  
pp. 525-529 ◽  
Author(s):  
R B Delston ◽  
M H Kothary ◽  
K A Shangraw ◽  
B D Tall
Keyword(s):  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Terminal Amino Acid ◽  
Isolation And Characterization ◽  
Vibrio Tubiashii ◽  
Strong Homology ◽  
Isoelectric Points ◽  
Terminal Amino

A Vibrio tubiashii hemagglutinin, a protease, was purified by ammonium sulfate precipitation, gel filtration, and hydrophobic interaction chromatography. It agglutinates sheep, chicken, bovine, rabbit, guinea pig, and human erythrocytes. It has a molecular mass of 35 kDa, isoelectric points of 3.5 and 3.7, and is inhibited by ortho-phenanthro line, phosphoramidon, and Zincov. The N-terminal amino acid sequence (Ala-Gln-Ala-Thr-Gly-Thr-Gly- Pro-Gly-Gly-Asn-Gln-Lys-Thr-Gly-Gln- Tyr-Asn-Phe-Gly) has strong homology to other Vibrio proteases.Key words: Vibrio tubiashii, metalloprotease, hemagglutinin.

scholarly journals Isolation and characterization of a 30 kDa protein with antifungal activity from leaves of Engelmannia pinnatifida

1996 ◽  
Vol 316 (3) ◽  
pp. 723-727 ◽  
Author(s):  
Q. Khai HUYNH ◽  
Jeffry R. BORGMEYER ◽  
Christine E. SMITH ◽  
Leslie D. BELL ◽  
Dilip M. SHAH
Keyword(s):  
Antifungal Activity ◽  
Plant Pathogens ◽  
Gel Filtration ◽  
Self Incompatibility ◽  
Terminal Amino Acid ◽  
Reverse Phase ◽  
Ammonium Sulphate Precipitation ◽  
Isolation And Characterization ◽  
Terminal Amino

During the course of screening plants for novel antifungal activity, we found that a high-molecular-mass fraction of an extract from leaves of Engelmannia pinnatifida exhibited potent and broad-spectrum antifungal activity. In this study a 30 kDa protein from E. pinnatifida leaves was purified to homogeneity by ammonium sulphate precipitation, gel filtration, Mono-Q and C18 reverse-phase column chromatographies. The purified protein showed potent antifungal activity against various plant pathogens with as little as 50 ng. The N-terminal amino acid sequence of the purified protein was determined as XXTKFDFFTLALQXPAXF, where X indicates an unidentified residue. This sequence showed 35–50% sequence identity with purified style glycoproteins associated with self-incompatibility from wild tomato, tobacco and petunia, a phosphate-starvation-induced ribonuclease from cultured tomato cells and the SIR 63.4 kDa protein from yeast.

Isolation and characterization of β-lipolytic hormone from porcine pituitary gland

1970 ◽  
Vol 48 (9) ◽  
pp. 1017-1021 ◽  
Author(s):  
C. Gilardeau ◽  
M. Chrétien
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Pituitary Gland ◽  
Amino Acid Composition ◽  
Biological Activities ◽  
Terminal Amino Acid ◽  
Isolation And Characterization ◽  
Pituitary Glands ◽  
Terminal Amino

A lipolytic substance was isolated from porcine pituitary glands. It's amino acid composition, molecular weight, N-terminal amino acid, isoelectric point, and biological activities are reported. These results are compared to the corresponding values of sheep β-lipolytic hormone.

scholarly journals Isolation and characterization of lung connective-tissue glycoproteins

1982 ◽  
Vol 203 (3) ◽  
pp. 593-601 ◽  
Author(s):  
C Lafuma ◽  
M Moczar ◽  
L Robert
Keyword(s):  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Lung Parenchyma ◽  
Isolation And Characterization ◽  
Isoelectric Points ◽  
And Function

1. Glycoproteins of hamster, rat and baboon lung parenchyma were investigated by using [14C]glucosamine incorporation in vitro followed by sequential extraction of the macromolecular components and characterization of the glycoproteins in the extracts. 2. Slices of lung parenchyma maintained in vitro incorporated [U-14C]glucosamine linearly with time into non-diffusible macromolecules for up to 5h. All the macromolecule-associated 14C label was present as [14C]glucosamine. 3. These 14C-labelled macromolecules were extracted from previously delipidated and salt-extracted lung by 5M-guanidinium chloride in the presence of dithiothreitol and proteinase inhibitors before (extract A1) and after (extract A2) hydrolysis of the collagen by collagenase. The [14C]glucosamine-labelled glycoproteins in extracts A1 and A2 contained 55 and 5% respectively of the total [14C]glucosamine incorporated in the lung of all three species studied. 4. The [14C]glucosamine-labelled glycoproteins were analysed by gel-filtration chromatography, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing. The major [14C]glucosamine-labelled glycoproteins of baboon lung parenchyma had apparent mol.wts. of about 400 000, 140 000 and 65 000 with isoelectric points respectively of 4.8, 5.4 and 5.4. The hamster lung glycoproteins with isoelectric points of 4.1 and 5.8 were devoid of hydroxyproline and contained galactose, mannose and N-acetylglucosamine. These experiments indicate that several distinct glycoproteins are synthesized in situ by the cells of pulmonary parenchyma and may well play a role in its structure and function.

Purification of Clostridium perfringens type C theta toxin

1973 ◽  
Vol 19 (8) ◽  
pp. 881-885 ◽  
Author(s):  
A. H. W. Hauschild ◽  
A. Lecroisey ◽  
J. E. Alouf
Keyword(s):  
Clostridium Perfringens ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Terminal Amino Acid ◽  
Single Protein ◽  
Dodecyl Sulfate ◽  
Type C ◽  
Terminal Amino ◽  
Anionic Polyacrylamide

Clostridium perfringens type C theta toxin was purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The purified toxin was free of alpha, beta, delta, and kappa toxins. Electrophoresis of the toxin in both the anionic polyacrylamide disc gel and in the polyacrylamide dodecyl sulfate gel yielded single protein bands. L-Lysine was determined as the sole N-terminal amino acid. The specific hemolytic activities of two purified preparations were 3.6 × 106 and 4.8 × 106 HU/mg N; the specific toxicities were 8.1 × 103 and 7.7 × 103 mouse MLD/mg N. The molecular weight determined by the polyacrylamide – dodecyl sulfate method was 74 000.

Isolation and characterization of myoglobin and its two major isoforms from sheep heart.

1989 ◽  
Vol 35 (5) ◽  
pp. 778-782 ◽  
Author(s):  
J T Wu ◽  
R K Pieper ◽  
L H Wu ◽  
J L Peters
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Cardiac Muscle ◽  
Ammonium Sulfate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Agarose Gel ◽  
Isolation And Characterization ◽  
Isoelectric Points

Abstract We isolated myoglobin from sheep heart by homogenizing cardiac muscle in 70%-saturated ammonium sulfate, followed by chromatography on a column containing carboxymethyl(CM)-Sephadex gel. Two major isoforms of myoglobin, designated Mb 7.9 and Mb 8.1, were separated by chromatofocusing and were distinguished by their different patterns seen on either isoelectrofocusing or on electrophoresis on polyacrylamide gel. The isoelectric points of the major bands of Mb 7.9 and Mb 8.1 were 7.4 and 7.16, respectively. Both isoforms were identical in size when examined by gel filtration chromatography but differed slightly when analyzed by polyacrylamide gradient gel in the presence of sodium dodecyl sulfate. The Mr of Mb 7.9 (15,900 Da) is slightly smaller than that of Mb 8.1 (18,400 Da). When reacted against rabbit anti-sheep myoglobin, two isoforms also appeared as two nonidentical precipitin lines on agarose gel.

scholarly journals Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum

1981 ◽  
Vol 199 (1) ◽  
pp. 9-15 ◽  
Author(s):  
M Janusz ◽  
K Starościk ◽  
M Zimecki ◽  
Z Wieczorek ◽  
J Lisowski
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Room Temperature ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Guanidinium Chloride ◽  
Terminal Amino Acid ◽  
Polyproline Ii ◽  
Terminal Amino

A proline-rich polypeptide isolated from sheep colostrum is described. The molecular weight of the polypeptide determined by gel filtration is 17 200. However, in the presence of guanidinium chloride the molecular weight found is about 6000. The polypeptide contains about 22% of proline, a high proportion of non-polar amino acids, a low percentage of glycine, and no alanine, arginine and cysteine residues. The only N-terminal amino acid found is leucine. C.d. spectra in water and in 50% (v/v) trifluoroethanol suggest the presence of block sequences of proline residues forming helices of polyproline II type. The proline-rich polypeptide is soluble at 4 degrees C but is reversibly precipitated on warming to room temperature. Maximal precipitation is observed at pH 4.6 and at ionic strength above 0.6. The precipitation depends on the concentration of the polypeptide. No effect of other proteins, Ca2+ and Zn2+ ions on the precipitation of the polypeptide was found. The proline-rich polypeptide is not an amphipathic protein. The lack of effect of the polypeptide on proteolytic enzymes ruled out the possibility that it is an inhibitor of proteinases.

scholarly journals Preparation and characterization of fragments from the N-terminal end of bovine serum albumin under native conditions

1993 ◽  
Vol 296 (2) ◽  
pp. 347-350 ◽  
Author(s):  
T Saeed ◽  
A Salahuddin
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Proteolytic Digestion ◽  
Thiol Groups ◽  
Optical Studies ◽  
Terminal Amino Acid ◽  
Hydrodynamic Parameters ◽  
Terminal Amino ◽  
Domain I

The domain I of BSA, containing residues 1-183 of the protein sequence, was isolated by CNBr treatment. It was further reductively cleaved into two subfragments, N1 and N2, in 8 M urea; the subfragments were regenerated in GSH and GSSG. The fragment N and subfragments N1 and N2 were found to be homogeneous with respect to size and charge. Results for amino acid composition, N-terminal amino acid sequence, thiol groups and M(r) suggested that the fragments N1 and N2 contain residues 88-183 and 1-87 of the intact BSA respectively. Optical studies, intrinsic-viscosity measurements, gel-filtration data and derived hydrodynamic parameters, taken together with the results on proteolytic digestion, showed that fragment N, as well as its subfragments N1 and N2, exist in compact and globular conformation and that the conformation of N2 fragment is more compact than that of the N1 fragment.

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 Isolation and characterization of novel lectins from Canavalia ensiformis DC and Dioclea grandiflora Mart. ex Benth. seeds

2005 ◽  
Vol 17 (3) ◽  
pp. 315-324 ◽  
Author(s):  
Luz Marina Melgarejo ◽  
Nohora Vega ◽  
Gerardo Pérez
Keyword(s):  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Canavalia Ensiformis ◽  
Human Red Blood Cells ◽  
Isolation And Characterization ◽  
Dioclea Grandiflora ◽  
Covalently Linked ◽  
Type 3

Two lectins were isolated from Canavalia ensiformis and Dioclea grandiflora seeds. Gel filtration produced a fraction corresponding to Con A or D. grandiflora lectin while erythroagglutination assays revealed a distinct fraction presenting a lectin that agglutinates human red blood cells (RBCs) but not rabbit RBCs. Hydrophobic interaction chromatography showed that the latter fraction yielded a protein that readily agglutinates human erythrocytes; the lectin was also purified by affinity chromatography on Lac-Sepharose showing similar properties to that of the Phenyl-Sepharose-purified lectin. Despite minor differences (carbohydrate content or A1%1cm), the two lectins showed similar molecular properties in that they consisted of two non-covalently linked monomers having a Mr of 29-30 kDa and their pI values indicated that both lectins were slightly acidic proteins. The C. ensiformis lectin (CEL-II) and D. grandiflora lectin (DGL-II) specifically recognised the H-type 2 blood group (alpha-L-Fuc (1-2)-beta-D-Gal (1-4)-beta-D-GlcNAc-O-R), while binding to H-type 1, H-type 3, H-type 4, Leª or Le y was weaker. Carbohydrate inhibition of erythroagglutination showed that simple sugars were weakly recognised by the lectins, if at all. The N-terminal region presented a unique sequence hitherto found only in some Diocleinae lectins (designated type II). The overall results confirmed the existence of a second distinct lectin type, phylogenetically close to Diocleinae species. The data indicate a functional similarity among lectins of this type which possesses distinctive characteristics differentiating them from "classical" Man/Glc lectins.

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