scholarly journals Panulirus interruptus hemocyanin. The amino acid sequence of subunit b and anomalous behaviour of subunits a and b on polyacrylamide gel electrophoresis in the presence of SDS

1988 ◽  
Vol 178 (2) ◽  
pp. 403-412 ◽  
Author(s):  
Peter A. JEKEL ◽  
Henk J. BAK ◽  
Nell M. SOETER ◽  
Johan M. VEREIJKEN ◽  
Jaap J. BEINTEMA
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Anomalous Behaviour ◽  
Panulirus Interruptus ◽  
Subunit B

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

16 BIOCHEMICAL ANALYSIS OF COMPONENT IN SEMINAL GEL SECRETED WITH BOAR SEMEN

2015 ◽  
Vol 27 (1) ◽  
pp. 100
Author(s):  
G. Takahashi ◽  
M. Maeda ◽  
Y. Kimura ◽  
H. Funahashi
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Sugar ◽  
Polyacrylamide Gel ◽  
High Fertility ◽  
Rp Hplc ◽  
Ms Analysis ◽  
Freeze Dried

Seminal gel (SG), a part of semen, of the boar originates from secretions from the Cowper's gland and has a high viscosity and water-holding capacity, preventing backflow of semen at natural mating. However, there are is little information available about biochemical and functional characteristics of boar SG. In this study, as a first step to elucidate the chemical features of the SG, we examined the structure of O-glycans and the primary structure of protein from the boar SG. Seminal gel was collected from ejaculated semen of a Berkshire boar with high fertility and freeze-dried. Samples were preserved in a refrigerator until experiments were conducted. For Exp. 1 the presence of O-glycans in SG was confirmed by detection of the amino sugar, galactosamine (GalNH2), from acid hydrolysis of GalNAc. The freeze-dried SG (1 mg) was hydrolyzed with 4N trifluoroacetic acid at 110°C for 2 h. The resulting amino sugar was labelled with phenyl isothiocyanate (PITC) and then analysed by RP-HPLC. The GalNAc was detected as a main amino sugar, suggesting that the SG contains O-glycosylated glycoprotein. For Exp. 2 the O-glycans were prepared from the freeze-dried SG (5 mg) by hydrazinolysis at 100°C for 2 h. After N-acetylation, the O-glycans were pyridylaminated. The structures were identified by anion-exchange HPLC, size-fractionation HPLC, glycosidase digestion, and ESI-MS and MS/MS analysis. Almost all glycans were digested by α2–3,6-sialidasae, indicating that these O-glycans are sialylated and give the glycoproteins viscosity. Furthermore, the MS analysis showed that the de-sialylated O-glycans consist of HexNAc-PA (m/z 300.0) and Hex-HexNAc-PA (m/z 462.0) and major glycans are di- or tri-saccharides. For Exp. 3 proteins in the SG were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition with 5% 2-mercaptoethanol. Proteins were stained with Coomassie Brilliant Blue R-250. Three bands (~160, 140, and 70 kDa) were found on 7.5% polyacrylamide gel, but two bands (160, 140 kDa) were converted to ~130 kDa after the sialidase digestion, indicating that native two proteins (160 and 140 kDa) may be highly sialylated. For Exp. 4 internal amino acid sequence was analysed using one of the peptic peptides. The freeze-dried SG (5 mg) was digested with porcine pepsin in 5% formic acid at 37°C for 3 h. The resulting peptides were separated by RP-HPLC. N-terminal sequence of one of the peptic peptides was WSEKYGIPGGKAH. The amino acid sequence showed a high homology with tyrosine-protein kinase ZAP-70. These results suggest that boar SG contains mucin-like glycoproteins carrying heavily sialylated O-glycans. Additionally, the current study suggests a possibility that some protein components of the boar SG derive from high concentration of the kinase in (dead) sperms.

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.

scholarly journals Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

1969 ◽  
Vol 113 (3) ◽  
pp. 489-499 ◽  
Author(s):  
C. R. Parish ◽  
G. L. Ada
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Cyanogen Bromide ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Lysine Residues

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as ∈-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ‘C,D’ complex and a presumed ‘AB’ fragment. 4. The sum of the amino acid analyses of fragments A and B and the ‘C,D’ complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 ∈-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [125I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ‘C,D’ complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B–A–D–C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

scholarly journals Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma

1987 ◽  
Vol 241 (3) ◽  
pp. 685-692 ◽  
Author(s):  
P Manjunath ◽  
M R Sairam
Keyword(s):  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Terminal Residue ◽  
Acidic Proteins

Three major acidic proteins of bovine seminal plasma, BSP-A1, BSP-A2 and BSP-A3, were purified to homogeneity, by employing fast protein liquid chromatography, gel filtration and h.p.l.c. The proteins were purified on the basis of their stimulatory effect on the basal release of gonadotropins by rat anterior-pituitary cells in culture. All three proteins migrated as distinct single bands in the presence or absence of 2-mercaptoethanol in SDS/polyacrylamide-gel electrophoresis. Their Mr values were estimated to be between 15,000 and 16,500 by SDS/polyacrylamide-gel electrophoresis. Similar Mr estimates were obtained when they were subjected to gel filtration on a calibrated column of Sephadex G-75 equilibrated in 0.05 M-acetic acid, pH 3.0. However, BSP-A1 and BSP-A2 were eluted as aggregated molecules (Mr 60,000-120,000) during gel filtration on Sephadex G-200 equilibrated in 0.05 M-NH4HCO3, pH 8.5, or phosphate buffer, pH 7.0, containing 0.15 M-NaCl. In the presence of 8 M-urea both BSP-A1 and BSP-A2 were eluted at positions corresponding to Mr values of 17,000-20,000. BSP-A1 and BSP-A2 had an identical amino acid composition, which differed largely from that of BSP-A3. All three proteins contained aspartic acid as the N-terminal residue, and cysteine was identified as the C-terminal residue. BSP-A1 and BSP-A2 are glycoproteins containing galactosamine, sialic acid and neutral sugars, but BSP-A3 did not contain any covalently attached sugars. Whereas BSP-A2 and BSP-A3 were eluted unadsorbed, BSP-A1 bound to wheat-germ lectin-Sepharose 6MB and could be eluted by the competing sugar N-acetyl-D-glucosamine. Treatment of BSP-A1 and BSP-A2 with trypsin resulted in complete loss of gonadotropin-release activity, but BSP-A3 retained full activity. Antibody raised against BSP-A1 did not cross-react with BSP-A3, or vice versa. All these properties indicated marked structural differences between BSP-A3 and BSP-A1 (or BSP-A2). On the basis of amino acid composition it was concluded that BSP-A1, BSP-A2 and BSP-A3 are the same as the gonadostatins [Esch, Ling, Bohlen, Ying & Guillemin (1983) Biochem. Biophys. Res. Commun. 113, 861-867].

scholarly journals Amelogenins. Purification and partial characterization of proteins from developing bovine dental enamel

1973 ◽  
Vol 131 (3) ◽  
pp. 471-484 ◽  
Author(s):  
F. Michael Eggert ◽  
Grania A. Allen ◽  
Ralph C. Burgess
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dental Enamel ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Amino Acid Compositions ◽  
Small Proteins ◽  
Equilibrium Ultracentrifugation

1. Procedures are described for the purification of amelogenin electrophoretic components and their analysis for homogeneity by polyacrylamide-gel electrophoresis at both acidic and alkaline pH values. 2. Most of these components belonged to two main groups, termed the J group and the C group after their major electrophoretic components. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis indicated that, within each group, proteins were of similar size, but the C-group proteins were larger than those of the J group. 3. By sedimentation-equilibrium ultracentrifugation and amino acid analysis, the four J-group components were found to be very small proteins (mol. wt. 5500–3000) and, except for one, similar in amino acid composition. The components of the C group were found to be proteins of moderate size (mol. wt. 16800–16100) with very similar amino acid compositions. A third minor amelogenin group of intermediate size was also found, but not further analysed. Details of the results of the ultracentrifuge studies are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50014 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. 4. Two of the J-group components were similar to amelogenins isolated by other workers. 5. All amelogenins analysed were rich in proline, glutamic acid, histidine and methionine, and contained no half-cystine. Their amino acid compositions, combined with their molecular weights, serve to distinguish the amelogenins from both collagens and keratins.

scholarly journals Purification and properties of a cellulase from Aspergillus niger

1977 ◽  
Vol 165 (1) ◽  
pp. 33-41 ◽  
Author(s):  
P L Hurst ◽  
J Nielsen ◽  
P A Sullivan ◽  
M G Shepherd
Keyword(s):  
Amino Acid ◽  
Aspergillus Niger ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Cellulolytic Enzyme ◽  
Protein Amino Acid ◽  
Ph Optimum

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.

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