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.

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.

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.

Cloning of a bovine renal epithelial Na+ channel subunit

1997 ◽  
Vol 272 (6) ◽  
pp. 1-1
Author(s):  
C. M. Fuller ◽  
M. S. Awayda ◽  
M. P. Arrate ◽  
A. L. Bradford ◽  
R. G. Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Na Channel ◽  
Terminal Portion ◽  
Human Sequence ◽  
Relative Molecular Weight ◽  
Epithelial Na Channel

Pages C641-C654: C. M. Fuller, M. S. Awayda, M. P. Arrate, A. L. Bradford, R. G. Morris, C. M. Canessa, B. C. Rossier, and D. J. Benos. “Cloning of a bovine renal epithelial Na+ channel subunit.” Page C642: Fig. 1 contains two errors in the published sequence of the cDNA agr-bENaC clone presented. A severe COOH compression at nucleotide positions 1750 and 1760 resulted in a double frameshift in the COOH-terminal portion of the sequence. Correction of the nucleotide sequence causes the termination codon to fall at position 1951 (as opposed to position 2092 as previously published), predicting a translated polypeptide of 650 amino acids as opposed to 697 residues as previously reported. This shortened protein has a calculated molecuar mass of 73.4 kDa, although it is observed to migrate with a relative molecular weight of ap80,000 on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The overall homology of the nucleotide sequence with the rat and human agr-ENaC clones is slightly increased by this sequence change to 80 and 84% identities, respectively. In the COOH-terminal region, the homology increases to 53% identity from 43% identity for the rat clone and to 64% identity from 51% identity for the human sequence. A revised nucleotide and amino acid sequence is given in the revised Fig. 1. The sites of the COOH insertion are underlined and the altered amino acid sequence is given in bold. However, this sequence revision does not affect the conclusions of this or subsequent papers from our laboratory concerning this cDNA clone. The amended sequence has been deposited with GenBank (accession no. U14944). The authors apologize for any inconvenience caused by this error. (See PDF)

scholarly journals The First Archaeal ATP-Dependent Glucokinase, from the Hyperthermophilic Crenarchaeon Aeropyrum pernix, Represents a Monomeric, Extremely Thermophilic ROK Glucokinase with Broad Hexose Specificity

2002 ◽  
Vol 184 (21) ◽  
pp. 5955-5965 ◽  
Author(s):  
Thomas Hansen ◽  
Bianca Reichstein ◽  
Roland Schmid ◽  
Peter Schönheit
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Divalent Cations ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Enzyme ◽  
Start Codon ◽  
Aeropyrum Pernix

ABSTRACT An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa. The apparent Km values for ATP and glucose (at 90°C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent V max was about 35 U/mg. The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (k cat/Km ), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg2+, which was most effective, could partially be replaced with Co2+, Mn2+, and Ni2+. The enzyme had a temperature optimum of at least 100°C and showed significant thermostability up to 100°C. The coding function of open reading frame (ORF) APE2091 (Y. Kawarabayasi, Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, M. Takahashi, M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui, Y. Nagai, K. Nishijima, H. Nakazawa, M. Takamiya, S. Masuda, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, and H. Kikuchi, DNA Res. 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A. pernix, was proved by functional expression in Escherichia coli. The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A. pernix. N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091. The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors. This is the first report of the characterization of an ATP-dependent glucokinase from the domain of Archaea, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses.

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.

scholarly journals Cloning and Expression of a Phloretin Hydrolase Gene from Eubacterium ramulus and Characterization of the Recombinant Enzyme

2004 ◽  
Vol 70 (10) ◽  
pp. 6131-6137 ◽  
Author(s):  
Lilian Schoefer ◽  
Annett Braune ◽  
Michael Blaut
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Propionic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolase Activity ◽  
Cloning And Expression ◽  
Putative Promoter Region ◽  
Stem Loop

ABSTRACT Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37°C and 7.0, respectively. The Km for phloretin was 13 ± 3 μM and the k cat was 10 ± 2 s−1. The enzyme did not transform phloretin-2′-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl2 to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively.

scholarly journals Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

2002 ◽  
Vol 68 (7) ◽  
pp. 3532-3536 ◽  
Author(s):  
María J. Benito ◽  
Mar Rodríguez ◽  
Félix Núñez ◽  
Miguel A. Asensio ◽  
María E. Bermúdez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Penicillium Chrysogenum ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Extracellular Protease ◽  
Collagenolytic Activity ◽  
Sds Page

ABSTRACT An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.

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