scholarly journals Strain-specific differences in the proline-rich proteins and glycoproteins induced in rat salivary glands by chronic isoprenaline treatment

1985 ◽  
Vol 230 (2) ◽  
pp. 369-378 ◽  
Author(s):  
M G Humphreys-Beher
Keyword(s):  
Amino Acid ◽  
Salivary Glands ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Difference ◽  
Marker Enzyme ◽  
Parotid Glands ◽  
Amino Acid Compositions ◽  
Submandibular Glands

Parotid and submandibular glands were isolated from five strains of rat after chronic injection of the β-adrenergic receptor agonist isoprenaline (isoproterenol). The glands were observed to have undergone a marked increase in wet weight, owing to hypertrophy and hyperplasia. The 100 000 g soluble fraction of gland cell lysates were extracted with 10% (w/v) trichloroacetic acid, and the soluble material subsequently analysed by SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis. By this procedure, evidence was obtained for the induction, in isoprenaline-treated parotid and submandibular glands, of proline-rich proteins with apparent Mr values ranging from 20 000 to 40 000. Heterogeneity was evident in the proteins produced for a specific gland between the rat strains, although the amino acid compositions were the same. Products from induced mRNAs translated in vitro had similar mobilities in SDS/polyacrylamide gels, despite the apparent difference in mobility of trichloracetic acid-extracted proline-rich proteins from the various strains. Strain-specific differences were noted for the proline-rich glycoproteins from control salivary glands as well as those induced as a consequence of isoprenaline treatment. Although the glycoproteins had similar amino acid compositions, there was considerable heterogeneity in the carbohydrate compositions for these proteins, suggesting that the differences were the result of post-translational modifications during glycosylation. Induction of the increased activity of the Golgi membrane marker enzyme UDP-galactose:2-acetamido-2-deoxy-D-glucosamine 4 β-galactosyltransferase (EC 2.4.1.22) occurred to the same extent in the parotid glands of all strains examined. There was no change in the specific activity of a second enzyme, UDP-galactose:N-acetylgalactosaminyl-protein 3 β-galactosyltransferase (no EC designation).

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.

Purification, characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme

2002 ◽  
Vol 69 (3) ◽  
pp. 419-431 ◽  
Author(s):  
SUBHADRA PRIYADARSHINI ◽  
VINOD K. KANSAL
Keyword(s):  
Antibacterial Activity ◽  
Amino Acid ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Buffalo Milk ◽  
Size Exclusion ◽  
Sds Page ◽  
Wide Range

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39·3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56·5% homology with bovine milk lysozyme and 30·4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.

scholarly journals Characterization of the d-Xylulose 5-Phosphate/d-Fructose 6-Phosphate Phosphoketolase Gene (xfp) from Bifidobacterium lactis

2001 ◽  
Vol 183 (9) ◽  
pp. 2929-2936 ◽  
Author(s):  
Leo Meile ◽  
Lukas M. Rohr ◽  
Thomas A. Geissmann ◽  
Monique Herensperger ◽  
Michael Teuber
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Guanosine Monophosphate ◽  
Bifidobacterium Lactis ◽  
Terminal Amino ◽  
Cloned Gene

ABSTRACT A d-xylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase (Xfp) from the probioticBifidobacterium lactis was purified to homogeneity. The specific activity of the purified enzyme with d-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme.Km values for d-xylulose 5-phosphate and d-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (namedxfp) encoding 825 amino acids. The xfp gene was identified on the chromosome of B. lactis with the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from the xfp gene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes, pta andguaA, were localized adjacent to xfp on theB. lactis chromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes inMycobacterium tuberculosis. However, xfp is transcribed in B. lactis as a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase.

scholarly journals Purification and characterization of a phospholipase A2 from the venom of the coral snake, Micrurus fulvius microgalbineus (Brown and Smith)

1979 ◽  
Vol 179 (3) ◽  
pp. 603-606 ◽  
Author(s):  
L D Possani ◽  
A C Alagòn ◽  
P L Fletcher ◽  
M J Varela ◽  
J Z Juliá
Keyword(s):  
Amino Acid ◽  
Phospholipase A2 ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Coral Snake ◽  
Phospholipase Activity ◽  
Crude Venom

A phospholipase A2 was purified from the Mexican coral snake Micrurus fulvius microgalbieus (Brown and Smith). Gel filtration of the soluble crude venom on Sephadex g-50 resolved five fractions, of which fraction II had 98% of the total phospholipase activity. This fraction was rechromatographed on a CM-cellulose column that resolved eight fractions, four of which had an important phospholipase activity. The first fraction (II-1) was homogeneous by polyacrylamide-gel electrophoresis and displayed a phospholipase specific activity of 920 units/mg of protein. The apparent molecular weight as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 14000. The amino acid analysis revealed the presence of 119 amino acid residues, with 12 half-cystines. the N-terminal sequence was shown to be Ser-Leu-Leu-Asx-Phe-Lys-Asx-Met-Ile-Glu-Ser-Thr..., which is homologous with that of phospholipases from other snake venoms.

scholarly journals Enzymatic Properties of a Novel Liquefying α-Amylase from an Alkaliphilic Bacillus Isolate and Entire Nucleotide and Amino Acid Sequences

1998 ◽  
Vol 64 (9) ◽  
pp. 3282-3289 ◽  
Author(s):  
Kazuaki Igarashi ◽  
Yuji Hatada ◽  
Hiroshi Hagihara ◽  
Katsuhisa Saeki ◽  
Mikio Takaiwa ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mass ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Amino Acid Sequences ◽  
Ph Optimum ◽  
Calculated Molecular Mass

ABSTRACT A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein−1, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)8 barrel motif in domain A.

scholarly journals Purification and properties of guinea-pig submandibular-gland kallikrein

1983 ◽  
Vol 209 (1) ◽  
pp. 125-134 ◽  
Author(s):  
F Fiedler ◽  
M J C Lemon ◽  
C Hirschauer ◽  
G Leysath ◽  
F Lottspeich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Ethyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Amino Acid Residues ◽  
Purification And Properties ◽  
Order Of Magnitude

Guinea-pig submandibular kallikrein has been purified from the glands to electrophoretic homogeneity by conventional procedures. The enzyme is active as a kininogenase, releasing kallidin at a rate of 462 micrograms/min per mg of protein from bovine kininogen, and proved potently hypotensive in the guinea pig and in the dog, properties which indicate its tissue kallikrein nature. The specific activity determined on the substrate N-alpha-benzoyl-L-arginine ethyl ester (11.1 mumol/min per mg of protein) is much lower than that measured with N-acetyl-L-phenylalanyl-L-arginine ethyl ester (483 mumol/min per mg of protein). The latter value is of an order of magnitude comparable with the specific activities of other tissue kallikreins determined with this sensitive kallikrein substrate. The enzyme is a glycoprotein consisting of 237 amino acid residues and containing three to four glucosamine molecules. Its amino acid composition is not identical with that reported for guinea-pig coagulating-gland kallikrein, but is remarkably similar to that of the porcine tissue kallikreins. Apparent Mr values are 29000 (sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) or 34000 (gel filtration). The amino acid sequence of the first 31 N-terminal residues was determined and was found to be closely homologous with that of other tissue kallikreins.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

scholarly journals The molybdenum–iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ‘mapping’

1976 ◽  
Vol 155 (2) ◽  
pp. 383-389 ◽  
Author(s):  
C Kennedy ◽  
R R. Eady ◽  
E Kondorosi ◽  
D K Rekosh
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Sodium Dodecyl Sulphate ◽  
Azotobacter Vinelandii ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Content ◽  
Rhizobium Japonicum ◽  
Fe Protein

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide ‘maps’ after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.

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