scholarly journals Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea

1999 ◽  
Vol 339 (3) ◽  
pp. 713-720
Author(s):  
Vincent HEALY ◽  
Shawn DOONAN ◽  
Tommie V. McCARTHY
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Cloning ◽  
Peptide Mapping ◽  
Gel Filtration ◽  
Significant Degree ◽  
Prolonged Incubation ◽  
Armillaria Mellea ◽  
Cloned Cdna ◽  
Basidiomycete Fungus

We have purified an endo-exonuclease from the fruiting body of the basidiomycete fungus Armillaria mellea by using an ethanol fractionation step, followed by two rounds of column chromatography. The enzyme had an apparent molecular mass of 17500 Da and was shown to exist as a monomer by gel-filtration analysis. The nuclease was active on both double-stranded and single-stranded DNA but not on RNA. It was optimally active at pH 8.5 and also exhibited a significant degree of thermostability. Three bivalent metal ions, Mg2+, Co2+ and Mn2+, acted as cofactors in the catalysis. It was also inhibited by high salt concentrations: activity was completely abolished at 150 mM NaCl. The nuclease possessed both endonuclease activity on supercoiled DNA and a 3ʹ-5ʹ (but not a 5ʹ-3ʹ) exonuclease activity. It generated 5ʹ-phosphomonoesters on its products that, after a prolonged incubation, were hydrolysed to a mixture of free mononucleotides and small oligonucleotides ranging in size from two to eight bases. Elucidation of its N-terminal amino acid sequence permitted the cDNA cloning of the A. mellea nuclease via a PCR-based approach. Peptide mapping of the purified enzyme generated patterns consistent with the amino acid sequence coded for by the cloned cDNA. A BLAST search of the SwissProt database revealed that A. mellea nuclease shared significant amino acid similarity with two nucleases from Bacillussubtilis, suggesting that the three might constitute a distinct class of nucleolytic enzymes.

scholarly journals A STUDY OF IMMUNOGLOBULIN STRUCTURE

1966 ◽  
Vol 124 (3) ◽  
pp. 307-330 ◽  
Author(s):  
C. Baglioni ◽  
D. Cioli
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Peptide Mapping ◽  
Gel Filtration ◽  
Bence Jones Protein ◽  
Type K ◽  
The Common ◽  
L Proteins ◽  
Peptide Maps ◽  
Bence Jones Proteins

Urinary proteins of patients with myeloma, prepared by precipitation with ammonium sulphate, have been separated by gel filtration on Sephadex G-100 after reduction and aminoethylation. Many specimens separated into a major peak of Bence Jones protein and into minor peaks of albumin, a protein tentatively identified with heavy chain and a smaller molecular weight protein corresponding to the variable portion of the corresponding Bence Jones protein. The Bence Jones protein purified by gel filtration was analyzed by electrophoresis and by peptide mapping after tryptic digestion. The peptide maps of 24 type K and 20 type L Bence Jones proteins were compared. A set of common peptides was identified in the peptide maps of the Bence Jones proteins of the same type; the common peptides of type K proteins were completely different from the common peptides of type L proteins. The patterns of distinctive peptides was compared; no similarities were found between distinctive peptides of type K and of type L proteins. Some similarities were observed in the distinctive peptides of proteins of the same type. The similarities involved in many cases peptides containing cysteine, whereas similarities in other peptides were limited. This observation suggested that the amino acid sequence around the cysteines of the variable NH2-terminal half of the Bence Jones proteins may show less variability than other sequences. A few proteins of the same type differed in all their distinctive peptides, an indication that multiple amino acid differences exist between individual Bence Jones proteins. The genetic mechanisms responsible for the variability in the amino acid sequence of the NH2-terminal half of the light chains of immunoglobulins are discussed in view of the results of the comparison by peptide mapping of the Bence Jones proteins.

scholarly journals Amino acid sequence of a porcine zona pellucida glycoprotein ZP4 determined by peptide mapping and cDNA cloning

Reproduction ◽  
1994 ◽  
Vol 100 (1) ◽  
pp. 245-255 ◽  
Author(s):  
A. Hasegawa ◽  
K. Koyama ◽  
Y. Okazaki ◽  
M. Sugimoto ◽  
S. Isojima
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Cloning ◽  
Zona Pellucida ◽  
Peptide Mapping

scholarly journals Streptococcus salivarius Fimbriae Are Composed of a Glycoprotein Containing a Repeated Motif Assembled into a Filamentous Nondissociable Structure

2001 ◽  
Vol 183 (9) ◽  
pp. 2724-2732 ◽  
Author(s):  
Céline Lévesque ◽  
Christian Vadeboncoeur ◽  
Fatiha Chandad ◽  
Michel Frenette
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Surface ◽  
Polyclonal Antibodies ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Spontaneous Mutant ◽  
Streptococcus Salivarius ◽  
Streptococcal Species ◽  
Repeated Motif

ABSTRACT Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivariusfimbriae did not dissociate when they were incubated at 100°C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 × 106 to 40 × 106Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 μm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/φ, where X represents a modified amino acid residue and φ represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis andStreptococcus constellatus.

Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA

1987 ◽  
Vol 143 (2) ◽  
pp. 740-748 ◽  
Author(s):  
Micheline Misrahi ◽  
Michel Atger ◽  
Luc d'Auriol ◽  
Hugues Loosfelt ◽  
Cécile Meriel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Progesterone Receptor ◽  
Amino Acid Sequence ◽  
Complete Amino Acid Sequence ◽  
Cloned Cdna ◽  
Human Progesterone Receptor

Physicochemical properties of a novel α-L-arabinofuranosidase fromRhizomucor pusillusHHT-1

2001 ◽  
Vol 47 (8) ◽  
pp. 767-772 ◽  
Author(s):  
A KM Shofiqur Rahman ◽  
Shinya Kawamura ◽  
Masahiro Hatsu ◽  
M M Hoq ◽  
Kazuhiro Takamizawa
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Rhizomucor Pusillus ◽  
Terminal Amino ◽  
Metal Cofactors

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

scholarly journals Purification from Placenta, Amino Acid Sequence, Structure Comparisons and cDNA Cloning of Human Glutaredoxin

1995 ◽  
Vol 227 (1-2) ◽  
pp. 27-34 ◽  
Author(s):  
C. Alicia Padilla ◽  
Emilia Martinez-Galisteo ◽  
J. Antonio Barcena ◽  
Giannis Spyrou ◽  
Arne Holmgren
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Cloning ◽  
Sequence Structure

Identification and cDNA cloning of a Xenopus nucleolar phosphoprotein, xNopp180, that is the homolog of the rat nucleolar protein Nopp140

1995 ◽  
Vol 108 (10) ◽  
pp. 3339-3347 ◽  
Author(s):  
C. Cairns ◽  
B. McStay
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Cloning ◽  
Cdna Clone ◽  
Xenopus Oocyte ◽  
Ribosome Biogenesis ◽  
Nucleolar Protein ◽  
Nuclear Localisation Signal ◽  
Cdc2 Kinase

The monoclonal antibody G1C7, recognises both Xenopus nucleolin and a protein of 180 kDa present in Xenopus oocyte nucleoli. This antibody was used to obtain a cDNA clone encoding the 180 kDa protein now called xNopp180 (Xenopus nucleolar phosphoprotein of 180 kDa). Analysis of the deduced amino acid sequence from this cDNA shows that xNopp180 is almost entirely composed of alternating acidic and basic domains. We show that xNopp180 is heavily phosphorylated and that it contains multiple consensus sites for phosphorylation by casein kinase II and cdc2 kinase. In addition we show that xNopp180 is the 180 kDa antigen recognised by the monoclonal antibody No-114, thus allowing reinterpretation of previous work with this antibody. xNopp180 appears to be the Xenopus homolog of the rat nucleolar protein Nopp140. Nopp140 is a nuclear localisation signal binding protein that shuttles on curvilinear tracks between the nucleolus and the cytoplasm. Possible roles for xNopp180/Nopp140 in ribosome biogenesis are discussed.

scholarly journals Primary structure of bovine complement activation fragment C4a, the third anaphylatoxin. Purification and complete amino acid sequence

1982 ◽  
Vol 207 (2) ◽  
pp. 253-260 ◽  
Author(s):  
M A Smith ◽  
L M Gerrie ◽  
B Dunbar ◽  
J E Fothergill
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Primary Structure ◽  
Gel Filtration ◽  
Complete Amino Acid Sequence ◽  
The Third ◽  
Human Sequence ◽  
Bovine Plasma ◽  
Bovine Sequence ◽  
C 50

Purification of C4a from heat-activated bovine plasma by elution from CM-Sephadex C-50 at pH 7.4 and gel filtration on Sephadex G-50 gives a 20% yield of pure C4a. The complete amino acid sequence of bovine C4a has been determined by automatic sequencer degradation of CNBr and enzymic fragments, and by carboxypeptidase digestion. The 77-residue bovine sequence shows 12 differences from the human sequence with five of these differences occurring in the C-terminal 11 residues. The sequence of C4a confirms earlier suggestions of homology with C3a and C5a: the three sequences show an almost equal number of identities with each other. The six cysteine residues of the ‘disulphide knot’ are conserved as well as seven other residues including the C-terminal arginine.

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