scholarly journals 2,4′-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) from Alcaligenes sp. 4HAP: a novel enzyme with an atypical dioxygenase sequence

1999 ◽  
Vol 344 (2) ◽  
pp. 397-402 ◽  
Author(s):  
David J. HOPPER ◽  
Mustak A. KADERBHAI
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Predicted Amino Acid Sequence ◽  
Sequencing Data ◽  
Amino Acid Sequencing ◽  
Terminal Amino Acid ◽  
Significant Similarity ◽  
Sds Page ◽  
C And N ◽  
Terminal Amino

2,4′-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) was purified to homogeneity from Alcaligenes sp. 4HAP grown on 4-hydroxyacetophenone. Measurements of the Mr of the native enzyme ranged from 81600 to 87000, whereas values of 21000 and 20379 were given by SDS/PAGE and electrospray MS respectively. The enzyme is a homotetramer and contains one atom of iron per molecule of enzyme. From C- and N-terminal analyses, primers for PCR were designed and the dad gene cloned and sequenced. The predicted amino acid sequence of dad, deduced from the nucleotide sequence, confirms the N-terminal amino acid sequencing data and contains the sequence of an internal tryptic peptide. It gave a calculated Mr of 20364. The gene was expressed in Escherichia coli and yielded active enzyme. The derived amino acid sequence does not show significant similarity to other dioxygenases or any strong similarity to protein sequences presently available in the databases.

High Expression of Human Amelogenin in E. Coli

1996 ◽  
Vol 10 (2) ◽  
pp. 187-194 ◽  
Author(s):  
D. Deutsch ◽  
E. Chityat ◽  
M. Hekmati ◽  
A. Palmon ◽  
Y. Farkash ◽  
...  
Keyword(s):  
Amino Acid ◽  
Western Blotting ◽  
Rt Pcr ◽  
Amino Acid Sequencing ◽  
Terminal Amino Acid ◽  
Expression Plasmid ◽  
Over Expression ◽  
E Coli ◽  
Sds Page ◽  
Terminal Amino

A human cDNA, encoding for the 175-aminoacid human amelogenin, was prepared by RT PCR from tooth bud mRNA and sub-cloned into pGEX-KG expression plasmid for over-expression in E. coli. The expressed protein was characterized by SDS-PAGE, Western blotting, and N-terminal amino acid sequencing.

Purification and characterization of three proteins isolated from the proteose peptone fraction of bovine milk

1993 ◽  
Vol 60 (2) ◽  
pp. 189-197 ◽  
Author(s):  
Esben S. Sørensen ◽  
Torben E. Petersen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Bovine Milk ◽  
Gel Chromatography ◽  
Major Protein ◽  
Terminal Amino Acid ◽  
Proteose Peptone ◽  
Sds Page ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

SummaryThree major proteins from the proteose peptone of bovine milk were purified by Sephadex G-75 gel chromatography, Q-Sepharose ion-exchange and additional Sephadex G-75 gel chromatography in the presence of urea. From their mobility in a gradient SDS-PAGE, the proteins were found to have molecular masses of 17, 28 and 60 kDa. The N-terminal amino acid sequence of the 17 kDa protein was found to be homologous with a camel whey protein. This protein has not previously been described in bovine milk. From the SDS-PAGE results, the 28 kDa protein was judged to be the major protein of proteose peptone, contributing ~ 25% of the total. The N-terminal amino acid sequence showed no homology to any known protein sequence, but the amino acid composition indicated that the 28 kDa protein is identical with the PP3 component from the proteose peptone fraction of bovine milk, or part of it. The 60 kDa protein was found to be bovine osteopontin, a very highly phosphorylated protein with an Arg-Gly-Asp sequence which mediates cell attachment.

C-Terminal amino acid sequence of chicken pepsinogen and its homology with sequences of other acid proteases

1980 ◽  
Vol 45 (4) ◽  
pp. 1144-1154 ◽  
Author(s):  
Miroslav Baudyš ◽  
Helena Keilová ◽  
Vladimír Kostka
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
The Other ◽  
Terminal Sequence ◽  
Terminal Part ◽  
Terminal Amino Acid ◽  
Tryptic Peptides ◽  
Terminal Amino ◽  
High Degree ◽  
Terminal Amino Acid Sequence

To determine the primary structure of the C-terminal part of the molecule of chicken pepsinogen the tryptic, chymotryptic and thermolytic digest of the protein were investigated and peptides derived from this region were sought. These peptides permitted the following 21-residue C-terminal sequence to be determined: ...Ile-Arg-Glu-Tyr-Tyr-Val-Ile-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ser-Pro-Leu-Ser.COOH. A comparison of this structure with the C-terminal sequential regions of the other acid proteases shows a high degree of homology between chicken pepsinogen and these proteases (e.g., the degree of homology with respect to hog pepsinogen and calf prochymosin is about 66%). Additional tryptic peptides, derived from the N-terminal part of the zymogen molecule whose amino acid sequence has been reported before, were also obtained in this study. This sequence was extended by two residues using an overlapping peptide. An ancillary result of this study was the isolation of tryptic peptides derived from other regions of the zymogen molecule.

scholarly journals The NH2- and COOH-terminal amino acid sequence of nuclear protein A24.

1976 ◽  
Vol 251 (19) ◽  
pp. 5901-5903 ◽  
Author(s):  
M O Olson ◽  
I L Goldknopf ◽  
K A Guetzow ◽  
G T James ◽  
T C Hawkins ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Nuclear Protein ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

scholarly journals Factor D̄ of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site

1980 ◽  
Vol 187 (3) ◽  
pp. 863-874 ◽  
Author(s):  
D M Johnson ◽  
J Gagnon ◽  
K B Reid
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Alternative Pathway ◽  
Amino Acid Sequences ◽  
Serine Residue ◽  
Amino Acid Sequence Analysis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat ‘group-specific protease’ [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined.

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