The Experts below are selected from a list of 9 Experts worldwide ranked by ideXlab platform
Sabine Otto - One of the best experts on this subject based on the ideXlab platform.
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Purification of the large mobilization protein of plasmid RSF1010 and characterization of its site-specific DNA-cleaving/DNA-joining activity
European journal of biochemistry, 1993Co-Authors: Eberhard Scherzinger, Volker Kruft, Sabine OttoAbstract:A site-specific and strand-specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this nicking at the duplex oriT in vitro using purified preparations of the three known RSF1010-mobilization proteins: MobA (78-kDa form of RSF1010 primase), MobB and MobC [Scherzinger, E., Lurz, R., Otto, S. & Dobrinski, B. (1992) Nucleic Acids Res. 20, 41–48]. In this study we report the purification of MobA to apparent homogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the single-stranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by single nucleotides at the 5′ or 3′ end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11-residue oligonucleotide AAGTGCGCCCT which is cleaved at the 3′ side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5′-phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3′ end at least the seven nucleotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a tyrosyl phosphate. Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with Lysine Carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22–30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex.
Eberhard Scherzinger - One of the best experts on this subject based on the ideXlab platform.
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Purification of the large mobilization protein of plasmid RSF1010 and characterization of its site-specific DNA-cleaving/DNA-joining activity
European journal of biochemistry, 1993Co-Authors: Eberhard Scherzinger, Volker Kruft, Sabine OttoAbstract:A site-specific and strand-specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this nicking at the duplex oriT in vitro using purified preparations of the three known RSF1010-mobilization proteins: MobA (78-kDa form of RSF1010 primase), MobB and MobC [Scherzinger, E., Lurz, R., Otto, S. & Dobrinski, B. (1992) Nucleic Acids Res. 20, 41–48]. In this study we report the purification of MobA to apparent homogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the single-stranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by single nucleotides at the 5′ or 3′ end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11-residue oligonucleotide AAGTGCGCCCT which is cleaved at the 3′ side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5′-phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3′ end at least the seven nucleotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a tyrosyl phosphate. Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with Lysine Carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22–30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex.
Randal A. Skidgel - One of the best experts on this subject based on the ideXlab platform.
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247 – Lysine Carboxypeptidase
Handbook of Proteolytic Enzymes, 2004Co-Authors: Randal A. SkidgelAbstract:Publisher Summary This chapter focuses on the structural chemistry and the biological aspects of Carboxypeptidase N (CPN) or Lysine Carboxypeptidase. CPN cleaves C-terminal Arg or Lys from a variety of synthetic and naturally occurring peptides. In general, the enzyme cleaves Lys faster than Arg and Ala is the preferred penultimate residue in most cases. Human CPN is a tetrameric enzyme of 280 kDa consisting of a dimer of heterodimers. Each heterodimer contains one catalytic subunit, with a reported molecular mass ranging from 44 to 55 kDa in SDS-PAGE, and one noncatalytic 83 kDa subunit. The gene for the catalytic subunit is on chromosome 10, whereas the gene for the regulatory subunit is on chromosome 8p22–p23. The genomic structure of the mouse 50 kDa subunit is very similar to that of CPE but is quite different from those of members of the more distantly related CPA/B subfamily (M14 subfamily A). CPN can be readily purified from outdated human plasma by a relatively simple two-step procedure involving DE-52 ion-exchange chromatography and affinity chromatography on arginine-Sepharose.
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247 Lysine Carboxypeptidase
Handbook of Proteolytic Enzymes (Second Edition)#R##N#Aspartic and Metallo Peptidases, 2004Co-Authors: Randal A. SkidgelAbstract:Publisher Summary This chapter focuses on the structural chemistry and the biological aspects of Carboxypeptidase N (CPN) or Lysine Carboxypeptidase. CPN cleaves C-terminal Arg or Lys from a variety of synthetic and naturally occurring peptides. In general, the enzyme cleaves Lys faster than Arg and Ala is the preferred penultimate residue in most cases. Human CPN is a tetrameric enzyme of 280 kDa consisting of a dimer of heterodimers. Each heterodimer contains one catalytic subunit, with a reported molecular mass ranging from 44 to 55 kDa in SDS-PAGE, and one noncatalytic 83 kDa subunit. The gene for the catalytic subunit is on chromosome 10, whereas the gene for the regulatory subunit is on chromosome 8p22–p23. The genomic structure of the mouse 50 kDa subunit is very similar to that of CPE but is quite different from those of members of the more distantly related CPA/B subfamily (M14 subfamily A). CPN can be readily purified from outdated human plasma by a relatively simple two-step procedure involving DE-52 ion-exchange chromatography and affinity chromatography on arginine-Sepharose.
Volker Kruft - One of the best experts on this subject based on the ideXlab platform.
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Purification of the large mobilization protein of plasmid RSF1010 and characterization of its site-specific DNA-cleaving/DNA-joining activity
European journal of biochemistry, 1993Co-Authors: Eberhard Scherzinger, Volker Kruft, Sabine OttoAbstract:A site-specific and strand-specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this nicking at the duplex oriT in vitro using purified preparations of the three known RSF1010-mobilization proteins: MobA (78-kDa form of RSF1010 primase), MobB and MobC [Scherzinger, E., Lurz, R., Otto, S. & Dobrinski, B. (1992) Nucleic Acids Res. 20, 41–48]. In this study we report the purification of MobA to apparent homogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the single-stranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by single nucleotides at the 5′ or 3′ end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11-residue oligonucleotide AAGTGCGCCCT which is cleaved at the 3′ side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5′-phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3′ end at least the seven nucleotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a tyrosyl phosphate. Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with Lysine Carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22–30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex.
