A simplified route to [<sup>14</sup>C] dimethylsuberimidate for polypeptide crosslinking studies

Packman, Leonard C.; Smith, David J.

doi:10.1016/0014-5793(78)81166-1

Cited by 3 publications

(4 citation statements)

References 13 publications

(7 reference statements)

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“…3). The deduced N-terminal sequence, Met-Asn-Tyr-Thr-Lys-Phe-Asp, corresponds to that determined by Edman degradation of type III CAT purified from E. coli J53 containing R387 (Fitton et al, 1978), and the overall deduced amino acid sequence is in agreement with the partial protein sequence determined by Packman (1978).…”

Section: Resultssupporting

confidence: 78%

Nucleotide sequence analysis and overexpression of the gene encoding a type III chloramphenicol acetyltransferase

Murray

Hawkins

Keyte

et al. 1988

Biochemical Journal

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The gene catIII, encoding a type III enterobacterial chloramphenicol acetyltransferase, was cloned from the transmissible plasmid R387 into pBR322 and bacteriophage M13 mp8. Nucleotide sequence analysis of 1160 bp of DNA identified an open reading frame encoding a protein of 213 amino acid residues and a calculated molecular mass of 24965 Da. The predicted N-terminal sequence is identical with that determined by Edman degradation of chloramphenicol acetyltransferase purified from Escherichia coli harbouring R387. Sequences equivalent to the consensus motifs for initiation and rho-factor-independent termination of transcription in E. coli occur 5' and 3' to the catIII open reading frame. In contrast with the catI gene, present on transposon Tn9 and many enterobacterial plasmids, expression of catIII is not subject to cyclic AMP-mediated catabolite repression in vivo and there is no sequence in the 5' non-coding DNA that resembles that deduced as the consensus for the binding of cyclic AMP receptor protein. Unique restriction-endonuclease cleavage sites were introduced adjacent to the catIII reading frame by using oligonucleotide-directed mutagenesis to facilitate insertion into E. coli expression vectors. Fully active chloramphenicol acetyltransferase represents 30-50% of the soluble protein component of cell-free extracts of E. coli containing the appropriate plasmids.

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Section: Resultssupporting

confidence: 78%

Nucleotide sequence analysis and overexpression of the gene encoding a type III chloramphenicol acetyltransferase

Murray

Hawkins

Keyte

et al. 1988

Biochemical Journal

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“…During the early stages of purification, recovery of enzyme activity was less than expected. Purification of chloramphenicol acetyltransferase from E. coli J53 containing plasmid R429 or plasmid R387 alone has given reproducibly typical recoveries of chloramphenicol acetyltransferase after the dialysis step of 95-100% (Packman, 1978). From E. coli J53 (R429, R387), however, only 73% of the enzyme activity is recovered under the same conditions (Table 1).…”

Section: Resultsmentioning

confidence: 95%

“…Protein in crude extracts was determined by the method of Lowry et al (1951), with bovine serum albumin as standard. Homogeneous preparations of enzyme were estimated by amino acid analysis by using the known amino acid composition of the enzymes concerned (Packman, 1978). Amino acid analysis Hydrolysis of protein was carried out in the presence of 6M-HCI, containing l0pM-phenol, in vacuo at 1050C for 24h.…”

Section: Protein Determinationmentioning

confidence: 99%

“…Although the purified hybrid chloramphenicol acetyltransferases were identified tentatively as a3fl and a2/32 with regard to subunit composition, it was necessary to confirm this presumptive assignment. From the amino acid composition of the a4 and /4 species of chloramphenicol acetyltransferase (Packman, 1978), it is possible to compute and predict the ratio of aspartic acid to glutamic acid expected for any enzyme comprising any combination of a and /3 polypeptide chains in a tetrameric association. The observed (duplicate samples) and predicted ratios were compared for parental and hybrid enzymes ( Table 3).…”

Section: Determination Of the Subunit Composition Of Hybrid Chloramphmentioning

confidence: 99%

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The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts

Packman¹,

Shaw²

1981

Biochemical Journal

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1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.

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Syntheses with isotopically labelled carbon. Methyl iodide, formaldehyde and cyanide

Finn¹,

Boothe²,

Vora³

et al. 1984

The International Journal of Applied Radiation and Isotopes

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A simplified route to [¹⁴C] dimethylsuberimidate for polypeptide crosslinking studies

Cited by 3 publications

References 13 publications

Nucleotide sequence analysis and overexpression of the gene encoding a type III chloramphenicol acetyltransferase

Nucleotide sequence analysis and overexpression of the gene encoding a type III chloramphenicol acetyltransferase

The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts

Syntheses with isotopically labelled carbon. Methyl iodide, formaldehyde and cyanide

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A simplified route to [14C] dimethylsuberimidate for polypeptide crosslinking studies

Cited by 3 publications

References 13 publications

Nucleotide sequence analysis and overexpression of the gene encoding a type III chloramphenicol acetyltransferase

Nucleotide sequence analysis and overexpression of the gene encoding a type III chloramphenicol acetyltransferase

The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts

Syntheses with isotopically labelled carbon. Methyl iodide, formaldehyde and cyanide

Contact Info

Product

Resources

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A simplified route to [¹⁴C] dimethylsuberimidate for polypeptide crosslinking studies