Pyruvate formate-lyase activating enzyme is an iron-sulfur protein

Journal of the American Chemical Society

Volumn 119, Issue 31, 1997, Pages 7396-7397

  Broderick, Joan B ^a   Duderstadt, Randall E ^b   Fernandez, Daniel C ^a   Wojtuszewski, Kristi ^a   Henshaw, Timothy F ^a   Johnson, Michael K ^b  

^a AMHERST COLLEGE   (United States)

^b UNIVERSITY OF GEORGIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

LYASE;

ARTICLE; ELECTRON SPIN RESONANCE; ENZYME ACTIVATION; ENZYME ACTIVITY; ENZYME ANALYSIS; ESCHERICHIA COLI; SPECTROPHOTOMETRY;

EID: 0030749454     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja9711425     Document Type: Article

References (35)

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13. 4c The cells were grown at 30°C to mid-log phase in Terrific Broth (Tartof, K. D.; Hobbs; C. A. Bethesda Res. Lab. Focus 1987, 86, 184) and then induced by increasing the temperature to 42 °C. Cells were harvested and lysed by sonication, and the soluble extract was treated with Polymin (0.45% final concentration) to precipitate nucleic acids. The clarified soluble extract was loaded onto a Sephacryl S-200 column (5 × 60 cm) and eluted with 50 mM Tris/200 mM NaCl/1 mM DTT, pH 7.2. Fractions were analyzed by SDS-PAGE, and those containing pure AE were pooled and concentrated. All purification steps were performed under an Ar atmosphere.
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15. Iron and sulfide analyses were carried out using the published procedures (Beinert, H. Methods Enzymol. 1978, 54, 435-445. Beinert, H. Anal. Biochem. 1983, 131, 373-378). Routine protein analyses were carried out using the published procedure (Bradford, M. Anal Biochem. 1976, 72, 248) and were converted to actual protein concentrations using a conversion factor of 0.65 derived from direct amino acid analysis carried out at the MCB Core Facility, University of Massachusetts.
16. Iron and sulfide analyses were carried out using the published procedures (Beinert, H. Methods Enzymol. 1978, 54, 435-445. Beinert, H. Anal. Biochem. 1983, 131, 373-378). Routine protein analyses were carried out using the published procedure (Bradford, M. Anal Biochem. 1976, 72, 248) and were converted to actual protein concentrations using a conversion factor of 0.65 derived from direct amino acid analysis carried out at the MCB Core Facility, University of Massachusetts.
17. 5
18. 4b The apparent discrepancy in the specific activities of recombinant and wild-type enzymes is currently under investigation.
19. + cluster and a slow-relaxing axial resonance, g = 2.01, 2.01, 1.97, that was still observable without broadening at 100 K. However, spin quantitations indicate that both are minor species accounting for <0.01 spins per monomer.
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