PHOSPHORYLATION COUPLED TO THE OXIDATION OF SULFITE AND 2-MERCAPTOETHANOL IN EXTRACTS OF THIOBACILLUS THIOPARUS

Abstract

The effect of 10⁻⁴ M 2, 4-dinitrophenol (DNP) on the production of high energy phosphate bonds during sulfite and 2-mercaptoethanol (2-ME) oxidation by cell extracts of Thiobacillus thioparus was determined. Phosphorylation was measured indirectly by¹⁴CO₂fixation and directly by³²PO₄esterification. DNP-sensitive phosphorylation was demonstrated by coupling sulfite oxidation with the concomitant phosphorylation of adenosine monophosphate (AMP) to¹⁴CO₂fixation beginning with ribose-5-phosphate. Esterification of³²PO₄was measured at pH values of 6.4, 7.2, and 8.0 with AMP and adenosine diphosphate (ADP) as the phosphate acceptor and sulfite as the electron donor. The optimal pH for the greatest DNP-sensitive phosphorylation was 7.2 with AMP. DNP at 10⁻⁴ M significantly reduced³²PO₄esterification at all pH values tested and with the three ADP concentrations employed. Maximum DNP-sensitive phosphorylation of ADP was demonstrated with 5 μmoles of ADP at pH 7.2. The maximum P:O ratio was 0.13. With 2-ME as the nonphysiological electron donor and AMP as the phosphate acceptor, no phosphorylation above the endogenous level was measured at the three pH values tested. With ADP as the phosphate acceptor and 2-ME as the electron donor,³²PO₄esterification significantly above the endogenous level was demonstrated at pH 6.4 with 5 μmoles of ADP; this phosphorylation was sensitive to 10⁻⁴ M DNP.