Printed in Great Britain Thymidine Metabolism in Mycoplasma horninis

1965) by Mycoplasma hominis suggested that this organism might use a scavenger path-way (Fig. I) to supply thymidine (TdR) nucleotides for deoxyribonucleic acid synthesis as has been suggested for M. Zaidlawii (Razin, 1962; Smith, 1964). To determine if this was a reasonable possibility we assayed in fractions of M. hominis the activity of several enzymes involved in the synthesis of thymidylate (dTMP) and its incorporation into DNA (as the triphosphate). The methods used to grow, harvest and fractionate the organisms have been described (Stock & Gentry, 1969). Briefly, washed cell pellets were lysed by osmotic shock and freezing and thawing, and then centrifuged. The supernatant was used as the cell-free extract. Thymidine (TdR) kinase (EC 2.7. I. 21) was assayed essentially as described by Takahashi, Ueda & Ogino (1966). The aqueous reaction mixture contained 22-7 p-moles KCl, 2.5 pmoles ATP, 3 pmoles 3-phosphoglycerate, 2.5 pmoles MgCl,, I-5 pmoles trishydroxymethylaminomethane (tris), pH 7.8, I 6.7 pmoles TdR [2-14C] (5 nCi), and 0.1 ml. enzyme preparation, all in 0.5 ml. final volume. DNA nucleotidyl transferase (EC 2.7.7.7) was assayed by the method of Magee (I 962). The reaction mixture contained 10 pg. highly polymerized, heat-denatured L-cell DNA as primer; 75 nmoles each of the triphosphates of TdR, deoxyadenosine, and deoxyguanosine; I-3 nmoles [3H]deoxycytidine triphosphate (dCTP) (2.5 pCi); 0.5 pmoles 2-mercaptoethanol; 15 pmoles tris, pH 7.8; 2.5 pmoles MgCl,; and 0.1 ml. enzyme preparation in 0.5 ml. final volume. Deoxycytidylate (dCMP) deaminase (EC 3.5.4.12), was assayed with the following reaction mixture: 2-5 pmoles tris, pH 7.4; 32 nmoles [2-14C]dCMP (12.5 nCi); and 0.1 ml. enzyme preparation in 0.25 ml. final volume. The reaction was stopped by freezing at- 20"; after thawing, 50 pl. of the mixture was spotted on Whatman no.3 paper, which then was developed in a mixture of 75 ml. 95 % ethanol+ 30 ml. I M