Yeasts divalent cation binding

Not all MIPS enzymes from thermophiles may have divalent metal ion-dependent activities. While crude protein extracts of A. fulgidus exhibited a MIPS activity that absolutely required divalent metal ions and was totally inhibited by 1 mM EDTA, the production of I-l-P from G-6-P (carried out >75 °C) by protein extracts from M. igneus and T. maritima was observed in the presence of 1 mM EDTA. This observation (Chen and Roberts, unpublished results) suggests that either the MIPS from these particular archaea is not divalent metal ion-dependent or that the metal ions bind very tightly and are not easily removed. None of the MIPS activities from thermophiles are affected by the addition ofNELt-1". Since the divalent cation in A. fulgidus MIPS has been suggested to aid in the aldol condensation and since this is supposedly done by NH4+ in the yeast enzyme, it is of interest how this step occurs in the other archaea and bacteria. 

RNA substrate. Nonspecific inhibitors may be commercial preparations of total RNA or tRNA from a bacterial or yeast source quantities used in assays may be as great as 1-10 pg per reaction, and should be empirically determined for each particular RNA-protein interaction. In all cases, these RNAs should be added from stocks prepared with the respective buffer for the binding reactions given that these will include divalent metal cations, these stocks should be freshly prepared so as to minimize RNA degradation during storage. 

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