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Highly purified preparations of the DNAdependent RNA polymerase obtained from Escherichia coli contain about 2 g-atoms of tightly bound zinc per mol (molecular weight 370,000) of enzyme. When the purified enzyme is fractionated on Sephadex G-150 or G-200, corre-lation is observed between the zinc and enzymic activity. Although some of the preparations examined also contain iron, copper, and magnesium, the content of these metal ions show no consistent correlation with RNA polymerase activity.Initiation of RNA synthesis is specifically inhibited by 1,10-phenanthroline. Less-effective inhibition is observed for other chelating agents or for a nonchelating phenanthroline analog. The analog also exhibits a pattern of inhibition differing from that characteristic of 1,10-phenanthroline. Binding of purine nucleoside triphosphates at the lower-affinity (Kd = 0.15 mM) site may also be prevented by the addition of 1,10-phenanthroline. One or both of the bound zinc atoms may, therefore, participate in the initiation of RNA synthesis.DNA-dependent RNA polymerase (EC 2.7.7.6) purified from Escherichia coli has been shown to possess a specific binding site for purine nucleoside triphosphates, with a dissociation constant of about 0.15 mM (1, 2). Interaction of nucleoside triphosphates at this site occurred in the absence of added divalent metal ion, and was blocked by the addition of rifamycin. It was proposed that this site might be responsible for binding the 5'-terminal nucleoside triphosphate that is involved in the initiation of RNA synthesis (2, 3). The absence of a metal requirement for binding at this site suggested that the enzyme might contain a bound metal ion and, hence, that chelating agents might inhibit RNA polymerase in the presence of added Mg2+, which is required both for RNA synthesis (1) and for binding of nucleoside triphosphates at a second site (Kd = 0.015 mM) (2). Our results indicate that RNA polymerase contains 2 atoms of bound zinc per molecule of enzyme, and that the chelating agent, 1,10-phenanthroline, specifically inhibits the initiation of RNA synthesis. Slater et al. (4)

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1. Cell-free extracts of Rhizopus arrhizus contain exclusively cytosolic pyruvate carboxylase and NADglutamate dehydrogenase, a single mitochondrial isoenzyme of NADP-isocitrate dehydrogenase, and both mitochondrial and cytosolic isoenzymes of NADP-malate dehydrogenase (decarboxylating). Other enzymes examined have sub-cellular localisations similar to those characteristic of mammalian liver.2. Purified preparations of R. arrhizus pyruvate carboxylase are subject to partial regulatory inhibition by L-aspartate and 2-oxoadipate. L-Glutamate acts as a less effective analogue of L-aspartate while 2-oxoglutarate is ineffective. Competition studies indicate the presence of separate inhibitory sites for L-aspartate and 2-oxoadipate.3. Under routine assay conditions R. arrhizus pyruvate carboxylase shows significant activation by acyl derivatives of coenzyme A with long chain acyl CoA being more effective than acetyl-CoA. This activation is no longer observed in the presence of high concentrations of pyruvate, MgATPZ-and HCO; . 5. The studies indicate that R. arrhizus possesses an entirely cytosolic pathway for the conversion of glucose to fumaric acid and that both the organisation of pyruvate metabolism and the regulation of pyruvate carboxylase differ significantly in this organism as compared to that proposed previously for Aspergillus nidulans.Eukaryotic pyruvate carboxylases which catalyse the Pyruvate + MgATPZ-+ HCO; -M g ' + , K t Oxaloacetate have similar catalytic properties and sub-unit organisation [2]. However, we have proposed that they can be divided into two subclasses on the basis of their localisation within the cell and their responses to regulatory effectors [3].Pyruvate carboxylases in vertebrate tissues are localised in the mitochondrial matrix [4-71. These enzymes show an absolute requirement for activation by a short-chain acyl derivative of CoA, e.g. acetyl-CoA, in order to express their maximal catalytic activity and are subject to inhibition by L-glutamate. Long-chain acyl derivatives of CoA are not effective as activators of these enzymes [8 -111. In contrast pyruvate carboxylases in higher fungi are localised exclusively in the cytosol and can readily express their maximal catalytic activity in the absence of an acyl derivative of CoA. However, when assayed under conditions where such activation can be observed these enzymes are most effectively stimulated by long-chain acyl derivatives of CoA, e.g. palmitoyl-CoA, reaction [l]:

The sub-cellular localisation of enzymes has been defined by latency analysis, and fractionation by differential centrifugation, in cell-free extracts prepared from the mycelium of Aspergillus nidulans by growth in the presence of 2-deoxyglucose followed by treatment with a mixture of P-glucuronidase, sulphatase and P-glucanase and exposure to N2 cavitation at 5.2 PMa. In such extracts pyruvate carboxylase and NAD-dependent and NADPdependent glutamate dehydrogenases are exclusively localised in the cytosol whereas all the other enzymes studied have sub-cellular localisation patterns similar to those described for mammalian liver. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for many of the enzymes, e.g. N A D -malate dehydrogenase, NADP -isocitrate dehydrogenase, glutamate/oxaloacetate transaminase, fumarase, which show a marked extent of incomplete latency and the presence of significant activity in the mitochondrial and cytosolic fractions prepared by differential centrifugation. A novel method is described for detection of citrate synthase activity following electrophoresis of the cell-free extract. Application of this method confirms the absence of a unique cytosolic isoenzyme of citrate synthase and hence shows that citrate synthase activity detected in the soluble fraction results from damage to the mitochondria during isolation.A scheme is proposed on the basis of these data to describe the organisation of lipid and amino acid synthesis from glucose in an organism which posessesses a cytosolic pyruvate carboxylase.