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A new non-functional modified form of milk xanthine oxidase is described. This contains molybdenum in a quinquivalent state, which is resistant to both oxidation and reduction. The new species is derived from the native enzyme in a two-step process. The first step is the conversion into the desulpho form, via loss ofthe 'persulphide' sulphur, and the second involves reaction with ethylene glycol or other reagents. The species gives a characteristic Mo(V) electron-paramagnetic-resonance signal, without proton splittings, designated Resting II. This is virtually identical with signals reported previously from resting turkey liver xanthine dehydrogenase and rabbit liver aldehyde oxidase. The possibility is discussed that species Resting II, prepared with ethylene glycol, contains a -COCH20H residue bound to a nitrogen ligand of molybdenum.

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The interaction between Escherichia coli translation-initiation factor IF-1 and ribosomes was studied in binding experiments by Airfuge centrifugation. IF-1 binds to the 30S, but not to the SOS, ribosomal subunit and its binding is strongly stimulated by IF-3 and IF-2, either alone or in combination. From the dependence of the Kd of the 30s-subunit -IF-1 complex on ionic strength, it can be concluded that IF-1 binds primarily via an ionic intcraction, most likely with the 16s rRNA, with the minimum number of ion pairs involved being 2.7-3.6. The 30S-subunit -IF-1 interaction is unaffected by temperature changes between 1 1 "C and 44°C and is thus accompanied by a negligible enthalpy change. It is concluded that the interaction is an entropy-driven process triggered mainly by the release of counter ions from the RNA phosphates.Titration of 30s-subunit -IF-1 complexes with 50s subunits causes the ejection of the factor indicating that IF-1 is released from the ribosomes during the subunit association step which marks the transition from a 30S-initiation-complex to a 70s initiation complex. Omission of IF-1 from reaction mixtures containing all purified components required for protein synthesis results in a much-reduced translational rate [8]. To define the mechanism by which IF-1 exercises its stimulation, structural, functional and genetic studies have been performed on this factor. By fluorescence stopped-flow kinetics, it was found that IF-1 produces a 2-fold or a 4-fold rate increase in 30s-initiationcomplex formation when IF-3 or IF-2-GTP or both are present [9]. By initial-rate kinetic analysis of 30s-initiationcomplex formation, it was found that IF-1 increases approximately 2.5-fold the limiting V,,, of this process. IF-1 was found not to affect the affinity of the ribosomes either for the initiator Met-tRNA or for the random poly(AUG) used as template. The kinetic effect of IF-I, on the other hand, was found to titrate roughly with the 30s ribosomal subunits suggesting that these represent the target of the IF-1 activity [lo].A thorough understanding of the functional role of IF-1 cannot be expected without a better knowledge of its way of interacting with ribosomes. Concerning this point, the data available so far are rather scarce and conflicting [ll]. In fact, the interaction between IF-I and ribosomes is very sensitive to the hydrostatic pressure generated by sucrose-gradient centrifugation, which makes this method unsuitable for obtaining quantitative results. To overcome the problem, lixation with glutaraldehyde has been used; this method is likely to produce artifacts, however, as suggested by the very low K, value estimated in these studies and by the fact that IF-1 was found to bind equally well to 30s and 50s subunits and 70s ribosomes [12]. The alternative approach of applying fluorescent spectroscopy to the study of the IF-1 -ribosome interaction unfortunately failed due to the inactivation of the factor brought about by the fluorescent labelling procedureIn the present study we have used cent...

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