Photoreactivating enzyme from Escherichia coli: isolated enzyme lacks absorption in its actinic wavelength region and its ribonucleic acid cofactor is partially double stranded when associated with apoprotein

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Abstract
Isolated photoreactivating enzyme (PRE) from E. coli exhibits some optical density at wavelengths > 300 nm. After correcting for the effects of light scattering, however, no true absorption was found in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60.degree. C and subsequent cooling to 4.degree. C release the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (.mu. = 0.01), heating to 15.degree. C or protease treatment at 4.degree. C results in irreversible loss of part of the double strandedness. The difference spectrum at 4.degree. C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. Both A-U and G-C base pairs are present in equal concentrations and about 20% are in a double-stranded conformation in the native enzyme.

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