Partial purification and characterization of nuclear ribonuclease P from wheat

Ribonuclease P (RNase P) from wheat nuclei has been purified over 1000-fold, using wheat germ extract as starting material and a combination of poly(ethylenglycol) precipitation and column chromatography. The enzyme was shown to be of nuclear origin by its characteristic ionic requirements; for optimum activity it requires 0.5-1.5 mM Mg2+, which can be partly replaced by Mn2+. With about 100 kDa, wheat nuclear RNase P has the lowest molecular mass reported so far for a eukaryotic RNase P. The enzyme has an isoelectric point of 5.0 and a buoyant density of 1.34 g/ml in CsCl, suggesting the presence of a nucleic acid component; it is, however, insensitive against treatment with micrococcal nuclease. Wheat germ RNase P requires an intact tertiary structure of the pre-tRNA substrate; its cleavage efficiency is also influenced by the presence of an intron, and by the nature of the 3' terminus of the substrate. The apparent K-m and V-max for an intronless plant pre-tRNA(Tyr) are 10.3 nM and 1.12 fmol/min, respectively.