Characterization of ribonuclease P isolated from rat liver cytosol.
Rat liver ribonuclease P was isolated from a cytosolic fraction and shown to have optimal activity in the presence of 1 mM MgCl2 and 150-200 mM KCl using Escherchia coli pre-tRNA(Tyr) as substrate. In cesium sulfate isopycnic density gradients, the enzyme had a buoyant density of 1.36 g/ml, indicating that it is a ribonucleoprotein complex. Analysis of the RNAs in the enzyme sample purified through two successive Cs2SO4 density gradient steps revealed the copurification of two major species of RNA (RRP1 and RRP2) along with several less abundant RNAs. Rat liver ribonuclease P activity was insensitive to micrococcal nuclease pretreatment. However, the nuclease-treated preparations contained several incompletely degraded RNA species that may have been sufficient to support the ribonuclease P activity. When RNase A was substituted for micrococcal nuclease, the ribonuclease P activity was diminished by greater than 90%, suggesting the requirement for an RNA subunit for activity.
Jayanthi
G P
GP
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298.
Van Tuyle
G C
GC
D000818:Animals; D002414:Cations, Monovalent; D002499:Centrifugation, Density Gradient; D002586:Cesium; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D015636:Magnesium Chloride / Q000494:pharmacology; D016337:RNA, Catalytic / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D043262:Ribonuclease P; D012259:Ribonuclease, Pancreatic
D002414:Cations, Monovalent; D016337:RNA, Catalytic; D015636:Magnesium Chloride; D002586:Cesium; C025407:cesium sulfate; D004722:Endoribonucleases; D043262:Ribonuclease P; D012259:Ribonuclease, Pancreatic