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Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion.

ABSTRACT: 1. The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0 degrees C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0 degrees C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0 degrees C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37 degrees C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37 degrees C. Re-activation was slow at 20 degrees C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, S20,W of the subparticle remained at 52+/- 1S until the sample was incubated at 37 degrees C when S20,W increased to 56 +/- 1S compared with the value of 58 +/- 1S for the subparticle as originally isolated.

SUBMITTER: Cox RA

PROVIDER: S-EPMC1164268 | biostudies-other | 1976 Dec

REPOSITORIES: biostudies-other

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Project description:Larger ribosomal subparticles (L-subparticles) of rabbit ribosomes were treated with either ribonucleases (I or T1) or proteinases (trypsin or chymotrypsin), and their capacity to function in poly(U)-directed polyphenylalanine synthesis and in the puromycin reaction was investigated. The effects of pretreatment of L-subparticles on the reconstruction of active subparticles from core-particles derived by treatment with 2.75 M-NH4Cl/69 mM-MgCl2 and split-protein fractions were also examined. The protein moiety of proteinase-treated L-subparticles was analysed by one-dimensional sodium dodecyl sulphate/polyacrylamide- and two-dimensional polyacrylamide-gel electrophoresis. The introduction of 16--100 scissions in the RNA moiety had no effect on the activity of the L-subparticles in polyphenylalanine synthesis, and there was no effect on the stability of L-subparticles to high-salt shock treatment and a marginal effect on the reconstruction of L-subparticles from high-salt-shock core-particles and split-protein fractions. In contrast, L-subparticles treated with low amounts of trypsin (0.56 ng of trypsin/microgram of L-subparticle) were inactive in polyphenylalanine synthesis, and their capacity to function in partial-reconstruction experiments was diminished. Activity in the puromycin reaction was increased by 70% as a result of trypsin treatment (280 ng of trypsin/microgram of L-subparticle). At least two of the acidic proteins implicated in the translocation function were not affected by trypsin treatment. Trypsin-treated L-subparticles had lost their capacity to bind the smaller ribosomal subparticle (S-subparticle). The protein(s) needed for S-subparticle binding were shown to be present in high-salt-shock cores. At least six proteins associated with the core-particles were attack during trypsin treatment of L-subparticles. An examination of L-subparticles isolated from trypsin-treated polyribosomes showed that the amount of trypsin necessary to decrease the activity of the subparticle by 50% was about twice that needed in the treatment of L-subparticles alone. The largest protein of rabbit L-subparticles (approx. 51 000 daltons) was cleaved in a stepwise fashion by trypsin to fragments of approx. 40 000 daltons. This protein was also cleaved by chymotrypsin.

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Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion.

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