Relaxation kinetics of E. coli ribosomes

• Published in: Biophysical chemistry 1977-11, Vol.7 (3), p.179-188
• Main Authors: Chaires, J.B., Tai, M., Huang, C., Kegeles, G., Infante, A.A., Wahba, A.J.
• Format: Article
• Language: English
• Subjects: Centrifugation, Density Gradient; Chemical Phenomena; Chemistry, Physical; Escherichia coli; Kinetics; Light; Magnesium; Ribosomes; Scattering, Radiation
• ISSN: 0301-4622; 1873-4200
• DOI: 10.1016/0301-4622(77)87020-8

Relaxation kinetics measurements on two types of ribosome preparations were parformed by the pressure-jump and temperature-Jump techniques, using light scattered at 90° as detector. For freshly prepared tibosomes isolated as 70S tight coupled from 26 000 RPM sucrose gradint sedimentation in 10 mM Mg 2+, surprisingly large reaction amplitudes were found in 10 mM Mg 2+ wilh both techniques, leading to an overall formation constant for 70S couples approximately three orders of magnitude smaller than that reported fot tight couples. For pelleted, two-tunes salt-washed ribosomes, amplitude titration versus Mg 2+ in the pressure-jump apparatus showed an amplitude maximum near 10 mM Mg 2+ with a relaxation time near 20 ms, and a second amplitude maximum near 2.5 mM Mg 2+ with a relaxation time near 25 s. Both types of preparation on reanalysis on sucrose gradients at 5 mM Mg 2+ showed approximately 15% of subunits, with a distinct zone in the 50S region. 70S light couples recovered from a sucrose density gradient separation at 5 mM Mg 2+ on pelleted two-times salt-washed ribosomes behaved in the same way as the original sample in pressure-jump experiments at 10 mM Mg 2+. These findings have been interpreted as follows (I) the processes observed at 10 mM Mg 2+ are due entirety to the relatively small loose couple content of the samples, even in the case of material isolated as 70S tight couples, (2) the processes observed at 2.5 mM Mg 2+ are due almost entirely to the preponderant tight couple population of the material, and (3) samples isolated as 70S tight couples from sucrose gradients at 5 mM Mg 2+ spontaneously revert within hours into micro-heterogeneous material containing about 15% loose couples, for both types of ribosomes.