Salt effects in the glutathione-facilitated reactivation of reduced bovine pancreatic ribonuclease.
The rate of regeneration of reduced RNase by glutathione was examined in the presence of several added substances: substrate, phospholipid, other proteins, bacterial ribosomes, and neutral salts. Of these, only neutral salts showed substantial effects. K2HOP4 and (NH4)2SO4 strongly accelerated regeneration, the alkali chlorides showed moderate acceleration or inhibition, while LiBr and KSCN strongly inhibited. The t1/2 for regeneration in 1 M Pi is 4 min compared to 75 min in the absence of Pi; in 0.5 M KSCN t1/2 greater than 100 min. The pattern of specific salt effects is similar to a Hofmeister series. There is a strong parallel between the pattern of specific salt effects on the kinetics of RNase regeneration and the pattern of effects of the same salts on the equilibrium stability of biopolymers. This suggests that the role of salts in the regeneration is to stabilize or destabilize rate-limiting folding intermediates. Pi-accelerated glutathione regenerations showed a broad temperature optimum from 30-37 degrees. In strong contrast with the virtual concentration independence of the Pi-free controls, with Pi = 1 M, both rates and yields of RNase activity were decreased markedly at [RNase] greater than 2 x 10(-6) M. Phosphate and pyrophosphate showed additive, and in some cases, synergistic accelerations. These results suggest that specific ion binding occurs in addition to general solvent effects.
D000818:Animals; D002417:Cattle; D004926:Escherichia coli / Q000502:physiology; D005978:Glutathione / Q000494:pharmacology*; D007700:Kinetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology*; D010743:Phospholipids / Q000494:pharmacology; D011489:Protein Denaturation; D011506:Proteins / Q000494:pharmacology; D012260:Ribonucleases / Q000378:metabolism*; D012270:Ribosomes / Q000502:physiology; D012492:Salts / Q000494:pharmacology; D013696:Temperature
D010743:Phospholipids; D011506:Proteins; D012492:Salts; D012260:Ribonucleases; D005978:Glutathione