Allotropism in aspartyl-tRNA synthetase from procine thyroid.

Abstract

Three forms of aspartyl-tRNA synthetase, PC-1, PC-2 and PC-3 (based on their order of elution from phosphocellulose columns), were purified from porcine thyroid utilizing fractional pH precipitations and column chromatography. Two forms, PC-1 and PC-2, eluted identically from gel filtration columns and migrated the same electrophoretically and in glycerol density gradients. They were estimated to have sedimentation coefficients of 7 S and molecular weights of about 150000. Both PC-1 and PC-2 were equally inhibited by KCl. Their elution positions from phosphocellulose were unchanged upon rechromatography, indicating that they were nonequilibrium forms and they were present in post-mitochondrial supernatants in roughly equal amounts. All three forms were present in the same ratios whether or not phenylmethylsulphonyl fluoride was present in homogenizing buffers. PC-3 had the characteristics of an aminoacyl-tRNA synthetase complex. It contained aminoacyl-tRNA synthetase activity for 16 of 18 amino acids tested and was about 4% low-molecular-weight RNA. The complex did not significantly penetrate 5% polyacrylamide gels, was excluded by Sephacryl S-200 and had a sedimentation coefficient greater than 20 S indicating a particles mass in excess of 500000 daltons. In addition, the PC-3 fraction contained a ribonuclease capable of degrading both aminoacyl-tRNA and tRNA. After storage, rechromatography of PC-3 on phosphocellulose showed that some dissociation of the enzymes had taken place. Aspartyl-tRNA synthetase activity was found in positions analogous to PC-1 as well as PC-3, and ribonuclease activity was found in two positions on the columns, one coincident with PC-3 and one preceding the position of elution of PC-1. Dissociation of the complex was also observed using glycerol density gradient centrifugation and by chromatography on Sepharose4B. When present in the PC-3 complex, the aspartyl-tRNA synthetase activity and the ribonuclease activity were both stimulated by KC1. However, when dissociated to their free forms, the aspartyl-tRNA synthetase activity was inhibited by KC1, whereas the ribonuclease remained stimulated. The effects on aspartyl-tRNA synthetase, appeared to be potassium-ion-specific but were more pronounced in the presence of phosphate dianion than in chloride ion. The differential effects of potassium ion on the free and complexed forms of aspartyl-tRNA synthetase and the prescence of a ribonuclease in the aminoacyl-tRNA complex that is active toward tRNA could have functional significance in the intact cell.