LOW-MOLECULAR WEIGHT ASPARTYL-TRANSFER RNA-SYNTHETASE FROM PORCINE THYROID - PURIFICATION, CHARACTERIZATION, AND HETEROGENEITY

Abstract

The total low MW aspartyl-tRNA synthetase activity of porcine thyroid is distributed among 4 distinct forms, all of which are identical in size, as determined by gel filtration. The predominant form was purified 25,000-fold to near homogeneity. A high concentration of glycerol (25%, vol/vol) was required throughout the procedure to maintain stability. The native enzyme was of the .alpha.2-type with a MW = 120,000 estimated by gel filtration. Its subunits were MW = 53,000 as determined using polyacrylamide gel electrophoresis under denaturing conditions. The enzyme had an isoelectric point of pH 5.4 and pH optimum that varied from pH 7.3 to 8.8 depending on the type of buffer present. The variation in pH optimum was related to a salt effect. All salts tested were inhibitory, with the degree of inhibition dependent on the anion present. PPi was a particularly powerful inhibitor; Km values for aspartate and tRNAAsp were significantly reduced in the presence of PPi. Evidence is presented that the allotropism of the low MW forms is not due to phosphorylation, proteolytic degradation or stable enzyme-substrate complexes.