Hymenolepis diminuta: Isolation, purification, and reconstruction in vitro of a cell-free system for protein synthesis

Abstract

The rat tapeworm (cestode) Hymenolepis diminuta was fractionated into its subcellular components essential for cell-free protein synthesis. Experiments with S-30 and the S-100 fractions indicated that they were functionally active, but the S-30 was not as active as the S-30 system obtained from bacteria. The S-30 was fractionated into its two major constituents, microsomes and soluble fractions. Ribosomes were purified from the microsomal fraction. When the microsomal and ribosomal fractions were added back to the S-30, a low stimulatory effect was observed. When these same fractions were added back to the S-100, reconstituting the S-30, there was enhancement of the synthesis of protein. Preincubation of the S-100 fraction increased its acylation activity twofold over the nonpreincubated S-100. The isolation and partial purification of the pH 5 fraction, tRNA and synthetases from the S-100, demonstrated that these fractions were functional in aminoacylation assays, but the synthetase fraction had a relatively low stimulatory effect on acylation of tRNA. Isolated tRNAs charged with individual amino acids indicated that tRNA phe was most active in transferring amino acids into peptides when directed by synthetic messenger. Puromycin inhibited the synthesis of protein by 70–98% in the reconstituted system. This study demonstrates the isolation of functionally active subcellular components for cell-free protein synthesis from a parasitic helminth. These procedures can be applied to other helminth species and to the investigation of the action of anthelminthics.