Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes

Anal Biochem. 1991 Nov 1;198(2):219-23. doi: 10.1016/0003-2697(91)90416-q.

Abstract

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).

MeSH terms

  • Animals
  • Binding Sites
  • Cell Fractionation
  • Cell-Free System
  • Humans
  • Peptide Biosynthesis
  • Peptides*
  • Placenta / chemistry*
  • Poly U
  • RNA, Ribosomal / isolation & purification*
  • RNA, Transfer, Phe / chemistry
  • Rabbits
  • Reticulocytes
  • Ribosomal Proteins / isolation & purification
  • Ribosomes / chemistry*
  • Ribosomes / physiology

Substances

  • Peptides
  • RNA, Ribosomal
  • RNA, Transfer, Phe
  • Ribosomal Proteins
  • Poly U
  • polyphenylalanine