Isolation and Electron Microscopic Characterization of the High Molecular Mass Aminoacyl-tRNA Synthetase Complex from Murine Erythroleukemia Cells

M T Norcum

doi:10.1016/s0021-9258(18)63808-6

Abstract

A high molecular mass aminoacyl-tRNA synthetase complex has been isolated from a murine erythroleukemia cell line. This multienzyme complex contains activities for the arginyl-, aspartyl-, glutamyl-, glutaminyl-, isoleucyl,- leucyl-, lysyl-, methionyl-, and prolyl-tRNA synthetases. This enzyme composition, the polypeptide pattern observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the relative stoichiometry of the component polypeptides are characteristic of high molecular mass complexes of aminoacyl-tRNA synthetases isolated from a variety of mammalian tissues and cell types. Negatively stained preparations of native complex and of glutaraldehyde-treated material have been examined by electron microscopy. In both cases, a distinctive particle is observed which appears in several orientations. The most common views are of two different projections of a squarish particle that measures approximately 27 x 27 nm. Other commonly observed views are of a "U" shape, a rectangle, and a triangle. All of these views are seen in both gradient-purified samples and those prepared directly from material as isolated. These data are consistent with a model for the multienzyme aminoacyl-tRNA synthetase complex as a "cup" or elongated U structure. These studies demonstrate that the high molecular mass complex of eukaryotic aminoacyl-tRNA synthetases does have a coherent structure that can be visualized by electron microscopy.

Highlights

Most of the attempts to decipher the structural organization of this complex of nine different synthetase activities have focused on the isolation and characterizatoiof nthe individual constituent polypeptides
The results of this study demonstrate that thehigh molecular mass aminoacyl-tRNA synthetase complex common to many eukaryotic cell types is presentin the murine erythroleukemia cell line DS-19E5
This multienzyme complex can be readily purified from these cells with a simple protocol involving only two high speed centrifugations and two affinity chromatographic steps(TableI)

Summary

Analytical Methods

Aminoacyl-tRNA synthetase activity was measured by the incorporation of ["CI-amino acids into tRNA. Samples containing about 100 pgof aminoacyl-tRNA synthetase were prepared by adding either electron microscopic grade aqueous 8%glutaraldehyde (Polysciences, Inc.) to give 0.4% (v/v) or an equal volume of the above buffer. Molecular mass marker proteins were diluted in the same buffer to a volume equal to that of the samples of aminoacyl-tRNA synthetase complex. These calibration mixtures contained two or three of the following proteins in various combinations: carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), catalase (220 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa). Grids were preparedwithin 10-15 min at room temperature.Electron micrographs were obtained with a Zeiss EM-10 microscope operated at 60 kV and absolute magnifications of 50,000 and 63,000

RESULTS

DISCUSSION

E Triangle