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)

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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
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