Resolution and Identification of Ribosomes from Mycobacteria

Abstract

Ribosomes were isolated from mycobacteria (BCG) by differential centrifugation by use of a modification of Nirenberg's procedure. Individual components of the ribosomal spectrum were resolved in linear sucrose density gradients. Sedimentation constants of the individual components were determined by analytical ultracentrifugation. Ribosomes from Escherichia coli labeled with '4C-uracil were used as markers for an independent confirmation of the identity of individual peaks. The typical ribosomal spectrum of 70S units and 50S and 305 subunits was obtained in tris(hydroxymethyl)aminomethane buffer with 0.01 M Mg(CH3COO)2. This corresponds to the ribosomal spectrum obtained in E. coli under comparable conditions. In 0.0001 M Mg', the 70S units were dissociated to 50S and 30S particles. Pancreatic ribonuclease produced no significant changes in main ribosomal components. The isolation techniques used in this work precluded the recognition of polyribosomes.

Delayed Hypersensitivity Reactions Provoked by Ribosomes from Acid-Fast Bacilli I. Ribosomal Isolation, Characterization, Delayed Hypersensitivity, and Specificity

“…In the present work, extreme care has been exercised in the preparation of ribosomal ma- terials to ensure their intact structure and to separate the ribosomes from cell-free membranes. The pressure chamber was chosen to disrupt the mycobacterium because this method gave good disruption and produced active ribosomal preparations, as confirmed by Ribi et al (27) and Trinka et al (34)(35)(36). Partial purification was accomplished by washing ribosomes in NH4C1 overnight, followed by centrifugation to pellet the ribosomes.…”

Ribosomes of Acid-Fast Bacilli: Immunogenicity, Serology, and In Vitro Correlates of Delayed Hypersensitivity

“…The CR gave 10 immune precipitin arcs and the RNA preparation gave 2 arcs when developed with this antiserum. DISCUSSION Although ribosomes from various mycobacteria (8,25,27,28) have been isolated, it was only recently reported that ribosomes or ribosomal protein was tested for the ability to provoke delayed hypersensitivity reactions. Ribosomal protein from BCG (16) and ribosomes or ribosomal subunits from BCG and M. smegmatis have been shown to contain antigens capable of provoking delayed skin reactions in sensitized animals (1).…”