Subcellular localisation and sedimentation behaviour of antigen 60 from Mycobacterium bovis BCG

Preparation, composition and immunological properties of A60 of Mycobacterium bovis BCG were previously described (Cocito and Vanlinden 1986). The present study focused on the intracellular distribution of this antigen. Fractionation of mycobacterial homogenates by ultracentrifugation indicated that most of A60 was present within the cytoplasm. Some of the antigen was located within the cell wall, from which it was released by extraction with alkali. Submission of cytoplasm to high speed centrifugation caused A60 to cosediment with ribosomes; however, dissociation of ribosomes in low-Mg buffer did not alter the sedimentation pattern of A60. Labelled A60, after ultracentrifugation in sucrose density gradients without Mg²⁺, was distributed throughout the entire gradient: treatment of (¹²⁵I)A60 with urea or detergents produced a peak of radioactivity located in the upper part of the gradient. It is concluded that A60 is represented by a heterogeneous family of molecules of increasing sizes: polymerization being enhanced by Mg²⁺ and reversibly prevented by urea. Some or all of the biological properties hitherto attributed to ribosomal particles may, in fact, be due to their contamination with cosedimented A60.Abbreviations CMN Group of Gram-positive microorganisms including the genera Corynebacterium, Mycobacterium and Nocardia - BCG Mycobacterium bovis, strain Calmette-Guérin - TMA Thermostable macromolecular antigen, a family of mycobacterial antigens to which A60 belongs - PPD Purified protein derivative, according to F. B. Seibert (cf. Daniel and Janicki 1978) - OT Old tuberculin, according to R. Koch (cf. Youmans 1979b) - NMT Buffer containing NH₄Cl, Mg acetate and Tris (see Materials) - PBS Phosphate-buffered saline (see Materials) - A60 Antigen 60 of BCG (according to the classification of Closs et al. 1980)