Abstract
Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Further studies on a labile immunogenic particulate substance isolated from Mycobacterium tuberculosis. J. Bacteriol. 87:278–285. 1964.—A particulate fraction which was highly immunogenic for mice was collected by ultracentrifugation from mycobacteria disrupted in 0.25 m sucrose buffer. The active immunogenic material was present in the gelatinous pellet obtained after centrifugation at 40,000 rev/min (144,000 × g) for 3 hr. This active material could be prepared free from whole cells and cell walls. This was done either by several centrifugations at lower speeds, or by filtering the supernatant fluid from the 10,000 rev/min centrifugation through a Millipore filter (porosity 0.5 μ). No microorganisms were found on slides or in cultures made from these filtrates. The immunogenic moiety in the particulate fraction was found to be very labile. Temperatures higher than 0 to 4 C inactivated the immunogenic activity. There was an irreversible linear decrease in activity as the temperature increased. If fractions were frozen or lyophilized, the activity remained as high as the original material for 4 weeks, and then rapidly decreased. The immunogenic material also was very sensitive to the hydrogen-ion concentration; the optimal activity was found at pH 6.8 to 7.0. The activity decreased rapidly at more acid or alkaline pH values. Also, particulate fraction prepared in sucrose buffer at pH 7.3 and 7.6 was much less active than that prepared in sucrose buffer at pH 7.0. Immunogenic activity was decreased if the particulate fraction was dialyzed overnight against 0.01 m phosphate buffer or distilled water at 4 C. The detergent sodium lauryl sulfate inactivated immunogenic activity. Moreover, the use of a Waring Blendor to blend the ruptured cell mass before centrifugation decreased the activity. Finally, a markedly lower activity resulted if both the 20,000 and 40,000 rev/min centrifugations were done the day after the rupture of the cells. Some refinements in technique which are used now in the preparation of the particulate fraction are detailed.
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- FREDERIC J. Action of various substances on the mitochondria of living cells cultivated in vitro. Ann N Y Acad Sci. 1954 Nov 17;58(7):1246–1263. doi: 10.1111/j.1749-6632.1954.tb45906.x. [DOI] [PubMed] [Google Scholar]
- HARMAN J. W., KITIYAKARA A. Studies on mitochondria. VI. The relationship between the structure, osmotic reactivity and ATPase activity of mitochondria from pigeon skeletal muscle. Exp Cell Res. 1955 Jun;8(3):411–435. doi: 10.1016/0014-4827(55)90119-1. [DOI] [PubMed] [Google Scholar]
- KANAI K., YOUMANS G. P. Immunogenicity of intracellular particles and cell walls from Mycobacterium tuberculosis. J Bacteriol. 1960 Nov;80:607–614. doi: 10.1128/jb.80.5.607-614.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
- KANAI K., YOUMANS G. P., YOUMANS A. S. Allergenicity of intracellular particles, cell walls, and cytoplasmic fluid from Mycobacterium tuberculosis. J Bacteriol. 1960 Nov;80:615–621. doi: 10.1128/jb.80.5.615-621.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
- KIELLEY W. W., KIELLEY R. K. A specific adenosinetriphosphatase of liver mitochondria. J Biol Chem. 1953 Jan;200(1):213–222. [PubMed] [Google Scholar]
- LEHNINGER A. L. Reversal of various types of mitochondrial swelling by adenosine triphosphate. J Biol Chem. 1959 Sep;234:2465–2471. [PubMed] [Google Scholar]
- LIPSETT M. N., CORWIN L. M. Studies on stability of rat liver mitochondria. I. Role of oxidative phosphorylation in swelling. J Biol Chem. 1959 Sep;234:2448–2452. [PubMed] [Google Scholar]
- YOUMANS A. S., YOUMANS G. P., MILLMAN I. Immunogenicity of particles isolated from Mycobacterium tuberculosis. Proc Soc Exp Biol Med. 1957 Dec;96(3):762–768. doi: 10.3181/00379727-96-23602. [DOI] [PubMed] [Google Scholar]
- YOUMANS G. P., MILLMAN I., YOUMANS A. S. The immunizing activity against tuberculous infection in mice of enzymatically active particles isolated from extracts of Mycobacterium tuberculosis. J Bacteriol. 1955 Nov;70(5):557–562. doi: 10.1128/jb.70.5.557-562.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
- YOUMANS G. P., YOUMANS A. S., KANAI K. The difference in response of four strains of mice to immunization against tuberculous infection. Am Rev Respir Dis. 1959 Nov;80:753–756. doi: 10.1164/arrd.1959.80.5.753. [DOI] [PubMed] [Google Scholar]
- YOUMANS G. P., YOUMANS A. S. The measurement of the response of immunized mice to infection with Mycobacterium tuberculosis va. hominis. J Immunol. 1957 May;78(5):318–329. [PubMed] [Google Scholar]