SEPARATION AND PROPERTIES OF THE MYCOBACTERICIDAL PRINCIPLE FROM LYSOSOMAL COMPONENTS OF TUBERCULOUS MICE

Abstract

Differential centrifugation of a sucrose homogenate of tuberculous mouse spleens was conducted to obtain sediments of cytoplasmic granules at 1, 000 g, 3, 900 g, 8, 400 g, 15, 000 g, and 25, 000 g for 20 min. Acid phosphatase activity of the Triton-extract of each sediment indicated that the 15, 000 g and 25, 000 g sediments were rich in lysosomes. A large-molecular mycobactericidal fraction associated with cathepsin and acid phosphatase activities could be separated from the detergent-extracts of the 8, 400 g, 15, 000 g and 25, 000 g sediments by gel-filtration on Sephadex G-150 column, but not from the 3, 900 g sediment. From the lungs of the same group of mice, such a mycobactericidal fraction was not obtained.
The active fraction from the spleens was further treated with one of the following three agents; 90% acetone, ethyl ether, and 2M urea. The mycobactericidal activity was found in the 90% acetone-soluble fraction and also in the small-molecular protein fraction released by ether-or urea-treatment. Both subfractions were free from the enzymatic activities. Their mycobactericidal activity was heat-stable at 100 C for 5 min. The difference between the two was that the 90% acetone-soluble subf raction was more effective with less Folin value and with less exposure time to the bacilli, but more dependent on acidic environment than the protein fraction.