| Abstract: | Differential centrifugation of homogenates of Harding-Passey melanoma demonstrated that aryl sulfatase A and β-glucuronidase sediment with particles (i.e., lysosomes) distinct from those particles bearing tyrosinase (i.e., melanosomes). The sedimentation curves for the lysosomal enzymes and tyrosinase, however, demonstrated that an adequate separation of these particle types could not be obtained by differential centrifugation. Isopycnic density gradient centrifugation was used to obtain the necessary resolution. The results of the density gradient studies demonstrated that lysosomes and melanosomes could be separated by this technique, as judged by enzyme distribution among the fractions recovered from the gradients and from electron microscopic examination of the melanosome fractions. It was further evident that the purified and washed melanosomes contained significant amounts of both acid hydrolase activities. Indeed 24% to 27% of the total acid hydrolase activities recovered from the density gradients were associated with the melanosome fractions. The acid hydrolases associated with the melanosomes could not be solubilized by treatment with 0.1% (v/v) Triton X-100, nor by exposure to hypo-osmotic shock. The melanoma lysosomes, however, did release most of both their hydrolase activities into soluble form after treatment with the same percentage of detergent. The lysosomes were, however, very resistant to rupture by exposure to hypo-osmotic conditions. |
