LVSOSOMKS, A NliW GROUP OK c;YTOPLA,SMl(; PARTICLES I3I 



ditions favor the autolysis ot the particles, so that additional enzyme molecules 

 may become free to participate in the reaction as the incubation proceeds. In the 

 second place, part of the free enzyme molecules present in the original prepara- 

 tion undergo secondary adsorption onto the surface ol mitochondria, microsomes 

 or other particles, or remain trapped within injured lysosomes, thus appearing 

 in the sediment after high-speed centrifugation. With acid phosphatase, some 

 50-60 per cent of the free activity has been found to sufTer this fate in sucrose 

 solutions, about half this amount in media of higher ionic strength ( 13). 



Assay of Lysosomal Enzymes. In view of the above considerations, it is 

 necessary to distinguish clearly between various assay measurements of lysosomal 

 enzymes. 



Total Lictivitics are measured after c]uantitati\e release by one of the treat- 

 ments listed in hgure i. The most con\cnienl method is to run the assay in the 

 presence of o.i jier cent Triton X-ioo, taking a few precautions to guard against 

 some artifacts caused by the detergent ( 103). 



Free activities are determined similarly but without activating treatment and 

 under conditions which preserve the integrity of the particles as much as possible; 

 i.e., by incubating at 37° for no longer than 10 minutes in a medium containing 

 0.25 M sucrose and at a pH no lower than 5. These conditions are essential to 

 keep the autolysis of intact particles at a minimum, but often furnish values 

 representing only a small relative rise above the blanks. Since, in addition, there 

 is always some uncertainty in such assays as to what is the true blank value, 

 determinations of free activity are frequently inaccurate and may in some cases 

 be almost impracticable. 



Unsedimentable activities are measured under the same conditions as the total 

 activities, and thus with the same degree of accuracy, on the supernatant obtained 

 after centrifuging for 30 minutes at 100,000 g. For the reasons mentioned above, 

 these values are lower than the free activities, but may serve to estimate the latter, 

 to which they are approximately proportional. 



Latent activities (termed 'bound' in our early publications) are obtained by 

 subtracting the free from the total activities. They represent an estimate of the 

 amount of intact particles present. 



Individuality and Homogeneity of Lysosomes. The existence of lysosomes as 

 an individual group, distinct from mitochondria and from microsomes, has been 

 deduced entirely from centrifugation experiments. These have also providctl the 

 information on which is based our estimate of the dimensions of the [)articles. 

 Here again, acid phosphatase has acted as our tracer enzyme. 



As shown in figure 2A, the distribution of bound acid phosphatase, as studied 

 by means of conventional fractionation tcchnic]ues (14), was found to differ 

 significantly from those found by Schneider and Hogeboom (86) for the mito- 

 chondrial cytochrome oxidase and by Hers, Berthet, Berthet and de Duve (44) for 



