56 Discussion 



pH optima calls to mind the old calculation of the number of hydrogen 

 ions that would be found in a high-power microscope field (Peters, R. A. 

 (1929). J. State Med., 37, 1), which would work out at about one per 

 particle of the size we are considering. I wonder whether the mere 

 switch of hydrogen ion concentration in a very small localized situation 

 might sometimes affect the activation or otherwise of some of these 

 enzymes. 



de Duve : We can safely say that these are two distinct enzymes, when 

 they are studied in disrupted systems. The alkaline one, which requires 

 magnesium, is completely inhibited by ethylenediamine tetra-acetic 

 acid, whereas the acid one is not affected by ethylenediamine tetra- 

 acetic acid, and requires no magnesium. On the other hand, the acid 

 DNAse is best studied at fairly high ionic strength, between 0-1 and 

 0-2, whereas the alkaline one is inhibited by high ionic strength. 



Coxon : Would either of them be active in living cells? 



de Duve: That is quite a different point, and we do not know that. 

 They could be entirely inactive. 



Estabrook: With reference to Prof. Slater's comment. Prof. Potter, 

 you published that heart muscle homogenates were at least 90 per cent 

 sensitive to antimycin. This was the difference between liver and heart 

 muscle ; the antimycin sensitivity of the DPN system. 



Potter : In the liver homogenates there was complete sensitivity when 

 no DPN was added. Insensitivity was observed on addition of external 

 DPN. 



Lehninger : In regard to lysosomes, in what different tissues have you 

 found these particles. Prof, de Duve? 



de Duve: The answer to this question depends on what type of 

 evidence is needed before one is allowed to conclude that lysosomes are 

 present in a tissue. In liver, for which we have the most complete case, 

 the following criteria are fulfilled. (1) The acid hydrolases of the lysoso- 

 mal group are present largely in particulate form and are therefore 

 sedimentable in a properly prepared homogenate. (2) When present 

 within intact particles, they are essentially unreactive towards external 

 substrates. (3) They are released in soluble and fully active form by a 

 variety of treatments which injure the particles. (4) By the use of 

 special techniques, the particles which contain these hydrolases can be 

 partly separated as a group both from true mitochondria and from 

 microsomes. (5) All lysosomal hydrolases are liberated in the same 

 proportion when the activating treatment is graded so as to cause only a 

 partial release of the enzymes. 



The next best case is on kidney, where Straus (1956, 1957, loc. cit.) has 

 clearly shown criteria 1 and 4 to be fulfilled and has obtained some evi- 

 dence, which we have confirmed, for criteria 2 and 3. Brain, which has 

 been extensively studied in my laboratory by Miss Berleur, answers 

 criteria 1, 2 and 3, but so far has failed to conform to criterion 4, pre- 

 sumably in view of the technical difficulties inherent in the fractionation 

 of such highly heterogeneous material. Finally, indications in favour 

 of criteria 1 and 2 have been obtained on spleen, thyroid and, according 

 to the literature, pancreas and pituitary, as well as on amoebae. We are 



