Acid Hydrolases, Lysosomes and Bone Resorption Induced 57 



closely parallel fashion (with the possible exception of part of the acid phenyl- 

 phosphatase) in preparations subjected to graded activating treatments, suggesting 

 that they are associated either with the same particle, or with particles possessing 

 similar properties (Vaes, in press b). These experiments allow the conclusion that the 

 acid hydrolases of bone cells are associated with particles having the main charac- 

 teristics of lysosomes. No data have been obtained so far on the intracellular locali- 

 zation of the coUagenase activity of bone cells, except for some preliminary experi- 

 ments (with C. M. Lapiere) showing that part of this activity could be associated 

 with cytoplasmic particles; in liver, a collagenase activity has been located by others 

 in the lysomoses (Frankland and Wynn, 1962). 



Further experiments have furnished evidence for an involvement of the acid 

 hydrolases of bone cells in the process of bone resorption: they are reported here 

 briefly; full details will be published elsewhere (see also as a preliminary note, Vaes, 

 in press a). 



Experiments in a tissue-culture system of resorbing bone 



Bone resorption was induced in cultures of calvaria from 18 — 19 day-old mouse 

 embryos by the addition to the medium of 1 U.S. P. unit/ml. of parathyroid extract 

 (PTE). Resorption lacunae first became macroscopically visible in the parietals after 

 1 — 2 days cultivation; they extended rapidly between the 2nd and the 4th day, 

 forming actual holes in the parietals. Lacunae appeared in the frontals around the 

 3rd day and progressed slowly thereafter (Fig. 1 in Vaes, in press a). 



During the development of resorption increasing amounts of /^-glucuronidase, 

 /?-galactosidase and y?-N-acetylaminodeoxyglucosidase were released into the medium 

 as compared to non-resorbing controls; this is true also for acid deoxyribonuclease 

 and possibly for cathepsin. Two non-lysosomal enzymes, alkaline phenylphosphatase 

 and catalase, were released in similar amounts in the medium of resorbing and of 

 control calvaria during the first days of culture and thereafter appeared in smaller 

 amounts in the medium of the resorbing calvaria (Fig. 2 in Vaes, in press a). The 

 medium of the resorbing calvaria was also found to be significantly more acid than 



Table 1. Balance of enzyme activies between the 2nd and 7th day of cultivation for control 



and resorbing (PTE) calvaria. Only means (mumoles/minute per calvarium) and p values 



(for the difference between C and PTE) are presented 



the medium of the control: this increased acidity was already detectable after 

 7 — 8 hours cultivation (Vaes, in press a). 



The average activities present in one calvarium on the 2nd and on the 7th day of 

 cultivation and those recovered from its cultivation medium between these 2 days, 

 are shown in Table 1. It is apparent from these data that both resorbing and control 



