PROPERTIES OF 3 PROTEINASES FUNCTIONING AT G1, S AND G2 PHASES IN HELA-CELLS AND THEIR INHIBITION BY GUANIDINO-ACID AND AMIDINO-ACID ESTERS

HeLa cells were synchronized by double thymidine-block and allowed to grow after removal of thymidine. Three proteinases, tryptase 17:17, pr oteinase In and late G2 proteinase, were prepared from the HeLa cells harvested at the time when each proteinase appeared in the cell cycle of the cells. All of them,were suggested to be trypsin-like serine pro teinases, because they hydrolyzed trypsin-specific fluorogenic substra tes and their activities were inhibited by benzamidine, soybean trypsi n inhibitor, leupeptin, tosyl-L-lysine chloromethan (TLCK) and diisopr opylfluorophosphate (DEP). However, the actions of these proteinases o n the substrates and inhibitors suggested that they were three differe nt proteinases. They were strongly inhibited by 4-tert-butylphenyl and biphenyl esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, amidinopiperidine-4-acetic and 4-propionic acids, which retard the se cond DNA synthetic (S) and mitotic (M) phases for 3h, 4-tert-butylphen yl ester of amidinopiperidin-4-carboxylic acid, which blocks initiatio n of S phase, the ester of amidinopiperidine-4-butyric acid, which sup presses the second S and M phases, and the esters of trans-4-amidinocy clohexanecarboxylic and 4-propionic acids which inhibit M phase.