Reinvestigation of the proteolytic activity of neocarzinostatin

Neocarzinostatin (NCS) is the most studied member of a family of chromoprot eins secreted by a range of actinomycetes species, It has been proposed tha t in addition to their antitumoral activity related to the bound chromophor es, this group of related proteins could be a secreted proteases superfamil y; With the aim of dissecting the molecular basis of the proteolytic activi ty of NCS, an expression system allowing efficient expression of apo-NCS in Escherichia coli was constructed. The recombinant protein was properly fol ded and functional. Its histone-specific proteolytic activity was similar t o the activity described for the natural protein. Further analyses unambigu ously demonstrated that the proteolytic activity could be physically separa ted from NCS. This activity is therefore due not to NCS itself but to minor contaminating proteases, the nature of which differed in the recombinant a nd natural NCS preparations. The histone degradation test commonly used to monitor proteolytic activity is extremely sensitive and may easily generate false-positive results. These results strongly suggest that the possible p roteolytic activity of the proteins of this family should be critically rec onsidered.