Reinvestigation of the proteolytic activity of neocarzinostatin.
Neocarzinostatin (NCS) is the most studied member of a family of chromoproteins secreted by a range of actinomycetes species. It has been proposed that in addition to their antitumoral activity related to the bound chromophores, this group of related proteins could be a secreted proteases superfamily. With the aim of dissecting the molecular basis of the proteolytic activity of NCS, an expression system allowing efficient expression of apo-NCS in Escherichia coli was constructed. The recombinant protein was properly folded and functional. Its histone-specific proteolytic activity was similar to the activity described for the natural protein. Further analyses unambiguously demonstrated that the proteolytic activity could be physically separated from NCS. This activity is therefore due not to NCS itself but to minor contaminating proteases, the nature of which differed in the recombinant and 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 proteolytic activity of the proteins of this family should be critically reconsidered.
Heyd
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Laboratoire de Modélisation et d'Ingénierie des Protéines, EP1088, Université de Paris-Sud, F-91405 Orsay Cedex, France.
Lerat
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Adjadj
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Minard
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P
Desmadril
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D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016477:Artifacts; D002384:Catalysis; D002417:Cattle; D002942:Circular Dichroism; D004340:Drug Contamination; D010450:Endopeptidases / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005189:False Positive Reactions; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D011480:Protease Inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013302:Streptomyces / Q000737:chemistry* / Q000201:enzymology*; D013379:Substrate Specificity; D009353:Zinostatin / Q000737:chemistry* / Q000378:metabolism
D001059:Apoproteins; D006657:Histones; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D009353:Zinostatin; D010450:Endopeptidases