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6Hh-C protein was stimulated by 12 μM cholesterol, but this cleavage was neither stimulated nor inhibited by the addition of jervine, cyclopamine, or tomatidine, even at 350 μM.
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Other sterols that participated in the reaction with similar efficiency to cholesterol are β-sitosterol, 5-androsten-3β-ol, and 7β-hydroxycholesterol. Sterols that participated with reduced efficiency are coprastan-3-ol, ergosterol, 4β-hydroxycholesterol, 19-hydroxycholesterol, 20α-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol. Epicholesterol, cholesterol acetate, α-ecdysone, 20-OH ecdysone, 3-keto-5-cholestene, and thiocholesterol (3(β)-thiol) were unable to participate in the reaction. The in vitro reaction thus requires an unhindered hydroxyl at the 3β position on a tetracyclic sterol nucleus, although neither the isooctyl side chain nor the number or positions of the double bonds in the sterol nucleus appear to affect autoprocessing critically.
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We thank R. I. Kelley, Y. Lange, D. Leahy, and R. K. Mann for comments on the manuscript; S. Morton and T. Jessell for antibodies; W. Gaffield for plant alkaloids; Wyeth-Ayerst for AY9944; and Hoechst Marion Roussel for triparanol. M.K.C. is a Physician Postdoctoral Fellow and P.A.B. is an investigator of the Howard Hughes Medical Institute.
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