Single cell endocrinology: analysis of P-450scc activity by fluorescence detection methods.
A mechanism-based, fluorogenic probe for the cytochrome P-450scc (cholesterol side chain cleavage) enzyme, rate-limiting for the conversion of cholesterol to steroid hormones, is introduced and its application to the study of enzyme activity and regulation in single steroidogenic cells by several fluorescence detection methods is demonstrated. Reaction of the probe with P-450scc gives pregnenolone and the highly fluorescent resorufin anion. Spectroscopic changes in probe fluorescence, indicative of P-450scc activity, were monitored by steady-state fluorescence spectroscopy, flow cytometry, and microspectrofluorometry. This unique probe provides a nonradiometric indicator for real-time measurement of P-450scc activity in single living cells.
Marrone
B L
BL
Life Sciences Division, Los Alamos National Laboratory, NM 87545.
Simpson
D J
DJ
Yoshida
T M
TM
Unkefer
C J
CJ
Whaley
T W
TW
Buican
T N
TN
D000818:Animals; D002469:Cell Separation; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes; D006107:Granulosa Cells / Q000378:metabolism; D006728:Hormones / Q000378:metabolism; D007834:Lasers; D008928:Mitochondria / Q000378:metabolism; D010053:Ovary / Q000166:cytology / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013256:Steroids / Q000378:metabolism
D005456:Fluorescent Dyes; D006728:Hormones; D010078:Oxazines; D013256:Steroids; C014180:resorufin; D002786:Cholesterol Side-Chain Cleavage Enzyme