Interaction of cytochrome P‐450scc with cytochrome b_s

Abstract

Received 26 No~rnb~r I~)0 ,~r.~trophcstometrl¢, ~lttntt)r ~,hromalOllr~phy and eros~,hnklnil exl~rm~nls provided e~M,~nu Ih~t cyh~hton~ P,4~0w¢ fron'~ b~ln¢ ~dren~ort). ¢al mtt~htmdrta farms a Iqlhl eumple~c with =yt~hrame ~ from r~bblt h~er rnlcrmomes l)~ the r~or, s0tuled system fholest=r¢~l aside chain acthrlly ol'c)'tuchrome P-4SOscc was ~nhanc~l b~' the atldlllOn of~yt~hrorne b, Cyt~hrome P.4~IO,~, Cytoehf~me b,, t.:~mplex formation, Arltnhy ~hromatollraph),, Chemical woss.hnklnll I, INTRODUCTIONCytochrom¢ P.4S0.dependent monooxygenases can be classified into two large groups [I}'. 0) 'ferredoxindependent' monooxygenases each consistmg of a t'lavoprotein, an iron.sulfur protein, and cytoehrome P.4S0; (h) 'm=crosomaP monooxygenases each containin8 only a flavoprotein and cytochrome P-450. Interrelation between these two groups of monooxygenases, regulat=on of the electron transfer in the membranes of endoplasmic reticulum and mitochondria, and interaction of the two groups of monooxygenases are unclear. Here we report that cytochrome P-4$0ssc from bovine adrenocort=cal mttochondria can form a tight complex wtth cytochrome bs from rabbtt hver mtcrosomes. Our results are consistent with the hypothesis that there =s an interactton between 'microsomal' and 'ferredoxmdependent' monooxygenases and that cytochrome b~ may function as a mobtle earner between microsomes and mttochondrta. MATERIALS AND METHODS 1 C/)emtca/$Thyopropyl-Sepharose, Scpharose 4B, CNBr activated Sepharose 4B, Sephadex G-25 (free) were f-orn Pharmacla Sodmrn cholate, cholesterol, pregnenolone, Tween 20, EDC from Serva P.rfftcatJon of mtcrosoma/and mttochondrlal enzymesCytochrome P-450scc was purified from bovine adrenocortlcal mttochondrta to electrophoretic homogenetty on immobilized adrenodoxm and cholate-Sepharose as described previously [2]. Intact cytochrome bs (d-bat containing the hydrophoblc C-terminal sequence was purified as a by-product of punflcatmn of cytochrome 220045, USSRAbbrevtatton" EDC, l.ethyl-3-(dlrnethylarnmopropyl)carbod.rntde 2,3 Immobih~tlotl of ¢ytochrom¢ P.4JO$ce add eytochrome bjCytoclname P.4S0~:e tva= Immobihzed o. CNBr.acttv=ted $<pharose 4B In 100 mM phosphate buffer, pH T,61T0-110 nmol of =ytochrome P.4S0sce were irnrnobihzed on each | 0 rnl of settled Eel, Immobdlzatton of eytochrome b) was i~rformed in ¢h¢ same way, aboul 100 nmol or cytochrom¢ bs were =mrnobll)xed on I ml of settled 1¢1 2,4, Spcctrophotometrw analysis of intemcoon o.f ¢¥tochrome P.4$Osee with eyroehrotne bsA, bsorpllOn spectra were recorded at room temperature in a Shlrnadzu UV-'~000 or Specord M.40 spectrophotomeler. The effect of cytochrome bs addition on the absorption spectrum ot'¢yto~hrome P.450s¢¢ was measured by addmi the same concentrat=on of cytochrorne bs m rite reference cuvetle to cancel the al~sorpllon due to ¢ytoehrome b~ 2 5 Cross.lu)king of e.vtoehrome P.4$Oscc c~,toehrome b~ complex Cylochrorne P.4$0scc and ¢ytochrorne bs were sep~ately dialyzed against 10 rnMI phosphate buffer, pH 7 4 To o solution containing eytochrome P-...

A pathway for the metabolism of vitamin D 3 : Unique hydroxylated metabolites formed during catalysis with cytochrome P450scc (CYP11A1)

“…3B. In agreement with earlier reports (27), the presence of b5 results in an Ϸ2-fold stimulation of CYP11A1. (25,26,28) with the 1D 1 H NMR spectra of D3 (Fig.…”

“…This problem prompted us to test ways of increasing the yield of D3 products. It has been reported that the addition of purified rat liver microsomal b5 to a reaction mixture containing the reconstituted CYP11A1 system increases the rate of cholesterol metabolism (27). Fig.…”

Interaction of Apo-cytochrome b5 with Cytochromes P4503A4 and P45017A: Relevance of Heme Transfer Reactions

“…Most controversial and least understood is the mechanism by which b 5 stimulates some P450-catalyzed reactions but not others (5). The direct interaction of b 5 with CPR (54-57) and different forms of microsomal P450s (58-65), as well as with mitochondrial and bacterial types of P450, such as P450cam (66, 67) and P450scc (68), has been shown. The primary structure of b 5 has been determined by direct protein sequencing (69)(70)(71) and confirmed by the deduced amino acid sequence determined from the nucleotide sequence of cDNA (72,73).…”

Olağanüstü Zamanaşimiyla Taşinmaz Mülki̇yeti̇ni̇n Kazanilmasi Ve İyi̇ni̇yet Şartinin Aranmayişi İle Anayasa Mahkemesi̇ Kararinin Geti̇rdi̇ği̇ Deği̇şi̇kli̇kleri̇n Değerlendi̇ri̇lmesi̇

“…Tescil davasında, zilyet, olağanüstü zamanaşımıyla kazanım şartlarını sağladığına ilişkin ispat yükü altındadır 79 . Zilyetliğin ispatı konusunda, tapulu taşınmazlar bakımından herhangi bir kısıtlama mevcut olmayıp, her türlü delille ispat söz konusu olabilmektedir 80 . Ancak, tapusuz taşınmazların ispatı, taşınmazın sulu toprak veya kuru toprak olmasına göre farklılık göstermektedir 81 .…”