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Mechanism of peroxisomal 24-hydroxylation of 3α,7α,12α-trihydroxy-5β-cholestanoic acid in rat liver
142
~~cco~d~~~to current mrtcepts, the last step in the ~~osy~th~sis of bile acids from cho~~st~r~~involves oxidative cleavage of the side3~~7~,~2~“trihydroxy_5~-cbolesta~oic aci dro~y~5~-chol~s~~oie ac {I]_ 24-~yd~~~y~~tio~ of 3~~7~~~~~-t~bydrox~- ~ho~es~~~~~~ acid has een demonstrated both with the ~toc~o~d~~a~ and the m~crosoma~ fraction of rat liver corn~~~e with cytosol f2,3]_ From the cofactor d~~~~~e~ of the ~tocbondr~~ system studied collaborators [2,4,5], it may be cone ~~ch~~~s~ of s~de-cb~~ ckavsnge is of &oxidation of fatty acids, and that the reactions are performed with the CoA 3a,7~,12cr-trihydroxy-5P-cbolestaraoi@. a ~o~v~~si~~ should thus involve particip CoA synthetase, of an acy1 dehydro~~~~s~ or a~
choiestanoic add into chohc acid than had the microsomal and the ~toch~~~~al fraction [6,7]. Previous co~t~~d~c~o~ results with respect to subce%&r &~&.iur~may he e?rtpfained by the ~ffere~t degree d ~~~a~~~M~ ~U~~~~~~~ of the micx5soma and mitoc~on~~al prep~ations used. It was shown that the peroxisomal conversion was absoiuteiydependent upon ATP, CoA, 8iAD’+ and W?+ 1 md that the substrate had to be activated by CoA prior to the conversion i7$_ Since smafi amounts of 3~7tu,12a,2Lf_tetr~ydro~~5~-c~o~~tanoic acid were isolated> it w&s believed tha,t this ~rnp~und was an interm~at.~ in the reaction. Attempts to remonstrate an ~saturat~ intermediate in the reactirm failed, however ‘Bat there is such a ~2~~~~~~~~~d ~~~~~~~~~ ia the peroxisomaL conversion of 3ar,7cu,J2a-trihydroxy-5~cholestanoic acid into 3a,%,I2q24-tetrahydroxy-
a commercial pellet &et, were used for Preparation of the peroxisomes. The liver was finely minced and ~~rnoge~~ed by one stroke in a Pott@ Ehehjekrn ~~rn~~~~~r. FrGrn the 10% Ever ~orno~e~ate in 0,25% sucrose and 1 m&B EDTA with Trizma Base (pN 6.5), a postnucfear supe~ata~~ was prepared by centrifugation at 2200 rev/mm (600 x g,, j for 10 min, Tie p&etwas rehomogenized and thhes~~~~s~o~ r~entrifug~. The combined supernatants were centr~~g~ at 65Qo rev/m& ~4~ X g,,) for 10 r&n. The 4900 x & s~~e~~at~~ was ~~t~f~g~ at 16 200 rq&nin (24 2138X g,,) for 10 min The resulting pdlet (light ~tocb~~d~al fraction) was washed once and resuspended in the horn~~~n~ medium, P~ro~soma~ en~ched fractions were separated OII a Linear sucrose gradient ~24-4~~~ w/w) with Dextran T 40 (2-3.8%, w/w) in a vertical rotor,
SR.nhnlaotor\r\;o Jp-~~l”ILmLcbAIIIc.
Thza I ua
oAA QClU
;c. 10
ek/*vx,n .TLlWWA1
’ A:rontlrrr Ia.IILwlly
:y. 111 :ki::
present study by performing the reaction in an ~s~~-a~o~~~~re and in the presence of a deuterated n~~~~”
I:mL+ U&uL
-:+T.nL,.n~c:nl uILL”L4ucuM.~IIal
iL,,r:.... IlzaLU”II
vl”...l WilJ
I....,.--1 uiycxcw
_.a “LA
the $5~ of the gradient and centrifuged at 20000 revJ%Iin (35 800 X gIly> [xp Fractions of 3 mi were collected, The fractioris with. the highest ~ero~sorn~ marker enzymes (7) were pooled. and diluted I : 3 with 0.25 mM sucrose/f5 mM Hepes fpH 7.4)+ The ~Ko~~rnes were su~~ue~t~~ ~e~~e~ted at 3rw,7~,12rr-T~hydrOXy~5~~t7~-~H~Ch~~eSta~OiC 75 QOBX g,, for 1 h. The resulting pellet was resusacid (02 rnC~~~~rn0~)as we13as the co~~p~~~~~~ pended in a minimal vohtme of the d&&on ~n~abe~~ steroid and 3n,7~tu,12ar,24-tetr~ydroxymedium to a concentration of IO-20 mg pro5&cholestanoic acid were prepared as described pret~ous$ /3], The 3&]&&~ rvww..nl*~il *I.?RO tein/ml. ~“Al,y”k+nu w ix3 purified ~rnrne~ate~~ before use by hjgh~~~rfor~ mance liquid G~r~M~~~~~~~y fi%%C], using ph~spbat~ buffer ia methanoI as eIutin# solvent The i~cub~t~o~~ mixtare costumed the Folfow[cf. below), The <25R)- and ~25~)~is~r~e~s of ing in a total volwme of 7.5 ml of 0.1 M Tris-HCf 3ff,7ff,l2~-t~~ydroxy-5~=cho~~stanoic acid sep buffer, fpH 8.0): 7,5 mM ATP/2 mM CoA/lO arated under the ~PLC-~~di~o~s used and the m&f Ii@$I,fS pm FAD/2 mM NAD_/l,S mg material was faund to consist of about 28% of the ~~~~~~~~rn~~ protein suspsd& in100pl s$o*25 @SR)- and about 72% of the ~25~~~s~rner. The M swxo~e 14 mM Hqxs buffer/~~H 7.4) two G2S isomers were not separated, since both 3~,~~~~2~-tr~hydrox~~~-cholest~oi~ acid, 30 ,ug forms are equally well converted into cholic acid unlabeled and 500000 cpm. labeled compound and 3a,7cr,12a,24-tetrahydroxy-5@cholestanoic dissolved in 20 ~1 of ethanol. The air in the acid in the peroxisomal system used 171. Deu~~cuba~~on vessel was removed and replaced with terated water (99% pure) was obtained from Merck an atmosphere consisting of about SQ% IQ, aand ~~~st~d~~ F.R.G.) and “W2 gas (98% pure) was 50% r802. In another experiment the incubation from Pro&em, B.O.C. {Deer Park Road, London, was ~~rforrn~ in an, but with sn incubation U.K.). me~i~rn ~nt~~~g more than 95% “H,O. The i~c~bat~ous were p~~f~~ed for 20 tin at 37 0 C and were term~na~cd by auction of 150 pt of 6 M
After 36) min of e was acidified an ethyl acetate. The ethyl acetate
was
eluted with 24% 25 m in methanol. Aliquots of the fractions were as“_.,_A F-F T,; 6,X,1, OorJe+U IUS rnrl;r\n&;,r:+.r laUlvaW1”lry ;, la1 a_ Dn*l,n‘-J i aL.na*L_n I ir-Lai v i:,..:FJ llyueer scintillation spectrometer after addition of countsolution (Packard a Gel HI). ecovelry of ioactivity from the IL-column was essentially complete. Combined
gas-liquid
ch~oma~og~a~hy-mass
spec-
0 Thus3 only a negiig tracing at m/e 753,
qereas a significant peak ~2s
2rometry
The appropriate chromatographic fractions were converted into the methyl ester-trimethylsilyl ether derivatives [9] and analyzed by combine atography-mass spectrometry using a 1.5% olumn at 280 ‘C and the LKB 9008 instruent (LKB Instruments Inc., Stockholm, ed with a multiple ion detector. experiments the instrument was focuse ions at m/e 753, m/e 754 and m/e 755 (M - 15 ions), in some experiments on the ions at r~/ie 588, 589 and 590 (M - (2 .90)ions) and in some experis on the ions at m/e I89 and 190 (cf. euterium contents ted according to
In agreement with the previous work [7], cholic was the major product (about 4%) and ,12a,24-tetrahydroxy-5fi-cholestanoic acid a minor product (about I%) after incubation of 3a,7ql2a-trihydroxy-5b-cbolestanoic aci peroxisomal fraction. It should be pointe under the gas chromatographic conditions employed, there was no separation between the (24R)and the (24S)-isomers of 3a,7cu,12a,24tetrahyrlsnw~.l_~R_rhnlprtonrr;r catA. UllU ~nrl alcn nnt ~IVAJ-~r-UII”IU~CUllVlV U_U UlU” IX”_ betweepB the (2X)and (25S)-isomers of 3a,7q12a-trihyroxy-QLcholestanoic acid. The synthetic stanard material contained a mixture of all the four possible isomers of 3a,7a,12a,24-tetrahydroxyS,kholestanoic acid.
between the height of the peak at m/e 585 and that at m/e 587 obtained in selected ion monita:~. t~i~et~y~s~~y~ether, methyl ester; it was IlO] that the mat ial contained 0.‘” euteriiam was pre-
same as that obtained in ahe corresponding ana@The fragment a’s m/e 253 is
cholestanoic acid. The fragment at m/e 189 Is bably due to From the rati in the tracing of the ion at m/e 189 of the ioiz and the height 0 hagmen% calculated that at m/e 190, it mntainm-l Qb atnmc nf ~,,I~IIIyAyIu “..,6; -..,II-” -* dPlalPrirlm lW1ll--l---~
Clear evidence is given in the uction of a Why
~~cco~d~~~to current mrtcepts, the last step in the ~~osy~th~sis of bile acids from cho~~st~r~~involves oxidative cleavage of the side3~~7~,~2~“trihydroxy_5~-cbolesta~oic aci dro~y~5~-chol~s~~oie ac {I]_ 24-~yd~~~y~~tio~ of 3~~7~~~~~-t~bydrox~- ~ho~es~~~~~~ acid has een demonstrated both with the ~toc~o~d~~a~ and the m~crosoma~ fraction of rat liver corn~~~e with cytosol f2,3]_ From the cofactor d~~~~~e~ of the ~tocbondr~~ system studied collaborators [2,4,5], it may be cone ~~ch~~~s~ of s~de-cb~~ ckavsnge is of &oxidation of fatty acids, and that the reactions are performed with the CoA 3a,7~,12cr-trihydroxy-5P-cbolestaraoi@. a ~o~v~~si~~ should thus involve particip CoA synthetase, of an acy1 dehydro~~~~s~ or a~
choiestanoic add into chohc acid than had the microsomal and the ~toch~~~~al fraction [6,7]. Previous co~t~~d~c~o~ results with respect to subce%&r &~&.iur~may he e?rtpfained by the ~ffere~t degree d ~~~a~~~M~ ~U~~~~~~~ of the micx5soma and mitoc~on~~al prep~ations used. It was shown that the peroxisomal conversion was absoiuteiydependent upon ATP, CoA, 8iAD’+ and W?+ 1 md that the substrate had to be activated by CoA prior to the conversion i7$_ Since smafi amounts of 3~7tu,12a,2Lf_tetr~ydro~~5~-c~o~~tanoic acid were isolated> it w&s believed tha,t this ~rnp~und was an interm~at.~ in the reaction. Attempts to remonstrate an ~saturat~ intermediate in the reactirm failed, however ‘Bat there is such a ~2~~~~~~~~~d ~~~~~~~~~ ia the peroxisomaL conversion of 3ar,7cu,J2a-trihydroxy-5~cholestanoic acid into 3a,%,I2q24-tetrahydroxy-
a commercial pellet &et, were used for Preparation of the peroxisomes. The liver was finely minced and ~~rnoge~~ed by one stroke in a Pott@ Ehehjekrn ~~rn~~~~~r. FrGrn the 10% Ever ~orno~e~ate in 0,25% sucrose and 1 m&B EDTA with Trizma Base (pN 6.5), a postnucfear supe~ata~~ was prepared by centrifugation at 2200 rev/mm (600 x g,, j for 10 min, Tie p&etwas rehomogenized and thhes~~~~s~o~ r~entrifug~. The combined supernatants were centr~~g~ at 65Qo rev/m& ~4~ X g,,) for 10 r&n. The 4900 x & s~~e~~at~~ was ~~t~f~g~ at 16 200 rq&nin (24 2138X g,,) for 10 min The resulting pdlet (light ~tocb~~d~al fraction) was washed once and resuspended in the horn~~~n~ medium, P~ro~soma~ en~ched fractions were separated OII a Linear sucrose gradient ~24-4~~~ w/w) with Dextran T 40 (2-3.8%, w/w) in a vertical rotor,
SR.nhnlaotor\r\;o Jp-~~l”ILmLcbAIIIc.
Thza I ua
oAA QClU
;c. 10
ek/*vx,n .TLlWWA1
’ A:rontlrrr Ia.IILwlly
:y. 111 :ki::
present study by performing the reaction in an ~s~~-a~o~~~~re and in the presence of a deuterated n~~~~”
I:mL+ U&uL
-:+T.nL,.n~c:nl uILL”L4ucuM.~IIal
iL,,r:.... IlzaLU”II
vl”...l WilJ
I....,.--1 uiycxcw
_.a “LA
the $5~ of the gradient and centrifuged at 20000 revJ%Iin (35 800 X gIly> [xp Fractions of 3 mi were collected, The fractioris with. the highest ~ero~sorn~ marker enzymes (7) were pooled. and diluted I : 3 with 0.25 mM sucrose/f5 mM Hepes fpH 7.4)+ The ~Ko~~rnes were su~~ue~t~~ ~e~~e~ted at 3rw,7~,12rr-T~hydrOXy~5~~t7~-~H~Ch~~eSta~OiC 75 QOBX g,, for 1 h. The resulting pellet was resusacid (02 rnC~~~~rn0~)as we13as the co~~p~~~~~~ pended in a minimal vohtme of the d&&on ~n~abe~~ steroid and 3n,7~tu,12ar,24-tetr~ydroxymedium to a concentration of IO-20 mg pro5&cholestanoic acid were prepared as described pret~ous$ /3], The 3&]&&~ rvww..nl*~il *I.?RO tein/ml. ~“Al,y”k+nu w ix3 purified ~rnrne~ate~~ before use by hjgh~~~rfor~ mance liquid G~r~M~~~~~~~y fi%%C], using ph~spbat~ buffer ia methanoI as eIutin# solvent The i~cub~t~o~~ mixtare costumed the Folfow[cf. below), The <25R)- and ~25~)~is~r~e~s of ing in a total volwme of 7.5 ml of 0.1 M Tris-HCf 3ff,7ff,l2~-t~~ydroxy-5~=cho~~stanoic acid sep buffer, fpH 8.0): 7,5 mM ATP/2 mM CoA/lO arated under the ~PLC-~~di~o~s used and the m&f Ii@$I,fS pm FAD/2 mM NAD_/l,S mg material was faund to consist of about 28% of the ~~~~~~~~rn~~ protein suspsd& in100pl s$o*25 @SR)- and about 72% of the ~25~~~s~rner. The M swxo~e 14 mM Hqxs buffer/~~H 7.4) two G2S isomers were not separated, since both 3~,~~~~2~-tr~hydrox~~~-cholest~oi~ acid, 30 ,ug forms are equally well converted into cholic acid unlabeled and 500000 cpm. labeled compound and 3a,7cr,12a,24-tetrahydroxy-5@cholestanoic dissolved in 20 ~1 of ethanol. The air in the acid in the peroxisomal system used 171. Deu~~cuba~~on vessel was removed and replaced with terated water (99% pure) was obtained from Merck an atmosphere consisting of about SQ% IQ, aand ~~~st~d~~ F.R.G.) and “W2 gas (98% pure) was 50% r802. In another experiment the incubation from Pro&em, B.O.C. {Deer Park Road, London, was ~~rforrn~ in an, but with sn incubation U.K.). me~i~rn ~nt~~~g more than 95% “H,O. The i~c~bat~ous were p~~f~~ed for 20 tin at 37 0 C and were term~na~cd by auction of 150 pt of 6 M
After 36) min of e was acidified an ethyl acetate. The ethyl acetate
was
eluted with 24% 25 m in methanol. Aliquots of the fractions were as“_.,_A F-F T,; 6,X,1, OorJe+U IUS rnrl;r\n&;,r:+.r laUlvaW1”lry ;, la1 a_ Dn*l,n‘-J i aL.na*L_n I ir-Lai v i:,..:FJ llyueer scintillation spectrometer after addition of countsolution (Packard a Gel HI). ecovelry of ioactivity from the IL-column was essentially complete. Combined
gas-liquid
ch~oma~og~a~hy-mass
spec-
0 Thus3 only a negiig tracing at m/e 753,
qereas a significant peak ~2s
2rometry
The appropriate chromatographic fractions were converted into the methyl ester-trimethylsilyl ether derivatives [9] and analyzed by combine atography-mass spectrometry using a 1.5% olumn at 280 ‘C and the LKB 9008 instruent (LKB Instruments Inc., Stockholm, ed with a multiple ion detector. experiments the instrument was focuse ions at m/e 753, m/e 754 and m/e 755 (M - 15 ions), in some experiments on the ions at r~/ie 588, 589 and 590 (M - (2 .90)ions) and in some experis on the ions at m/e I89 and 190 (cf. euterium contents ted according to
In agreement with the previous work [7], cholic was the major product (about 4%) and ,12a,24-tetrahydroxy-5fi-cholestanoic acid a minor product (about I%) after incubation of 3a,7ql2a-trihydroxy-5b-cbolestanoic aci peroxisomal fraction. It should be pointe under the gas chromatographic conditions employed, there was no separation between the (24R)and the (24S)-isomers of 3a,7cu,12a,24tetrahyrlsnw~.l_~R_rhnlprtonrr;r catA. UllU ~nrl alcn nnt ~IVAJ-~r-UII”IU~CUllVlV U_U UlU” IX”_ betweepB the (2X)and (25S)-isomers of 3a,7q12a-trihyroxy-QLcholestanoic acid. The synthetic stanard material contained a mixture of all the four possible isomers of 3a,7a,12a,24-tetrahydroxyS,kholestanoic acid.
between the height of the peak at m/e 585 and that at m/e 587 obtained in selected ion monita:~. t~i~et~y~s~~y~ether, methyl ester; it was IlO] that the mat ial contained 0.‘” euteriiam was pre-
same as that obtained in ahe corresponding ana@The fragment a’s m/e 253 is
cholestanoic acid. The fragment at m/e 189 Is bably due to From the rati in the tracing of the ion at m/e 189 of the ioiz and the height 0 hagmen% calculated that at m/e 190, it mntainm-l Qb atnmc nf ~,,I~IIIyAyIu “..,6; -..,II-” -* dPlalPrirlm lW1ll--l---~
Clear evidence is given in the uction of a Why