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Hydrolysis of sphingomyelin to ceramide with hydrofluoric acid
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!!ence,Bangalove-S60012, Ind~
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ide neat ~ ~quanti~tively:on treatment with 40% i yelin ~ t h HFis much slower than phosphoglye fattVacid comvosition orthe stereochemical
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!lipid in mann nalian tissues , is usually clmracterized id composition. In such studies, sphingomyelin rochloric acid in anhydrous methanol or in methatemperature. These procedures of acid methaph0rylation of the base [2] and also to the foroducts of sphingosine [3]. Alkaline hydrolysis, on, the otherhand, does n~,Itproduce secondary products but appears unsuitable for -the isolation:of!Sp~gosin~ bases in high yield [4]. To obviate these limitations, e with bacterial g-chain base has been found ~ichoic acids ~ort on the reacthe quantitative
A, Preparatfon of spMngo1~oids "
methanol (2 : 1) and
Minn. 55912, USA. 373 •
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Hydrolysis of sphingomyelin with phospholipase C:
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.F.‘Ahulyticut methods _
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‘.Fai&acid methyl esters were analydd on a 6 ft. column of 15% polyethylene &cd %&ate.ai 2kk in a gas chramaiograph equipped with a flame ionization Fh&horus was d&rmined b$ Bartlett’s procedure [ 151. 7_ ’ d&d& . , 1 I1 III. R&&S 1I Sptigomyelin when treated with 4&GHF at 40°C yielded a single lipid soluble product with an Rf of 0.45 on TLCp]ai:esdeveloped with chloroform : methanol (95 : 5, V/V).me product did not stain ,tith phospholipid spray reagent 1161 and showed ‘atic&i&y identical to ceramidi: formed by hydrolysis of sphingomyefin i _.~ 14th CL ive&hii &haspholipase C (fig. 1,1. With the more polar solvent system chbro’ ,‘.f&m_,: r&h&~1 I i-i@ @i’S : 25 : 3, by v/d.), c&amide moved with an 12f of 0.93 and _’ ‘..~~~~is~f&$t &&phoius positive spot w@r an Rf of 0.48, probably ceramide phos-_ ~‘~.~p~~~, wgs no&ed.Hawev&, ceramide:phosphate was observed only in the beginning ‘. .“’ &f$!@&kitient (12 hr) and was not piesent at later time periods indicating that -:.‘&ehyd&ytic product of sphingamyelbnis essential.&ceramide.As could be ex1 “‘$e@ed cer&kside and gangk~de did j&t react wjth HF (fig. I).
ethanolamine [S] and bacterial phosbut a strdngerreagent (S&60% of sphingomyelin with HF at U*C about 3@&washydrolyzed even in almost complete hydrolysis occurred in 8 hr with CT.welchlii to 0.6 d did not alter the j.
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~ed. either by ~s isolated by sp~ngo~e . _
....
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......
~-,-~i~imanolicHcl,giVes+ordy about 50---60~ of the e b~se [2]. Besides, acidic conditions lead to the forproducts from allylic,group transformations, methyl tsar hydrOxyl: carrymg carbon atoms [3]. The most enzyma~(IiCcleavage ofsphingomyelins . . . . . to ceramides ts.,It:iS Sihown herel that hydrolysis of sphingomyelin !,O°Cisalmost completein 72 hr and the ceramide its fatty addcomposition or in the stereochemistry ~+with:4~: HFmav therefore be a suitable method
+'!=
:A ~ o W l ~ g e m e n t .
.
Financial support from the ~dian Council of Medical Research is gratefully ack-
.
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,•
,
- -++~.[ 2
[3
++++i++~.!4 ..,i-+ U s
+ [+
:C Sweeley J, Am OitC~emists Soc. 42 (196) 294 ~; K0~k~m,J. Chromates,+31 (1967) 632 i~0thfuLs~andR,Gigl~,~J' Lipid.Rex,2 (1961) 228 hm~tog+ 31(1967) 643 ictaq:hen~iScand. . . . . ~2 (i96m 3050 eio chem. J. 127(1972) 399 ~wn. Biochem./$ ,.143 stea'~[atJ: Baddj~ey' (1;974) 46 1 1975) 464 d M; es, Bi,~;chim.Biophys~Acta 398 ( s~i[G.H.-Slr.,.~n~tanle~. J. BioL Chem, 226(1957) 497 ++.+A
++++] ~e++iiC+~nij+,`Biochem.Physiol, 37+(1959) 91+1 i,E:i~;~m-and,:G.O,Steen, Bio~im.Biophys. Acta 316 (1973) 317 [d+p,~ iS~:iS i(J~964), i132
-
.
Bi0L:~++'~'rni~L23.4(1959)466 ~diF~¥~ Kmtetsky, J, LipidRes. 9(1968) 396 ++
+
+
It6] v
:!?i. : .
-
!!ence,Bangalove-S60012, Ind~
'~:"'~:'i!
_
. ~: ..
_
,
.
ide neat ~ ~quanti~tively:on treatment with 40% i yelin ~ t h HFis much slower than phosphoglye fattVacid comvosition orthe stereochemical
~: -
"
.
"
-
!lipid in mann nalian tissues , is usually clmracterized id composition. In such studies, sphingomyelin rochloric acid in anhydrous methanol or in methatemperature. These procedures of acid methaph0rylation of the base [2] and also to the foroducts of sphingosine [3]. Alkaline hydrolysis, on, the otherhand, does n~,Itproduce secondary products but appears unsuitable for -the isolation:of!Sp~gosin~ bases in high yield [4]. To obviate these limitations, e with bacterial g-chain base has been found ~ichoic acids ~ort on the reacthe quantitative
A, Preparatfon of spMngo1~oids "
methanol (2 : 1) and
Minn. 55912, USA. 373 •
i
L
:
Hydrolysis of sphingomyelin with phospholipase C:
I
~.
~:~[
i
r
.
1
.F.‘Ahulyticut methods _
.s I
-_
_I.’
L .
;
‘.Fai&acid methyl esters were analydd on a 6 ft. column of 15% polyethylene &cd %&ate.ai 2kk in a gas chramaiograph equipped with a flame ionization Fh&horus was d&rmined b$ Bartlett’s procedure [ 151. 7_ ’ d&d& . , 1 I1 III. R&&S 1I Sptigomyelin when treated with 4&GHF at 40°C yielded a single lipid soluble product with an Rf of 0.45 on TLCp]ai:esdeveloped with chloroform : methanol (95 : 5, V/V).me product did not stain ,tith phospholipid spray reagent 1161 and showed ‘atic&i&y identical to ceramidi: formed by hydrolysis of sphingomyefin i _.~ 14th CL ive&hii &haspholipase C (fig. 1,1. With the more polar solvent system chbro’ ,‘.f&m_,: r&h&~1 I i-i@ @i’S : 25 : 3, by v/d.), c&amide moved with an 12f of 0.93 and _’ ‘..~~~~is~f&$t &&phoius positive spot w@r an Rf of 0.48, probably ceramide phos-_ ~‘~.~p~~~, wgs no&ed.Hawev&, ceramide:phosphate was observed only in the beginning ‘. .“’ &f$!@&kitient (12 hr) and was not piesent at later time periods indicating that -:.‘&ehyd&ytic product of sphingamyelbnis essential.&ceramide.As could be ex1 “‘$e@ed cer&kside and gangk~de did j&t react wjth HF (fig. I).
ethanolamine [S] and bacterial phosbut a strdngerreagent (S&60% of sphingomyelin with HF at U*C about 3@&washydrolyzed even in almost complete hydrolysis occurred in 8 hr with CT.welchlii to 0.6 d did not alter the j.
,:.
,
. >1
.
“, !
,2:-
-~ ?-. ......
.--"
,%
no~ SI~!I:,s i .~ .
v~ ,
; !~.7 !~
.
~ !!iiii~¸.,~ ~i ~i~/¸~ii~,~~,,~..... ~~,~, ~ .... ~
!~ ~:!7 i~I
•-
2 2
8
.
i i~i:i~!i~i:~i ,i~,~!~i!i~i!~iii: 9&6
~ii~i~!i~'i
+,o,+eadd+. .
"
+
+++ - -+
.
.-
~ed. either by ~s isolated by sp~ngo~e . _
....
. . , . •
•
:
:
::-{ !.!-i~
i
,
-
" ~
~..
-.
.
. +
.
......
~-,-~i~imanolicHcl,giVes+ordy about 50---60~ of the e b~se [2]. Besides, acidic conditions lead to the forproducts from allylic,group transformations, methyl tsar hydrOxyl: carrymg carbon atoms [3]. The most enzyma~(IiCcleavage ofsphingomyelins . . . . . to ceramides ts.,It:iS Sihown herel that hydrolysis of sphingomyelin !,O°Cisalmost completein 72 hr and the ceramide its fatty addcomposition or in the stereochemistry ~+with:4~: HFmav therefore be a suitable method
+'!=
:A ~ o W l ~ g e m e n t .
.
Financial support from the ~dian Council of Medical Research is gratefully ack-
.
-.
,•
,
- -++~.[ 2
[3
++++i++~.!4 ..,i-+ U s
+ [+
:C Sweeley J, Am OitC~emists Soc. 42 (196) 294 ~; K0~k~m,J. Chromates,+31 (1967) 632 i~0thfuLs~andR,Gigl~,~J' Lipid.Rex,2 (1961) 228 hm~tog+ 31(1967) 643 ictaq:hen~iScand. . . . . ~2 (i96m 3050 eio chem. J. 127(1972) 399 ~wn. Biochem./$ ,.143 stea'~[atJ: Baddj~ey' (1;974) 46 1 1975) 464 d M; es, Bi,~;chim.Biophys~Acta 398 ( s~i[G.H.-Slr.,.~n~tanle~. J. BioL Chem, 226(1957) 497 ++.+A
++++] ~e++iiC+~nij+,`Biochem.Physiol, 37+(1959) 91+1 i,E:i~;~m-and,:G.O,Steen, Bio~im.Biophys. Acta 316 (1973) 317 [d+p,~ iS~:iS i(J~964), i132
-
.
Bi0L:~++'~'rni~L23.4(1959)466 ~diF~¥~ Kmtetsky, J, LipidRes. 9(1968) 396 ++
+
+
It6] v