! " # $%Mg 2+ "&'( )* "# ' $ %& "#! # $%&!" + / ' ( )'*+% "#$ %&!PAP #!PAP 1,6')' '/'5$ ) #"#)123 &-#4/.-,-!PAP Mg 2+ 2 7)'<#)'/'5$ ' 7'.8,9")$ 65#:;,)*+,$6 /#'Mg 2+ 7'$"#=8,98>? 8%=#Signal transduction#-8 @A',BC '=7')D"# '1EF G/5$PAP 2b PAP 2a ) #"#PAP 2 '6)+H1#/###? PAP 1 $8&Mg 2+ $7E8*E PAP 2 %BE@ 4'L %,#4B$/8I #)J7 6")6$2K=IB4" )$ "'.E8*EMg 2+ *B?%E=)$$7"MD<<N 8,9" )$O 1 7")6'$I+'#8,9)$)*- 4+ I "4@56=)P Mg 2+ $7 /5$ Q '*R)1'23 ''&-)+' )-*S)4.NGPAP 2b >5 # ")6'$(Hexagonal)H II (Lamellar)L a "' )'L )K'#PAP 2b '%'B/#)+ "'4@'56=)'P 7')P'/'5)$X-176)U *6R"8J*TS# #";,'T'3 $L a )/5)$' H II $L a )%& )$-V /5)$Mg 2+ 7U *6R" 8J* 4@56=TS 4'+ I "'4@'56='/'.- 'L )K')6$L a )PAP 2b : WD" 6 -'S";,T%2SZ6/#)+8,9:Y E$L a )X$$)*- '[\'1# H II '$L a )%& /#$-V"4@56=TS#L a )X$ /8)+2)69)X_]^^76)" S\1$8&X_]^^76) ' %>'Y 'E$-*E)V,.- L )KL a )PAP 2b >")+ H6 "'4@'56=%'`*'Ea @E)$ ##)+ 8,9:-Y E$##)+ H II $L a )%& Mg 2+ 7'7'N/'#)+ ''8,9:Y E$L a )X$8*9$)*- 4+ I 8')+'H67 V,##)6$L a )$ #5)6$H II )#H%>Y E$ /##)+ >7\@*$#Mg 2+ 7$">.- H II L a.- *-+," E-mail: heidarian46@yahoo.com ()*+,- +).
4'+ I "'4@'56= Mg 2+ 7'-*B? /5$7")6$I+#)*- ( ") '''''#''''$''&EI#''''6#''#'' -N!DTT ('6)"#!"#n'* '' Soyabean I'..->46;D',!NEM 'B,%' 46'';D8''D%''''- 4''.$tripsin ''''''''-''*+%'''''6--n''''''4'''''; EDTA 2d!Sigma,USA S 3 %'''''''''')'-'' " ''''+4'''''''&,)'''' ( )''''*+EGTA 3 /!Merck,Germany "'&-">,*X PAP 2b $%5 6#43 " :'@B Fleming3 o ')'$)1'23 ' 3 '$8'D%'''- '*S)\ #)+Q *R )1'2">'5#'9/!h 8')+HBatch wise /#$)+cf^c^^i# 1# #E]e if&'['-'5 O'&G $%88 9: ; < 47 /!q #)+")+ I:H.)$#8I5I##p)''5? /8#3 6)-5 6#-">4= '. 8)"B # 8 8"@8#+8A e #5I# 9BH"6=2$r # H II '''!Lammelar L a s''''''''''''- '$6= #4C =@.$)'J5$!Hexagonal \''>Q R' "'?7'@8' 6 #4'-O )G4$84@BB5@">5 #'[#c^''$ C "'##A)6'\' 4'-,r 5?6S$]f^^g I'7#'('*1I#6$ I5?8[#$##)+ #''?!(''@*+4*D )''D)''6, ''*]^^#)''+^th B # H II '''812*-u$##)+ : 1.Phosphatidate phosphohydrolase 2. Ethylene diamine tetra acetic acid 3. Ethylene bis (Oxyethylene nitrilo) tetra acetic acid 4. Aggregated 2 )1) ]!PAP, C 3.1.3.4 ''' ' ( )'*+ % "#$ ''''''%& %2S( )*+%"#/!].-,-!P i 9 )' )'*+")'6.'#':D7.9$:.- 4 %' 4,'-%'%'`b '-)";, #4/!c ] #)+ )'[I#''6#4( #)' ''5$') #"#)123 &- '6+PAP 1 7'$ ###'? ' )'@ ( 6' 7''$'#'=@,$'-8,98>?)4/#5 ''>!NEM ''B,%'' N*''$ ##Mg 2+ %'=#'';,' ;,)'*+,$6# #5 '6)+"?BF D6X#-)<#)/!e d 8 Signal ##'''5 '''''''6''+PAP 2 7'''$ '>'B,% N\ ##:transduction 7'fmM)'$"'8'J*#!f d #'5B #PAP g #5>Mg 2+ 2 -I5&Pi&[96/!h PAP 2b PAP 2a )' #"#)1'23 ''&- $' PAP 2a kd^i'&)'j'1,-5$ " )'$"# '11/!h f #%@ PAP 2b 8'*E$/86)+2PAP 2b PAP 2a ") Q *R4<B4l )D 2$7.- PAP 2a @. '6)<H7" )$" JSF %$[18I -'SPAP 2b " )'$'6)+'H'1/!h 8 '" 4Zn 2+ Mn 2+ Ca 2+ ">">)P 4)'D"# 4<.4 )D)*-(,)D )D%` * "'>)'P<<N @C K=#/!h 5$ /!h 8'6)<21<m-V">- "'8,98>? PAP 2b -8I5Q R4.mB #'5 >( )*+%"#\ #;,$ /!h PAP 2 "'.'E8*EC K=#17.- 1#)<#L )G/86)<2Mg 2+ 7$ ")6'$2K'=I'B4" )$6)+H : W'D4'L 'V', /8I #)J7 6 %E=)$$: MD<<N 8,9)$
H 8 "I+"- H/A J K" 4 8B # " )""* 'I )D)w)$8>?.H II L a?# ]^]^te^t]^"'8'j*'hu$h II $L a ) "'8J* I 4''@'5'6=*f^e^c^ #)*- 4+*]f ]^htffc]^ #)+#H8,9:H.)$# 7, '%2#dfgnmx('G#"'r V?)w "'#B'2'$Z'6!]^ 5I=" [# /#)+"r V?%$#7 8 Mg 2+ 3 6)-MK"4L @ 8&) H II L a -"88?#8 88N8: ; < @88 &)8 89: ;< ',,"#'E8'6'# #. X-188888# 7'.E$)<#6# 57.E$6# B: 8',9:H.'\'1'5")#/#)+r R68 ^tc^"'8'j* '^tdfmm" 'S 8")#/#$ MgCl 2 *]^ ec]^te X-17'6)'['8',9:H.'\'1 MgCl 2 '*]^ec]^te^tc^"8j* '$,,Ba ;/#)+#H^tdfmM ##'',,'Bi'&[96/'5'$@'[#]^ L a '"'618)D'' A)6 c #]f^^g "'61",, #)+?#$ H II "61-r '$Z6/!]] ]^.6)+)[";,T#L a #MgCl 2 8'J*%'$#L a )''2#2 /#)+7[ X-176)" S:H.\1 : OP- #"'r V'?)'w 7''u$]IB5#B 7',H II )''$L a )'%& ")$dfgnm 'I8,9:H.\1# r V'?7'785V+$-#7#B4/#5 '$':'L a )H II {%@%,#$" :'-\1# H II {%@9$$6[#, /$ 7''.E'$'I'8'#$ (*1/##)'+&P7 /!v #56)+)J#L a,")6$ H II 8L a 88 C#8)D"4 8'>?.$88%8 9: ; <! " # # ''7,#H II $L a )w)$ ^tdfmm8'j*#ha D8,9:H.)$ ('G#"'r V'?)w:H.)$# '[# '%2#H II %@")$dfgnmx "'r V'?%'$#7'#B2$H6 8y)[ /!]^ v #)+ FG88) PAP 2b $%88?88E#884+ H II )''>8'>?.! " # 8 H II L a - ^tdfmm8'j* r R6B:,,8)6$ '[X-17'6)'[:H.'\1#)6$ H II )''$)6$L a )%&")$ #)+#H 8''''J*#''''H\''''1#!Phase transition '5'$@[#I#$ #)+ Ca 2+ 7]^^mM ]^ml'$ '#)+'?x.$o$uh II r a ;/!]^ I#6'$654S# 565$R$)u$ #r I'$ # I'5#)'=iF '-r '"'5'* ' )'u'$ #r '$'$f%[s%be4 #)+A)6 '$-H.)$#r *S)4)=#/#)+)@ '*]^ qgec]^tf^"'8'j*"61b ) ',,8'a ;'/!]^ 5%S#$X-17 6) )''$'^tdfmm8j*"#<b-)<# ]^ qgec]^tf^"''''61:h.'''')$#l a '*-'$a ;' #'H#$X-176)' * '$Z'6 '5")+I>8,9 z,, 7'6')U *6R"8 ''J*%$#8,9.1.2 /#)+H II L a ")")$X-1 H 8 "I+"- H/A J K" 4 "' 8'J*.8 PAP 2b 9"* ''' '''*^]cfhq^f^e^c^]^u '''*6R #'HG$)*- 4+ I "4@56= ")'+I8,9 #H8,9:H.\1 '4@'56=8'J*%'$#8,92$Z6 /#)+
')*- 4'+' *c^ I * #'H'5$8&8,9:H.\1#)P4)6$ /..- T'3 $L a )IB[$2#gIB5#B# T'S#H II L a ''"' '?a 'D";,'' 4'+ I "'4@5'''6=-V'"'8'J* '7'%@'5'-uB/#5I)*- X'$'2#-V'"4@56=8J*:''$# /$:\1#L a ) T'S#L a )'$)6$X$2#hIB5#B 8,'''9:H.')'$# Mg 2+ U '*6R"'8'J* %@54/#7''X-176)['' "'61 X',Y 'E$ Mg 2+ 7'8'J*:'-#7 #89z 4,##)+L a :- H II %@ )6'$8'X-17'''6)''['-H.\1 /5$.4 L )K'#PAP 2b '%'BcI'B5#'''B 7#B4/# 7H II L a ") ")6'$L )K''$*'BPAP 2b ''-# #'X-17'6)TS#6SH II )$ #%'''''B4' ##)6$L a )L )K$%B )'$)$ctf'emm8'''j*#x-17'6)''ts )'Pb ')'$edib5#b#/#5 )1)''6$ %'& )$)*- 4+ I "''4@56= #'-u'''b#'5'i'#''h II '$L a #4@56=8J*:$#5I#B %,#$-#56-"r V?5:H.\1 /5$\''1#H II {%@")+*? 4'+ I U '*6R"'8'J*)'PfIB5#B /'#'7'' PAP 2b 8',9)$)*- Y 'E$-V'"' 4@'56=T'S#'B4'O $u ]^"''8J*@''u$#)+ 8,''9:.3.2.1 2 4 6 8 1 12 Time (min) "! /#$dfgnmx(g 8,9:H.)$# *^tdf 8J* Absorbance
12 Activity, nmol P i/ m in/m g protein 1 8 6 4 2 2 4 6 8 1 12 TritonX-1,mM "((- H II, L a )*+(((' PAP 2b $%& # "'8'J*T'S#! H II! L a "')''$)6$"618,9:H.)$#!^tf µ g PAP 2b 8,'9 /#)+")+IX-176)U *6R.35.3 Absorbance.25.2.15.1.5 2 4 6 8 1 12 Time (min) " H II 5L a!/,12-34. ^"'8''J* 'H#8,9:H.)''$#''*^tdf '''7',"'r V''?)'w /5")+II *! f^! e^! c^! ]^! ]! ^te! ^t]!
.35.3 Absorbance.25.2.15.1.5 1 2 3 4 5 6 7 8 9 1 11 12 Time (min) H II 5 L a!78,)912-34 6! c! ]! ^"'''8J*'H'#8,9)'$#*^tdf 7,"r V?)w /5")+I4+)*- *! ]f! ]^! htf! f,activity, nmol Pi/ min/ mg protein 25 2 15 1 5 1 2 3 4 5 6 7 8 Conc (mm) PAP 2b ; <78,)9,/,4 : '$! ')*- 4'+! I U *6R" 8J*TS#8,9:H.)$#!^tf µ g PAP 2b 8,9 /5")+I+'?'G
PA in La form, % 25 2 15 1 5 5 1 15 2 25 3 35 4 45 5 55 Conc (mm) C 7-D) ; A7B ><? @ L a 125 = 78,) 9,/, '7',$! )*- 4+! I U *6R"8J*7# a DL a %@5$IB[$ '\'"'T')'P#7I5#P i #)+"? H II r 8,9:H.)$#^tdfmM /5")+Ikh^ )*-)D 12 P A i n L a fo rm, % 1 8 6 4 2 2 4 6 8 1 12 MgCl2, mm X-1- B,FGL a 125 Mg 2+ C 7-D); A7B4 E H I J #! X-176)''''['! X-176)"61:H.)''''$#L a %@5$I''B[$ ''' kh^ ')*-)D'\'"'T')'''P#7I'5#P i #)+"?H II r Mg 2+ U *6R"8'J* /5")+I - I; Q &
a 'D'-#'5I#''8,''9:'H.\1# 8'$Pi'&) H II '$L a )%& [#885V+ %'E H II $L a )%& #(S)>$/##)+U [6 s'7''%e4*b?/#8, =#"#96$ #7#'$s&'5)s&5>I)H(Gr )N" 8',9'#['<B'-!g #)'$' '' '#['-)'<#%'E/'.#)'[)P81PAP 2 #''5$)PH II $L a )%& " )$ <6'$'>)P-8I#6#:H.)$#I5%S #'=)'P(*1C =)w$ ##>7@ 8J*$ 4@'56=7''#4*B?/!]v..-(BE b ''''&''-#)''$''I '')*- 4''+ "#B#/!]v #5L a "''w"''''d %'''&' )$-V"4@56=)'Pe dib5 '-#'5''6)+H6 #5I H II $L a ' L a )''%'&' :H.\1#&-)4TS /..-89B H II $ "'8'J*#-V'"'4@56=)''Pf#'B# I'%@54#/#5I8,9)$U *6R -V'"4@56=8J*:$b.6-#5 #'='-'$':8,9:H.\1# '$7''E \L a )L )K)$")<# T'%'2SZ'6gIB'5#'B#/8 Mg 2+ 7 )'%'& #-V"4@56=)P";, 6)'''+'H6#'''B4 #5I H II $L a {%@' ''4@'56=4#? -#5 # \'1#L a )'X'$ 'SY E$ H II 4)$'.$/'$:8,9>TS#H6 PAP 2 '")'$)<#"4%2SZ6 '@.'$'?$ 5$L a )L )K8>?!]^ #'5')'6'$H II )''X,Y ''E$Mg 2+ 7 '8,9)$">")P74-#)-x6.67 /#7$" ## #)''6'$L a )'I'B[$'2#hIB5#B# X-7'6)''[ " S:H.\1#Mg 2+ ''TS #'-#''5 '&.6%@54/#5I1 %''&''' X 17'6)'[:''H.\'1 "'>'$'#'=8',9")'$;,''=)$ ')1 Mg 2+ *'$'PAP 1 8,9#6)} # /!]c 8'Mg 2+ '$6'$ ) PAP 2 @,S##5 Ca 2+ %'`6)''}#">)P)$%2SZ6 "''>)''P-S Zn 2+ Mg 2+ PAP 2b " )'''''$ 1#',/!g f 5$PAP 2b 8'',''9)$> /8'I7,B6S@>s$"I5-V )' PAP 2b -#5IcIB5#'''B# Y 'E$'-*'E)'''V',.-L )K)6$L a '$##)+ H II $L a )%& %> :''-Y 'E$ '#")6$L a )7#)-x= #6)} #">)P)<#L )G/##)+ 8,9 '>4$ 8I''mD#''$"';,*D '>4'.Ni'B9/!]e ]d ###'''? '''' ';,'")'I )'+$ (K%B")6-6j'1, #'? ''>~ '"8J*#6S%B4 # 8' '$'$'>(K'"'8'$P@.%,$##!]f_]h #)+H II X,Y E$!]e ]d ''$'u$#/#)'+ 'B)'[' L )'K'#'- 'S#'.'$46'5#8*9$-I#)+&P ~ ')#'TS# ##>-$(K$%B") #')'$I F'E/!]q ##)'+ 'X',7#H II >6- ''$U '*6R">-(K[-I5&P-V '['$B6'u$-##*6R?# -V''b,''u'$'?'$V', /!]e ## H II {#H )''6''$'L a )''PAP 2b 7N#)-(67 %'E -Mg 2+ 7$"4)$.$.-L )'K' ".8*E$u$#/#5$H II )I..-X, :@B S8,98''>'?Mg 2+ 7''$PAP 1 '-u S)PAP 1 -#)-&P L )K'##)'+#H H II $L a )%& G# =)$Mg 2+ 7'''.%'',#4B$!]^.- %'& X',Y 'E$'5$4D"8J*#> '#\' 'S)'%@'7.-# H II $L a ) /!]^ #5PAP 1 )'''6$ H II $ L a )w ]IB5#'B#
1u'8'[#'HO')G)6$' H II $L a ) i'> I##:'-9BH"a @*;B-%@89B /##)+\1#)6$L a )X$Y E$ 7'6)"'%iB6S/##)+ H II $L a )'' ")6$46)+)[.8.?)#)' X-1 '$ I#)''-'>E.K'"X4# %'&' 7'@#@%@5)w#'H References 1. Smith SW, Weiss SB, Kennedy EP/ Enzymatic dephosphorylation of phosphatidic acid/ J Biol Chem 1957, 228: 915-22. 2. Brindley DN/ Intracellular translocation of phosphatidate phosphohydrolase and its possible role in the control of glycerolipid synthesis/ Prog Lipid Res 1984, 115-33. 3. Jamal Z, Martin A, Munoz AG, et al. Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol; J Biol Chem 1991, 266: 2988-96. 4. Martin A, Comez- Munoz A, Jamal Z, et al. Charactrization and assay of PAP; Methods Enzymol 1991, 197: 553-63. 5. Waggoner DW, Martin A, Dewald J, et al. Purification and characterization of a novel plasma membrane phosphatidate phosphohydrolase from rat liver; J Biol Chem 1995, 27: 19422-19429. 6. Kanoh H, Imai SI, Yamada K, et al/ Purification and properties of phosphatidic acid phosphatase from procine thymus membrane; J Biol Chem 1992, 267: 2539-25314. 7. Fleming IN, Yeaman SJ/ Purification and characterization of N- ethylmaleimideinsensitve phosphatidic acid phosphohydrolase (PAP 2 ) from rat liver; Biochem J 1995, 38: 983-989 *H/)123 &- @6,-8,9#4,Q R/.7S/q /fq_f^c ]dqe>$( IB5#I #F @5D*EI<#B*E 9. Seddon JM. Structure of the inverted hexagonal (H II ) phase, and nonlamellar phase transition of lipids; Biochim Biophys Acta 199, 131: 1-69 1. Haghighi B, Yari M, Tori S. The relationship between cation induced substrate configuration and enzymatic activity of phosphatidate phosphohydrolase from human liver; Iranian Biomed J 2, 4: 13 19 11. Comfurius P, Zwaal RFA. The enzymatic synthesis of phosphatidylserine and purification by CM- cellulose column chromatography; Biochim Biophys Acta 1977, 488: 36-42. 12. Butterwith SC, Hopewell R, Brindley DN. Partial purification and characterization of the phosphatidate phosphohyrolase of rat liver; Biochem J 1984, 22: 828-833. 13. Farren SB, Hope MJ, Cullis PR. Polymorphic phase preference of phosphatidic: A 31 p and 2 H NMR study; Biochem Biophys Res Commun 1983, 111: 675-682. 14. Papahadjopoulos D, Vail WJ, Pangborn WA, et al. Studies on membrane fusion. II) Induction of fusion in pure phospholipids membranes by calcium ions and other divalent metals; Biochim Biophys Acta 1976, 448: 265-283. 15. Hope MJ, Cullis PR. Effects of divalent cations and ph on phosphatidylserine model membranes. A 31 P NMR study; Biochem Biophys Res Commun 198, 92: 846-852.
16. Jacobson K, Papahadjopoulos D. Phase transition and phase separation in phospholipids membrane induced by changes in temperature, ph and concentration of bivalent cations; Biochemistry 1975, 14: 153-161 17. Hauser H, Friner E, Darke A. Crystalline anhydrous Ca_phosphatidylserine bilayer; Biochem Biophys Res Commun 1977, 76: 276-274. 18. Ito T, Ohnishi SI. Ca 2+ - induced lateral phase separations in phosphatidic acid phosphatidylcholine membranes; Biochim Biophys Acta 1974, 325: 29-37. 19. Marsh D, Watts A, Smith ICP/ Dynamic structure and phase behavior of dimyristoylphosphatidylethanolamine bilayers studied by deuterium nuclear magnetic resonance; Biochemistry 1983, 22: 323 326.
Rat Membrane Phosphatidate Phosphohydrolase and the Reason for its Mg+ Independence Heidarian E 4 *, Haghigh B 2. 1) Dept of Biochemistry, Faculty of Medicine, Ilam University of Medical Sciences. 2) Dept of Biochemistry, Faculty of Pharmacy, Esfahan University of Medical Sciences. Abstract Introduction: phosphatidate phosphohydrolase (PAP) catalyzes the dephosphorylationof phosphatidic acid to yield Pi and diacylglycerol.two forms of PAP in rat hepatocyte have been reported.a cytosolic form (PAP 1 ) that is responsible for glycerolipid metabolism and 2+ requires Mg for its activity. Another form (PAP 2 ) is primarily involved in lipid signaling pathways which dose not need Mg 2 +. It has two isoforms; PAP 2a and PAP2b. Little information is known about enzymological characterization of PAP 2 especially its substrate. We investigated the enzyme behavior against Mg 2+ structure- breaking agents (urea and guanidine HCl) with respect to its substrate. Materials & Methods: PAP 2b was purified from rat hepatocyte membrane using a multi stage chromatography. The enzyme activity was determined against L a (lamellar) and H II (Hexagonal) forms of substrate in the presence of triton X-1. The effect of Mg 2+ concentration and urea and guanidine HCl (structure- breaking agents) were examined on L a to H II phase transition and enzyme activity. Results: PAP 2b consumes L a form of phosphatidate. Cations such as Mg 2+ result in H II from L a phase of substrate and therefore reduce enzyme activity. Urea and guanidine HCl increase enzyme activity due to prevention of H II formation. Both can also reduce the effect of cations on L a to H II transition of phosphatidate. Conclusion: Since PAP 2b consumes L a form of substrate parameters can induce L a to H II phase transition which result in low enzyme activity. In contrast, factors such as urea and guanidine HCl increase activity due to prevention of L a to H II phase transition. As Mg 2+ stimulates H II formation and the enzyme needs L a form substrate, it can be concluded that, Not only does the enzyme need no Mg 2+,but it is controlled by it too. Key words: Membrane phosphatidate phosphohydrolase, phosphatidic acid, L a and H II phosphatidate. *** * Corresponding Author: Dept of Biochemistry, Faculty of Medicine, Ilam University of Medical Sciences.