Characterization of Cysteine Residues in Cabbage Phospholipase D by Sulfhydryl Group Modifying Chemicals

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1 Journal of the Korean Chemical Society Printed in the Republic of Korea v x yw w s s q D l» p š * w yw ( ) Characterization of Cysteine Residues in Cabbage Phospholipase D by Sulfhydryl Group Modifying Chemicals Eun-Hie Koh* Department of Chemistry, Duksung Woman s University, Seoul , Korea (Received August 12, 2006). s s q D(PLD) 8 l» p q w» w v (SH)» w ƒ yw w. 5,5 - p (2- p ) (DTNB) l» SH» w» w w, 412nm y DTNB l k PLD 1 4 SH»ƒ ùkû ù, 8 M 3 k PLD 8 SH»ƒ. l» (4 ) š ù ü ƒ š w. SH» x p-j j (PCMB), p, p, š N- e PLD y y g. p m(dtt) w w PCMB w y y PLD ƒ y z. w» q w SH» y mw wù w q w k z PLD y y j ùkû. l» y-y y PLD y e w y w mw. y y w 70% PLD y DTT w. l PLD l» SH» w mw y w, w 4 SH» PLD y w w eš ùkû. : s s q D, v», 5,5'- p (2- p )(DTNB), p-j j (PCMB) ABSTRACT. Several SH group modifying chemicals were used to characterize the eight cysteine residues of cabbage PLD. 5,5 -dithiobis(2-nitrobenzoate)(dtnb) was used to titrate the SH group of cysteine residues. Based on the optical density at 412nm due to the reduced DTNB, 4 SH groups are found to be present in a native PLD while 8 SH groups in the denatured PLD whose tertiary structure was perturbed by 8M urea. The results imply that among the 8 cysteine residues of PLD, the half(4) are exposed on the surface whereas the other half are present at the interior of the enzyme tertiary structure. The PLD was inactivated by SH modifying reagents such as p-chloromercuribenzoate(pcmb), iodoacetate, iodoacetamide, and N-ethylmaleimide. At the addition of dithiothreitol(dtt) only the PCMB inhibited PLD activity was recovered reversibly. The micro-environment of the exposed SH group of cysteine residues was examined with various disulfide compounds with different functional groups and we found that anionic or neutral disulfides appear to be more effective than the positively charged cystamine for inactivating the PLD activity. The effect of redox state of 362

2 s s q D l» 363 cysteine residues on the PLD activity was further explored with H 2O 2. The oxidation of SH groups by H 2O 2 inhibited the PLD activity more than 70%, which was mostly recovered by DTT. From these results, we could confirm chemically that all the cysteine residues of PLD are present as in their reduced SH forms and the 4 SH groups exposed on the surface of the enzyme may play important roles in the regulation of PLD activity. Keywords: Phospholipase D, Sulfhydryl Group, 5,5'-dithiobis(2-nitrobenzoic acid)(dtnb), p-chloromecuribenzoate(pcmb) s s q D(Phospholipase D(PLD); EC ) ƒ ww s qp (phosphatidic acid(pa)) OH» w z. z y y s sp g (phosphatidylcholin(pc)) ƒ w j lecithinase ù z Ÿ w s z x. 1 PLD w s wz ƒ s s s q s» w, y z j ƒ š. PLD 2, y (ROS) w 3,4, 5 y x 6 Cu +2 w7 w. w wš PLD f ù x w. PLD cdna x 8 castor bean v w, 9, 10», 11»12 PLD m w š. d, l» l» š. l» q w 3 y k š 13 w, y k v (SH)» z y w»» w š. wì 14 l»» sü w w wš š š. 15 ù w CXXC š 16 glutaredoxin protein disulfide isomerase v - q y y k y y š š. l» w w œw» w. 17 PLD v (SH)» x j p-j j (PCMB) w PLD y w, 18 z l» ƒ y š v ƒ w p» x. ù 19 ƒ, PLD αƒ cdna l w kda PLD 812 l» 8 q š CXXC š ùkû. 8 l» SH» wš»ƒ q w ÿƒ j. PLD p ƒ MALDI-TOF» w l y v (SH)» w. 20 x PLD SH»ƒ PLD s ù ƒ v» x j yw w mwš w. PLD SH» 5,5'- p (2- p ) (DTNB) w yw y wš, ù ƒ ƒ q w t SH» mw SH» y w š w. SH» y-y y PLD y w e w š w.. w w (savoy)t w. Sigma z (U.S.A.) l phosphatidylcholin(pc), sodium dodecyl

3 364 š sulfate(sds), 5,5'-dithiobis(2-nitrobenzoate)(DTNB), N-ethylmaleimide(NEM), dithiothreitol(dtt), iodoacetic acid, iodoacetamide 1,4-piperazinediethanesulfonic acid (PIPES) w š, ethyl disulfide, cystamine, 3',3'-ditiodipropionic acid 2-hydroxyethyl disulfide Aldrich(U.S.A.) w. Octyl sepharose CL- 4B Pharmacia Biotech(Sweden) t, w w PC y j m v w w. p-chloromercuribenzoate (PCMB) hydrogen peroxide TCI(Japan) w w w. s q q D. PLD Lee Choi 21 w. m ƒ 55 C o w z þ g. m ƒ Lambrecht and Ulbrich-Hoffman mm CaCl 2 sww 20 mm PIPES buffer(ph 6.2) s x k octylsepharose CL-4B column š +2 Ca y j g. 400, PLD SDS-PAGE ùkû. s s q D y d. PLD y d PC» w Ÿw ww. Appleton w š Jung 23 w z wù g» š. PLD PC j s 5mmole PC g l x» k,» PC 2:1 25 mm SDS 0.1 ml š q» k z 10 mm MES (ph 6.3) 0.9 ml g. PC-SDS yw 125 mm CaCl ml z 0.01 ml š 37 C 15 g.» ice-water bath jš, 5%(w/v) (BSA) 0.1 ml 20%(v/v) perchloric acid 0.15 ml. yw vortex mixer z 15 w. d 1.0 ml x»š 0.2 ml potassium triiodide choline periodide e. e 1,2-dichloroethane 5 ml z 365 nm Ÿ d w. PLDy 1 choline mol w. DTNB w SH». v w stock yw PLD ƒ 3.04 µm š DTNB 0.1 mm w. š CaCl 2 EGTA ƒƒ ƒ 5mM 1mM w. ph M KH 2PO 4. TNB Ÿ 412 nm (ε=13,600 M 1 cm ) 1 Uvikon-930 spectrophotometer w d w.»»» w PLD w yw blank w PLDƒ yw (25 C) w w» w 10 z Ÿ d w. PLD SH» k k w +2 Ca w mw. PLD w k wš x ww w. x SH» x 10% ü w. SH» x x. SH» x ph M KH 2PO4 š PCMB Boyer 24 w. PLD x 37 C k z û PLD y d w. DTT w PLD y z x p w wš 2 yw ƒw ww. x x x w ù s ³e ùkü.. PLD w Bradford 25 ww. š PLD l». Ellman DTNB v (SH)» w y q x wš y TNB - ü. x w DTNB SH» l» j Journal of the Korean Chemical Society

4 Table 1. Titration of SH group in PLD with DTNB. SH»ƒ 26 w ÿ š. PLD l» cdna l 8 10, ù ƒ MALDI- TOF» w PLD l l» y k SH» š. y w 20» w x PLD DTNB w. TNB ph,» DTNB w» x 0.01 M KH 2PO 4 buffer(ph 6.5) w š DTNB 0.1 mm kw. d TNB Ÿ 412 nm l PLD SH» Table 1 w. k PLD 4 SH»ƒ. wr k SH» yƒ ƒ» w Ca 2+, SDS, w. Ca 2+ DTNB w, 0.5% SDS j w e w ùkû. ù urea guanidine-hcl w PLD g 8 ƒ SH»ƒ. PLD e x š w MALDI-TOF» w ew š j ƒ. PLD l y k yw y w. š ù ƒ l» (4 ) š ù ü ƒ œw š. SDS w SH»ƒ ƒw PLD 3 ƒ w w, PLDy d PC-SDSy» wš mw. s s q D l» 365 Reaction condition Reagent added Absorbance at 412 nm [SH] a (µm) [PLD] b (µm) [SH]/[PLD] c native PLD Ca +2 effect denaturing CaCl 2 5 mm EGTA 1 mm SDS 0.5 % urea 8 M guanidine-hcl 6 M a SH concentration was calculated from absorbance of TNB 1 (ε=13600 M 1 cm 1 ). b Protein was determined by Bradford protein assay. c The ratio means the number of SH group per PLD molecule SH» x yw w. PLD v (SH)» x j PCMB w y w ù, 18,19 PLD 10,20 w 8 l»ƒ y k PLD SH» p v jš. Fig. 1 PCMB w PLD y y š. PLD y d» w PC-SDSy» w, t PLD y 3.75 nmol/min/mg protein. PCMB w y y y (percent of control activity) ùkü. PLD y y ùkû 20 µm PCMB 15 y 80%¾ y yƒ w Fig. 1. Time-course of PLD inactivation by PCMB. Effect of PCMB was examined with 0.52 µm PLD for different periods of incubation time in 0.01 M KH 2PO 4 buffer (ph 6.5) at 37 o C. The concentration of PCMB: control ( ); 5 µm ( ); 10 µm ( ); 20 µm ( ). At the end of 15 min incubation period the mixtures were treated with 0.5 mm dithiothreitol for 5 min. The residual activity was determined as described in Experimental section.

5 366 š. y y PLD 0.5 mm DTT w y. t SH»ƒ ƒ PCMB w w š š. SH» x p p Fig. 2, š NEM Fig. 3 ƒƒ w. ky j PLD y w ù DTT w y z w. p 2.5mM l PLD y 75% y y g, NEM 5mM, 60 60% y y z. p NEM š w w y z ù küš. Yang 18 NEM z ƒ š š w, Lee mm NEM 50%¾ z w š šwš. x x y Fig. 2. Effect of iodoacetate and iodoacetamide on the PLD activity. The reaction was carried out with 0.52 µm PLD for 30 min in 0.01 M KH 2PO 4 buffer (ph 6.5) at 37 o C. Iodoacetate ( ); iodoacetamide ( ). Fig. 3. Effect of N-ethylmaleimide on the PLD activity. All reactions were performed under the same conditions as previous experiment. The varied time of pre-incubation was examined: 30 min ( ); 60 min ( ). PLD w w. PLD SH»ƒ z y v ƒ w SH» x PLD w y y z ƒ w. t SH» y w š ƒ q w mw. Table 2 w. m 5 q DTNB sw ethyl disulfide, cystamine, 2-hydroxyethyl disulfide, 3,3'-dithiodipropionic acid PLD y w g. 30% w z k wš q 50% ƒ y y z. x w w k wù w q PLD w š. y w SH» w» ƒ w š. Table 2. Effect of various disulfides on the activity of PLD. a RSSR Molecular formula [RSSR] Inhibition(%) 5,5 -dithiobis(2-nitro-benzoic acid) [SC 6H 3(NO 2)CO 2H] 2 1 mm 52.5 cystamine (SCH 2CH 2NH 2) µm b ,2 -hydroxyethyl disulfide (SCH 2CH 2OH) 2 1 mm ,3 -dithiodipro- pionic acid (SCH 2CH 2 CO 2H) 2 1 mm 52.6 ethyl disulfide (SCH 2CH 3) 2 1 mm 54.3 a The reaction was performed in 0.01 M KH 2PO 4 buffer (ph 6.5) for 15 min at 37 o C. The amount of PLD used was 0.52 µm. b The maximum inhibition was obtained at this concentration. Journal of the Korean Chemical Society

6 s s q D l» 367 Fig. 4. Effect of H 2O 2 on the PLD activity. H 2O 2 was preincubated for 15 min and after that DTT was treated for another 15 min. The remaining PLD activity was determined as previously described. SH» y w. PLD y SH» ƒ SH» y-y y PLD y w e ƒ H 2O 2 w w. Fig. 4 PLD y w H 2O 2 w w. dw y PLD y 5mM PLD y 70% ùkû. SH» x ew x SH»ƒ PLD y j w w wù š w. H 2O 2 y w y y PLD DTT w k. x PLD 8 SH» w MALDI-TOF» w PLD 20 yw y w ƒ š w. š ù ƒ k PLD 4 SH» (Table 1) l» (4 ) š, SH»ƒ PLD y y wš ƒ f w. ew PCMB DTT w PLD y ƒ y y x(fig. 1) l» y -y y PLD y e y w (Fig. 4). ù PLD f w œw» jš. t SH» ƒ w ¾? w ƒ l» w p (site-specific mutagenesis) x ƒ w. š PLD y w SH»» d ÿ» ÿ ƒ û. x PLD PLD r ƒ w p - - qp (HKD) ƒ»10 p»ƒ s qp -z w ƒ. 27 PLD y w SH»»» k w. SH» w PLD y ¾? š 3,4 š y (ROS) w 5 ƒ ƒ š. PLD 4 SH»ƒ PLD y w w eš s»ƒ w w w x ½. x 1. Heller, M. Adv. Lipid Res. 1978, 16, Peng, Z.; Beaven, M. A. J. Immunol. 2005, 174, Wang, X.; Dyer, H. J.; Zheng, L. Arch. Biochim. Biophys. 1993, 306, Wang, X. Plant Physiol. 2005, 139, Zhang, W.; Yu, L.; Zhang, Y.; Wang, X. Biochim. Biophys. Acta, 2005, 1736, Ryu, S. B.; Wang, X. Plant Physiol. 1995, 108, Navari-Izzo, F.; Cestone, B.; Cavallini, A.; Natali, L.; Giordani, T.; Quartacci, M. F. Phytochemistry, 2006, 67, Wang, X.; Xu, L.; Zheng, L. J. Biol. Chem. 1994, 269, Ueki, J.; Monoka, S.; Komeri, T.; Kumashiro, T. Plant cell Physiol. 1995, 36, Kim, D. U.; Roh, T.; Lee, J.; Noh, J. Y.; Jang, Y. J.; Hoe, K. L.; Yoo, H. S.; Choi, M. -U. Biochim. Biophys. Acta 1999, 1437, Qin, C.; Wang, X. Plant Physiol. 2002, 128, 1057.

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