- Email: [email protected]
Thioesterase and protein deacylase activities of porcine pancreatic phospholipase A2
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•
,
, r
:
ELSEVIER
Biochimica et Biophysica Acta 1299 (1996) 17-22
Biochi f ic~a A~ta etBiophysica
Thioesterase and protein deacylase activities of porcine pancreatic phospholipase A 2 Mercedes Nocito, Garbifie Roy, Luisa M. Villar, Carmen Palacios, Antonio Serrano, Jose C. Alvarez-Cermefio, Pedro Gonzfilez-Porqu6 * Department of Immunology, Hospital Ramdn y Cajal, Madrid, Spain Received 25 January 1995; revised 17 July 1995; accepted 3 August 1995
Abstract
The thioesterase activity of porcine pancreatic phospholipase A 2 has been investigated with non-phospholipid substrates. The acyl-CoA hydrolase activity towards acyl-CoA derivatives is specific for long chain fatty acids (14 C, 16 C) but is unable to hydrolyze short chain acyl-CoA compounds (below 8 C). The same enzyme also shows protein deacylase activity liberating [3H]palmitic acid from [3H]palmitoyl-acyl carrier protein. Keywords: Thioesterase; Phospholipase A2; Acyl-protein esterase
1. Introduction
Phospholipases A 2 (EC 3.1.1.4) are calcium-requiring esterases that hydrolyze specifically the sn-2 fatty acyl ester bond of phosphoglycerides, to produce a fatty acid and a lysophospholipid. Phospholipase A 2 enzymes are widespread in nature, playing crucial roles in normal cellular functions by participating in the metabolism and turnover of phospholipids [1] and are involved in signal transducing mechanisms [2], inflammatory reactions [3] and platelet function [4-6]. Different PLA 2 isoenzymes have been found in extracellular fluids [7], associated with cell membranes [8], or in the cytosol of most cells studied [91. The Group I pancreatic PLA 2 is secreted as proenzyme into the extracellular space where it is converted to fully active enzyme [10] serving mostly a digestive function, although its involvement in the synthesis of prostaglandins has also been reported [11]. Increased levels of secretory Group I and Group II PLA 2 are found in rheumatoid arthritic synovial fluid [12] and serum from patients with septicemia, pancreatitis, acute respiratory distress syndrome, or other severe diseases [13]. Although PLA 2 is considered an acyl-esterase, Dennis
* Corresponding author. Fax: +34 1 3369016. 0005-2760/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 1 7 6 - X
and Goodenough have demonstrated that porcine pancreatic PLA 2 shows thioesterase activity towards acyl-thioester bonds in the sn-2 position of phosphoglycerides [ 14,15]. In the present study we report that the same enzyme shows thioesterase activity towards non-phospholipid substrates, catalyzing the hydrolysis of the thioester bond of acylSCoA derivatives (acylCoA-hydrolase activity) or palmitoyl-acyl carrier protein (acyl-protein deacylase activity).
2. Materials and methods
Porcine pancreatic phospholipase A 2 and kits for the determination of phospholipase A and free fatty acids (Boehringer Mannheim, Germany). Polystyrene microplates containing 96-wells were purchased from Nunc (Netherlands). Acetyl coenzyme A, n-butyryl coenzyme A, n-octanoyl coenzyme A, palmitoyl coenzyme A, myristoyl coenzyme A, Triton X-100, 5,5-dithiobis (2-nitrobenzoic acid), hydroxylamine, ATP, calcium chloride, lithium chloride, MgCI2, dithiotreitol (DTT), Mes, acyl carrier protein (ACP) and acyl-ACP synthetase from Escherichia coli were from Sigma (USA). Silica-gel for thin-layer chromatography (HPTLC), Tris base, isopropyl alcohol, chloroform, methanol, acetic acid (Merck, Germany); DEAE-cellulose (DE52), No. 3MM filter paper (Whatman, USA); [1-J4C]palmitoyl CoA (spec. act. 57 Ci/mol), [1-
18
M. Nocito et al. / Biochimica et Biophysica Acta 1299 (1996) 17-22
3H]palmitic acid (spec. act. 54 C i / m m o l ) (Amersham, UK); n-hexane (BDH, UK); FPLC superose 12 (Pharmacia, Sweden); isogel agarose IEF plates pH range 3 - 1 0 (FMC, USA); optiphase high safe liquid scintillation cocktail (LKB, Sweden).
for the determination of the free fatty acids liberated in the reaction. In this system, free fatty acids are, in the presence of acyl-CoA synthetase, ATP, and CoA, converted into acyl-CoA derivatives, wich are subsequently oxidized to enoyl-CoA by acyl-CoA oxidase. The H202 resulting in the reaction is determined colorimetrically (A540 nm) by means of 2,4,6 tribromo-3 hydroxy benzoic acid and 4 amino antipyrine [16]. A standard curve with palmitic acid was used in order to calculate the amount of free fatty acid liberated in the reaction. All steps were performed as described by the manufacturers using the original reagents included in the kits and adjusting the final volume to 0.3 ml.
2. I. Determination of thioesterase activity using palmitoyl CoA as a substrate Microplate assay. 50 /zl of the reaction mixture (2 × ) (0.1 M Tris-HC1 buffer (pH 7.0), 0.4 mM CaCI 2, 1 mM palmitoyl-CoA, 2 mM DTNB and 50 ~1 of porcine pancreatic phospholipase A 2 at the appropiate dilution were incubated in a microplate well at 37°C. The CoA-SH liberated in the reaction was followed continously by means of the DTNB present in the reaction mixture. The colour developed was read at 405 nm. using a Titertek Multiscan 8-channel spectrophotometer. Standard curve obtained with CoA-SH was used to calculate the amount of CoA-SH liberated in the reaction. Radioactive assay. 0.075 /~Ci of [14C]palmitoyl-CoA was incubated for 1 h at 37°C in the presence of 0.05 M Tris-HC1 buffer (pH 7.0), 1 mM CaCI 2 and enzyme at the appropiate dilution (final volume 25 /zl). Then, the free [14 C]palmitic acid released in the reaction was extracted by adding 500 /zl of n-hexane. After shaking and centrifugating, 200 ~1 of the upper phase was withdrawn and counted in 3 ml of scintillation solution.
2.3. Determination of protein-deacylase activity of phospholipase A 2 Palmitoyl3H-ACP synthesis. Palmitoyl3H-ACP was synthetized as already described [17]. 130/zCi [3H]palmitic acid, 1 mg ACP and 60 /~g Acyl-ACP synthetase was incubated at room temperature in a standard mixture which contained 5 mM ATP, 10 mM MgC12, 2 mM DTT, 0.4 M LiC1, 2% Triton X-100 and 0.1 M Tris-HC1 (pH 8.0) in a final volume of 500 /zl. Every hour, 10 /zl of the assay mixture was withdrawn and deposited on a Whatman No. 3MM filter disc and washed with two changes of methanol/chloroform/acetic acid (6:3:1, v / v ) to remove unreacted fatty acid. The filter papers were dried and counted in 3 ml of scintillation solution. After 5 h incubation, a plateau in the incorporation of [3H]palmitic acid to ACP was reached (9.106 c p m / m g protein). The mixture was then diluted 10-fold in 10 mM Mes (pH 6.0) and absorbed onto a l-ml DEAE-cellulose
2.2. Determination of phospholipase A 2 activity using lecithins as substrate Commercial 'phospholipase A' and 'free fatty acids, half micro-tesr kits from Boehringer Mannheim were used
,t~o6nm
B ~14
7 1,4
300
/~.~4onm
cpm (Thousands)
350
16
250
~2oo
lO
m
0,8
:I:150
,6
2'2~ C /~125 1,,
/
/.
!1oon-Wm
..7
8
oi
D nl
0 <
6
0,4
:tS0
0,2 0
5
10
15
/~M PEA2
20
0
~
0
,
100
200
300
nM PLA2
, 0
400
5 10 15 20 25 30
nM PLA2
Fig. 1. Sensitivityof the different assays for thioesterase and phospholipaseactivities of porcine pancreatic phospholipaseA 2. (A) Thioesterase activity using the microplate assay. (B) Thioesterase activity using the radioactive assay. (C) Phospholipase activity using the commercial assay. All the assays were performedas described in Section 2, with incubations of 1 h at 37°C.
19
M. Nocito et a l . / Biochimica et Biophysica Acta 1299 (1996) 17-22
500-
cpra (Thousands) 30~
400
25-
c p m (Thousands)
A ~
\
20 300
15--
lo!
10~ ~
", .oo-
o
// ¢/
5~ ) 0
100
i
5
O
t-
10
IB
20
0
/JM PI.~
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\
5
10
15
0,4
016
0,8
Fig. 3. Protein-deacylase activity of porcine pancreatic phospholipase A 2. (A) Deacylation of palmitoyl [3H]ACP by phospholipase A~. (B) Deacylation of palmitoyl [3H]ACP by hydroxylamine.
20
Pt~ 0JU)
tivity was obtained. Palmitoyl [3H]ACP was eluted with 0.6 M LiC1 10 mM Mes (pH 6.0). Deacylation by phospholipase A 2. 30000 cpm of palmitoyl [3H]ACP (spec. act. 9000 cpm//xg protein) was incubated for 30 min at 37°C with varying concentrations of phospholipase A: in 50 mM Tris-HCl, 2 mM CaC12 (final volume 100 /xl). Then 5 /xl of the mixture was withdrawn and deposited on a Whatman No. 3MM filter disc, washed to remove the fatty acids released by the
Fig. 2. Effect of the acyl-group chain length on the thioesterase activity of porcine pancreatic phospholipase A 2. The substrates were ( • ) acetyl-CoA (2:0), ( 0 ) n-butyryl-CoA (4:0), (*) n-octanoyl-CoA (8:0), (11) miristoyl-CoA (14:0) and (©) palmitoyl-CoA (16:0) at a final concentration of 1 mM in the microplate assay. Incubation time of 3 h at 37°C.
column equilibrated with 10 mM Mes (pH 6.0) to remove the unreacted fatty acid, which was eluted with 80% isopropyl alcohol, 10 mM Mes (pH 6.0) until no radioac-
A21snm
A40s and 540nm
2,6 KD
-~ OD215nm
4 9 , 5 t>
~ PE-PLA2
t
2 7 , 5 t> 1 8 . 5 t> 6,5t>
1,8
0,2
Hydroxylamine (M)
THIOESTERASE
-
1,B
-1 ,
0,8
0~B -
0¢
0
-~- -
I
t
I
10
20
30
Fraction
I
40
10
v w V
50
60
Number
Fig. 4. SDS-PAGE and Superose 12 gel filtration of porcine pancreatic phospholipase A 2. Coelution of thioesterase and phospholipase activities. Column: Superose 12 1.5 × 30 cm. equilibrated in 10 mM Tris-HC1, 0.15 M NaCI, pH 7.5 (TBS). Rate: 25 ml/h. Fractions: 450 ~1. Protein was detected by reading the absorbance at 215 nm (A:m5) with 200 /xl of each fraction diluted with 800 /.tl of 10 mM Tris-HCl (pH 7.5), 150 mM NaC1 (TBS). The phospholipase activity was determined with 5 /xl of each fraction diluted in TBS after incubation of 15 min in the presence of lecithins as substrate (see Section 2). Thioesterase activity was determined with 20 /zl of each fraction after incubation of 1 h in the presence of palmitoyl-CoA and DTNB (see microplate assay under Section 2).
M. Nocito et al. / Biochimica et Biophysica Acta 1299 (1996) 17-22
20
effect of the e n z y m e , dried and counted as previously described. Deacylation by hydroxylamine. 30 000 cpm of palmitoyl [3H]ACP was incubated for 1 h at room temperature with increasing concentrations of h y d r o x y l a m i n e (pH 9.0) in distilled water (final v o l u m e 100 /xl). Then 5 /zl of the mixture was deposited on a W h a t m a n No. 3 M M filter disc, washed, dried and counted as described above. Identification of released fatty acids by TLC. An extraction of the fatty acids released in both deacylations was made by adding 500 /xl of hexane to the mixtures. After shaking and centrifugating the samples, 25 /zl of the top phase was withdrawn and chromatographed on silica-gel
plates ( H P T L C ) in c h l o r o f o r m / a c e t i c acid (96:4, v / v ) . Palmitoyl [3H]ACP and [3H]palmitic acid were used as markers. Radioactive bands were located and counted with a Berthold radioactivity linear analyzer.
3. Results 3.1. Thioesterase and phospholipase activities of porcine pancreatic phospholipase A 2 Fig. I A shows the thioesterase activity ( p a l m i t o y l - C o A hydrolase) of porcine pancreatic P L A 2 incubated under the conditions described for the microplate assay in the pres-
A54onm
A4o5nm
2--
0,7 PE-PLA2 O- THIOESTERASE '0,6
1,5 0,5 >... I.--
>-
I---
0,4
/
1 LLI <~ tl, .._1 O "1-
LId
0,3
/j
CO O "r13_
I--o
Ct~ LId I--
"0,2
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"-I0,1
5 6 7 8 9 10 11 12 13 14 15 Fraction
pH
3.6
5.5
Number
J
6.1
A
A
7.4
10.2
Fig. 5. Isoelectrofocusing of porcine pancreatic phospholipase A2. The phospholipase activity was detected with 5/xl of each fraction after incubating for 15 rain with lecithins (see Section 2). The thioesterase activity was determined with 50/xl of each fraction after incubation in the microplate assay in the presence of DTNB (Section 2). Isoelectric focusing was performed on commercial agarose IEF 3-10 plates for 1 h at 5 W. Two samples of 25 /xg of PLA 2 were run in parallel, one of them was stained with Coomassie and the other sample was sliced in fractions of 0.5 cm. Each slice was eluted with 500 /xl of 10 mM Tris-HCl pH 7.8 overnight. Phospholipase A 2 (in the presence of lecithins) and thioesterase activity (in the presence of palmitoyl-CoA and DTNB) were performed as described under Section 2. pl markers were: cytochrome c 10.2; myoglobin (horse) major band 7.4, minor band 7.0; carbonic anhydrase 6.1; fl-lactoglobin (A,B) 5.4 and 5.5; ovalbumin 4.8; glucose oxidase 4.2 and amyloglucosidase 3.6.
M. Nocito et a L / Biochimica et Biophysica Acta 1299 (1996) 17-22
ence of DTNB. As can be seen, in order to obtain reliable results, a high amount of enzyme should be added. However, the sensitivity can be increased over 100 times by using the radioactive assay described in Section 2 (B) or the commercial assay using lecithin as substrate (phospholipase A 2 activity) (C). 3.2. Effect of the acyl-group chain length on the thioesterase actit'i O' of PLA 2 We have compared the thioesterase activity towards acyl-CoA compounds with different chain length. The compounds were acetyl-CoA, n-butyryl-CoA, n-octanoylCoA, miristoyl-CoA and palmitoyl-CoA. Fig. 2 shows a clear preference of PLA 2 for acyl-coA compounds with long chain length (palmitoyl and miristoyl-CoA), and is unable to cleave the thioester bonds in acyl-CoA compounds of shorter chain length than 8 C. 3.3. Protein-deacvlase actir'i~ of phospholipase
A2
Acylation of proteins by thioester bonds shows a clear preference for palmitic acid over shorter as well as longer acyl chains [18]. In order to study the possible deacylating activity of the phospholipase A 2, an acylprotein containing a thioester bond (palmitoyl3H-ACP) was synthetized by means of acyl-ACP synthetase as described in Section 2. The acylprotein was then incubated with increasing amounts of porcine pancreatic PLA 2 (Fig. 3A) or hydroxylamine, which can release the palmitate from thioester bonds [19] (Fig. 3B). A single peak with the same R E as [3H]palmitic acid run as a control was found when the product of the reaction was analyzed by thin-layer chromatography (see Section 2). 3.4. Thioesterase and phospholipase are different activities of the same enzyme Fig. 4 shows the SDS-PAGE of the commercial PLA 2 used in this study (inset) and the gel filtration on Superose 12 where the majority peak of protein coincides with the PLA 2 and thioesterase activities. The isoelectric focusing behaviour of PLA 2 is shown in Fig. 5. A major protein band with a p l of about 6.3 and two minor impurities (pl 6.1 and 5.8) were observed when the gel was stained with Coomassie Brilliant Blue R-250. A parallel strip of the agarose gel was sliced and phospholipase and thioesterase activities were assayed after elution of the protein from the slices. As can be observed, both activities had the same p l as the major stained protein band.
4. Discussion
Acyl thioesters in the sn-2 position of phospholipids have been described as alternative substrates of natural
21
occurring phospholipids for the determination of PLA 2 activity from different origins [14]. This assay, although less sensitive than the radioactive assays, allows the use of non-radioactive substrates and the continuous monitoring of the PLA: activity by titration of the SH compounds formed in the reaction by means of DTNB [14]. Among other enzymes, porcine pancreatic PLA~ has shown thioesterase activity towards these synthetic phospholipids [15]. In this study, we have investigated the thioesterase activity of the enzyme towards non-phospholipid thioesters. As shown in Fig. 1, porcine pancreatic PLA2 hydrolyzes palmitoyl-CoA and the activity can be measured by titrating the CoA-SH liberated in the reaction (Fig. 1A) or [~4C]palmitic acid (Fig. 1B), the radioactive assay being about 100 times more sensitive than the colorimetric assay. The enzyme is specific for long chain fatty acids, and hydrolyzes with the same efficency miristoyl-CoA or palmitoyl-CoA, being much less active towards octanoyl-CoA, and does not hydrolyze butyryl-CoA or acetyl-CoA (Fig. 2). However, PLA 2 shows a clear preference for phospholipids substrates. The same amount of enzyme releases about 200 times more product when using lecithin as substrate (Fig. 1C) than when using palmitoyl-CoA as substrate (Fig. 1A or IB). PLA 2 from bee venom, however, does not show this thioesterase activity (data not shown). Although the commercial porcine pancreatic PLA 2 shows a high degree of homogeneity as judged by SDSPAGE (over 95%), the possibility was investigated that the thioesterase activity found was due to the presence of a contaminating enzyme in the PLA 2 preparation. The gel filtration under non-denaturating conditions (Fig. 4) and isoelectricfocusing (Fig. 5) shows in both cases that PLA 2 (with lecithins as substrate) and thioesterase activity (with palmitoyl-CoA as substrate) coincided with the major protein peak, indicating that both activities reside in the same molecule. Also, both activities show the same thermal inactivation curve (50% inactivation after 30 min at 100°C, 75% after 1 h, 100% after 2 h) and the same dependence for Ca 2+ in the reaction (data not shown). Finally, the activity of porcine pancreatic PLA 2 towards [3H]palmitoyl-acyl carrier protein was investigated. As shown in Fig. 3A, PLA 2 deacylated the protein as efficiently as hydroxylamine (Fig. 3B). The protein deacylase activity of PLA 2 could have important physiological consequences since acylation/deacylation of proteins are important processes regulating the activity and cellular localization of many proteins. This process has been involved in the fusion between viral and cellular membranes [20] and in the release of virus particles from infected cells [21], showing a decrease in both activities when proteins are deacylated by hydroxylamine [22,23]. Since PLA 2 is a widespread enzyme both intra- and extracellular, it could be one of the deacylating enzymes in the organism providing a natural defense to the host against viral infections.
22
M. Nocito et al. / Biochimica et Biophysica Acta 1299 (1996) 17-22
Acknowledgements This work was supported by Research Grant 91/118 and 93/397 from the Fondo de Investigaciones Sanitarias de la Seguridad Social. References [1] Van den Bosch, H. (1980) Biochim. Biophys. Acta 604, 191-246. [2] Asaoka, Y., Yoshida, K., Sasaki, Y., Nishizuka, Y., Murakami, M., Kudo, I. and Inoue, K. (1993) Proc. Natl. Acad. Sci. USA 90, 716-719. [3] Pruzanski, W. and Vadas, P. (1988) J. Rheumatol. 15, 1601-1603. [4] Wong, B., Tang, W. and Ziboh, V.A. (1992) FEBS Lett. 305, 213-216. [5] Domin, J. and Rozengurt, E. (1993) J. Biol. Chem. 268, 8927-8934. [6] Symer, D.E., Paznekas, W.A. and Shin, H.S. (1993) J. Exp. Med. 177, 937-947. [7] Nevalainen, T.J., Kortesuo P.T., Rinttala E. and M~irki, F. (1992) Clin. Chem. 38, 1824-1829. [8] Kanda, A., Ono, T., Yoshida, N., Tojo, H. and Okamoto, M. (1989) Biochem. Biophys. Res. Commun. 163, 42-48. [9] Rehfeldt, W., Resch, K. and Goppelt-Struebe, M. (1993) Biochem. J. 293, 255-261.
[10] Borgstrom, A., Erlanson-Albertsson, C. and Borgstrom, B. (1993) Scand. J. Gastroenterol. 28, 455-459. [11] Tohkin, M., Kishino, J., Ishizaki, J. and Arita, H. (1993) J. Biol. Chem. 268, 2865-2871. [12] Pruzanski, W., Vadas, P., Stefanski, E. and Urowitz, M.B. (1985) J. Rheumatol. 12, 211-216. [13] Chang, J., Musser, J.H. and McGregor, H. (1987) Biochem. Pharmacol. 36, 2429-2436. [14] Yu, L. and Dennis, E.A. (1991) Methods in Enzymol. 197, 65-75. [15] Bhat, M.K., Mueller-Harvey, I., Summer, I.G. and Goodenough, P.W. (1993)Biochim. Biophys. Acta 1166, 244-250. [16] Shimizu, S., Tani Y., Yamada, H., Tabata, M. and Murachi, T. (1980) Anal. Biochem. 107, 193-198. [17] Cooper, C.L., Hsu, L., Jackowski, S. and Rock, C.O. (1989). J. Biol. Chem. 264, 7384-7389. [18] Schmidt, M.F.G. (1989) Biochim. Biophys. Acta 988, 411-426. [19] Kaufman, J.F., Krangel, M.S. and Strominger, J.L. (1984) J. Biol. Chem. 259, 7230-7238. [20] Schmidt, M.F.G. (1982) Trends Biochem. Sci. 7, 322-334. [21] Schmidt, M.F.G. (1983) Curr. Top. Microbiol. Immunol. 102, 101129. [22] Schmidt, M.F.G. and Lambrecht, B. (1985) J. Gen. Virol. 66, 2635-2647. [23] Lambrecht. B. and Schmidt, M.F.G. (1986) FEBS Lett. 202, 127132.
•
,
, r
:
ELSEVIER
Biochimica et Biophysica Acta 1299 (1996) 17-22
Biochi f ic~a A~ta etBiophysica
Thioesterase and protein deacylase activities of porcine pancreatic phospholipase A 2 Mercedes Nocito, Garbifie Roy, Luisa M. Villar, Carmen Palacios, Antonio Serrano, Jose C. Alvarez-Cermefio, Pedro Gonzfilez-Porqu6 * Department of Immunology, Hospital Ramdn y Cajal, Madrid, Spain Received 25 January 1995; revised 17 July 1995; accepted 3 August 1995
Abstract
The thioesterase activity of porcine pancreatic phospholipase A 2 has been investigated with non-phospholipid substrates. The acyl-CoA hydrolase activity towards acyl-CoA derivatives is specific for long chain fatty acids (14 C, 16 C) but is unable to hydrolyze short chain acyl-CoA compounds (below 8 C). The same enzyme also shows protein deacylase activity liberating [3H]palmitic acid from [3H]palmitoyl-acyl carrier protein. Keywords: Thioesterase; Phospholipase A2; Acyl-protein esterase
1. Introduction
Phospholipases A 2 (EC 3.1.1.4) are calcium-requiring esterases that hydrolyze specifically the sn-2 fatty acyl ester bond of phosphoglycerides, to produce a fatty acid and a lysophospholipid. Phospholipase A 2 enzymes are widespread in nature, playing crucial roles in normal cellular functions by participating in the metabolism and turnover of phospholipids [1] and are involved in signal transducing mechanisms [2], inflammatory reactions [3] and platelet function [4-6]. Different PLA 2 isoenzymes have been found in extracellular fluids [7], associated with cell membranes [8], or in the cytosol of most cells studied [91. The Group I pancreatic PLA 2 is secreted as proenzyme into the extracellular space where it is converted to fully active enzyme [10] serving mostly a digestive function, although its involvement in the synthesis of prostaglandins has also been reported [11]. Increased levels of secretory Group I and Group II PLA 2 are found in rheumatoid arthritic synovial fluid [12] and serum from patients with septicemia, pancreatitis, acute respiratory distress syndrome, or other severe diseases [13]. Although PLA 2 is considered an acyl-esterase, Dennis
* Corresponding author. Fax: +34 1 3369016. 0005-2760/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 1 7 6 - X
and Goodenough have demonstrated that porcine pancreatic PLA 2 shows thioesterase activity towards acyl-thioester bonds in the sn-2 position of phosphoglycerides [ 14,15]. In the present study we report that the same enzyme shows thioesterase activity towards non-phospholipid substrates, catalyzing the hydrolysis of the thioester bond of acylSCoA derivatives (acylCoA-hydrolase activity) or palmitoyl-acyl carrier protein (acyl-protein deacylase activity).
2. Materials and methods
Porcine pancreatic phospholipase A 2 and kits for the determination of phospholipase A and free fatty acids (Boehringer Mannheim, Germany). Polystyrene microplates containing 96-wells were purchased from Nunc (Netherlands). Acetyl coenzyme A, n-butyryl coenzyme A, n-octanoyl coenzyme A, palmitoyl coenzyme A, myristoyl coenzyme A, Triton X-100, 5,5-dithiobis (2-nitrobenzoic acid), hydroxylamine, ATP, calcium chloride, lithium chloride, MgCI2, dithiotreitol (DTT), Mes, acyl carrier protein (ACP) and acyl-ACP synthetase from Escherichia coli were from Sigma (USA). Silica-gel for thin-layer chromatography (HPTLC), Tris base, isopropyl alcohol, chloroform, methanol, acetic acid (Merck, Germany); DEAE-cellulose (DE52), No. 3MM filter paper (Whatman, USA); [1-J4C]palmitoyl CoA (spec. act. 57 Ci/mol), [1-
18
M. Nocito et al. / Biochimica et Biophysica Acta 1299 (1996) 17-22
3H]palmitic acid (spec. act. 54 C i / m m o l ) (Amersham, UK); n-hexane (BDH, UK); FPLC superose 12 (Pharmacia, Sweden); isogel agarose IEF plates pH range 3 - 1 0 (FMC, USA); optiphase high safe liquid scintillation cocktail (LKB, Sweden).
for the determination of the free fatty acids liberated in the reaction. In this system, free fatty acids are, in the presence of acyl-CoA synthetase, ATP, and CoA, converted into acyl-CoA derivatives, wich are subsequently oxidized to enoyl-CoA by acyl-CoA oxidase. The H202 resulting in the reaction is determined colorimetrically (A540 nm) by means of 2,4,6 tribromo-3 hydroxy benzoic acid and 4 amino antipyrine [16]. A standard curve with palmitic acid was used in order to calculate the amount of free fatty acid liberated in the reaction. All steps were performed as described by the manufacturers using the original reagents included in the kits and adjusting the final volume to 0.3 ml.
2. I. Determination of thioesterase activity using palmitoyl CoA as a substrate Microplate assay. 50 /zl of the reaction mixture (2 × ) (0.1 M Tris-HC1 buffer (pH 7.0), 0.4 mM CaCI 2, 1 mM palmitoyl-CoA, 2 mM DTNB and 50 ~1 of porcine pancreatic phospholipase A 2 at the appropiate dilution were incubated in a microplate well at 37°C. The CoA-SH liberated in the reaction was followed continously by means of the DTNB present in the reaction mixture. The colour developed was read at 405 nm. using a Titertek Multiscan 8-channel spectrophotometer. Standard curve obtained with CoA-SH was used to calculate the amount of CoA-SH liberated in the reaction. Radioactive assay. 0.075 /~Ci of [14C]palmitoyl-CoA was incubated for 1 h at 37°C in the presence of 0.05 M Tris-HC1 buffer (pH 7.0), 1 mM CaCI 2 and enzyme at the appropiate dilution (final volume 25 /zl). Then, the free [14 C]palmitic acid released in the reaction was extracted by adding 500 /zl of n-hexane. After shaking and centrifugating, 200 ~1 of the upper phase was withdrawn and counted in 3 ml of scintillation solution.
2.3. Determination of protein-deacylase activity of phospholipase A 2 Palmitoyl3H-ACP synthesis. Palmitoyl3H-ACP was synthetized as already described [17]. 130/zCi [3H]palmitic acid, 1 mg ACP and 60 /~g Acyl-ACP synthetase was incubated at room temperature in a standard mixture which contained 5 mM ATP, 10 mM MgC12, 2 mM DTT, 0.4 M LiC1, 2% Triton X-100 and 0.1 M Tris-HC1 (pH 8.0) in a final volume of 500 /zl. Every hour, 10 /zl of the assay mixture was withdrawn and deposited on a Whatman No. 3MM filter disc and washed with two changes of methanol/chloroform/acetic acid (6:3:1, v / v ) to remove unreacted fatty acid. The filter papers were dried and counted in 3 ml of scintillation solution. After 5 h incubation, a plateau in the incorporation of [3H]palmitic acid to ACP was reached (9.106 c p m / m g protein). The mixture was then diluted 10-fold in 10 mM Mes (pH 6.0) and absorbed onto a l-ml DEAE-cellulose
2.2. Determination of phospholipase A 2 activity using lecithins as substrate Commercial 'phospholipase A' and 'free fatty acids, half micro-tesr kits from Boehringer Mannheim were used
,t~o6nm
B ~14
7 1,4
300
/~.~4onm
cpm (Thousands)
350
16
250
~2oo
lO
m
0,8
:I:150
,6
2'2~ C /~125 1,,
/
/.
!1oon-Wm
..7
8
oi
D nl
0 <
6
0,4
:tS0
0,2 0
5
10
15
/~M PEA2
20
0
~
0
,
100
200
300
nM PLA2
, 0
400
5 10 15 20 25 30
nM PLA2
Fig. 1. Sensitivityof the different assays for thioesterase and phospholipaseactivities of porcine pancreatic phospholipaseA 2. (A) Thioesterase activity using the microplate assay. (B) Thioesterase activity using the radioactive assay. (C) Phospholipase activity using the commercial assay. All the assays were performedas described in Section 2, with incubations of 1 h at 37°C.
19
M. Nocito et a l . / Biochimica et Biophysica Acta 1299 (1996) 17-22
500-
cpra (Thousands) 30~
400
25-
c p m (Thousands)
A ~
\
20 300
15--
lo!
10~ ~
", .oo-
o
// ¢/
5~ ) 0
100
i
5
O
t-
10
IB
20
0
/JM PI.~
0
\
5
10
15
0,4
016
0,8
Fig. 3. Protein-deacylase activity of porcine pancreatic phospholipase A 2. (A) Deacylation of palmitoyl [3H]ACP by phospholipase A~. (B) Deacylation of palmitoyl [3H]ACP by hydroxylamine.
20
Pt~ 0JU)
tivity was obtained. Palmitoyl [3H]ACP was eluted with 0.6 M LiC1 10 mM Mes (pH 6.0). Deacylation by phospholipase A 2. 30000 cpm of palmitoyl [3H]ACP (spec. act. 9000 cpm//xg protein) was incubated for 30 min at 37°C with varying concentrations of phospholipase A: in 50 mM Tris-HCl, 2 mM CaC12 (final volume 100 /xl). Then 5 /xl of the mixture was withdrawn and deposited on a Whatman No. 3MM filter disc, washed to remove the fatty acids released by the
Fig. 2. Effect of the acyl-group chain length on the thioesterase activity of porcine pancreatic phospholipase A 2. The substrates were ( • ) acetyl-CoA (2:0), ( 0 ) n-butyryl-CoA (4:0), (*) n-octanoyl-CoA (8:0), (11) miristoyl-CoA (14:0) and (©) palmitoyl-CoA (16:0) at a final concentration of 1 mM in the microplate assay. Incubation time of 3 h at 37°C.
column equilibrated with 10 mM Mes (pH 6.0) to remove the unreacted fatty acid, which was eluted with 80% isopropyl alcohol, 10 mM Mes (pH 6.0) until no radioac-
A21snm
A40s and 540nm
2,6 KD
-~ OD215nm
4 9 , 5 t>
~ PE-PLA2
t
2 7 , 5 t> 1 8 . 5 t> 6,5t>
1,8
0,2
Hydroxylamine (M)
THIOESTERASE
-
1,B
-1 ,
0,8
0~B -
0¢
0
-~- -
I
t
I
10
20
30
Fraction
I
40
10
v w V
50
60
Number
Fig. 4. SDS-PAGE and Superose 12 gel filtration of porcine pancreatic phospholipase A 2. Coelution of thioesterase and phospholipase activities. Column: Superose 12 1.5 × 30 cm. equilibrated in 10 mM Tris-HC1, 0.15 M NaCI, pH 7.5 (TBS). Rate: 25 ml/h. Fractions: 450 ~1. Protein was detected by reading the absorbance at 215 nm (A:m5) with 200 /xl of each fraction diluted with 800 /.tl of 10 mM Tris-HCl (pH 7.5), 150 mM NaC1 (TBS). The phospholipase activity was determined with 5 /xl of each fraction diluted in TBS after incubation of 15 min in the presence of lecithins as substrate (see Section 2). Thioesterase activity was determined with 20 /zl of each fraction after incubation of 1 h in the presence of palmitoyl-CoA and DTNB (see microplate assay under Section 2).
M. Nocito et al. / Biochimica et Biophysica Acta 1299 (1996) 17-22
20
effect of the e n z y m e , dried and counted as previously described. Deacylation by hydroxylamine. 30 000 cpm of palmitoyl [3H]ACP was incubated for 1 h at room temperature with increasing concentrations of h y d r o x y l a m i n e (pH 9.0) in distilled water (final v o l u m e 100 /xl). Then 5 /zl of the mixture was deposited on a W h a t m a n No. 3 M M filter disc, washed, dried and counted as described above. Identification of released fatty acids by TLC. An extraction of the fatty acids released in both deacylations was made by adding 500 /xl of hexane to the mixtures. After shaking and centrifugating the samples, 25 /zl of the top phase was withdrawn and chromatographed on silica-gel
plates ( H P T L C ) in c h l o r o f o r m / a c e t i c acid (96:4, v / v ) . Palmitoyl [3H]ACP and [3H]palmitic acid were used as markers. Radioactive bands were located and counted with a Berthold radioactivity linear analyzer.
3. Results 3.1. Thioesterase and phospholipase activities of porcine pancreatic phospholipase A 2 Fig. I A shows the thioesterase activity ( p a l m i t o y l - C o A hydrolase) of porcine pancreatic P L A 2 incubated under the conditions described for the microplate assay in the pres-
A54onm
A4o5nm
2--
0,7 PE-PLA2 O- THIOESTERASE '0,6
1,5 0,5 >... I.--
>-
I---
0,4
/
1 LLI <~ tl, .._1 O "1-
LId
0,3
/j
CO O "r13_
I--o
Ct~ LId I--
"0,2
LId
C)
"-I0,1
5 6 7 8 9 10 11 12 13 14 15 Fraction
pH
3.6
5.5
Number
J
6.1
A
A
7.4
10.2
Fig. 5. Isoelectrofocusing of porcine pancreatic phospholipase A2. The phospholipase activity was detected with 5/xl of each fraction after incubating for 15 rain with lecithins (see Section 2). The thioesterase activity was determined with 50/xl of each fraction after incubation in the microplate assay in the presence of DTNB (Section 2). Isoelectric focusing was performed on commercial agarose IEF 3-10 plates for 1 h at 5 W. Two samples of 25 /xg of PLA 2 were run in parallel, one of them was stained with Coomassie and the other sample was sliced in fractions of 0.5 cm. Each slice was eluted with 500 /xl of 10 mM Tris-HCl pH 7.8 overnight. Phospholipase A 2 (in the presence of lecithins) and thioesterase activity (in the presence of palmitoyl-CoA and DTNB) were performed as described under Section 2. pl markers were: cytochrome c 10.2; myoglobin (horse) major band 7.4, minor band 7.0; carbonic anhydrase 6.1; fl-lactoglobin (A,B) 5.4 and 5.5; ovalbumin 4.8; glucose oxidase 4.2 and amyloglucosidase 3.6.
M. Nocito et a L / Biochimica et Biophysica Acta 1299 (1996) 17-22
ence of DTNB. As can be seen, in order to obtain reliable results, a high amount of enzyme should be added. However, the sensitivity can be increased over 100 times by using the radioactive assay described in Section 2 (B) or the commercial assay using lecithin as substrate (phospholipase A 2 activity) (C). 3.2. Effect of the acyl-group chain length on the thioesterase actit'i O' of PLA 2 We have compared the thioesterase activity towards acyl-CoA compounds with different chain length. The compounds were acetyl-CoA, n-butyryl-CoA, n-octanoylCoA, miristoyl-CoA and palmitoyl-CoA. Fig. 2 shows a clear preference of PLA 2 for acyl-coA compounds with long chain length (palmitoyl and miristoyl-CoA), and is unable to cleave the thioester bonds in acyl-CoA compounds of shorter chain length than 8 C. 3.3. Protein-deacvlase actir'i~ of phospholipase
A2
Acylation of proteins by thioester bonds shows a clear preference for palmitic acid over shorter as well as longer acyl chains [18]. In order to study the possible deacylating activity of the phospholipase A 2, an acylprotein containing a thioester bond (palmitoyl3H-ACP) was synthetized by means of acyl-ACP synthetase as described in Section 2. The acylprotein was then incubated with increasing amounts of porcine pancreatic PLA 2 (Fig. 3A) or hydroxylamine, which can release the palmitate from thioester bonds [19] (Fig. 3B). A single peak with the same R E as [3H]palmitic acid run as a control was found when the product of the reaction was analyzed by thin-layer chromatography (see Section 2). 3.4. Thioesterase and phospholipase are different activities of the same enzyme Fig. 4 shows the SDS-PAGE of the commercial PLA 2 used in this study (inset) and the gel filtration on Superose 12 where the majority peak of protein coincides with the PLA 2 and thioesterase activities. The isoelectric focusing behaviour of PLA 2 is shown in Fig. 5. A major protein band with a p l of about 6.3 and two minor impurities (pl 6.1 and 5.8) were observed when the gel was stained with Coomassie Brilliant Blue R-250. A parallel strip of the agarose gel was sliced and phospholipase and thioesterase activities were assayed after elution of the protein from the slices. As can be observed, both activities had the same p l as the major stained protein band.
4. Discussion
Acyl thioesters in the sn-2 position of phospholipids have been described as alternative substrates of natural
21
occurring phospholipids for the determination of PLA 2 activity from different origins [14]. This assay, although less sensitive than the radioactive assays, allows the use of non-radioactive substrates and the continuous monitoring of the PLA: activity by titration of the SH compounds formed in the reaction by means of DTNB [14]. Among other enzymes, porcine pancreatic PLA~ has shown thioesterase activity towards these synthetic phospholipids [15]. In this study, we have investigated the thioesterase activity of the enzyme towards non-phospholipid thioesters. As shown in Fig. 1, porcine pancreatic PLA2 hydrolyzes palmitoyl-CoA and the activity can be measured by titrating the CoA-SH liberated in the reaction (Fig. 1A) or [~4C]palmitic acid (Fig. 1B), the radioactive assay being about 100 times more sensitive than the colorimetric assay. The enzyme is specific for long chain fatty acids, and hydrolyzes with the same efficency miristoyl-CoA or palmitoyl-CoA, being much less active towards octanoyl-CoA, and does not hydrolyze butyryl-CoA or acetyl-CoA (Fig. 2). However, PLA 2 shows a clear preference for phospholipids substrates. The same amount of enzyme releases about 200 times more product when using lecithin as substrate (Fig. 1C) than when using palmitoyl-CoA as substrate (Fig. 1A or IB). PLA 2 from bee venom, however, does not show this thioesterase activity (data not shown). Although the commercial porcine pancreatic PLA 2 shows a high degree of homogeneity as judged by SDSPAGE (over 95%), the possibility was investigated that the thioesterase activity found was due to the presence of a contaminating enzyme in the PLA 2 preparation. The gel filtration under non-denaturating conditions (Fig. 4) and isoelectricfocusing (Fig. 5) shows in both cases that PLA 2 (with lecithins as substrate) and thioesterase activity (with palmitoyl-CoA as substrate) coincided with the major protein peak, indicating that both activities reside in the same molecule. Also, both activities show the same thermal inactivation curve (50% inactivation after 30 min at 100°C, 75% after 1 h, 100% after 2 h) and the same dependence for Ca 2+ in the reaction (data not shown). Finally, the activity of porcine pancreatic PLA 2 towards [3H]palmitoyl-acyl carrier protein was investigated. As shown in Fig. 3A, PLA 2 deacylated the protein as efficiently as hydroxylamine (Fig. 3B). The protein deacylase activity of PLA 2 could have important physiological consequences since acylation/deacylation of proteins are important processes regulating the activity and cellular localization of many proteins. This process has been involved in the fusion between viral and cellular membranes [20] and in the release of virus particles from infected cells [21], showing a decrease in both activities when proteins are deacylated by hydroxylamine [22,23]. Since PLA 2 is a widespread enzyme both intra- and extracellular, it could be one of the deacylating enzymes in the organism providing a natural defense to the host against viral infections.
22
M. Nocito et al. / Biochimica et Biophysica Acta 1299 (1996) 17-22
Acknowledgements This work was supported by Research Grant 91/118 and 93/397 from the Fondo de Investigaciones Sanitarias de la Seguridad Social. References [1] Van den Bosch, H. (1980) Biochim. Biophys. Acta 604, 191-246. [2] Asaoka, Y., Yoshida, K., Sasaki, Y., Nishizuka, Y., Murakami, M., Kudo, I. and Inoue, K. (1993) Proc. Natl. Acad. Sci. USA 90, 716-719. [3] Pruzanski, W. and Vadas, P. (1988) J. Rheumatol. 15, 1601-1603. [4] Wong, B., Tang, W. and Ziboh, V.A. (1992) FEBS Lett. 305, 213-216. [5] Domin, J. and Rozengurt, E. (1993) J. Biol. Chem. 268, 8927-8934. [6] Symer, D.E., Paznekas, W.A. and Shin, H.S. (1993) J. Exp. Med. 177, 937-947. [7] Nevalainen, T.J., Kortesuo P.T., Rinttala E. and M~irki, F. (1992) Clin. Chem. 38, 1824-1829. [8] Kanda, A., Ono, T., Yoshida, N., Tojo, H. and Okamoto, M. (1989) Biochem. Biophys. Res. Commun. 163, 42-48. [9] Rehfeldt, W., Resch, K. and Goppelt-Struebe, M. (1993) Biochem. J. 293, 255-261.
[10] Borgstrom, A., Erlanson-Albertsson, C. and Borgstrom, B. (1993) Scand. J. Gastroenterol. 28, 455-459. [11] Tohkin, M., Kishino, J., Ishizaki, J. and Arita, H. (1993) J. Biol. Chem. 268, 2865-2871. [12] Pruzanski, W., Vadas, P., Stefanski, E. and Urowitz, M.B. (1985) J. Rheumatol. 12, 211-216. [13] Chang, J., Musser, J.H. and McGregor, H. (1987) Biochem. Pharmacol. 36, 2429-2436. [14] Yu, L. and Dennis, E.A. (1991) Methods in Enzymol. 197, 65-75. [15] Bhat, M.K., Mueller-Harvey, I., Summer, I.G. and Goodenough, P.W. (1993)Biochim. Biophys. Acta 1166, 244-250. [16] Shimizu, S., Tani Y., Yamada, H., Tabata, M. and Murachi, T. (1980) Anal. Biochem. 107, 193-198. [17] Cooper, C.L., Hsu, L., Jackowski, S. and Rock, C.O. (1989). J. Biol. Chem. 264, 7384-7389. [18] Schmidt, M.F.G. (1989) Biochim. Biophys. Acta 988, 411-426. [19] Kaufman, J.F., Krangel, M.S. and Strominger, J.L. (1984) J. Biol. Chem. 259, 7230-7238. [20] Schmidt, M.F.G. (1982) Trends Biochem. Sci. 7, 322-334. [21] Schmidt, M.F.G. (1983) Curr. Top. Microbiol. Immunol. 102, 101129. [22] Schmidt, M.F.G. and Lambrecht, B. (1985) J. Gen. Virol. 66, 2635-2647. [23] Lambrecht. B. and Schmidt, M.F.G. (1986) FEBS Lett. 202, 127132.