Biochimica et Biophysica Acre. 1085( 1991~77-81
77
© 1991 ElsevierScience PublishersB.V. 0b~5-2760/91/$03.50 ADONIS 000527609100232S
Calcium regulation of the human PMN cytosolic 15-1ipoxygenase R a l p h C. N i c h o l s a n d J a c k Y. V a n d e r h o e k Department of Biochemh'try and Molecular Biology, The George Washington Unicersity-Schcol of Medicine and Heahh Sciences, Washingtvn, DC (U.S.A.)
(Received4 April 19011 Key words: Calcium;Cytosol;Lipoxygenase Addition of tracer (pg) amounts of [aHlaraehidonic acid to the 120000 × g ojtosolie fraction of h u m a n polymerphonuclear leukocytes (PMNs) produced [3H]-IS-HETE, the product of the 15-1ipox~fgenase, as the m ~ o r metaboIRe. in the presence of nanomolar and low micromolar amounts of calcium, PH]-IS-HETE formation was increased as much as IS-fold which correspon6ed to 17% conversion of added substrate. This enhancement of the cytosolic 1S-lipoxygenase activity, which was reversible by EGTA, was inhibited by phosphatidyl serine and phosphatidyl choline. Millimolar levels of calcium iuhiLited the cytosolic 15-1ipoxygenase and the 5-1ipox~genase product 5-HETE could reverse this inhibition. These results indicate that calcium is an important modulator of the PMN l$-IipoxTgenase when the enzyme is in a cytosolic milieu.
Introduction H u m a n leukoq, tes contain both 5- and 15-1ipo ~ g e n a s e s [1]. These enzymes catalyze the oxygenation of arachidonie acid to biologically active metabolites including leukotrienes, lipoxins, HPETEs and HETEs which have been implicated as mode!at.ors of inflammatory and immunological reactions [2,3]. Although the pure 5.1ipoxygenase activity is controlled by several factors such as calcium and ATP [4], there ai'e additional factors controlling the activity of the 5-1ipoxygenase in the cellular environment. These include both cytosolic and membrane-associated stimulatory factors such as the recently described 5-1ipoxygenase activating factor [4-6]. In view of these results, it is quite possible that cellular factors also regulate the 15-1ipo~genase when the enzyme is located in a cellular milieu. The purpose of the present study was to examine the effects of several cellular components on
Abbreviations: HEEDTA, N-hydro~ethylethylanediaminetriacehc acid; H(P)ETE, hydro(pe:o)xyeicosatetracnoic acid; PC, phosphatidyl choline; PMN, polymorphonuclear leukocyte; PS, phosphatidyl serin¢; RP($P)-HPLC, reverse phase (straight phase)-bigh pressure liquid chromatography.
Correspondence:J.Y. Vanderhoek, Department of Biochemistry and Molecular Biology, The George Washington University-Schoolof Medicine, 2300 Eye Street, N.W., Washington,DC 20037, U.S.A.
the 15-1ipoxygenase activity present in unfractionated cytosolic PMN preparations. Materials and Methods Leukocytes were isolated from heparinized blood from human donors who had not taken aspirin-like drugs for the previous 2 weeks. PMNs were separated on Ficoll-Hypaque cushions and washed with Dulbecco's phosphate-buffered saline (10 ~iM dextrose (pH 7.4), GIBCO, Grand Island, NY) [7]. Contaminating red cells were l y ~ d by hypotonic shock. Cell viability, evaluated with Trypan blue, was consistently greater than 98%. For intact cell experiments, cells were kept at room temperature until assayed. If homogenized, cells were cooled to 0 ° C . For calcium studies, H e p e s / E G T A / H E E D T A (HEH) buffers (pH 7.2) were prepared at ~lecled calcium concentrations using the system described by Fabioto and Fabioto [8]. Calcium concentrations of stock solutions were determined by atomic absorption. Cells were suspended at 2 - 1 0 7 / m l in the appropriate buffer and were homogenized (Ultrasonics (Plainview, N.Y.) model W-140 cell disrupter at a power setting of 3.5) at 0 ° C after the addition of the proteinase inhibitors phenylmethyl sulfonyl fluoride (1 raM) and leupeptin (1 /~g/ml). Homogenization was per,~?:~ed in three times 15 s pulses at 1 rain intervals. Intac: cells and cell debris were removed by lowspeed ( 5 0 0 × g ) centrifugation for 20 min at 4 o C. Cytosolic and micro-
somal fractions were separated by high-speed centrifugation (120000 × g ) for 1 h at 4°C. Supernatants were removed and kept at 0 ° C until assayed the same day. Microsomes were resolubilized in the appropriate buffer by sonication at 0 ° C. In the exogenously added-calcium experiments, a calcium stock solution was prepared in HEH (pH 7.8) with enough MgCI 2 added to give a final concentration of 1 mM when this solution was added to calcium-free HEH. In the reversibility studies, excess EGTA was added from a 60 mM stock solution to give a final concentration of 1 mM. The pH decreased (from 7.2 to 7.10) in the 5 p,M calcium samples. This change did not exceed the buffering range of the solution nor the pH range of the en~'me (6.5-7.5 [9]). In separate experiments, enzyme activity was maximal at pH values as low as 6.5 in HEH (data not shown). Phospholipid vesicles were prepared by evaporating the solvent from the phospholipid stock solution under nitrogen, adding the appropriate amount of calcium-free H E H buffer to produce a concentration of 4 m g / m l and sonicating at 0 ° C for three times 15 s. Effects of phospholipids were determined by adding the phospholipid vesicles to the cytosol, vortexing the mixture and incubating at 4 ° C for 30 mix prior to the addition of 420 pg [3H]arachidonic acid. To assay lipoxygenase activity in intact PMNs and homogenatcs, 500 /~1 aliquots were placed in a 2 3 ° C water bath. The reaction was initiated by the addition of 1 /~1 [3H]arachidonic acid (New England Nuclear, 2.8 T B q / m m o l in ethanol or dimethyl sulfoxide; 420 pg) and was terminated after 30 mix by addition of 1.65-fold volume excess of M e O H / w a t e ; / f o r m i c acid (750:90:10) to which internal standards (a mixture of monoHETEs [I0]) had been added. Samples were stored at - 20 o C until lipids were extracted. Samples were extracted into chloroform [7], dried under nitrogen and dissolved in 30 p,I acetonitrile/ water/acetic acid (65:35:0.035). RP-HPLC separation was performed on a Zorbax (Mac Mud, Chadd's Ford, PA) C18 0.46 x 25 cm column coupled to a C18 pre-column which was attached to a Perkin Elmer Series 4 liquid chromatograph. [3H]-HETEs were separated with an acetonitrile/water (0.1% acetic acid)/ methanol (40:23:37) solvent program run isocratically at a flow rate of 1.0 m l / m i n . Eluate was monitored at 235 nm (LC 85B variable wavelength UV detector) and collected with an LKB (Bromma, Sweden) 2211 fraction collector. Retention times and peak areas were monitored with a Hewlett-Packard 3396A plotter/ integrator. The identities ot radioactive products were eonfirmeo by comparison of SP-HPLC retention times with authentic standards. Radioactive lipoxygenase product formation was determined by scintillation counting using 3a70B as scintiIlant (Research Products Incorporated, Mt. Prosp~,ct, IL). Sample recovery
ranged from 70-100%. Protein was determined by a modified Lowry (Pierce, Rockford, IL). The total 120000 × g cytosolic protein content obtained from 107 PMNs was 0.34 + 0.02 mg (n = 36) and product formation after 30 mix was expressed as mass of radioactive HETE formed per 0.34 mg prottdn ill the sample (unless noted differently). HPLC solvents were from Fisher Scientific. 5-HETE was purchased from Cayman Chemical (Ann Arbor, MI). All reagents, unless otherwise stated, were from Sigma (St. Louis, MO). Results
In view of our previous report that a free, nonesterifled pool of arachidonic acid was associated with isolated human leukocytes [10], it was decided to determine whether PMNs could metabolize tracer amounts of exogenously added [3H]arachidonic acid. Addition of 2.6 nM (420 pg) [3H]arachidonic acid to intact PMNs (107 cells suspended in 0.5 mi Dulbecco's saline buffer which contains 1 m M calcium) produced low amounts (0.1 + 0.02 pg, n = 5) of [3H]-15-HETE, the product of the 15 lipoxygenase (Table I). Using a cell-free supernatant preparation, obtained by homogenization of 107 PMNs in Dulbeeco's buffer and centrifugaticn at 120000 x g , resulted in the formation of 1.3 + 0.68 pg [3H]-15-HETE (n = 6). Adding the calcium chelator E G T A to the 1 2 0 0 0 0 × g cytosolic preparation produced 13 + 3.4 pg [3H]-15-HETE (n = 7) after 30 mix which represented a 3% conversion of added araehidonic acid substrate. Although [3H]-15H E T E production in cell-free supernatant preparations increased only 30% when the incubation times were increased from 5 to 30 mix, the latter reaction time was chosen to ensure complete product formation (data not shown). These results also suggested that millimolar levels of calcium inhibited the activity of the
TABLE 1 15-Lipoxygenase activity in unstimulated intact and broken cell preparations
Human PMNs (2' 107/mDwere suspended in Dulbeeco's phosphate saline buffel or in buffer containing 3 mM EGTA. Subcellular ~npernatan`' fractions were prepared as described under Materials and Methods and 15-1ipoxygenaseactivitywas measured as 13H]-15HETE production after 30 mix from exogenously added tracer [aH]arachidonic acid (2.6 riM, 420 pg). Product formation is per 107 intact cells et ,'he equivalentsupernatant protein content of 0.34 mg. Results are expressedas the mean±S.E, from 3-7 different donors. PMN preparation Intac`'cells 500×g supernatant 120000×8 supernatant 120000×g supernatant+EGTA
[3H]-I5-HETE (pg) 0.10±0.02 0.82±0.44 1.3 ±0.68 13 ±3.4
79 TABLE II Rerersal of Ca-"~-stimulated 15-1ipoxygenasein PMN cytosol
I0 0 "
0
//
0.1
. . . .
1
10
m,~_..
100 1000 I0.000
CALCIUM O,M)
Fig. 1 Calcium.dependent modulation of the cytosolic PMN 15-1ipoxygenase. Human PMNs (2"(07/ml) in HEH buffers containing 0-5000/zM calcium were homogenized and centrifuged to produce 120000× g cytosolic fractions. The 15-hpoxygenaseactivity of these cytnsolic preparations was measured as [aHI-15-HETE formation from exogenously added tracer [3H]arachidonic acid (2.6 nM). Data are presented as the mean 4- S.E. from 4 + 7 different donors. 15-1ipoxygenase present in cytosolic preparations. Since previous reports had d e m o n s t r a t e d that millimolar levels of calcium s t i m u l a t e d partially purified 15-1ipoxygeuase p r e p a r a t i o n s [9,11,12], it was decided to carefully examine the effect of calcium on the PMN 15-1ipoxygenase in cytosolic preparations. Fig. 1 illustrates a calcium titration curve in which the effects of n a n o m o l a r to millimolar concentrations of calcium were tested on the 15-1ipoxygenase activity in the 1 2 0 0 0 0 × g s u p e r n a t a n t fraction from h u m a n P M N s s u s p e n d e d in H E H buffer. A 6-fold increase in [aH]-15-HETE formation (38 + 15 pg, n = 7) was observed at 1 / z M free calcium relative to control without calcium (6.0 + 4.0 pg, n = 7) and the optimal calcium concentration for 15-1ipo~genase stimulation (77 4- 29 p g [3H]-15-HETE, n = 7) was produced at 5 0 / z M . In experiments involving micromolar (or less) concentrations of calcium, [3H]-15-HETE was the major metabolite formed and exceeded [ 3 H ] - 5 - H E T E ' p r o d u c t i o n by at least a 10-fold factor. W h e n the calcium concentration was increased further, inhibition of 15-1ipoxygenase activity was observed, A't 5 m M calcium, [3H]-15-HETE production was significantly inhibited below control values (for four m a t c h e d experiments: control, 4.4 + 1.8 pg [3H]-15-HETE; 5 m M calcium, 0.88 4- 0.31 p g [3H]-15-HETE). The observed inhibition of the cytoso;ic 15-1ipoxygenase at 0.5 and 5 m M calcium could not be explained by calcium precipitation of [aH]arachidonic acid since 99% of the radioactivity r e m a i n e d in the 120000 x g cytosol at the e n d of these incubations (results not showr.). Sonication of cells results in the formation of complex vesicles [13]. Since the 120000 × g cytosolie fractien,~ were p r e p a r e d from cells s u s p e n d e d in buffers of selected calcium concentrations prior to homogenization, it was possible that calcium did not directly influence the 15-1ipoxygenase but affected vesicle formation. This possibility was exa m i n e d by d e t e r m i n i n g the effects of a d d i n g calcium to
Human PMN cytosol (120000x g) was prepared in 0, 1 and 5 .aM calcium-comaining buffers as described under Materials and Methotis. The samples were incubated with EGTA (2 mM final concentration) for 5 m;.~ at ,! °C ~r':or to the z~ditio~ ~f ['~H]arachidonicacid (2.6 nM). [aH]-I5-HETE formation after 30 rain was monitored as a measure of 15-1ipoxygenaseactivity. Each experiment indicates a different donor. Experiment [3H]-I5-HETE formation(pg) 0~M Ca2+ I~M Ca2+ A B C D
5~MCa 2+
control +EGTA control +EGTA 5.0 (.8 28 3.0 6.3 3.7 26 6.2 16 4.8 ~ 6.1 81 30 ~ 18
1.1 2.1 4.7 12
120000 X g supernatant fractions which had been prep a r e d from cells suspended in calcium-free buffer. It was found that exogenous addition of 1 and 5 /~M calcmm produced 34 + 7.8 pg (2-fold increase, n = 3) and 84 +_ 24 pg (6-fold increase, n = 3) [aH]-15-HETE. The calcium-dependent stimulation of the 15-1ipoxygenase was reversible since 1 mM E G T A inhibited this process (Table ll). Calcium also a p p e a r s to induce a partial m e m b r a n e association of active 15-1ipoxygenase. A s shown in Fig. 2, the 15-1ipoxygcnase activity in the microsomal meb r a n e fraction at 50 and 500 p.M calcium increased to 8.8 4- 4.1 and 12 4- 9.2 pg [3H]-15-HETE (n = 3), respectively, from that observed in calcium-free m e d i u m 0.88 _+ 0.24 pg [aH]-15-HETE (n = 4). It was previously reported that 5 - H E T E stimulates the 15-1ipoxygenase in intact P M N s suspended in Duibecco's phophate-buffered saline containing 1 m M calcium [10] and preliminary experiments indicated that 8
.~
120
0
1
r~ _ 0
(i
_
5
= ~o~ mcaos0~
~J 50
~1~
_,~
500
5000
~t.ClUM Oaa)
Fig. 2. 15-Lipoxygenascactivity in human PMN cytosolic and microsomal fractions at different calcium concentrations. PMN cytosolic (120000x g supernatant) and microsomal-membrane (resuspended 120000x g pellet) fractions were prepared in buffers containing the indicated calcium concentrations. 15-Lipoxygenaseactivity of thesc fractions was assayed as described under Materials and Methods. Data arc from three experiments except 5 mM calcium (n = 2).
8O TABLE Ill Effect of 5-HETE on calcium.treated 15-1ipoxygenase in PMN cytosol
PMNs were homogenizedin Dulbecco'sPBS which contains 1 mM calcium or in calcium-free medium (Dulbecco's PBS with 3 mM EGTA or HEH buffer without calcium) and the 120000x g supernatants prepared as described under Materials and Methods. Sampies were treated with 8 p.M 5-HETE immediatelyprior to initiating the assayby addition of 2.6 nM [3H]arachidonate. After 30 min the assay was terminated and [3H]-15-HETE formation quantified. Results are expressed as mean+S.E, from at least three different donors
Control + 5-HETE(8 #.M)
[3H]-15-HETEformation(pg) calcium-free 1 mM calcium 5.1+3.0 1.1 + 0.44 5.8±2.0 20 ± 10
#M 5-HETE produced optimum conversion of arachidonic acid to [3H]-15-HETE by the 15-1ipoxygenase in 500 x g supernatants (prepared in Dulbecco's PBS) (results not shown). In view of the above results, the effect of calcium on the 5-HETE stimulatable 15-1ipoxygenase activity in 120000 × g eytosol was examined (Table III) In calcium-free buffer, 8 p.M 5-HETE did not affect i5-1ipoxygenase activity. However, 5-HETE completely overcame the inhibitory (79%) effect of 1 mM calcium on the 15-1ipoxygenase. Since phospholipids have been reported to affect lipoxygenase activities [11], the effects of various phospholipids on the micromolar calcium-stimulatable activity of cytosolic 15-1ipoxygenase were examined. In the presence of 5 ttM calcium, both PS and PC vesicles (100 /zg/ml) inhibited the calcium stimulatable 15lipcxygenase activity (75 and 55%, respectively)whereas neither PI nor PE had ant effect (Table IV). No appreciable inhibition was observed with PS-vesicles at 20 t~g/ml and at higher concentrations (up to I m g / m l ) all other phospholipids inhibited although the pattern remained the same. When the effects of these phosTABLE 1V Effects of different phospholipids on the cytosolic PMN 15.1ipoxygenase
Phospholipid liposomes(10 #.g in 2.5 #l calcium-free buffer)were preincubated with cytosolic fractions (0.1 ml. prep~lred from 107 cells/ml of 0 or 5/J,M calcium-containingbuffers)at 4 o for 30 min. 15-Lipoxygenase activity was measured as [3H]-IS-HETEformation 30 min after the addition of 2.6 nM [3H]arachidonicacid. The results (mean+ S.E.) ate from three separate experiments. Phospholipid (100/~g/ml) -
PS PC PE P1
Ca2+ (p.M) 0 5 5 5 5 5
[3HI-15-HETE formation (dpm/107 cells) 8500+-7800 51400:1:.6000 194002:6800 27600+4400 48600+ 1300 52500:[=7700
pholipids on the cytosolic 15-1ipoxygenase in the presence of 0.5 mM calcium were investigated, no s:i:nulation of the enzyme activity was observed (results not shown). Discussion
Several reports in the literature indicate a possible paradox regarding the calcium requirement of the leukocytic 15-1ipoxygenase. Intact human PMNs, in buffers containing 1 mM calcium, exhibited very low 15-1ipoxygenase acivity which could be markedly stimulated to utilize endogenous arachidonic acid by various HETEs [10]. The 15-1ipoxygenase in partially purified preparations from human and rabbit PMNs as well as human eosinophils reportedly required millimolar levels of calcium for maximal activity but pure leukocytic 15-1ipoxygenase did not exhibit any calcium dependancy [9,11,12,14]. Since the 15-1ipoxygenase is a cytosolic enzyme, we decided to examine the effects of nanomolar to millimolar concentrations of calcium on the 15-1ipoxygenase activity in unfractionated 120000 × g cytosolic preparations of PMNs so as to approximate the native intraceihlar environment of the enzyme. In addition, tracer (nanomolar) concentrations of arachidonic acid substrate were used in these studies rather than m~cromolar levels used by other investigators since the former are more physiologically relevant. Under these conditions, a significant increase in 15-1ipoxygenase activity was evident at 1 / z M calcium, maximal activity was observed at 50/~M calcium but at 0.5-5 raM, calcium appeared to strongly inhibit the enzyme activity. These results indicate that physiologically relevant (nanomohr end low micromolar) levels o f calcium can stimulate the 15-1ipoxygenase. A recent lepo~ indicates that this range of calcium concentrations affected the 5-1ipoxygenase from RBL-1 cells in an identical manner [15], suggesting that calcium can simultaneously influence both lipox'ygenases in an identical fashion. Since various cellular fact~,rs appear necessary for cellular 5-1ipoxygenase activity [4-6], it is quite possible that there is also a calcium-dependant factor(s) which is essential for 15-1ipoxygenase activity in a cellular milieu. This could explain the apparent paradox regarding the different calcium requirements of impure and pure leukoc~t~e 15-1ipoxygenase preparations. Moreover, inhibition of the cytosolic 15-1ipoxygenase at high calcium levels could be rationalized by removal or inactivation of this putative factor by calcium. The ability of 5-HETE to overcome the inhibitory effects of high calcium levels does not appear to be due to the complexing of calcium to the 5-HETE carboxylate group since 5-HETE methyl ester could also stimulate the cellular enz~.'m.e (results not showrt). However, this stimulatory effect could be due to some, as yet undefined, interaction of 5-HETE with tbe puta-
tive 15-1ipoxygenase stimulatory factor. The reversal of the calcium inhibition of the cytosolic 15-1ipoxygenase by 5-HETE and the presence of proteinase inhibitors in the cytosolic preparation make it unlikely that this calcium inhibition is due to inactivation of the 15-1ipoxygenase by proteinases. One explanation for the differences observed in calcium requirements needed to stimulate the leukocytic 15-1ipoxygenase may be the different concentrations of arachidonic acid used, i.e., nanomolar in the present study and micromolar in the other cited studies [9,11,12]. Comparison of the 15-1ipoxygenase activities of the cytosolic and microsomal-membrane fractions as a function of calcium concentration indicate that there was a progressive increase in the association of active 15-1ipoxygenase with microsomal membranes which reached a maximum at 500 ~,M calcium. At this level, the microsomai enzyme activity exceeded the cytosolic activity but amounted to only 10% of the total activity observed at 50 IzM calcium. In the presence of 1 mM calcium, PC stimulated partially purified eosinophilie 15-1ipo~genase preparations [11]. The effect of various phospholipids on the cytosolic PMN 15-1ipoxygenase were examined at 5/z M calcium, a condition that partially stimulated the enzyme. None of the phospholipids tested further enhanced the 15-1ipoxygenase but both PS and PC inhibited the enzyme. This inhibition could not be reversed by prior treatment with 5-HETE or removal of vesicles by centrifugation prior to the addition of substrate (results not shown). It is possible PS and PC interfere with the putative cytosolic calcium-dependent factor so that this factor can no longer stimulate the 15-1ipoxygenase. In conclusion, the present studies indicate that nanomolar and mieromolar concentrations of calcium stimulate the 15-1ipo~genase present in a cytosolic environment to rnet~SotLze tracer (nanomolar) amounts of arachidonic acid. The inhibitory effect of 1 mM calcium on :he 15-1ipox'ygenase can be overcome by the
presence of exogenously added 5-HETE. Pat't of the cytosolic 15-1ipoxygenase activity can be inhibited by phosphatidyl serine and phosphatidyl choline. Acknowledgements The authors thank Dr. Gary Fiskum and Becky Vonakis for their helpful advice during the course of this work. This work was supported by a grant from the National Institutes of Health. References ! Borgeat,P. and Samuelsson,B. (1979)Proc. Natl. Acad.Sci. USA 76, 3213-3217. 2 Samuelssoa,B. (1983)Science 220, 568-575. 3 Samuelsson,B., Dahlen, S,E., Lindgren,J.A., Rouzer, C.A. and Serhan, C.N. (1987) Science237, 1171-1176. 4 Rouzer, C.A. and Samuelsson, B. (1985) Proc. Natl. Acad. Sci. USA 82, 6040-6044. 5 Rouzer, C.A., Shimizu,T. and Samuelsson, B. (1985) Proc. Natl, Aead. Sci. USA 82, 7505-7509. 6 Miller, D.K., Gillard, J.W., Vickers, PJ., Sadowski,S., Leveille, C.. Mancini, J,A., Chatleson, P,, Dixon, R.A.F., Ford-Hutchinson, A.W., Fonin, R., Gauthier, J.Y., Rodkey, J., Rosen, R,, Rouzer, C., Sigel, I.S,, Strauder, C.D. and Evans, J.F. (1990) Nature 343, 278-281. 7 Vanderhoek,J.Y., Ka.,anin,M.T. and Ekborg, S.L (1985)J. Biol. Chem. 260, 15482-15487. 8 Fabioto, A. and Fabioto, F. (1979)J. Physiol.(Paris) "/5,463-505. 9 S~berrnan, R.J., tlarpcr, T.W., Betteridge,D., Lewis, R.A. and Au~ten, K.F. (1985)J. Biol. Chem. 260, 4508-4515. I0 Nichols, R.C. and Vanderhoek, J.Y. (1990) J. Exp. Med. 17!, 367-375. II Sigal,E., Grunberger, D., Cashman, J.R., Craik, C.S., Caughey, G.H. and Nadel, J.A. (1988) Biochim. Biophys. Res. Commun. 150, 376-383. 12 Narumiya,S., Salmon. J.A., Cottee, F.H., Weatherley,B.C. and Flower, RJ. (1981)2. :'liol.Chem. 256, 9583-9592. 13 Francis,J.W., Smolen,J,E. Balazovich,KJ., Sandborg, R.R. and Boxer, L.A. (1990)Biochim.Biophys.Acta 1025,1-9. 14 Sigal,E., Grunberger, D., Craik.,C.S., Caughey,G.H. and Nadel, J.A. (1988)J. Biol. Chem. 263, 5328-5332. 15 Cochran, F.R. and Finch-Ariena,M.B. (1989)Bi~¢hem.Biophys. Res. Commun. 161,1327-1332.
77
© 1991 ElsevierScience PublishersB.V. 0b~5-2760/91/$03.50 ADONIS 000527609100232S
Calcium regulation of the human PMN cytosolic 15-1ipoxygenase R a l p h C. N i c h o l s a n d J a c k Y. V a n d e r h o e k Department of Biochemh'try and Molecular Biology, The George Washington Unicersity-Schcol of Medicine and Heahh Sciences, Washingtvn, DC (U.S.A.)
(Received4 April 19011 Key words: Calcium;Cytosol;Lipoxygenase Addition of tracer (pg) amounts of [aHlaraehidonic acid to the 120000 × g ojtosolie fraction of h u m a n polymerphonuclear leukocytes (PMNs) produced [3H]-IS-HETE, the product of the 15-1ipox~fgenase, as the m ~ o r metaboIRe. in the presence of nanomolar and low micromolar amounts of calcium, PH]-IS-HETE formation was increased as much as IS-fold which correspon6ed to 17% conversion of added substrate. This enhancement of the cytosolic 1S-lipoxygenase activity, which was reversible by EGTA, was inhibited by phosphatidyl serine and phosphatidyl choline. Millimolar levels of calcium iuhiLited the cytosolic 15-1ipoxygenase and the 5-1ipox~genase product 5-HETE could reverse this inhibition. These results indicate that calcium is an important modulator of the PMN l$-IipoxTgenase when the enzyme is in a cytosolic milieu.
Introduction H u m a n leukoq, tes contain both 5- and 15-1ipo ~ g e n a s e s [1]. These enzymes catalyze the oxygenation of arachidonie acid to biologically active metabolites including leukotrienes, lipoxins, HPETEs and HETEs which have been implicated as mode!at.ors of inflammatory and immunological reactions [2,3]. Although the pure 5.1ipoxygenase activity is controlled by several factors such as calcium and ATP [4], there ai'e additional factors controlling the activity of the 5-1ipoxygenase in the cellular environment. These include both cytosolic and membrane-associated stimulatory factors such as the recently described 5-1ipoxygenase activating factor [4-6]. In view of these results, it is quite possible that cellular factors also regulate the 15-1ipo~genase when the enzyme is located in a cellular milieu. The purpose of the present study was to examine the effects of several cellular components on
Abbreviations: HEEDTA, N-hydro~ethylethylanediaminetriacehc acid; H(P)ETE, hydro(pe:o)xyeicosatetracnoic acid; PC, phosphatidyl choline; PMN, polymorphonuclear leukocyte; PS, phosphatidyl serin¢; RP($P)-HPLC, reverse phase (straight phase)-bigh pressure liquid chromatography.
Correspondence:J.Y. Vanderhoek, Department of Biochemistry and Molecular Biology, The George Washington University-Schoolof Medicine, 2300 Eye Street, N.W., Washington,DC 20037, U.S.A.
the 15-1ipoxygenase activity present in unfractionated cytosolic PMN preparations. Materials and Methods Leukocytes were isolated from heparinized blood from human donors who had not taken aspirin-like drugs for the previous 2 weeks. PMNs were separated on Ficoll-Hypaque cushions and washed with Dulbecco's phosphate-buffered saline (10 ~iM dextrose (pH 7.4), GIBCO, Grand Island, NY) [7]. Contaminating red cells were l y ~ d by hypotonic shock. Cell viability, evaluated with Trypan blue, was consistently greater than 98%. For intact cell experiments, cells were kept at room temperature until assayed. If homogenized, cells were cooled to 0 ° C . For calcium studies, H e p e s / E G T A / H E E D T A (HEH) buffers (pH 7.2) were prepared at ~lecled calcium concentrations using the system described by Fabioto and Fabioto [8]. Calcium concentrations of stock solutions were determined by atomic absorption. Cells were suspended at 2 - 1 0 7 / m l in the appropriate buffer and were homogenized (Ultrasonics (Plainview, N.Y.) model W-140 cell disrupter at a power setting of 3.5) at 0 ° C after the addition of the proteinase inhibitors phenylmethyl sulfonyl fluoride (1 raM) and leupeptin (1 /~g/ml). Homogenization was per,~?:~ed in three times 15 s pulses at 1 rain intervals. Intac: cells and cell debris were removed by lowspeed ( 5 0 0 × g ) centrifugation for 20 min at 4 o C. Cytosolic and micro-
somal fractions were separated by high-speed centrifugation (120000 × g ) for 1 h at 4°C. Supernatants were removed and kept at 0 ° C until assayed the same day. Microsomes were resolubilized in the appropriate buffer by sonication at 0 ° C. In the exogenously added-calcium experiments, a calcium stock solution was prepared in HEH (pH 7.8) with enough MgCI 2 added to give a final concentration of 1 mM when this solution was added to calcium-free HEH. In the reversibility studies, excess EGTA was added from a 60 mM stock solution to give a final concentration of 1 mM. The pH decreased (from 7.2 to 7.10) in the 5 p,M calcium samples. This change did not exceed the buffering range of the solution nor the pH range of the en~'me (6.5-7.5 [9]). In separate experiments, enzyme activity was maximal at pH values as low as 6.5 in HEH (data not shown). Phospholipid vesicles were prepared by evaporating the solvent from the phospholipid stock solution under nitrogen, adding the appropriate amount of calcium-free H E H buffer to produce a concentration of 4 m g / m l and sonicating at 0 ° C for three times 15 s. Effects of phospholipids were determined by adding the phospholipid vesicles to the cytosol, vortexing the mixture and incubating at 4 ° C for 30 mix prior to the addition of 420 pg [3H]arachidonic acid. To assay lipoxygenase activity in intact PMNs and homogenatcs, 500 /~1 aliquots were placed in a 2 3 ° C water bath. The reaction was initiated by the addition of 1 /~1 [3H]arachidonic acid (New England Nuclear, 2.8 T B q / m m o l in ethanol or dimethyl sulfoxide; 420 pg) and was terminated after 30 mix by addition of 1.65-fold volume excess of M e O H / w a t e ; / f o r m i c acid (750:90:10) to which internal standards (a mixture of monoHETEs [I0]) had been added. Samples were stored at - 20 o C until lipids were extracted. Samples were extracted into chloroform [7], dried under nitrogen and dissolved in 30 p,I acetonitrile/ water/acetic acid (65:35:0.035). RP-HPLC separation was performed on a Zorbax (Mac Mud, Chadd's Ford, PA) C18 0.46 x 25 cm column coupled to a C18 pre-column which was attached to a Perkin Elmer Series 4 liquid chromatograph. [3H]-HETEs were separated with an acetonitrile/water (0.1% acetic acid)/ methanol (40:23:37) solvent program run isocratically at a flow rate of 1.0 m l / m i n . Eluate was monitored at 235 nm (LC 85B variable wavelength UV detector) and collected with an LKB (Bromma, Sweden) 2211 fraction collector. Retention times and peak areas were monitored with a Hewlett-Packard 3396A plotter/ integrator. The identities ot radioactive products were eonfirmeo by comparison of SP-HPLC retention times with authentic standards. Radioactive lipoxygenase product formation was determined by scintillation counting using 3a70B as scintiIlant (Research Products Incorporated, Mt. Prosp~,ct, IL). Sample recovery
ranged from 70-100%. Protein was determined by a modified Lowry (Pierce, Rockford, IL). The total 120000 × g cytosolic protein content obtained from 107 PMNs was 0.34 + 0.02 mg (n = 36) and product formation after 30 mix was expressed as mass of radioactive HETE formed per 0.34 mg prottdn ill the sample (unless noted differently). HPLC solvents were from Fisher Scientific. 5-HETE was purchased from Cayman Chemical (Ann Arbor, MI). All reagents, unless otherwise stated, were from Sigma (St. Louis, MO). Results
In view of our previous report that a free, nonesterifled pool of arachidonic acid was associated with isolated human leukocytes [10], it was decided to determine whether PMNs could metabolize tracer amounts of exogenously added [3H]arachidonic acid. Addition of 2.6 nM (420 pg) [3H]arachidonic acid to intact PMNs (107 cells suspended in 0.5 mi Dulbecco's saline buffer which contains 1 m M calcium) produced low amounts (0.1 + 0.02 pg, n = 5) of [3H]-15-HETE, the product of the 15 lipoxygenase (Table I). Using a cell-free supernatant preparation, obtained by homogenization of 107 PMNs in Dulbeeco's buffer and centrifugaticn at 120000 x g , resulted in the formation of 1.3 + 0.68 pg [3H]-15-HETE (n = 6). Adding the calcium chelator E G T A to the 1 2 0 0 0 0 × g cytosolic preparation produced 13 + 3.4 pg [3H]-15-HETE (n = 7) after 30 mix which represented a 3% conversion of added araehidonic acid substrate. Although [3H]-15H E T E production in cell-free supernatant preparations increased only 30% when the incubation times were increased from 5 to 30 mix, the latter reaction time was chosen to ensure complete product formation (data not shown). These results also suggested that millimolar levels of calcium inhibited the activity of the
TABLE 1 15-Lipoxygenase activity in unstimulated intact and broken cell preparations
Human PMNs (2' 107/mDwere suspended in Dulbeeco's phosphate saline buffel or in buffer containing 3 mM EGTA. Subcellular ~npernatan`' fractions were prepared as described under Materials and Methods and 15-1ipoxygenaseactivitywas measured as 13H]-15HETE production after 30 mix from exogenously added tracer [aH]arachidonic acid (2.6 riM, 420 pg). Product formation is per 107 intact cells et ,'he equivalentsupernatant protein content of 0.34 mg. Results are expressedas the mean±S.E, from 3-7 different donors. PMN preparation Intac`'cells 500×g supernatant 120000×8 supernatant 120000×g supernatant+EGTA
[3H]-I5-HETE (pg) 0.10±0.02 0.82±0.44 1.3 ±0.68 13 ±3.4
79 TABLE II Rerersal of Ca-"~-stimulated 15-1ipoxygenasein PMN cytosol
I0 0 "
0
//
0.1
. . . .
1
10
m,~_..
100 1000 I0.000
CALCIUM O,M)
Fig. 1 Calcium.dependent modulation of the cytosolic PMN 15-1ipoxygenase. Human PMNs (2"(07/ml) in HEH buffers containing 0-5000/zM calcium were homogenized and centrifuged to produce 120000× g cytosolic fractions. The 15-hpoxygenaseactivity of these cytnsolic preparations was measured as [aHI-15-HETE formation from exogenously added tracer [3H]arachidonic acid (2.6 nM). Data are presented as the mean 4- S.E. from 4 + 7 different donors. 15-1ipoxygenase present in cytosolic preparations. Since previous reports had d e m o n s t r a t e d that millimolar levels of calcium s t i m u l a t e d partially purified 15-1ipoxygeuase p r e p a r a t i o n s [9,11,12], it was decided to carefully examine the effect of calcium on the PMN 15-1ipoxygenase in cytosolic preparations. Fig. 1 illustrates a calcium titration curve in which the effects of n a n o m o l a r to millimolar concentrations of calcium were tested on the 15-1ipoxygenase activity in the 1 2 0 0 0 0 × g s u p e r n a t a n t fraction from h u m a n P M N s s u s p e n d e d in H E H buffer. A 6-fold increase in [aH]-15-HETE formation (38 + 15 pg, n = 7) was observed at 1 / z M free calcium relative to control without calcium (6.0 + 4.0 pg, n = 7) and the optimal calcium concentration for 15-1ipo~genase stimulation (77 4- 29 p g [3H]-15-HETE, n = 7) was produced at 5 0 / z M . In experiments involving micromolar (or less) concentrations of calcium, [3H]-15-HETE was the major metabolite formed and exceeded [ 3 H ] - 5 - H E T E ' p r o d u c t i o n by at least a 10-fold factor. W h e n the calcium concentration was increased further, inhibition of 15-1ipoxygenase activity was observed, A't 5 m M calcium, [3H]-15-HETE production was significantly inhibited below control values (for four m a t c h e d experiments: control, 4.4 + 1.8 pg [3H]-15-HETE; 5 m M calcium, 0.88 4- 0.31 p g [3H]-15-HETE). The observed inhibition of the cytoso;ic 15-1ipoxygenase at 0.5 and 5 m M calcium could not be explained by calcium precipitation of [aH]arachidonic acid since 99% of the radioactivity r e m a i n e d in the 120000 x g cytosol at the e n d of these incubations (results not showr.). Sonication of cells results in the formation of complex vesicles [13]. Since the 120000 × g cytosolie fractien,~ were p r e p a r e d from cells s u s p e n d e d in buffers of selected calcium concentrations prior to homogenization, it was possible that calcium did not directly influence the 15-1ipoxygenase but affected vesicle formation. This possibility was exa m i n e d by d e t e r m i n i n g the effects of a d d i n g calcium to
Human PMN cytosol (120000x g) was prepared in 0, 1 and 5 .aM calcium-comaining buffers as described under Materials and Methotis. The samples were incubated with EGTA (2 mM final concentration) for 5 m;.~ at ,! °C ~r':or to the z~ditio~ ~f ['~H]arachidonicacid (2.6 nM). [aH]-I5-HETE formation after 30 rain was monitored as a measure of 15-1ipoxygenaseactivity. Each experiment indicates a different donor. Experiment [3H]-I5-HETE formation(pg) 0~M Ca2+ I~M Ca2+ A B C D
5~MCa 2+
control +EGTA control +EGTA 5.0 (.8 28 3.0 6.3 3.7 26 6.2 16 4.8 ~ 6.1 81 30 ~ 18
1.1 2.1 4.7 12
120000 X g supernatant fractions which had been prep a r e d from cells suspended in calcium-free buffer. It was found that exogenous addition of 1 and 5 /~M calcmm produced 34 + 7.8 pg (2-fold increase, n = 3) and 84 +_ 24 pg (6-fold increase, n = 3) [aH]-15-HETE. The calcium-dependent stimulation of the 15-1ipoxygenase was reversible since 1 mM E G T A inhibited this process (Table ll). Calcium also a p p e a r s to induce a partial m e m b r a n e association of active 15-1ipoxygenase. A s shown in Fig. 2, the 15-1ipoxygcnase activity in the microsomal meb r a n e fraction at 50 and 500 p.M calcium increased to 8.8 4- 4.1 and 12 4- 9.2 pg [3H]-15-HETE (n = 3), respectively, from that observed in calcium-free m e d i u m 0.88 _+ 0.24 pg [aH]-15-HETE (n = 4). It was previously reported that 5 - H E T E stimulates the 15-1ipoxygenase in intact P M N s suspended in Duibecco's phophate-buffered saline containing 1 m M calcium [10] and preliminary experiments indicated that 8
.~
120
0
1
r~ _ 0
(i
_
5
= ~o~ mcaos0~
~J 50
~1~
_,~
500
5000
~t.ClUM Oaa)
Fig. 2. 15-Lipoxygenascactivity in human PMN cytosolic and microsomal fractions at different calcium concentrations. PMN cytosolic (120000x g supernatant) and microsomal-membrane (resuspended 120000x g pellet) fractions were prepared in buffers containing the indicated calcium concentrations. 15-Lipoxygenaseactivity of thesc fractions was assayed as described under Materials and Methods. Data arc from three experiments except 5 mM calcium (n = 2).
8O TABLE Ill Effect of 5-HETE on calcium.treated 15-1ipoxygenase in PMN cytosol
PMNs were homogenizedin Dulbecco'sPBS which contains 1 mM calcium or in calcium-free medium (Dulbecco's PBS with 3 mM EGTA or HEH buffer without calcium) and the 120000x g supernatants prepared as described under Materials and Methods. Sampies were treated with 8 p.M 5-HETE immediatelyprior to initiating the assayby addition of 2.6 nM [3H]arachidonate. After 30 min the assay was terminated and [3H]-15-HETE formation quantified. Results are expressed as mean+S.E, from at least three different donors
Control + 5-HETE(8 #.M)
[3H]-15-HETEformation(pg) calcium-free 1 mM calcium 5.1+3.0 1.1 + 0.44 5.8±2.0 20 ± 10
#M 5-HETE produced optimum conversion of arachidonic acid to [3H]-15-HETE by the 15-1ipoxygenase in 500 x g supernatants (prepared in Dulbecco's PBS) (results not shown). In view of the above results, the effect of calcium on the 5-HETE stimulatable 15-1ipoxygenase activity in 120000 × g eytosol was examined (Table III) In calcium-free buffer, 8 p.M 5-HETE did not affect i5-1ipoxygenase activity. However, 5-HETE completely overcame the inhibitory (79%) effect of 1 mM calcium on the 15-1ipoxygenase. Since phospholipids have been reported to affect lipoxygenase activities [11], the effects of various phospholipids on the micromolar calcium-stimulatable activity of cytosolic 15-1ipoxygenase were examined. In the presence of 5 ttM calcium, both PS and PC vesicles (100 /zg/ml) inhibited the calcium stimulatable 15lipcxygenase activity (75 and 55%, respectively)whereas neither PI nor PE had ant effect (Table IV). No appreciable inhibition was observed with PS-vesicles at 20 t~g/ml and at higher concentrations (up to I m g / m l ) all other phospholipids inhibited although the pattern remained the same. When the effects of these phosTABLE 1V Effects of different phospholipids on the cytosolic PMN 15.1ipoxygenase
Phospholipid liposomes(10 #.g in 2.5 #l calcium-free buffer)were preincubated with cytosolic fractions (0.1 ml. prep~lred from 107 cells/ml of 0 or 5/J,M calcium-containingbuffers)at 4 o for 30 min. 15-Lipoxygenase activity was measured as [3H]-IS-HETEformation 30 min after the addition of 2.6 nM [3H]arachidonicacid. The results (mean+ S.E.) ate from three separate experiments. Phospholipid (100/~g/ml) -
PS PC PE P1
Ca2+ (p.M) 0 5 5 5 5 5
[3HI-15-HETE formation (dpm/107 cells) 8500+-7800 51400:1:.6000 194002:6800 27600+4400 48600+ 1300 52500:[=7700
pholipids on the cytosolic 15-1ipoxygenase in the presence of 0.5 mM calcium were investigated, no s:i:nulation of the enzyme activity was observed (results not shown). Discussion
Several reports in the literature indicate a possible paradox regarding the calcium requirement of the leukocytic 15-1ipoxygenase. Intact human PMNs, in buffers containing 1 mM calcium, exhibited very low 15-1ipoxygenase acivity which could be markedly stimulated to utilize endogenous arachidonic acid by various HETEs [10]. The 15-1ipoxygenase in partially purified preparations from human and rabbit PMNs as well as human eosinophils reportedly required millimolar levels of calcium for maximal activity but pure leukocytic 15-1ipoxygenase did not exhibit any calcium dependancy [9,11,12,14]. Since the 15-1ipoxygenase is a cytosolic enzyme, we decided to examine the effects of nanomolar to millimolar concentrations of calcium on the 15-1ipoxygenase activity in unfractionated 120000 × g cytosolic preparations of PMNs so as to approximate the native intraceihlar environment of the enzyme. In addition, tracer (nanomolar) concentrations of arachidonic acid substrate were used in these studies rather than m~cromolar levels used by other investigators since the former are more physiologically relevant. Under these conditions, a significant increase in 15-1ipoxygenase activity was evident at 1 / z M calcium, maximal activity was observed at 50/~M calcium but at 0.5-5 raM, calcium appeared to strongly inhibit the enzyme activity. These results indicate that physiologically relevant (nanomohr end low micromolar) levels o f calcium can stimulate the 15-1ipoxygenase. A recent lepo~ indicates that this range of calcium concentrations affected the 5-1ipoxygenase from RBL-1 cells in an identical manner [15], suggesting that calcium can simultaneously influence both lipox'ygenases in an identical fashion. Since various cellular fact~,rs appear necessary for cellular 5-1ipoxygenase activity [4-6], it is quite possible that there is also a calcium-dependant factor(s) which is essential for 15-1ipoxygenase activity in a cellular milieu. This could explain the apparent paradox regarding the different calcium requirements of impure and pure leukoc~t~e 15-1ipoxygenase preparations. Moreover, inhibition of the cytosolic 15-1ipoxygenase at high calcium levels could be rationalized by removal or inactivation of this putative factor by calcium. The ability of 5-HETE to overcome the inhibitory effects of high calcium levels does not appear to be due to the complexing of calcium to the 5-HETE carboxylate group since 5-HETE methyl ester could also stimulate the cellular enz~.'m.e (results not showrt). However, this stimulatory effect could be due to some, as yet undefined, interaction of 5-HETE with tbe puta-
tive 15-1ipoxygenase stimulatory factor. The reversal of the calcium inhibition of the cytosolic 15-1ipoxygenase by 5-HETE and the presence of proteinase inhibitors in the cytosolic preparation make it unlikely that this calcium inhibition is due to inactivation of the 15-1ipoxygenase by proteinases. One explanation for the differences observed in calcium requirements needed to stimulate the leukocytic 15-1ipoxygenase may be the different concentrations of arachidonic acid used, i.e., nanomolar in the present study and micromolar in the other cited studies [9,11,12]. Comparison of the 15-1ipoxygenase activities of the cytosolic and microsomal-membrane fractions as a function of calcium concentration indicate that there was a progressive increase in the association of active 15-1ipoxygenase with microsomal membranes which reached a maximum at 500 ~,M calcium. At this level, the microsomai enzyme activity exceeded the cytosolic activity but amounted to only 10% of the total activity observed at 50 IzM calcium. In the presence of 1 mM calcium, PC stimulated partially purified eosinophilie 15-1ipo~genase preparations [11]. The effect of various phospholipids on the cytosolic PMN 15-1ipoxygenase were examined at 5/z M calcium, a condition that partially stimulated the enzyme. None of the phospholipids tested further enhanced the 15-1ipoxygenase but both PS and PC inhibited the enzyme. This inhibition could not be reversed by prior treatment with 5-HETE or removal of vesicles by centrifugation prior to the addition of substrate (results not shown). It is possible PS and PC interfere with the putative cytosolic calcium-dependent factor so that this factor can no longer stimulate the 15-1ipoxygenase. In conclusion, the present studies indicate that nanomolar and mieromolar concentrations of calcium stimulate the 15-1ipo~genase present in a cytosolic environment to rnet~SotLze tracer (nanomolar) amounts of arachidonic acid. The inhibitory effect of 1 mM calcium on :he 15-1ipox'ygenase can be overcome by the
presence of exogenously added 5-HETE. Pat't of the cytosolic 15-1ipoxygenase activity can be inhibited by phosphatidyl serine and phosphatidyl choline. Acknowledgements The authors thank Dr. Gary Fiskum and Becky Vonakis for their helpful advice during the course of this work. This work was supported by a grant from the National Institutes of Health. References ! Borgeat,P. and Samuelsson,B. (1979)Proc. Natl. Acad.Sci. USA 76, 3213-3217. 2 Samuelssoa,B. (1983)Science 220, 568-575. 3 Samuelsson,B., Dahlen, S,E., Lindgren,J.A., Rouzer, C.A. and Serhan, C.N. (1987) Science237, 1171-1176. 4 Rouzer, C.A. and Samuelsson, B. (1985) Proc. Natl. Acad. Sci. USA 82, 6040-6044. 5 Rouzer, C.A., Shimizu,T. and Samuelsson, B. (1985) Proc. Natl, Aead. Sci. USA 82, 7505-7509. 6 Miller, D.K., Gillard, J.W., Vickers, PJ., Sadowski,S., Leveille, C.. Mancini, J,A., Chatleson, P,, Dixon, R.A.F., Ford-Hutchinson, A.W., Fonin, R., Gauthier, J.Y., Rodkey, J., Rosen, R,, Rouzer, C., Sigel, I.S,, Strauder, C.D. and Evans, J.F. (1990) Nature 343, 278-281. 7 Vanderhoek,J.Y., Ka.,anin,M.T. and Ekborg, S.L (1985)J. Biol. Chem. 260, 15482-15487. 8 Fabioto, A. and Fabioto, F. (1979)J. Physiol.(Paris) "/5,463-505. 9 S~berrnan, R.J., tlarpcr, T.W., Betteridge,D., Lewis, R.A. and Au~ten, K.F. (1985)J. Biol. Chem. 260, 4508-4515. I0 Nichols, R.C. and Vanderhoek, J.Y. (1990) J. Exp. Med. 17!, 367-375. II Sigal,E., Grunberger, D., Cashman, J.R., Craik, C.S., Caughey, G.H. and Nadel, J.A. (1988) Biochim. Biophys. Res. Commun. 150, 376-383. 12 Narumiya,S., Salmon. J.A., Cottee, F.H., Weatherley,B.C. and Flower, RJ. (1981)2. :'liol.Chem. 256, 9583-9592. 13 Francis,J.W., Smolen,J,E. Balazovich,KJ., Sandborg, R.R. and Boxer, L.A. (1990)Biochim.Biophys.Acta 1025,1-9. 14 Sigal,E., Grunberger, D., Craik.,C.S., Caughey,G.H. and Nadel, J.A. (1988)J. Biol. Chem. 263, 5328-5332. 15 Cochran, F.R. and Finch-Ariena,M.B. (1989)Bi~¢hem.Biophys. Res. Commun. 161,1327-1332.
