Mapping within the Carboxyl-terminal Domain of Small. GTP-binding Proteins ... article were defrayed in part by the payment of page charges. This article must ...
T H E JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269,No. 46, Issue of November 18,pp. 29024-29031, 1994 Printed in U.S.A.
Inhibition of NADPH Oxidase Activationby Synthetic Peptides Mapping within the Carboxyl-terminal Domain of Small GTP-binding Proteins LACK O F AMINO ACID SEQUENCE SPECIFICITY AND IMPORTANCE OF POLYBASIC MOTIF* (Received for publication, June 28, 1994, and in revised form, September 6, 1994)
Gili Joseph, Yara Gorzalczany, VasilijKoshkin, and EdgarPick$ From the Laboratory of Zmrnunopharmacology, the Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
The small GTP-binding protein (G protein) Racl is an membrane-localized, two-subunit flavocytochrome, known as obligatory participant in theassembly of the superoxide cytochrome b,,, (reviewed in Refs. 1-3). Electron transport by (Okbgenerating NADPH oxidasecomplexofmacrocytochrome b,,, is activated following its interaction with two phages. We investigated the effect of synthetic peptides, cytosolic proteins ( ~ 4 7 ~and ~ "p67Phox),which do not possess a mapping within the near carboxyl-terminal domainsof redox function, and the resultingmulticomponent aggregate is Racl and of related G proteins, on the activity of NADPH commonly known as the 0,-forming NADPH oxidase complex. oxidase in a cell-free system consisting of solubilized Identification of all NADPH oxidase components was made guinea pig macrophage membrane,a cytosolic fraction possible principally by the development of a cell-free activation enriched in p47pha"and p67ph0"(or total cytosol), highly system in which NADPH-dependent 0; production is elicited in purified Racl-GDP dissociationinhibitor for Rho (Rho mixtures of membranesand cytosol, derivedfrom resting GDI) complex, and the activating amphiphile, lithium phagocytes, by anionic amphiphiles suchas arachidonic acid(4) dodecyl sulfate. Peptides Racl-(178-188)and Racl-(178or SDS (5). 191), but not Rac2-(17&188),inhibited NADPH oxidase Failure t o fully restore NADPH oxidase activity in cell-free activity in a Racl-dependent system when addedprior to or simultaneously with the initiation of activation. assays consisting of membranes andcytosolic fractions containHowever, undecapeptides corresponding to the near ing p47ph" and p67ph" pointed toward the existence of an adcarboxyl-terminal domains of RhoA and RhoC and, most ditional component. This waspurified from cytosol to near honotably, a peptide containing the same amino acids as mogeneity (6) and shown to represent a complex of the small proRacl-(17&188), but in reversed orientation, were also GTP-binding protein (G protein) Racl with the modulator inhibitory. Surprisingly, 0; production in a Rac2-de- tein GDP dissociation inhibitor for Rho (Rho GDI) (7, 8). Racl pendent cell-free system was inhibited by Racl-(17& was found to be the active component in the Racl-Rho GDI 188)but not by Rac2-(17&188). Finally, basic polyaminocomplex, as demonstratedby the abilityof recombinant (7)or acids containing lysine, histidine, or arginine, also in- purified (8) Racl to support0, production in the cell-free syshibited NADPH oxidase activation. We conclude that in- tem. Several investigators reported that in human neutrophils hibition of NADPH oxidase activation by synthetic pep- the additionalNADPH oxidase componentwas Rac2, a small G tides mapping within the carboxyl-terminal domain of protein 92% homologous to Racl (9-11). Human neutrophil certain small G proteins is not amino acid sequencecytosol was, indeed, found to contain only Rac2 (12, 13).What specific but related to the presence of a polybasic motif. appears tobe clearlyestablished is that none of the Ras-related It has been proposed that such a motif serves as a small G proteins other than Racl and Rac2 are capable of plasma membranetargeting signal for a number of small NADPH oxidaseactivation. Thus, H-Ras, Ki-Ras, RaplA, G proteins (Hancock, J.F., Paterson, H., and Marshall, RaplB, RhoA, and CDC42Hs were all found to be inactive C.J. (1990)Cell 63, 133-139). (9-11, 14).' Dissociation of Rac from Rho GDI appears tobe an essential step in NADPH oxidase activation, by making monomeric Rac Oxygen-derived radicals are importanteffector agents in the free for interaction with another component of the NADPH microbicidal activity of phagocytic cells. The primordialoxygen oxidase complex. Recent evidence suggests that this could be radical, superoxide(O,)', is generated by the NADPH-depend- p67ph" (15). Several authors have reported thatRae2 transloent one-electron reduction of molecular oxygen by a specialized cates from the cytosol to the plasma membrane in course the of cell-free activation by anionic amphiphiles and in intact cells was supported by Grant 202/91 from the Israel Science * This work Foundation; a donation from Mr.and Mrs. J. Saltzman, Mr. and Mrs. L. stimulated by phorbol esters or chemotactic peptides (12, 13, 16-18).The carboxyl-terminal domain of Rac, corresponding t o Randall, and Mr. and Mrs. J. Garbell; the Basic Research Fund of Tel Aviv University; andthe David and Natalie Roberts Chair in Immuno- the hypervariable region of small G proteins, is of special inpharmacology (Tel Aviv University). The costs of publication of this terest because of its potential importance in determining the article were defrayed in part by the payment of page charges. This specificity of interaction with target molecules. In parallel situarticle must therefore be hereby marked "aduertisernent" in accordance ations, it was found that the carboxyl-terminal domain actedas with 18 U.S.C. Section 1734 solely to indicate this fact. f Incumbent of the David and Natalie Roberts Chair in Immuno- a targeting signalfor the association of Rab proteins withspepharmacology (Tel Aviv University). Tu whom correspondence shouldbe cific membranes in the endocytic pathway (19) and for the addressed. Tel: 972-3-6409078; Fax: 972-3-6429119. localization of the Ras-related G protein Yptl to theGolgi apThe abbreviations used are: O,, superoxide; G protein, GTP-binding protein; Rho GDI, GDP dissociation inhibitor for Rho; LiDS, lithium E. Pick, P. Kieselstein, and Y. Gorzalczany, manuscript in dodecyl sulfate; lp47+p67)Ph",cytosolic fraction enriched in p4TPh" and preparation. p67Ph".
29024
Inhibition of NADPH Oxidase by Synthetic Peptides paratus in yeast (20). Of relevance is also the finding that an antibody raised against a peptide corresponding to this region of Rac2 inhibited NADPH oxidase activation in the cell-free system (9). In the present study we investigated the effect of synthetic peptides correspondingto the near carboxyl-terminal domainof Racl and of related small G proteins of the Rho subfamily on NADPH oxidase activation in macrophage-derived a 0;generating cell-free system dependent on Racl. We found that a number of peptides werepotent inhibitorsof NADPH oxidase activation. However, this effect was not related t o amino acid sequence identity with the carboxyl-terminal domain of Racl but to the presence of a polybasic motif, shared by all inhibitory peptides. This conclusion is in disagreement with a recent report by Kreck et al. (21), which suggested that inhibition of NADPH oxidase activation by a carboxyl-terminal Racl peptide was sequence-specific. EXPERIMENTAL PROCEDURES Synthetic Peptides and Polyamino Acids-The peptides used in this of study are listed Fig. in 1.The locationof the peptides in the sequence the respective proteins is indicated by numbers in parentheses, representing thepositions of the amino- and carboxyl-terminal amino acids, and by the framed domains in the carboxyl-terminal segments of the sequences illustrated in the bottom half of the figure. Peptides were obtained from three commercial sources. On some occasions, in order to ascertain results, the same peptide was purchased from two or three manufacturers. The sourcesof the peptides and their degree of purity are described in thelegend of Fig. 1.All peptides were prepared witha free amine NH, terminus and a free acid COOH terminus, with the exception of Racl "antisense" (188478), which was synthesized as a COOH-terminal cysteine amide. Peptides purchased from Santa Cruz Biotechnology were delivered as a 200 pg/ml solution in 25mM sodium phosphate buffer, pH 7.4, 150 mM NaCl, 1.5 mM NaN,, and 200 pg/ml bovine serum albumin and were stored at"C. 4 All other peptides were obtained in powder form and first dissolved at a concentration of 1mM in 17.5mM acetic acid and stored in small aliquots a t -75 "C in polypropylene tubes. The following polyamino acids (homopolymers) were obtained from Sigma: poly-L-proline ( M , = 5850); poly-L-lysine hydrobromide ( M , = 3085); poly-L-histidine ( M , = 17,4501, and poly-L-arginine hydrochloride ( M , = 7450). All polymers were dissolved in distilled water to a concentration of 1 mM and stored frozen a t -75 "C in polypropylene tubes. Before use, both synthetic peptides and homopolymers were diluted to a concentration of 100400 p~ in 20 mM Tris-HC1, pH 7.5, 5 mM MgCl,, and 1 mM dithioerythritol. Preparation of Subcellular Fractionsfrom Macrophages-Guinea pig macrophages were obtained from the peritoneal cavity of animals injected with mineraloil 5-6 days before cell harvest. Cytosol and membranes were obtainedas described earlier(8), with twomodifications in the composition of the homogenization buffer; 5 mM MgCl, was added and 1 mM phenylmethyl-sulfonylfluoridewasreplaced by 20p~ 4-amidinophenylmethanesulfonylfluoride (pAPMSF). Human neutrophil cytosol, prepared as described inRef. 22, was a generous giftfrom Dr. I. Aviram (Tel Aviv University). Solubilization of Membranes and Purification of Cytochrome b559Membranes were washed in 1 M NaCl in solubilization buffer (6) and solubilized in 40 mM n-octyl-P-D-glucopyranoside (octyl glucoside) as described in the past(23). Before use in the cell-free assay, the solubilized membrane was diluted in detergent-free solubilization buffer in order t o reduce the concentration of octyl glucoside to 4-5mM. Cytochrome b,,, was purifiedfrom solubilized membranes and relipidated by a modification (24) of our original procedure (25). Preparation of Cytosolic Fraction Enriched in p47Ph" and p67PhoXThis was obtained by precipitation of cytosol by ammonium sulfate at 37% saturation, as described before (26). It is designated, throughout this paper, as (p47+p67Yh". Purification ofRacl-Rho GDZ Dimer-This was basedon the method described by us recently(8), subject toa number of modifications. Thus, on the occasions that cytosol was designated to serve aassource for the purification of Racl-Rho GDI dimer, the homogenization buffer usedfor cell disruption was supplemented with 10 p~ GDP. The ammonium sulfate precipitationof cytosol and hydrophobic interaction chromatography on phenyl-Sepharosestepswereperformed as originallydescribed.TheRacl-RhoGDI-containingfractions from the phenylSepharose column were pooled, supplemented with 10 p~ GDP, and
29025 CPPPVKKRKRK
Racl(178-188)
V,
W
Racl(178-191)
CPPPVKKRKRKCLL
Rac2 (178-188)
CPQPTRQQKRA Y L E C S A LT
Racl/Rac2(154-166)
0 RhoA (179-189) H
a w a
Q RG L K
LQARRGKKKSG LQVRKNKRRRG
RhoC (179-189) CDC42Hs (177-187)L
E P
P E PK
K S RR
N V F D E A I L A A L E PP
COC42Hs (167-183) RaplA (170-180) P
V E K K K P KK
(188-178) Racl
E PK
K S
KRKRKKVPPPC
( 'antisense' peptide )
Racl
Rac2
z
RhoA
155 G Y H E C S A K T K D G V R E V F E H A T R A 177
=)
RhoC
155 G Y L E C S A K T K E G V R E V F E H A T R A 177
cDC4as
153 K Y V E C S A L T Q K G L K I N V F D E A I L A 175
RaplA
1%
Racl
176 V LllC P P P V K K R K R K]C L L I L -COOH
W
(A
F Y D L V R Q I N R K T ~ P V E K K K P K K K S ~ I ~
z H
z
0 Rac2
176 V
LLC P Q P T R Q Q K R A1C S L L "N)H
H
+
RhoA
H
(A
-
192
Q A R R G K K K S GlC L V L -CDDH
193
178 G I L - Q V R K N K R R R GIC P I L -COCUi
193
178 A I L
RhoC COC42Hs 176 A ~ EL P P E P K]K
R
RaplA
-
Y
192
s 181 - - - - - - - - - -
RI-c v L L -cm --c
L LL - C m
191 184
FIG.1. Listof synthetic peptides used in the inhibition of NADPH oxidase experiments and the position of the corresponding amino acid sequences in the carboxyl-terminal domains of small G proteins. Gaps introduced to optimize the alignments are denoted by dashes. The commercial suppliers of peptides were Santa Cruz Biotechnology (A); Chiron Mimotopes, Australia (B); and Miniti Research Products, U. K. (C). The sources and degrees of purity of the peptides were as follows: Racl-(178-188) (A, B, C), >95%; Racl-(178-191) (A), >95%; Rac2-(178-188) (A, B), >95 to >98%; Racl/ Rac2-(154-166) (A), >95%; RhoA-(179-189) (B, C), >98 to >99%;RhoC(179-189) (C), >96%; CDC42Hs-(177-187) ( C ) , >95%; CDC42Hs-(167183) (A), >95%; RaplA-(170-180) (C), >99%; and Racl-(188+178) (C), >82%. dialyzed against 100 volumes of 20 mM Bis Tris-HC1, pH 6.0, 5 mM MgCI,, 1mM EGTA, 1mM dithioerythritol, 20 PM4-amidinophenylmethanesulfonyl fluoride, 2 PM leupeptin, and 10 PM GDP. The pool was applied to a 5-ml Econo-Pac CM cation exchange cartridge (Bio-Rad) linked in tandem with a 20-ml Hiload Q Sepharose high performance anion exchange column (Pharmacia Biotech Inc.) equilibrated with the type of buffer used for dialysis but lacking GDP. The column pair was washedwithequilibrationbufferuntilbase-lineabsorbancewas reached, the Econo-Pac CM column was dismantled, and a linear gradient of NaCl(0-0.5 M) in the equilibration buffer was applied toQthe Sepharosecolumn.EluatefractionscontainingRacl-Rho GDI were pooled, supplementedwith 10 GDPanddialyzedagainst 100 volumes of 20 mM Tris-HC1,pH 7.5,5mM MgCl,, 1mM dithioerythritol, and 10 p~ GDP. The pool was applied to a 20-ml Hiload Q Sepharose high performance column equilibrated with the type of buffer used for dialysis but lacking GDP. After washing the column with equilibration buffer until base-line absorbance was reached, a linear gradient of NaCl (0-0.5 M) was applied. Racl-Rho GDI eluted as a symmetrical peak of absorbance at 280 nm and was found tobe essentially free of contaminating proteinsby SDS-polacrylamide gel electrophoresis analysis with silver staining. The identities of Racl and Rho GDI were confirmed by immunoblotting with specific antibodies, as described (8). Highly purified preparations of the dimer were supplemented with PM 10 GDP and 100 pg/ml bovine serum albumin and stored a t -75 "C in polypropylene tubes. Cell-free NADPH Oxidase Assay-NADPH oxidase activity was assayed in a cell-free system, as described in Ref. 8. The assay mixture contained40pl of solubilized anddilutedmacrophagemembrane (10-12 pg of protein, corresponding to 2.5-3.0 pmol of cytochrome b,,,) and either 40 plof (p47+p67YhoX (50-100 pg of protein) combined with
29026
Inhibition of NADPH Oxidase
by Synthetic Peptides
10 pl of purified Racl-Rho GDI dimer (adjusted to contain 10 pmol 120Racl) or 30 pl of total cytosol in a total volume of 1 ml. 0, production was elicitedby lithium dodecyl sulfate (LiDS),followed after 90 s by the addition of 200 PM NADPH. In each experiment, the concentration of 100LiDS leading to maximal activation was determined and was, usually, b .in the 110-130 p~ range. Assays were r u n a t25 "C. In the majority of 42 experiments, 0, production was quantifiedby the initial rateof super2 80a oxide dismutase inhibitable ferricytochrome c reduction, as described in * '0 cytochrome b,,, turnRef. 5. NADPH oxidase activity was expressed as over rate (mol of Oz/s/mol of cytochrome b,,,) (25). In some experiments, I 60B NADPH oxidase activity was measured as LiDS-dependent NADPH U z oxidation, as described in Ref. 27. Finally, in other experiments, 0, 40production by purified relipidated cytochrome b,,, in the absence of x 0 Racl(178-188) cytosolic NADPH oxidase components was assayed as recently reported (24). Kinetic parameters (Lineweaver-Burk plots) were calculated using 2o 0 Racl(178-191) \ m\Rac2 Racl/Rac2 (178-188) O (154-166) " a n InPlot version 4.04 program (Graphpad Software). Experimental Design-The effect of Racl peptideanalogues on 0' ' NADPH oxidase activation was studied two in types of cell-free assays. 0 1 2 3 4 5 6 0, production was elicited in a mixture of solubilized memIn the first, Concentration of Peptide (UM) brane, (p47+p67Yh", and highly purified Racl-Rho GDI complex. The FIG.2. Inhibition of NADPH oxidase activation in a Racl-depeptide was added in two ways, preincubation and coincubation. For pendent cell-free system by Racl- but not by Rac2-relatedsynto a mixture of membrane, preincubation, peptide was added (p47+p67YhoX, and LiDS, followed, after 60s, by the addition of purified thetic peptides. Peptides were preincubated with a mixture of soluRacl-Rho GDI dimer and, after a further90 s, by the initiation of the bilized membrane, (p47+p67Yh", and LiDS, followed, after 60 s, by the addition of purified Racl-Rho GDI; after a further 90 s, 0,production reaction by NADPH. For coincubation, peptide was added toa mixture of membrane, (p47+p67Yh", purified Racl-Rho GDI dimer, and LiDS, was initiated by the addition of NADPH. Quantities for all oxidase followed, after 90 s, by the addition of NADPH. In the second type of components are detailed under "Experimental Procedures." NADPH oxidase activity was monitored by cytochrome c reduction. The peptides assay, 0, production was elicitedin a mixture of solubilized membrane used in these experiments were from Santa Cruz Biotechnology. For and total cytosol. Again, the peptide was added in two ways, preincueach peptide, the percentageof NADPH oxidase activities in the presbation and coincubation. For preincubation, peptide was added to a ence of various concentrationsof peptide was related to the activity in mixture of membrane and LiDS, followed after 60 s by the addition of the absence of peptide (72.7 2 5.2 mol of O;/s/mol of cytochrome b,,,; cytosol and, after a further90 s, by NADPH. For coincubation, peptide mean S.E.), which was considered to be 100%. Data points represent was added to a mixture of membrane, cytosol, and LiDS, followed after means of three t o seven experiments for eachpeptide; S.E.values 90 s by NADPH. Incubation with peptides was performed a t 25 "C and, varied from 4.5 to 11.5% of their respective means and are not shown in each experiment, individual measurements were done in duplicate. graphically. Addition of peptides, a t various concentrations, tothe assay mixture did notcause a change in pH,whichremainedstable at 7.0. Control amino acids upstream and partially overlapping with samples were supplemented with an equal volume of the buffer in which CDC42Hs-(177-187). These small G proteins are members of the peptides weredissolved. Y
"
the Rho subfamily, to which both Racl and Rac2 belong, and they arenot capable of activating NADPH oxidase (10,11, 14h2 RESULTS CDC42Hs is most closely related to Rac proteins, exhibiting Znhibition of NADPH Oxidase Activationby Racl approximately 70% overallidentity. We also tested RaplACarboxyl-terminal Peptides (170-180), corresponding to the near-carboxyl-terminal region Addition of peptides Racl-(178-188) or Racl-(178-191) to a of the Ras-related small G protein RaplA,which is not capable macrophage-derived, LiDS-activated, cell-free system resulted of activating NADPH oxidase (14), and Racl-(188-.178), conin a concentration-dependent inhibitionof 0; production. Pep- taining the same amino acids as Racl4177-178) but in the tides Rac2-(178-188), corresponding to the near carboxyl-ter- reverse order ("antisense" peptide). The initial experiments, minal domainof Rac2, and Racl/Rac2-(154-166) had no inhib- summarized in Fig. 2, were performed with synthetic peptides itory effect. Inhibition by Racl-(178-188) and Racl4178-191) obtained from one supplier (SantaCruz Biotechnology). Therewas evident when peptides were preincubated with membranefore, as an additional control, some of the experiments summaand (p47+p67)phoX and NADPH oxidase activation was initiated rized in Fig. 2 (those utilizing Racl-(178-188) and Rac24178188))were repeated with peptides obtained from two additional by purified Racl-Rho GDI (Fig. 2) and when peptides were preincubated with membrane followed by initiation of activa- suppliers (Chiron Mimotopes and Affhiti Research Products). tion upon addition of cytosol (results not shown graphically). Fig. 3 illustrates resultsof experiments inwhich peptides were The degree of inhibition was somewhat more marked in the preincubated with membrane and (p47+p6?Y"hox,followed by a separate seriesof experiments, the peptides the addition of Rad-Rho GDI. It is apparentthat Racl4178first situation. In were tested by using the coincubation method. We found that 188), obtained from two different suppliers, inhibits NADPH peptides that were inhibitory when using the preincubation oxidase activation ina dose-dependent manner, confirming the method wereequally effectivewhen added simultaneously with initial find. The lack of inhibitory activity of Rac2-(178-188) the othercomponents of the cell-free assay (results not shown). was alsoconfirmed. However, RhoA4179-189) and RhoC4179This was true whether membrane was combined with a mix- 189),two peptides possessinglittle sequence homology with the carboxyl-terminal region of Racl, both caused concentrationture of (p47+p67Yh" and Racl-Rho GDI or with total cytosol. dependent inhibitionof 0, production. The "antisense" peptide Lack of Sequence Specificity of Peptide-mediated Racl-(188+ 178) also caused a marked, concentration dependInhibition ent inhibitionof 0; production. Identical results were obtained when the peptides were preincubated with membrane followed The purpose of the next experiments was to find out whether inhibition of NADPH oxidase activation by Racl carboxyl-ter- by the addition of cytosol, but the degree of inhibition was minal peptides was aminoacid sequence-specific. For this pur- somewhat less pronounced (results not shown graphically). ICs, values for the inhibitory peptides are listed inTable I. pose, six additional peptides were tested. These included RhoA(179-189), RhoC-(179-189), and CDC42Hs-(177-187), corre- We have no ready explanationfor the difference in IC,, between sponding tothe near-carboxyl-terminal domains of RhoA, Racl-(178-188)produced by Santa Cruz Biotechnology and RhoC, and CDC42Hs, and CDC42Hs-(167-183), mapping 10 that synthesized by Chiron Mimotopes and Affhiti Research
29027
Inhibition of NADPH Oxidase by Synthetic Peptides
Rad (l78-lsS) R d (178-188) Rad (188-178) CDC42Hs (177-187) (167-183) 0 RhoA (179-189) * CDC42k RaplA (170-180) 0 RhoC (179-189) 0
A
A-A”824-6-
2
0
4
6
8
10
12
14
16
18
20
22
24
Concentration of Peptide (uH)
oL
0 Racl(178-188) RacP (178-188)
~~~~
0
1
2
3
4
5
6
7
8
Concentration o f Peptide (DM)
FIG.3. Inhibition of NADPH oxidase activation in a Racl-dependent cell-free system by synthetic peptides with minimal or no amino acid sequence identity with Racl. The experimental design was identical to that detailed in the legend Fig. to 2 and under “Experimental Procedures.” The peptides used in these experiments were from Chiron Mimotopes and AfTiniti Research Products, and results of experiments performed with peptides from these two sources were amalgamated. Data points represent means of three to four experiments for each peptide;S.E. values varied from 0.9 11.8% to of their respective means and are not shown graphically. NADPH oxidase activity in the absence of peptides was 85.6 * 6.0 mol ofOz/s/molof cytochrome (mean * S.E.).
FIG.4 . 0 ; production in a cell-free system composed of guinea pig macrophage membranes and human neutrophil cytosol is inhibited by Racl-(178-188)but not by Rac2-(178-188). Peptides (Santa Cruz Biotechnology) were preincubated with solubilized guinea pig macrophage membranes and LiDS for 60 s before the addition of human cytosol, whichwas followed, after90 s, by the addition of NADPH. 0, production was monitored by cytochrome c reduction. Oxidase components were present in amounts detailed under “Experimental Procedures.” Data points represent means of three experiments.S.E. values did notexceed 6.1% of their respective means and are not shown graphically. NADPH oxidase activity of guinea pig membranes combined withhuman cytosol, in theabsence of peptides, was 42.3* 4.7 mol of O@/mol of cytochrome b,,, (mean * S.E.).
TABLEI IC,, ualues for synthetic peptides capable of inhibiting cell-free activation of NADPH oxidase (p47+p67Yh” and Racl-Rho GDI had no effect on NADPH oxThe peptides were preincubated with either membrane, idase activity. (p47+p67Yh”, and LiDS for60 s before the additionof Racl-Rho GDI or with membrane and LiDS 60 fors before the additionof cytosol. I n both Peptide-mediated InhibitionIs Unrelated to the Nature of cases, NADPH was added 90 s after the addition of either Racl-Rho the G Protein Involved in NADPH Oxidase Activation GDI or cytosol. IC, values were calculated from mean percentage of be well established that, whereas both Racl and NADPH oxidase activitydata, derived fromthree to seven experiments, It appears to for each peptide. S.E. values did not exceed 10%of the mean percentage Rac2 can sustain 0, production in cell-free systems, human of NADPH oxidase activity data. The commercial suppliers of peptides neutrophil cytosol contains only Rac2 (12, 13) and guinea pig were Santa CruzBiotechnology (A), Chiron Mimotopes (B), a n d m i n i t i macrophage cytosol contains only Racl(8). We made useof this Research Products (C). IC.” Peptide
Supplier
Racl-(178-188) Racl-(178-188) Racl-(178-191) Racl-(188 C 178) RhoA4179-189) RhoC-(179-189) 2.17
A B,C A
Preincubation with Preincubation with membrane, (p47+67Yh”, membrane and and LiDS followed by LiDS followed Rad-Rho GDI by cytosol
Wf
-
B,C C
2.12 4.90 1.71 1.18 8.41
3.06 10.58 2.59 1.25 16.32
fact andof the findingthat a fully activecell-free system canbe assembled from guinea pig macrophage membranes and human neutrophil c y t 0 ~ 0 1 ~ order i n to examine the specificity of peptide-mediated inhibition. Using the preincubationprotocol, we investigated the effect of Racl-(178-188) and Rac2-(178188)on 0, production by a cell-free system consisting of solubilized guinea pig macrophage membrane and human neutrophil cytosol. As apparentin Fig. 4, Racl-(178-188) caused concentration-dependent inhibition of 0, production, with an IC,, of close t o 2.5 p,whereas Rac2-(178-188) was inactive. Should Rac peptides act by competing with native Rac, one would have expected Rac2-(178-188) to be the only one exerting an inhibitory effect or, at least, to be more active than Racl-(178-188).
Products (these two latter products exhibited equal activities). One possibility is that the actual concentration of the Santa Mechanism of Peptide-mediated Inhibitionof Cruz Biotechnology peptide, which was provided as a readyNADPH Oxidase made solution inbuffer also containingbovine serum albumin, The purpose of this seriesof experiments wasto elucidate the was in fact higher than that indicated by the manufacturer (the mechanism by which Racl-(178-188) and two undecapeptides, presence of albumin made checking the peptide concentration having little sequence homology with Racl (RhoA-(179-189) impossible). The IC,, values of Racl-(178-188), Racland RhoC-(179-189)) inhibit 0; production in a Racl-depend(1884 178), RhoA-(179-189) and RhoC-(179-189), produced by ent cell-free system. Chiron and Miniti, all fell in the
