34, Issue of December 5, pp. 2068&20696,1989. Printed in U. S.A.. G Protein Subunit Interactions. STUDIES WITH BIOTINYLATED G PROTEIN SUBUNITS*.
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1989 hy The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 264, No. 34, Issue of December 5,pp. 2068&20696,1989 Printed in U.S.A.
G Protein Subunit Interactions STUDIESWITH
BIOTINYLATEDG
PROTEIN SUBUNITS* (Received for publication, May 8,
1989)
Russell E. KohnkenS and JohnD. Hildebrandtt From the Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545
Modification of bovine brain G proteins by an N - binding proteins, or G proteins’ (Birnbaumer, 1987;Casey hydroxysuccinimide ester of biotin has been studied. and Gilman, 1988; Gilman, 1987; Hildebrandt et al., 1985; In the presence of GDP, but in the absence of Mg2+, Neer and Clapham, 1988; Rosenthal and Schultz, 1987; Stryer neither guanine nucleotide binding nor GTPase activ- and Bourne, 1986; Ui, 1984; Wessling-Resnick et al., 1987). ity of the protein was altered by modification using Among these signals are light and numerous hormones, neuless than 1.25 mM biotin derivative with 1 mg/ml G rotransmitters, andchemotactic agents. The G proteins are a protein. Under these conditions the a subunit wasmod- diverse family which includes Gi and G., named for their ified more extensivelythan the@ and y subunits. How- abilities to inhibit and stimulate adenylyl cyclase, respecever, biotinyl-a was less readily bound to streptavidin- tively, Gt, which activates retinal cGMP phosphodiesterase, agarose than was the less modified @ subunit. Biotinyl- and Go, an abundant component of brain membranes whose @rwas isolated fromthe modified, intact G protein and definitive function is yet to be identified. They consist of further characterized to determineif biotinylation al- three nonidentical subunits. The a subunits, which contain ters its functional properties. Isolated biotinyl-& and the GTP binding site, are unique to each protein and are unmodified @y were equivalent based upon: 1) inhibi- believed to be responsible for their functional specificity. The tion of the 549 cell membrane adenylyl cyclase, 2) P and y subunits arealso heterogeneous, but the significance changes in hydrodynamic parameters after being re- of this is not clear. combined with isolated a and treated with guanine G protein subunit dissociation is believed to be an impornucleotides or complexes of fluoride and aluminum, tant step in theiractivation and mechanism of action. With and 3)competition for isolated a binding to biotinyl-By persistent activators, such as GTPyS and AMF, the hydroimmobilized previously on streptavidin-agarose. Bio- dynamic properties of G proteins canbe altered (Sternweiset tinyl-@yprebound tostreptavidin-agarosewas 70al., 1981; Codina et al., 1984a; Huff and Neer, 1986; Wessling100% functional, basedupon binding of isolated a sub- Resnick and Johnson, 1987), and a can be resolved from a units. Estimatesof the affinityof a binding to biotinylP-y complex in vitro (Northup et al., 1983; Fung et al., @rindicate that bovine brain adl has a 10-16-fold stable 1981). Isolated G protein subunits, both a and P-y, have been higher affinity for by than does ass. Nonhydrolyzable implicated in the regulation of biological functions. Thus, guanine nucleotidesand complexes of fluoride and aluisolated a, stimulates adenylyl cyclase (Northup et al., 1983), minum decreased binding of either a39 or 1x41 to biotiai,also referred to as ak,opens K’ channels (Codina et al., nyl-@r, and these effects were dependent upon the 1987), and at stimulatesretinal cGMP phosphodiesterase amount of Mg2+present. GTP decreased binding of a39, (Fung et al., 1981). On the other hand, isolated has been but not all, to biotinyl-@y. Theseresults indicate that proposed to act directly on phospholipase A2 (Jelsema and GTP can affect G protein subunit interactions and that its effects do not necessarily require an intact mem- Axelrod, 1987; Kim et al., 1989) andto directly mediate brane environment or the participation of activating inhibition of adenylyl cyclase (Katada et aE., 1986a). Substoichiometric Py stimulates stoichiometric GTP binding to at in receptors orother membrane-associatedproteins. the presence of photolyzed rhodopsin, an activity thought to They further indicate that biotinylation of By does not alter its functional properties and that it can beused require the intact trimer, suggesting that a single Py can interact with more than one at (Fung, 1983). Reconstitution for studying G protein subunit interactions. studies suggest that Gi inhibits adenylyl cyclase, at least in part, through the liberation of a free Py complex which suppresses the activation of free a. in intactmembrane prepMany extracellular signals exert their intracellular effects aration (Katadaet al., 1984a, 1984b). Most recently, evidence through the activation of one or more guanine nucleotide has been presented which suggests that bothnonhydrolyzable GTP analogs (Iyengar et al., 1988) and GTP itself (Ransnas * This work was supported by United States Public Health Service Grant DK37219.A portion of the datapresented here were presented in abstract form to thecombined 1989 The American Society for Cell Biology and American Society for Biochemistry and Molecular Biology meetings. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked“aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Present address: Dept. of CellBiology, Northwestern Medical School, Chicago, IL 60611. J To whom correspondenceshould be addressed. Tel. 508-8428921.
The abbreviations used are: G proteins, signal-transducing guanine nucleotide-binding regulatory proteins with aP-y subunit composition; G8 and G,, the G proteins that mediate stimulation and inhibition of adenylyl cyclase; G,, the predominantGprotein in retinal rod outer segments; Go, the predominant G protein of bovine brain; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfoni~ acid bis-Tris, bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane; BSA, bovine serumalbumin; GTP-yS, guanosine-5’-(3-0-thio)triphosphate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; NHS-LC-biotin, sulfosuccinimidyl 64biotinamido)hexanoate.
20688
G Protein Biotinyl-By
20689
and Insel, 1988)cause dissociation of G, in intact membranes. In spite of the important postulatedrole for subunit dissoI ciation, G protein subunit interactions have been difficult to I 94.-. investigate directly. They havebeen studiedprimarily by changes in hydrodynamic parameters, determined by sucrose 67- 0 gradient centrifugation and gel filtration (Sternweis et al., 1981; Codina et al., 1984a, Huff and Neer, 1986; WesslingResnick and Johnson, 1987), or inferred from studies of G protein properties thought require to subunit association, such as ADP-ribosylationby pertussis toxin (Huff and Neer, 1986) or stimulation of G T P binding and hydrolysis (Fung, 1983). Such approaches have not allowed a detailed analysis of the 30regulation of subunit dissociation and have left unresolved a number of mechanistic issues. For example, they have failed to demonstrate formost of the G proteins that GTP, the physiological regulator, promotes subunit dissociation (Huff 1 2 3 4 and Neer, 1986; Kahn and Gilman, 1984). Described is the FIG. 1. Bovine brain G proteins. A, G protein subunits obtained preparation and characterization of a biotinylated P-y complex from the major fraction of the G protein recovered from chromatogwhich can be used to directly study G protein subunit inter- raphy on Accell QMA. Lane I , molecular weight markers. Lane 2, 3 actions. Here it has been used to demonstrate that GTP can pg of the major G protein pool from Accell QMA. Lane 3, 1.5 pg of effect (Y subunit binding toP y . the mixed n subunit fraction obtained by treatment of the G protein
I 1
-
EXPERIMENTALPROCEDURES
Materials-[y-"P]GTP was obtained from Amersham Corp. ["SI GTPyS was obtained from Du Pont-New England Nuclear. NHSLC-biotin and bicinchoninic acid protein assay reagent were from Pierce Chemical Co. GTPyS was from Boehringer Mannheim. Silicotungstic acid was from J . T. Baker. All other reagents were from commercial suppliers andwere the highest purityavailable. Protein Purification-G proteins were isolated from bovine brain according to a published procedure (Sternweis and Robishaw, 1984) with the following modifications.Homogenized membranes were washed once with 10 mM Tris-CI, pH 7.8, 200 mM sucrose and then withthesame buffer plus 50 mM NaCI. This resulted inbetter recovery of GTPyS binding activity (data not shown). Extraction with cholate and chromatography on DEAE Sephacel (Pharmacia LKB Biotechnology Inc.) and Ultrogel AcA34 (LKB) were as described (Sternweis and Robishaw, 1984). Hydrophobic chromatography was as described (Sternweis and Robishaw, 1984), except that octyl-agarose (Pharmacia LKB Biotechnology Inc.) was substituted for heptylamine-Sepharose. A typical preparation from two brains (250 g of tissue) yielded 70-80 mg of G protein with a specific activity of GTPyS binding of 13.7 pmol/pg. These preparations were a mixture of G proteins of approximately 80% Go (ans)and 10% each of two G, proteins (nJaand nrl).The n:p stoichiometry on the basis of Coomassie Blue staining was approximately 1:l. An additional step, ultimately required for the separation of nR9and nJI,was chromatography on Accell QMA after 5-fold dilution of the octyl-agarose pool, which was in cholate-containing buffers, into 20 mM bis-Tris-HCI, pH 7.0, 1 mM EDTA, 1 mM dithiothreitol, and 0.04% Lubrol. Typically, a 0.9 X ll-cm column was used which was eluted with a 1-liter gradient of 75-200 mM NaCl in the same buffer as above. The bulk of the protein (Fig. 1, lane 2) was a mixture of G proteins as above, whereas the leading edge (Fig. 1, lane 6 ) , 10-205 of total protein applied, contained only 39- and 41-kDa n subunits. Apparently homogeneous 39- (Fig. 1, lane 7) and 41-kDa (Fig. 1, lane 8) n subunits were obtained by a second pass over the Accell QMA column after separation of n and By as described below. Chromatography on w-amino-octyl-a~arose(0.9 X 14-cm column) was used to separate n and p'y (Fig. 1, lanes 3 and 4, respectively). G protein was incubated at30 "C for 15 min in HEDLN (30mM sodium Hepes, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 0.05% Lubrol-PX, and 100 mM NaCI) and AMF (20 p M AICln, 10 mM MgCI?, 10 mM NaF, and 50p~ GDP). After cooling to 4 "C, the sample was diluted with 5 volumes of TEDC (20 mM Tris-HCI, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 0.25s cholate) containing100 mM NaCl and AMF and loaded on the column. The column waseluted with two sequential gradients. first with a 200-ml gradient of 100-300 mM NaCl in TEDC plus AMF and then with a 200-ml gradient of 200 mM NaCI, AMF, TEDC to 50 mhl NaCI, 1.35 cholate, AMF, TED (20 mM Tris-HCI, pH 8.0, 1 mM EDTA, and 1 mM dithiothreitol). The protein subunit peaks were pooled, Mg" chelated with EDTA, concentrated on an Amicon YM-10 or YM-30 membrane, and stored at -80 "C or used for separation of the 11 subunits as described above. Unless indicated
I
(lane 2) with AMF and chromatography on octyl-Sepharose. Lane 4, 1.S pg of the By fraction obtained as in lane 3. R, preparation of and c y I I from G protein recovered from the leading edge of chromatography of Accell QMA. Lane 5, molecular weight markers. Lane 6, 3 pg of the starting G protein pool. Lane 7, 1 pg of an*.IAne 8, 1 p g of mal. otherwise, isolated n subunit used in the experiments reportedrefers to the mixed CY preparation represented in Fig. 1, lane 3. Modification of G Protein-Unless indicated otherwise, G protein was modified at 1 mg/ml protein in 100 mM sodium Hepes, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 0.05% Lubrol, 50 pM GDP with 300 p~ NHS-LC-biotin (from a 20 mM stock in dimethyl sulfoxide) for 30 min a t 21 'C. The reaction was terminated with ethanolamine, pH 8.0, added to a final concentration of 10 mM. Modified protein was desalted on AcA 202 into 100 mM NaCl in TEDC plus AMF and then incubated in that buffer a t 30 "C for 15 min. After cooling to 4 "C, the sample was applied to a w-amino-octyl-agarose column (2 ml) equilibrated in 100 mM NaCl in TEDC plus AMF. The column was eluted sequentially with 5 ml of each of the following solutions: 1) 300 mM NaCI, AMF, TEDC; 2) 500 mM NaCI, AMF, TEDC; 3) 200 mM NaCI,0.4% cholate, AMF, TED;and4)50 mM NaCI, 1.3% cholate, AMF, TED. Subunit fractionswere identified by SDS-PAGE, pooled, andconcentratedonan Amicon YM-10membrane. The concentrated protein was diluted 10-fold into 20 mM sodium Hepes, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 0.01% Lubrol, and 100 mM NaCI, reconcentrated, and stored a t -80 "C. Sucrose Gradient Sedimentation-G protein (30-40 p g ) was mixed with 10 pg of biotinyl-By and 10 pg of unmodified no in a total volume of 140 pl with HEDLN plus MgC12 and nucleotides as indicated in the individual experiments. These samples were incubated a t 30 "C for 20 min, cooled to 20 "C, mixed with 10 pl of 2 mg/ml RSA, 2 mg/ ml c-ytochrome c in HEDLN, and loaded on 4.8-ml 5-20?; sucrose gradients in HEDLN with or without M f l and nucleotides. Centrifugation was performed at 20 "C in a Beckman SW 50.1 rotor a t 40,000 rpm for 13 h. Centrifugation a t 4 "C gave qualitatively similarresults. Gradients were fractionated and analyzed by SDS-PAGE. The Effects of By on S49 Cell Membrane Adenylyl Cyclase ActidvWild t.yp S49 cells were grown and membranes prepared from them as described previously (Codina et al., 1984b). Effects of isolated By on forskolin-stimulated adenylyl cyclase activity were determined as follows. S49 cell membranes were diluted on ice to 5 pg protein/30 pI with HER (10 mM sodium Hepes, pH 7.5, 1 mM EDTA, and 0.1 mg/ ml BSA) plus 0.167 mM ATP, 50 p~ GTP, 5 mM MgCL 33.4 mM creatine phosphate, 33.4 pg/ml creatine phosphokinase, and 3.34 pg/ ml myokinase. Diluted S49 cell membranes (30 pl) were mixed with 10 pl of varying [Py concentrations in HER containing0.02.55 Luhrol PX. Samples of By containing cholate as a detergent were diluted to less than 0.1% final cholate concentration. Samples were incubated at 4 "C for 30 min and then for 15 min a t 30 "C.Forskolin-stimulated adenylyl cyclase activity was assayed by adding 10 pl of HER containing 1 pCi of [n-:'2P]ATP, 10,000 cpm [.'HICAMP, 5 mM CAMP, and 100 p~ forskolin andincubating for 15 min at 30 "C. The
20690
G Protein Biotinyl-py
reactions were terminated and CAMP produced determined as described previously (Codina et al., 198413). SDS-PAGE-SDS-PAGE on 11 or 12% polyacrylamide gels was performed according to Laemmli (Laemmli, 1970). Protein was detected by Coomassie Blue staining. Biotin incorporationwas detected following transfer to nitrocellulose (Schleicher and Schuell BA 85) (Towhin et al., 1979) with 1:lOOO avidin-peroxidase (Hyclone) in 50 mM Tris-CI, pH 8.0, 150 mM NaCl, 0.05% Nonidet P-40, 2.5 mg/ml BSA. Color development was performed in 40 mM Tris-C1, pH 8.0, 120 mM NaC1, 20% methanol with 5 ~ L 4-chloronaphthol M and 5 ~ L M HzO,. Immobilization of Subunits on Streptauidin-Agarose-A 50% slurry of streptavidin-agarose (Sigma)was diluted 10-fold in 2.5 mg/ml BSA or casein in 50 mM Tris-C1, pH 8.0, 150 mM NaCl and incubated for 1 h at 4 "C. The agarose was collected by centrifugation and washed two times with HEDLN.When biotinyl-py was to be bound to streptavidin-agarose, it was added in HEDLN and incubated at 4 "C for 1 h. Unbound P-y was removed by centrifugation, and the agarose was then washed two times with HEDLN. The specific conditions for measuring 01 binding to biotinyl-(37 are indicated in the individual experiments. Following separation of bound and free subunits, agarose pellets were resuspended with 40 pl of Laemmli sample buffer (Laemmli, 1970), heatedfor 5 min at 90 "C, andthen centrifuged for 2 min at 10,000 rpm in an Eppendorf microcentrifuge. Supernates were applied to 11 or 12% polyacrylamide gels. Theamount of a presentinthe agarose pellet was quantitated by scanning densitometry of Coomassie Blue-stained gels using a Kontesfiber optic scanner with aHewlett-Packard integrator. Measurement of GTPase Activity-GTPase activity was measured in 1.5-ml microcentrifuge tubes containing HEDLN, 10 mM MgC12, 20 p~ [y-3ZP]GTP(100 cpm/pmol), and 3 pg of G protein in a total volume of 50 pl. Samples were incubated for 16 min a t 30 "C and terminated by adding 300 p1 of 20 mM silicotungstic acid in 10 mM sulfuric acid at 4 "C. All subsequent steps were carried out at 4 "C. In preliminary experiments, silicotungstic acid was found to give lower blanks than either trichloroacetic or perchloric acid. Reaction rates were linear for at least 30 min, and the turnover number of the G protein preparation used in these experiments was 0.15 rnin". Free nucleotide was precipitated by adding 300 p1 of a 25% (v/v) Norit-A suspension. Samples were allowed to stand for 10 min and then 100 pl of 1 mg/ml BSA was added and Norit-A and protein precipitated by centrifugation in an Eppendorf microcentrifuge for 2 min at 10,000 rpm. Aliquots of the supernatants (75 ~ 1 were ) counted for ["PI phosphate in 2.5 ml of Ecolume (ICN Radiochemicals) in a Packard scintillation counter. Blanks (no G protein) were from 1 to 2% of total added radioactivity. Miscellaneous Assays-GTPyS binding was performed according to Sternweis and Robishaw (Sternweis and Robishaw, 1984) using immobilization of protein-bound nucleotide on nitrocellulose paper. Standard assay conditions, in a total volume of 40 pl, included 1 p~ [35S]GTP-yS(5000 cpm/pmol), 20 mMMgC12, and 12.5 pmol of G protein (1 pg of heterotrimer). Samples were incubated for 40 min at 30 "C, which measured equilibrium binding. Equivalent blanks, approximately 1%of total protein-bound radioactivity, were obtained with excess GTPyS (0.1 mM), no added G protein or heat-inactivated G protein. Values reported are not corrected for the fraction of G protein immobilized (approximately 70%), so that these underestimate actualbinding. Protein was determined using bicinchoninic acid according to themanufacturers' (Pierce Chemical Co.) specifications. Values obtained in this assay routinely fell between those obtained using the Lowry method (Lowry et al., 1951) or the Bradford method (Bradford, 1976).
RESULTS AND DISCUSSION
Preparation of Modified Subunits-Initial experiments were designed to determine condkions for modifying G protein subunits withoutloss of function. Attempts to modify isolated subunits while preserving function were not successful (data not shown), andthereforethisstrategy was not pursued. Instead, conditions were sought for modifying the intact trimer that would allow subsequent isolation of modified, but otherwise functionalsubunits.Preliminarystudiesonthe effects of modificationwith NHS-LC-biotinon G protein GTPase and G T P r S binding are summarized in Table I. In the absence of Mg2+, 1 mM NHS-LC-biotin did not affect
eitheractivity, whereasin the presence of Mg2+, GTPase activity was slightly inhibited by 14%. Inclusion of GDP or G T P during modification reduced this effect. In addition, in studiesusing an alternativeamino-reactivereagent, 2,4,6trinitrobenzenesulfonic acid, or a different N-hydroxysuccinimide derivative, Bolton-Hunter Reagent, the adverse efeffects of guanine nucleotides fects of M$+ and the protecting were more pronounced than those seenwith NHS-LC-biotin (data not shown). Analogous results in the presence of magnesium have also been reported with another amino-modifying reagent,fluorescein 5"isothiocyanate (Hingorani and Ho, 1987).Consequently, subsequent modifications wereroutinely performed in the absence of M e and presence of GDP in order to minimize any possible functional impairment of the modified proteins. When G protein was modified withdifferentNHS-LCbiotin concentrations up to 10 mM, in the presence of GDP and in the absenceof M e , there was no effect on GTPase activity or GTPyS binding a t concentrations below 1.25 mM. Even at 10 mM there was only 15% inhibition of GTPase activityand no inhibition of GTPySbinding(datanot shown). By staining of electroblotted protein with avidinperoxidase, a modification was detectable a t 10 FM and increased with the amount of reagent added (Figs. 2 and 3A). In general, modification of either (3 or y required more NHSLC-biotin than didmodification of a. The fraction of (37 modified was estimated by measuring the amountof protein that could be immobilized on streptavidin-agarose (Fig. 3B). Binding of G protein subunits to streptavidin-agarose was completely dependent upon biotinylation, since unmodified protein was not retained on the matrix. In the presence of GTPyS andM$+ to dissociate the subunits, immobilized /3y reached a plateau at 70-75% of the total(3y present, based on comparison of densitometric scansof SDS-PAGE gels of total andprecipitated samples. Itisunclear why (3 bindingto streptavidin-agarose does not reach 100%. Half-maximal immobilizationoccurred a t approximately 300 p M NHS-LCbiotin. These results suggest that most By was modified a t least once at less than 1mM NHS-LC-biotin. Thisconclusion was supported in subsequent experiments in which the subunits were separated after modification but prior to immobilization. In six independent experiments with 300-400 FM NHS-LC-biotin, 30-93% of isolated (37 became immobilized. In comparison to Pr,the behavior of a after modification with NHS-LC-biotinwas more complex. Immobilization of a (Fig. 3B) increased up to 50% of total added protein in the presence of GTPyS, at 2.5 mM NHS-LC-biotin, butdecreased at higher levels of modification. Half-maximal immobilization required approximately 600 PM NHS-LC-biotin. In addition, a isolated after modification with 300 FM NHS-LC-biotin was only 35% immobilized, as a fraction of total recovered cy protein, on streptavidin-agarose (data not shown). Thus, even though it was more extensively modified than was @ (Figs. 2 and 3A), cy was less likely to become immobilized (Fig. 3B). The difference in the behavior of a and /3 after modification can be seen most clearly in Fig. 3C. Here, the fractionof each subunit which could be immobilized on streptavidin-agarose is plotted asa function of biotin staining intensityof electroblotted proteins. In thecase of 0,fraction bound is plotted as a function of the summed staining intensities of p and y, since these subunits do not separate under nondenaturing conditions. The fractionof (3 bound increased almost linearly with amount of biotin incorporated, at low levels of modification, and then more slowly at higher levels. On the other hand, cy could be substantially modified without it being able to bind streptavidin-agarose.Subsequentexperimentsconcentrated
Biotinyl-By
G Protein
20691
TABLE I GTPase and GTPyS binding activities of biotin-modified G proteins G protein was incubated in the presence (modified) or absence (control) of 1 mM NHS-LC-biotin as described under “Experimental Procedures.” Samples contained 20 mM MgCl2, 50 p~ GDP, and/or 50 p~ GTP asindicated in the table. After incubation for 30 min a t 21 “C, the reactionwas terminated with ethanolamine and diluted 1:s with HEDLN. G protein was assayed for GTPase activity (3 pg) and for GTPyS binding (1 pg), each in triplicate. Carryover of GDP or GTP into the assayswas 3 p~ for GTPase activity and 1 p~ for GTPyS binding. The G protein preparation used in this experiment, when assayed directly without any pretreatment, had a GTPase turnover number of 0.20 min”. Values are expressed mean f standard error. Inhibition refers to the percent change in themodified protein as comparedto its corresponding control. Control hydrolyzed/pmol pmol
None GDP GTP Magnesium Magnesium Magnesium
+ GDP
+ GTP
GTPyS binding
GTPase
Additions
0.158 f 0.007 0.157 f 0.002 0.148 f 0.003 0.150 f 0.005 0.152 f 0.002 0.153 f 0.002
Modified
Inhibition
G proteinlmin 0.153 f 0.007 0.150 f 0.005 0.155 f 0.010 0.130 f 0.002 0.142 f 0.002 0.136 f 0.002
%
2
4 -5 14 7 11
Control
Modified
pmol GTPyS bound
7.5 f 0.6 7.9 f 0.2 8.1 f 0.2 6.3 f 0.2 6.7 f 0.2 6.7 f 0.1
7.3 f 0.1 7.4 f 0.4 7.6 f 0.4 6.1 f 0.1 6.8 -3 f 0.3 6.3 f 0.1
Inhibition %
3 6 7 3 6
modification impair subunit dissociation.Because of these results, andbecause evenat high levels of biotin incorporation less than 100% of modified By bound streptavidin-agarose “Experimental (Fig. 3C), the protocol for biotinylation (see Procedures”) generally resulted in 50-70% of the isolated By pa being bindable by streptavidin-agarose. Under these condi”P tions only a very small fraction of the biotinylated (Y could not be resolved from By (Fig. 4). The isolated biotinyl-By was retained by streptavidin-agarose and was resistent to washing (Fig. 4, lane 91,whereas ” unmodified By was not retainedby the matrix(Fig. 4, lane 7). Y DF Pretreatment of the streptavidin-agarosewith 100 p~ biotin 0 .01 .02 .04 .08 .17 .33 .63 1.25 2.5 5.0 10.0 completely prevented biotinyl-By binding (Fig. 4, lane 8). On the other hand, once biotinyl-By was bound to streptavidin[NHS-LC-biotin],mM agarose it did not dissociate from it duringa subsequent 24-h FIG.2. Biotin incorporation into G protein as a function of incubation in the presence of 100 p~ biotin (Fig. 4, lane IO). NHS-LC-biotin concentration. G protein (50 pg) was modified as Unmodified a did not bind to streptavidin-agarose either (Fig. described under “Experimental Procedures” and desalted on AcA 202 (LKB) to remove unbound biotin. Approximately 3 pg of modified G 4, lane 12) unless biotinyl-By had been bound previously to protein from each sample were applied to duplicate SDS-polyacryl- the matrix (Fig. 4, lane 13). These experiments demonstrate amide gels. Following electrophoresis, one gel was analyzed for biotin that biotinyl-By bound to streptavidin-agarose has the approincorporation using avidin-peroxidase as described under “Experipriate properties to make it a potentially useful reagent for mental Procedures” and the other stained with Coomassie Blue. This studying G protein subunit interactions. figure shows the distribution of biotinylated protein on the electroSimilar Behavior of Modified and Unmodified Py Subblotted gel. The identity of the material labeled as the y subunit is intactheterotrimer GTP-yS bindingand based solely on its coincident migration with protein on analogous units-Although gels stained with Coomassie Blue, also assumed to be the y subunit. GTPase activitieswere unaffected by modification under the We have not attempted to verify the identity of this biotinylated conditions investigated, the contribution of B-y to these acmaterial by any other means.DF, dye front. tivities is complex. Consequently, the function of biotinyl-By after its separation from a was also investigated. The adenylyl on characterizing biotinyl-By since the behavior of a could cyclase activity of S49 mouse lymphoma cell membranes has not be fully explained and it is not clear that it would be of been reported to be inhibited by exogenously added By. This use in future studies on subunit interactions. has been attributedtoits blocking activation,or causing Separation of Modified Subunitsand Requirements for deactivation, ofG, (Katada et al., 1984a,1984b) and is a Binding to Streptauidin-Agarose-Following modification potential mechanism for hormone-mediated inhibition of adwith NHS-LC-biotin, a and By were separated on w-amino- enylyl cyclase. Biotinyl-By inhibited forskolin-stimulated adoctyl-agarose as described under “Experimental Procedures.” enylyl cyclase activity in S49 membranes to the same extent, The results of one such separation are shown inFig. 4; lanes and with the same concentration dependence, as unmodified 3 and 4, show the Coomassie Blue-stained gel for starting By (Fig. 5). These data suggest that biotinylation does not protein and resolved P-y, respectively, whereas lanes 1 and 2 alter the function of By and that could it be used in membrane show the corresponding blots for biotin incorporation using reconstitution assays forG protein function. avidin-peroxidase. As before, a was more extensivelymodified The dissociation of a from P-y in thepresence of GTPyS or than was p (Fig. 4, lane I ) . Although the Coomassie Blue- AMF results ina characteristic shiftin the apparentsedimenstained gelof resolved By had only slight a contamination tation coefficient of thesubunits(Sternweis et al., 1981; (Fig. 4, lane 4 ) , by staining for biotin there appeared to be Codina et al., 1984a; Huff and Neer, 1986; Wessling-Resnick proportionally more a present (Fig. 4, lane 2). It thus appeared and Johnson, 1987). Modified and unmodified subunits were that a copurifying with biotinyl-By was very heavily biotin- examined by sucrose density centrifugation in order todirectly ylated. It is possible, but unproven, that high levels of (Y verify that modification had noeffect on subunit interactions.
-
20692
G Protein Biotinyl-py
,001
.oi
!l+d!!d 0.0 0.2 .001
1 .1
10
[NHSLC-biotin],mM
10
. 0. 1
l’iic
0.4 0.2 1
10
INHSLC-biotin], mM
100
0.0 0
1
2
3
4
5
6
intensity staining biotin
FIG. 3. The relationship betweenNHS-LC-biotin concentration and G protein biotinylation.A, biotin staining intensity of a,6, and y as a function of NHS-LC-biotin concentration. Densitometry of biotin-containing bands on the Western blot shown in Fig. 2 was performed as described under “Experimental Procedures.” Control experimentsindicated that scandensity of these Western blots was linearly proportional to the amount of biotinylated protein present except at very low levels of modification. These values were normalized by the scan density of the Coomassie Blue-stained subunits on an analogous polyacrylamide gel and the ratio plotted as the biotin staining intensity. The biotin staining intensities for y were normalized using the p subunit staining intensitieson the Coomassie Blue-stained gel, since and y do not separateunder the conditions used for modification. Open circles, a modification; closed circles, p modification; opentriangles, y modification. B, immobilization of a and p on streptavidin-agarose after modification with NHS-LC-biotin. G protein (3 Kg), modified and desalted as in Fig. 2, was incubated in 100 p1 of HEDLN containing20 mM MgC12and 40 p M GTPyS for 20 min at 30 “C. Samples were then added to 10 p1 streptavidin-agarose in 1 ml of HEDLN containing 20 mM MgCl, and 40 p~ GTPyS at 2 1 “C for 60 min. Protein bound to the agarose was collected by centrifugation, washed once with HEDLN, and resuspended in Laemmli sample buffer (Laemmli, 1970) for SDS-PAGE. Bound a and p were quantitated by densitometry and these values divided by the scan density of total a and 0 from identical samples on another gel. The ratio of protein bound/total protein was plotted as fraction bound. Control experiments indicated that thescan densityof Coomassie Blue-stained gels was linearly proportional to the amount of protein throughout the ranges of densities observed. Open circles, a subunit; closed circles, p subunit. C, fraction bound as a function of biotin staining intensity. The fraction of a or p bound from B was plotted as a function of the biotin staining intensity in A . Since p and y do not separate under the conditions used for binding to streptavidin agarose, the fraction of p bound was plotted as a function of the sum of the /3 and y biotin staining intensities. Open circles, a subunit; closed circles, /3 subunit.
Fig. 6 shows the distribution of unmodified G protein and biotinyl-@y estimated to be bound to the agarose ineach biotinyl-py on sucrose gradients relative to BSA and cyto- sample. This further suggests that not only are modified and chrome c. It isapparentthatbothMgGTPySandAMF unmodified @y equivalent,butthatbiotinyl-pyboundto induced a change in the sedimentation rate such that the G streptavidin-agarose and @yin solution behave similarly. protein migrated closer to the cytochrome c peak. On the Binding of a to Biotinyl-By-Previous studies have sugother hand, MgGTP had effect no on sedimentationbehavior, gested that the bovine brain a 4 1 has a higher affinity for Py or M$+. compared withthe control sample with nucleotide no than does a39 based upon isolation of a39as free a subunit The effect of AMF was completely reversed by incubation and aqlas a trimer (Neer et al., 1984; Sternweis andRobishaw, with EDTA in excess over Mg2+ prior to sedimentation. These1984) and on the concentrationdependence for /3y to support results suggest that MgGTPyS and AMF can induce a/Py pertussis toxin ADP-ribosylationof each a subunit (Huff and dissociation in vitro, but that MgGTPdoes not. This may be Neer, 1986; Katada et al., 1986b). The affinity and capacity due to a genuine lack of dissociation or may merely reflect of By for binding a41and a39were estimated here by binding favoring the associated state, over of free a subunit to biotinyl-pyimmobilized on streptavidinhydrolysis of GTP to GDP, the lengthy time course of sedimentation. Similar resultshave agarose (Fig. 8). In the presence of 65 nM biotinyl-By, the been reported by others (Huff and Neer, 1986; Kahn and maximum specific binding of a41was 51 nM, whereas that of Gilman, 1984). aS9was 44 nM. These values are equivalent to 68-79% of the As shown in Fig. 6, under all of the conditions examined, biotinyl-py concentrationused in the experiment. The apparbiotinyl-@y comigrated withthe unmodified protein.This ent KO for a41bindingto biotinyl-By, assuming asingle provides a direct demonstration that the modified @y does binding site,was estimated at 24 nM. In a second experiment, interact with unmodified a , for if it did not, it would have a 4 1 had maximal binding of 89% of total biotinyl-py added traveled closer to the cytochrome c peak than did the native withanapparent KU of 19 nM. Inthe case of 10-fold protein. Since biotinyl-@yalways migrated with the unmodihigher concentrations of a had tobe used to obtain saturable fied protein, these data indicate that itundergoes changes in sedimentation rate thought to reflect subunit dissociation and binding. The higher concentration of total a resulted in a nonspecific binding,althoughno re-association in response to Mg2+and nucleotides in a man- proportionalincreasein more than would be expected from thefree a subunit trapped ner identical to thatof the unmodified subunit. Based upon a binding tobiotinyl-By immobilized on strep- within the streptavidin-agarose pellet. The apparent KU for binding to biotinyl-@y in this experiment was 390 nM. In tavidin-agarose, another test of the functional equivalence of a second experiment, bound 79% of total biotinyl-@y with modified and unmodified @ywas devised. In the experiment an apparentKU of 340 nM. shown in Fig. 7, biotinyl-@ywas prebound to streptavidinIn these experiments, and in several others with different agarose, and then streptavidin sites without protein bound a subunit preparations, (Y bindbiotinyl-@y preparations and were blocked by a subsequent incubation with biotin. The ability of modified and unmodified by in solution to block a ing to biotinyl-@y prebound to streptavidin-agarosewas satbinding to the matrix was then tested. As shown in Fig. 7, urable with a maximum stoichiometry of0.7-1.1:l.O. These biotinyl-@y and unmodified @yinhibited a binding similarly. results were independent of the type or purity of a subunit In addition, the half-maximal concentration of either required used and the fraction of the biotinylated preparation that suggest that biotinto block a binding was approximately equal to the 1.8 Kg of would bind to streptavidin-agarose. They
G Protein Biotinyl-By i
ii I
I
iji
-
-
-
94
67 43
-
-
“ d l
iv I
- 94
1 2
20693
- 20 -- df -
67
-p
--
43-
#a
-P
df --
30
3 4 5 6 7 8 9 IO I 1 12 13 FIG. 4. Isolation of biotinyl-fly and the interaction of G protein subunits with streptavidin-agarose.
G protein was modified with NHS-LC-biotin and the subunits separated on w-amino-octyl-agarose as described under “Experimental Procedures.” Upper panel, shown in the figure are fractions from the column analyzed for hiotinylated subunits as described under “Experimental Procedures.” The column was eluted with the following solutions: i, TED, 0.25% cholate, 300 mM NaCI, AMF; ii, TED, 0.25% cholate, 500 mM NaCI, AMF; iii, TED, 0.4% cholate, 200 mM NaCI, AMF; iv, TED, 1.3% cholate, 50 mM NaCI, AMF. Lowerpanels, lanes 1 and 2, protein blot for biotin of starting G protein preparation after biotinylation (lane 1 ) and the resolved biotinyl-Sy fraction (lane 2). Lanes 3 and 4, the Coomassie Blue-stained gel corresponding to the starting G protein preparation (lune 3 ) and the resolved biotinyl-fly fraction (lane 4 ) . Lanes 5 and 6, molecular weight markers. Lanes 7-10, interaction of By with streptavidin-agarose. Lane 7, binding of unmodified By to streptavidin-agarose. Two pg of unmodified By were diluted into 500 pl of HEDLN with 10 pl of streptavidin-agarose and incubated for 1 h a t 4 “C. The agarose pellet was washed twice withHEDLN and then run on SDS-PAGEdescribed as under “ExperimentalProcedures.” Lane 8, biotin inhibition of biotinyl-By binding to streptavidin-agarose. Two pg hiotinyl-By were treated as for lane 7 but after preincubating the streptavidin-agarose with 100 p~ biotin for 30 min a t 4 ‘C. Lane 9, binding of biotinyl-fly to streptavidin-agarose. Twopg biotinyl-By were treated as for lune 7. Lane 10, stability of biotinyl-By binding to streptavidin-agarose. A sample equivalent tothat in lane9 was washed twice withHEDLN, resuspended in 500 pl of HEDLN with 100 p~ biotin, and incubated for 24 h a t 4 “C. Lane 11, molecular weight standards. Lanes 12 and 13, binding of a to streptavidin-agarose. Mixed a subunit (Fig. 1, lane 3 ) a t 50 pg/ml in a total volume of 250 pl of HEDLN was incubated for 1 h a t 4 “C with 10 pI of streptavidin-agarose containing either no biotinyl-py (lane 12) or 2 pg of biotinyl-By (lane 13). Samples were washed once with HEDLN and processed as above.
and Neer, 1986; Katada et al., 1986b). The apparentKOof aD9 for By, however, is a t least 3-fold higher than the ECsofound in these earlier studies. In part, this might be explained by .-c 70 differences in the methodsused to determine these estimates. E A In an assay where the endpoint, ADP-ribosylation, is essen2 E 60tially an irreversible step, the ECso for By will be influenced U 0 by the time of incubation and would be expected to indicate ahigher affinity than the actual K D forbinding. Go is a ;50 relatively abundant protein in bovine brain, about 0.5% of E 0. total particulate protein ina crude membrane preparation (Sternweis and Robishaw, 1984). Although it is hard to ex0 ,001 .01 .1 1 10 100 trapolate this to thepresumed intracellular concentration of [ Pr complex], P g/ml Go, it is almost certainly in the micromolar range. If the FIG. 5. Inhibition of forskolin-stimulated adenylyl cyclase regulation of subunit dissociation does have role a in G protein activity by By and biotinyl-By. Inhibition of S49 cell membrane function, a KD for a39 bindingto of0.3-0.4 I.~Mdoes not adenylyl cyclase hy !j-y (open circles) or hiotinyl-by (closed circles) was seem unreasonable. If the affinity of binding was much tighter assaved as described under “Experimental Procedures.” The hiotinylb-y preparation used in this experiment was 6 5 5 immobilizable on than this, it might notdissociate even upon activation. Howstreptavidin-agarose. Duplicate samples were assayed a t each By ever, whether or not, and under what conditions, G proteins concentration, and the average of the two samples is plotted in the are dissociated in intact membranes, or in cells, is still not figure. Oppn triangle. the activity in theabsence of any added By. clear. The results reportedhere for aR9may indicate that this G protein, in brain or in particular in other tissues where its levels may be considerably lower, is partially or fully dissoylated By is 70-100% functional and that it contains a single ciated even in its unactivated state. This is difficult to state CY subunit binding site. Our results suggest that ma,has a 10-15-fold higher affinity with much certainty from these data alone, since the local for By than does N : ~ In ~ . the case of ad,, its apparent affinity concentrations of the subunits in membranes of intact cells for By is similar to that determinedindirectly by its require- are hard to estimate. ment for By for ADP-ribosylation by pertussis toxin (Huff Guanine Nucleotide and Magnesium Regulation of Subunit
. E
20694
G Protein Biotinyl-/3y py
biotinyl- py
9 ” ” ” ”
.
.
A
-
0.2
u)
C
g
0.1
0.0 IO
FIG. 7. Competition of j3y and biotinyl-j3y for CY binding to biotinyl-j3y immobilized on streptavidin-agarose. Streptavidinagarose (200 pl packed volume) was incubated at 4 “C for 60 min in a final volume of ROO pl containing HEDLN, 50 p~ GDP, and 75 pg of biotinyl-[j-y (60% immohilizahle on streptavidin-agarose). Streptavidin sites withoutbiotinyl-By bound were then blocked by addition of 80 pl of a 0.5 mM solution of biotin and continuing the incuhation for 60 min. Thesuspension was then washed threetimes with HEDLN with 50 p~ GDP to remove unbound biotinyl-fly and hiotin and resuspended to a final volume of 500 pl in HEDLN with 50 pM GDP. This suspension was used to determine the ability of hiotinylBy or unmodified By to compete for n binding to the immobilized biotinyl-By. In this assay, 20 pl of the above suspension (estimated to contain 1.5-2.0 pg of bound biotinyl-/j-y) was added to 20 p1 of varying amounts of /;lyin HEDLN, 50 p~ GDP, 0.1 mg/ml RSA at 4 “C. The assay was started hy adding 20 pl of HEDLN, 50 pM GDP with 150 pg/ml isolated n suhunit. Samples were incubated for 60 Eonom TOP min at 4 “C and were vortexed a t 5 - to 10-min intervals. At the end Gradient Fractions of this incuhation, samples weredilutedwith 1 ml of incubation FIG. 6. Behavior of G protein on sucrose gradients.G protein buffer, centrifuged, and thenwashed once with the same buffer. The (30 p g ) was mixed with 10 p g of biotinyl-@y (3:3%immobilizable on final pellets were resuspended with Laemmli sample buffer (Laemmli, streptavidin-agarose) and 10 pg of unmodified no; and preincubated 1970), boiled for 5 min, and applied to an 11% polyacrylamide gel. as described under “Experimental Procedures” with: A, no additions The graphshows the ratio of the Coomassie Blue staining intensities (control); R, 10 p~ GTP-ySwith 10 mM MgCI?; C, AMF; D,AMF for n and 0 in each sample, as a function of total added /j-y (open followed by 1.5 mM EDTA; or E, 50 pM G T P with 10 mM MgCI,. circles) or biotinyl+y (solid circbs) in solution. Triangles indicate the Samples were layered onto sucrose gradients prepared containing the ratio of n/B staining intensity withnoadded f l y . The gel inserts same reagents as in the preincubation, except for the sample preabove the graph show the n (upper band) and [f (lower band) regions treated with AMF followed by EDTA, whichwaslayered onto a of the polyacrylamide gel from the experiment. Indicated at the left gradient containing only HEDLN. Gradient composition and sedi- is the @y preparation added in solution. The amounts of By added mentation are described under “Experimental Procedures.” Approxare, from left to right, 0, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0, 10.0, and 20.0 imately 40 fractions were collected from eachgradientandevennumhered fractions were analyzed by CoomassieBlue Staining of SDS-polyacrylamide gels and by Western blot analysis for biotincontaining protein. Densitometry of Coomassie Blue-stained SDS- tion. Fig. 9 shows the results of one of four experiments polyacrylamide gels was performed to quantitate the distribution of demonstrating that GTPdoes affect m 9 binding to biotinylC. protein (open circles), RSA (small open circles, hmuy line) and By in the absenceof any added magnesium. Fig. 10 showsthe cytochrome c (solid triangles, heaL:v line) and of Western blots for results of an experiment examining the interaction of magbiotinyl-ijy (open squares). The n and @ subunits of the G protein nesium and guaninenucleotides in affectingaR9or ad,binding comigrated under all conditions and were scanned simultaneously. to biotinyl-By. GTPyS and AMFdecreased binding of either The results for each gradient constituent were scaled so as to best show their relative positions on the gradient. The five plots were then N subunit to biotinyl-By and more magnesium was required alignedsuch that the BSA and cytochrome c peaks,indicated by for AMF than for GTPyS effects. Increasing M e blunted dashed,certical linen, were coincident. The arrous indicate the G the effects of G T P on wl9binding, perhaps due to stimulation protein and biotinyl-$7 peaks in each gradient.
of the GTPaseassociated with the protein. In contrast to m 9 , G T P did not appear toaffect a d ,binding tobiotinyl-By. Int~ractions-Previous studies investigating G protein subThe results reportedhere using cy association with biotinylunit interactions in detergent solution (Huff and Neer, 1986; By confirm previous results on theeffects of nonhydrolyzable Kahn and Gilman,1984) and analogous experiments reported guanine nucleotides and complexes of fluoride and aluminum here (Fig. 6 ) have failed to demonstrate that GTP, the phys- on G protein subunit interactions. They further substantiate iological regulator of G protein activation, affects G protein the utility of using this modified By complex for studying G subunit interactions. Whereas in a reconstituted system with protein function. In addition,however, they demonstrate that G, and rhodopsin (Fung, 1983) or in intact membranes in the G T P is a regulator of G protein subunit interactions,a t least case ofG, (Ransnasand Insel, 1988), evidence has been for N : ~ and ~ , that GTP effects do not necessarily require an presented suggesting that GTP can cause subunit dissocia- intact membrane environment or the participation of activat-
G Protein Biotinyl-By
20695 0.5
B
A
0.4 h
5 0.3 I
'"1
I
.-0
1
;;; 0.2
111
c
0.1
80
0.0
GCP
GTF '! S
GTP
Nucleotide FIG. 9. Effects of guaninenucleotides on a s s linding to bio-
0
100
200
3
Total u4, Added (nM)
0
1000
2000
3000
Total u3g Added (nM)
FIG. 8. Concentration dependence of binding of a38 and a41 to biotinyl-fly immobilized on streptavidin-agarose. Varying concentrations of or n4, were incubated for 1 h at 4 "C in 500 p1 of HEDLN, 10 p~ GDP containing10 pI of streptavidin-agarose with (Total Binding) or without (nonspecificbinding, N S R ) 1.2 pg (65 nM) of bound biotinyl-dy. At the end of the incubation, samples were pelleted in an Eppendorf microcentrifuge and the supernates aspirated. Sampleswere not washed, but resuspended directly in Laemmli sample buffer (Laemmli, 1970) and processed as described under "Experimental Procedures." Amount of n recovered in the pellets was determined from densitometric scans of the Coomassie Rlue-stained polyacrylamide gels. Each gel alsocontained a dilutionseries of known amounts of n or ijy to construct a standard curve of scanning density with amount of protein. Maximum binding and the apparent KI, were estimated by fitting bounduersus free n to a one-site binding model using the computer program ENZFITTER (Elsevier-Biosoft). A and C , hinding of nI1to biotinyl-By. I? and D , binding of nR9to biotinyl-By. Open circles, total n subunit recovered in pellets (Total Binding). Open squares, (Y subunit recovered in pellets not containing hiotinyl-ijy, nonspecific binding ( N S B ) .Closed circles, specific binding to biotinyl-By (Total Binding-NSR).
ingreceptors orothermembrane-associatedproteins.The lack of an effect of GTP on a4,binding is difficult to fully evaluate without a detailed kinetic analysis of G T P binding and hydrolysis, along with the effects of variable magnesium concentration and their correlation with G protein subunit interactions.This lack of an effect isnot simply dueto inability of the guanine nucleotide to bind, perhaps due to tightly bound GDP, since GTPyS doesaffectbinding. Whatever the explanationfor this difference between cyD9 and cyd1, however, we have been able to use it to resolve aR9 and c y l l from mixed cy subunit pools on abiotinyl-By affinity column eluted with G T P or AMF.* Such an affinity column will undoubtedly be useful for isolation of G protein N subunits from small quantities of starting material and for separation of cy subunits which are otherwise difficult to resolve. The most important single result from these experiments is that GTP can effect cyR9 binding to Pr, an effect which several others (Huff andNeer, 1986; Kahn and Gilman,1984), as well as ourselves (Fig. 6), have not been able to demonstrate by other techniques such as sucrose density centrifugation. These data support the idea that GTP causes subunit dissociation along withactivation of the G proteins (Gilman,1987). well as the lack of an The lack of an effect of G T P on as effect onat physiological Mg'+ concentrations,areconsistent with these proteins having a low basal activity due to R. E. Kohnken and J. D. Hildehrandt, unpublished observations.
tinyl-fly. Four pg of u : l g were incuhated in the presence or ahsence (nonspecific binding) of 2 pg of biotinyl-13y bound previously to 10 pl of streptavidin-agarose in a total volume of 200 pl of HEDLN for 30 rnin at 37 "C. Samples also contained30 p~ of the indicated guanine nucleotide. At the end of the incubation sampleswere pelleted in an Eppendorf microcentrifuge and processed as described in the legend of Fig. 8. The ratio of the Coomassie Blue staining intensity of t.he (Y subunit band to that of the B subunit band is plotted on the y axis. Nonspecific binding, expressed as the ratioof n subunit recovered in the pellets in the absence of biotinyl-(fy to the average biotinyl-By recovered in the other samples, was 0.05 and was subtracted from all of the samples. Error bars indicate the standarderror of the mean of triplicate determinations.
06-
a
'a
0 0.4._
m U
0.2-
-
GTPyS rn
0.0 < 01
1
Estimated Mg'
1
10
(mM)
< 01
1
1
10
Estimated Mg" (mM)
FIG. 10. Effects of guanine nucleotides, AMF. and magnesium on the binding of as,,and ad,to biotinyl-fly. Duplicate samples of n subunit were incubated in HEDLN with 1 pg of biotinylf l y , prebound to streptavidin-agarose, in the presence of GDP (30 p ~ )GTP , (30 p ~ ) GTPyS , (30 p ~ )or, AICln (30 p ~ plus ) NaF (10 mM). All samples contained 1 mM EDTA and total MgCI, to give approximately the indicated M e concentrations; no added MgCI, (
