Heterotrimeric G Proteins

560 Pages·1994·10.055 MB·English

by  Abelson J.N., Simon M.I. (eds.)

Preface
Heterotrimeric (a/33,) G proteins function as cell surface signal trans-
ducers for a large number of hormones, neurotransmitters, and for
autocrine and paracrine factors. It is now known that a hundred or so (not
counting the olfactory) receptors are coupled to various effectortsh rough
G proteins. This large number of receptors couple to members of one of
the four families of G proteins to transmit their signals. The specificity of
the receptor-G protein interactions determines the transmission of the
signal to different downstream pathways. As with all real life situations,
the specificity of interactions between receptors and G proteins is varied
and complex. Consequently, signals from a single receptor may be trans-
mitted through several pathways simultaneously. Each of the four fami-
lies of G proteins has many members. Cloning studies have indicated that
there are twenty a, four/3, and six ~/subunits. It has been suggested that
very large numbers of heterotrimeric G proteins with defined subunit
compositions can be generated from these individual subunits. "Knock-
out" experiments indicate that G proteins of defined subunit composition
communicate signals from different receptors. However, at this time
there is not sufficient general information to indicate that the functional
identity of a G protein is defined by the molecular identity of all of its
subunits. Consequently, in spite of the molecular heterogeneity of the/3
and ~/subunits, the different G proteins are still classified by the identity
of their ~o subunits. This classification is useful because it indicates which
intracellular messenger pathway is used. In this volume the nomenclature
of G proteins is based on the molecular identity of the a subunit. Hence it
should be noted that native purified G proteins may have a single molecu-
lar species of ~ subunits, but multiple forms of/3 and ~/subunits.
In the past decade there has been a substantial increase in our under-
standing of signal-transducing G proteins. This advance has been brought
about by a combination of molecular biological and biochemical tech-
niques. Both approaches are represented in this book. Two types of chap-
ters are included. The first presents techniques unique to the field of
signal-transducing G proteins, the second general techniques that have
been applied to the study of G-protein systems. Chapters of the latter type
may be useful even to researchers who do not work on signal-transducing
G proteins.
The field of heterotrimeric G proteins has been covered in part in
Volumes 901 and 591 of Methods in Enzymology. However, the subject
has not been covered in a systematic fashion. Consequently, in planning
XV
xvi ECAFERP
this volume, an attempt was made to include all or at least most of the
techniques in one volume. Thus techniques such as cholera and pertussis
toxin labeling as well as some techniques for the purification of native G
proteins are covered but by different authors. It should be noted that in G
protein research many laboratories use different protocols for the same
overall experiments. Since a single experimental protocol does not always
work for the same G protein in different systems it can be useful to have
more than one experimental procedure available to tackle the same ques-
tion. Several, but not all subjects, are thus addressed by multiple labora-
tories in this volume.
I thank Lutz Birnbaumer, Henry Bourne, and Buzz Brown for their
useful suggestions during the initial planning of this volume. I also thank
Ms. Lina Mazzella for her valuable assistance. Last, but not least, I thank
the authors for their contributions and for complying with my suggestions
for change in a timely fashion.
IVAR RAGNEYI
Contributors to Volume 237
Article numbers are in parentheses following eht seman of contributors.
Affiliations listed are .tnerruc
JANMEET S. ANANT (40), Jules Stein Eye DRAHCIR A. CERIONE (31), Department of
Institute, University of California at Los Pharmacology, Cornell University,
Angeles School of Medicine, Los Ithaca, New York 35841
Angeles, California 42009 CRAM ERBAHC 1( l, 34), Institut de Pharma-
ANNA M. YAGARA (26), Department of -loiB cologie mol~culaire et cellulaire, Unit~
ogy, California Institute of Technology, propre 114 ud Centre National de al Re-
Pasadena, California 52119 cherche Scientifique, 06560 Valbonne,
SEVY AUDIGIER (19), UMR Universitd 9925, France
Paul Sabatier, 31062 Toulouse Cedex, GNAIQNAIJ CHEN (35), Department of Phar-
France macology, Mount Sinai School of Medi-
ERIC A. BALCUEVA (39), Weis Center for cine, New York, New York 92001
Research, Geisinger Clinic, Danville, JUAN CODINA (9), Department of Biol- Cell
Pennsylvania 22871 ogy, Baylor College of Medicine, Hous-
JOgLLE BIGAY (11, 34), Institut de Pharma- ton, Texas 03077
cologie moldculaire et cellulaire, Unitd YELDARB M. DENKER (18), Department of
propre 114 du Centre National de al Re- Medicine, Brigham and Women's Hospi-
cherche Scientifique, 06560 Valbonne, tal and Harvard Medical School, Boston,
France Massachusetts 51120
LUTZ REMUABNRIB (9, 29), Departments of N. NARAKESANAHD (7), Department of Bio-
Biology, Cell Medicine, Molecular Physi- chemistry and Fels Institute for Cancer
ology, and Biophysics, and Division of and Molecular Biology, Temple Univer-
Neurosciences, Baylor College of Medi- sity School of Medicine, Philadelphia,
cine, Houston, Texas 03077 Pennsylvania 04191
NAHTANOJ L. BLANK (14), Division of Basic JANE DINGUS (36), Department of Cell and
Sciences, National Jewish Center for Im- Pharmacology Molecular and Experimen-
munology and Respiratory Medicine, tal Therapeutics, Medical University of
Denver, Colorado 60208 South Carolina, Charleston, South Caro-
SAMOHT NOLLIUOB (2), Department of -nilC lina 52492
ical Pharmacology, University of G6t- WILLIAM J. DUNN III (8), Departments of
tingen, 37075 Gfttingen, Germany Medicinal Chemistry and Pharmacog-
Jose. L. BOYER (15), Department of Phar- nosy, University of Illinois College of
macology, University of North Carolina Pharmacy, Chicago, Illinois 21606
School of Medicine, Chapel Hill, North JOHN H. EXTON (14), Department of Molec-
Carolina 99572 ular Physiology and Biophysics, Vander-
ALLAN YELDARB (29), Institute for Molecu- bilt University School of Medicine, Nash-
lar Genetics and Howard Hughes Medi- ville, Tennessee 23273
Institute, cal Baylor College of Medicine, ROnERT A. FIGLER (17), Departments of
Houston, Texas 03077 Molecular Physiology and Biological
ANNOD J. CARTY (4, 6, 35), Department of Physics, University of Virginia Health
Pharmacology, Mount Sinai School of Sciences Center, Charlottesville, Virginia
Medicine, New York, New York 92001 80922
ix
X CONTRIBUTORS TO VOLUME 732
LEAHCIM FORTE (33), Vollum Institute, Or- Blood Institute, National Institutes of
egon Health Sciences University, Port- Health, Bethesda, Maryland 29802
land, Oregon 10279 JOHN R. HEPLER (16), Department of Phar-
DRANREB K. -K. FUNG (40), Jules Stein Eye macology, University of Texas South-
Institute, University of California at Los western Medical Center, Dallas, Texas
Angeles School of Medicine, Los 53257
Angeles, California 90024 UMOTUST AMIJIHSAGIH I (3), Department of
Pharmacology, University of Texas
ENITSIRHC GALLAGHER (37), Departments
Southwestern Medical Center, Dallas,
of Anesthesiology and Genetics, Wash-
Texas 53257
ington University School of Medicine, St.
Louis, Missouri 63110 JOHN D. TDNARBEDLIH (36), Department of
Cell and Molecular Pharmacology and
JAMES C. GARRISON (17), Department of
Experimental Therapeutics, Medical Uni-
Pharmacology, University of Virginia
versity of South Carolina, Charleston,
Health Sciences Center, Charlottesville,
South Carolina 52492
Virginia 22908
INANOBE ATSUSHI (10), Department of Life
NAHMISARAN GAUTAM (37, 38), Depart-
Science, Tokyo Institute of Technology,
ments of Anesthesiology and Genetics,
Yokohama, Kanagawa 227, Japan
Washington University School of Medi-
cine, St. Louis, Missouri 63110 RAVI RAGNEYI (4, 35), Department of Phar-
macology, Mount Sinai School of Medi-
OLBAP V. GEJMAN (24), Unit on Molecular cine, New York, New York 92001
Clinical Investigation, Clinical Neuro-
KARL H. SBOKAJ (1, 2), lnstitutfiir Pharma-
genetics Branch, National Institute of
kologie, Universitiitsklinikum Essen,
Mental Health, National Institutes of
22154 Essen, Germany
Health, Bethesda, Maryland 29802
GARY L. NOSNHOJ (25), Division of Basic
RETEP KIHCSREIG (2), Department of Phar-
Sciences, National Jewish Center for Im-
macology and Toxicology, University of
munology and Respiratory Medicine,
Ulm, 89069 Ulm, Germany
Denver, Colorado 80206
ALFRED G. GILMAN (12, 16), Department of
TOSHIAKI KATADA (10), Department of
Pharmacology, University of Texas
Physiological Chemistry, Faculty of
Southwestern Medical Center, Dallas,
Pharmaceutical Sciences, University of
Texas 53257
Tokyo, Bunkyo-ku, Tokyo 113, Japan
NEHPETS G. GRABER (17), Department of
ENAITSIRHC KLEUSS (27), Bereich Moleku-
Pharmacology and Toxicology, West Vir-
larbiologie und Biolnformatik, Institut fiir
ginia University, Morgantown, West Vir-
Molekularbiologie und Biochemie, Freie
ginia 26505
Universitiit Berlin, D-14195 Berlin, Ger-
OTREBOGAD GRENET (9), Department of many
Cell Biology, Baylor College of Medicine,
ORIHCI IHSAYABOK (10), Department of Life
Houston, Texas 03077
Science, Tokyo Institute of Technology,
HEIDI E. HAMM (32), Department of Physi- Yokohama, Kanagawa 227, Japan
ology and Biophysics, University of Illi-
LLESSUR KOHNKEN (36), Molecular Geriat-
nois at Chicago, Chicago, Illinois 21606
rics, Libertyville, Illinois 60048
T. KENDALL HARDEN (15), Department of KENJI KONTANI (10), Department of Life
Pharmacology, University of North Caro- Science, Tokyo Institute of Technology,
lina School of Medicine, Chapel Hill, Yokohama, Kanagawa 227, Japan
North Carolina 27599
YDNAR S. HAUN (5), Laboratory of Cellular
Metabolism, National Heart, Lung, and Deceased.
CONTRIBUTORS TO VOLUME 732 xi
(16), KOZASA TOHRU Department of Phar- 1HSORIH (10), NISH1NA Department of Life
macology, University of Texas South- Science, Tokyo Institute of Technology,
western Medical Center, Dallas, Texas Yokohama, Kanagawa 227, Japan
53257 STEFAN SNNAMREFFO (22), Department of
(22), LAUGWITZ KARL-LUDWIG Institut fiir Biology, California Institute of Technol-
Pharmakologie, Freie Universitiit Berlin, ogy, Pasadena, California 52119
59141 Berlin, Germany URAHIHSOY OHOKA (10), Department of
ETHAN LEE (12), Department of Pharma- Life Science, Tokyo Institute of Technol-
cology, University of Texas Southwestern ogy, Yokohama, Kanagawa 227, Japan
Medical Center, Dallas, Texas 53257 OLIVIA C. ONG (40), Jules Stein Eye Insti-
FANG-JEN SCOTT LEE (5), Laboratory of tute, University of California at Los
Cellular Metabolism, National Heart, Angeles School of Medicine, Los
Lung, and Blood Institute, National Insti- Angeles, California 42009
tutes of Health, Bethesda, Maryland IoK-Hou PANG (13), Department of Glau-
29802 coma Research, Alcon Laboratories, Fort
WuN-CHEN LIN (40), Jules Stein Eye Insti- Worth, Texas 43167
tute, University of California at Los S. Russ PRICE (5), Renal Division, Emory
Angeles School of Medicine, Los University Hospital, Atlanta, Georgia
Angeles, California 42009 22303
MAURINE E. LINDER (12, 20), Department YEXELA N. PRONIN (38), Departments of
of Cell Biology and Physiology, Washing- Anesthesiology and Genetics, Washing-
ton University School of Medicine, St. ton University School of Medicine, St.
Louis, Missouri 01136 Louis, Missouri 01136
(23), LYONS JOHN Department of Drug Dis- NILKNARF (33), QUAN Vollum Institute, Or-
egon Health Sciences University, Port-
covery, Onyx Pharmaceuticals, Rich-
land, Oregon 10279
mond, California 60849
HELEN M. RARICK (32), Department of
EMEARG MILHGAN (21), Molecular Phar-
Physiology and Biophysics, University of
macology Group, Departments of Bio-
Illinois at Chicago, Chicago, Illinois
chemistry and Pharmacology, University
21606
of Glasgow, 21G 8QQ Glasgow, Scot-
MARK M. (8), RASENICK Departments of
land, United Kingdom
Physiology and Biophysics, University of
DRAHCIR M. NESNETROM (28), Department Illinois College of Medicine, Chicago, Illi-
of Medicine, Brigham and Women's Hos- nois 21606
pital and Harvard Medical School, Bos-
ODNANREF RIBEIRO-NETo (9), Department
ton, Massachusetts 51120
of Pharmacology, Duke University Medi-
(5), MOSS JOEL Laboratory of Cellular Me- cal Center, Durham, North Carolina
tabolism, National Heart, Lung, and 01772
Blood Institute, National Institutes of
JANET D. RontsnAw (39), Weis Center for
Health, Bethesda, Maryland 29802
Research, Geisinger Clinic, Danville,
ENNASUS M. (20), MUMBY Department of Pennsylvania 22871
Pharmacology, University of Texas ELLIOTT M. (3), ROSS Department of Phar-
Southwestern Medical Center, Dallas, macology, University of Texas South-
Texas 53257 western Medical Center, Dallas, Texas
EVA J. (18), NEER Department of Medicine, 53257
Brigham and Women's Hospital and Har- UWE RUDOLPH (29), Institute of Pharma-
vard Medical School, Boston, Massachu- cology, University of Zurich, CH-8057
setts 51120 Zurich, Switzerland
xii
CONTRIBUTORS TO VOLUME 237
ARNOLD E. RUOHO (7), Department of SAMOHT C. SAMOHT (18), Alexion Pharma-
Pharmacology, University of Wisconsin ceuticals, Inc., New Haven, Connecticut
Medical School, Madison, Wisconsin 11560
60735
Su-CHEN TSAI (5), Laboratory of Cellular
ENAURAM (25), RUSSELL Division of Basic Metabolism, National Heart, Lung, and
Sciences, National Jewish Center for Im- Blood Institute, National Institutes of
munology and Respiratory Medicine, Health, Bethesda, Maryland 29802
Denver, Colorado 60208
DRAHClR R. TRUOCNALLIAV (7), Division of
LRAC J. (18), SCHMIDT Department of Med- Basic Sciences, Department of Pediat-
icine, Brigham and Women's Hospital rics, National Jewish Center for Immu-
and Harvard Medical School, Boston, nology and Respiratory Medicine, Den-
Massachusetts 51120 ver, Colorado 80206
GONTER SCHULTZ (22, 27), lnstitut far PETER J. M. VAN TRETSAAH (30), Depart-
Pharmakologie, Freie Universitdt Berlin, ment of Biochemistry, University of Gr6-
59141-D Berlin, Germany ningen, 9747 AG Gr6ningen, The Nether-
J. G. (28), SEIDMAN Department of Genet- lands
ics, Harvard Medical School, Boston,
AHTRAM NAHGUAV (5), Laboratory of Cel-
Massachusetts 51120
lular Metabolism, National Heart, Lung,
MELVIN I. (26), SIMON Department of Biol- and Blood Institute, NationaIln stitutes of
ogy, California Institute of Technology, Health, Bethesda, Maryland 29802
Pasadena, California 52119
YRAG L. (15), WALDO Department of Phar-
ALAN V. AKCRMS (13), Department of Phar- macology, University of North Carolina
macology, University of Texas South- School of Medicine, Chapel Hill, North
western Medical Center, Dallas, Texas Carolina 27599
53257
A. JOHN NOSTAW (26), Department of Biol-
B. EWA AKSLAGAJ-RAANS (30), Cell Biology ogy, California Institute of Technology,
and Genetics Unit, Clusius Laboratory,
Pasadena, California 52119
Leiden University, 2333 AL Leiden, The
Netherlands LEE S. (24), WEINSTEIN Molecular Patho-
physiology Branch, National Institute of
NETSRAK REHCIPS (22), lnstitutfiir Pharma-
Diabetes and Digestive and Kidney Dis-
kologie, Freie Universitiit Berlin, 59141
eases, National Institutes of Health, Be-
Berlin, Germany
thesda, Maryland 29802
PAUL C. (13), STERNWEIS Department of
Pharmacology, University of Texas ENIREHTAC F. WELSH (5), Department of
Cell Biology and Anatomy, University of
Southwestern Medical Center, Dallas,
Miami, Miami, Florida 33136
Texas 53257
UBONUSTAK IHSAHAKAT (10), Department SAMOHT (1), WIELAND Institutfiir Pharma-
of Life Science, Tokyo Institute of Tech- kologie, Universitiitsklinikum Essen,
nology, Yokohama, Kanagawa 227, Ja- 22154 Essen, Germany
pan
LEAHCIM D. (36), WILCOX Department of
MADHAVI TALLURI (8), Departments of Cell and Molecular Pharmacology and
Physiology and Biophysics, University of Experimental Therapeutics, Medical Uni-
Illinois College of Medicine, Chicago, Illi- versity of South Carolina, Charleston,
nois 60612 South Carolina 52492
CONTRIBUTORS TO VOLUME 237 xiii
SAMOHT M. WILKIE (26), Department of TDRAHGRUB WITTIG (27), Bereich Moleku-
Pharmacology, University of Texas larbiologie und Biolnformatik, Institut fiir
Southwestern Medical Center, Dallas, Molekularbiologie und Biochemie, Freie
Texas 53257 Universitiit Berlin, Berlin, D-14195 Ger-
many
SIM WINITZ (25), Division of Basic Sci- YEVRAH K. (40), YAMANE Jules Stein Eye
ences, National Jewish Center for Immu- Institute, University of California at Los
nology and Respiratory Medicine, Den- Angeles School of Medicine, Los
ver, Colorado 60208 Angeles, California 42009
]1[ DETALUMITS-ROTPECER GTPyS GNIDNIB YB G SNIETORP 3
[1] Measurement of Receptor-Stimulated Guanosine
5'-O-(y-Thio)triphosphate Binding by G Proteins
By SAMOHT DNALEIW and KARL H. SBOKAJ
Introduction
Many transmembrane signaling processes caused by extracellular hor-
mones and neurotransmitters are mediated by receptors interacting with
heterotrimeric (a/3y) guanine nucleotide-binding proteins (G proteins)
attached to the inner face of the plasma membrane. Agonist-liganded
receptors apparently initiate activation of G proteins by catalyzing the
exchange of guano sine 5'-diphosphate (GDP) by guanosine 5'-triphosphate
(GTP) bound to the a subunits.l,2 In membrane preparations and reconsti-
tuted systems, this activation process is frequently monitored by studying
agonist stimulation of high-affinity GTPase, an enzymatic activity of
G-protein a subunits) However, the measurement of G-protein GTPase
activity reflects steady-state kinetics of the overall G-protein activity cycle
and not only the first step in the signal transduction cascade (i.e., the
GDP/GTP exchange reaction). Furthermore, with regard to the molecular
stoichiometry of receptor-G-protein interactions, only qualitative but not
quantitative data can be obtained.
To study the initial steps of G-protein activation by agonist-liganded
receptors in a quantitative manner, the binding of radiolabeled GTP ana-
logs, which are not hydrolyzed by the GTPase activity of G-protein a
subunits, to G proteins is determined. Of these GTP analogs, guanosine
5'-O-(y-[35S]thio)triphosphate ([35S]GTPyS) is most frequently used. This
nucleotide has a high affinity for all types of G proteins and is available
with a relatively high specific radioactivity (1000-1400 Ci/mmol; physical
half-life 87.4 days). Here we describe the measurement of receptor-
induced binding of [35S]GTPyS to membranous and detergent-solubilized
G proteins and how this method can be adapted to different G proteins
for an optimal response to receptor stimulation.
Materials
The [3SS]GTPyS (1000-1400 Ci/mmol) is obtained from Du Pont New
England Nuclear (Bad Homburg, Germany). The substance is delivered
I A. G. Gilman, Annu. Rev. Biochem. 56, 615 (1987).
2 L. Birnbaumer, J. Abramovitz, and A. M. Brown, Biochim. Biophys. Acta 1031, 361 (1990).
3 D. Cassel and Z. Selinger, Biochim. Biophys. Acta 452, 538 (1976).
Copyright @ 1994 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 732 All rights of reproduction in any form reserved.
4 Ga STINUBUS ]1[
in a buffer containing 01 mM N-tris(hydroxymethyl)methylglycine-
NaOH, pH 7.6, and l0 mM dithiothreitol (DTT). To minimize decomposi-
tion, the solution is diluted 100-fold in this buffer and stored in aliquots
at or below - ° 70 before use. If the reagent is not stored at these recom-
mended conditions, and after repeated freezing and thawing, chemical
decomposition is rather high.
Unlabeled nucleotides and 3-[(3-cholamidopropyl)dimethylammonio]-
1-propane sulfonate (CHAPS) are from Boehringer Mannheim (Mann-
heim, Germany). N-Ethylmaleimide, N-formylmethionyUeucylphenyl-
alanine (fMet-Leu-Phe), isoproterenol, and carbachol are from Sigma (St.
Louis, MO). Glass fiber filters (GF/C) are from Whatman (Clifton, NJ),
and nitrocellulose filters (pore size 0.45 /xm) are from Schleicher and
Schuell (Keene, NH).
Membranes of various cells and tissues are prepared as previously
described 7-4 and stored in aliquots at -70 .° Before use in the binding
assay, the membranes are thawed, diluted with l0 mM triethanolamine
hydrochloride, pH 7.4, containing 5 mM EDTA, centrifuged for I0-30
min at 30,000 g, and resuspended in 01 mM triethanolamine hydrochlo-
ride, pH 7.4, at the appropriate membrane protein concentration.
Equipment
Incubator or water bath
Filtration funnel with vacuum pump
Cooled centrifuge °, (4 up to 30,000 g) for membrane preparation
Ultracentrifuge with fixed-angle and swing-out rotors for preparation
of membranes, solubilized proteins, and sucrose density gradient
centrifugation
Shaker to equilibrate the filters with the scintillation cocktail
Liquid scintillation spectrometer
Freezer (preferably -70 ° or lower) for storage of membranes and
[35S]GTPyS
Measurement of Agonist-Induced [35S]GTPTS Binding to G Proteins
in Membranes
The assay is performed in 3-ml plastic reaction tubes. The assay volume
is 100/zl. The final concentrations of the reaction mixture constituents
4 p. Gierschik, M. Steisslinger, D. Sidiropoulos, E. Herrmann, and K. H. Jakobs, Eur. J.
Biochem. 283, 97 (1989).
5 G. Hilt" and K. H. Jakobs, Eur. J. Pharmacol. 172, 551 (1989).
6 D. S. Papermaster and W. J. Dreyer, Biochemistry 13, 2438 (1974).
7 G. Puchwein, T. Pfeuffer, and E. J. M. Helmreich, J. Biol. Chem. 249, 3232 (1974).
]1[ RECEPTOR-STIMULATED S,3PTG BINDING YB G PROTEINS 5
are as follows: triethanolamine hydrochloride (pH 7.4), 50 mM; MgCI2,
5 raM; EDTA, 1 mM; DTT, 1 mM; NaCl, 0-150 mM; GDP, 0-100/zM;
[35S]GTPyS, 0.3-0.5 nM (-50 nCi). The incubation temperature and mem-
brane concentration as well as the concentrations of NaCl and GDP have
to be adjusted to the individual cell type and the G-protein subtype acti-
vated by the receptor under study.
.1 The reaction mixture (40/zl) together with the receptor agonist or
its diluent (10/zl) are thermally preequilibrated for 5 min at the desired
reaction temperature.
2. The binding reaction is started by addition of the membrane suspen-
sion (50/.d) and vortexing.
3. Samples are incubated for the appropriate incubation time, for ex-
ample, 60 min at .° 03
4. The incubation is terminated by the addition of 2.5 ml of an ice-
cold washing buffer 05( mM Tris-HC1, pH 7.5, 5 mM MgCI2).
5. This mixture is passed through the filtration funnel. For systems
containing only membrane-bound G proteins, Whatman GF/C glass fiber
filters areu sed. In systems containing soluble G proteins (e.g., transducin),
nitrocellulose filters are required.
6. The reaction tube is washed two times with 2.5 ml of the washing
buffer, and this solution is also passed through the same filter.
7. The filter is additionally washed two times with 2.5 ml of thwea shing
buffer and then dried at room temperature.
8. The dried filters are put into 5-ml counting vials and equilibrated
with 4 ml of a scintillation cocktail for 20 rain at room temperature by
moderate shaking. Any commercially available scintillation cocktail suit-
able for counting of S53 can be used. Also a self-made cocktail consisting
of 2 liters toluene, 1 liter Triton X-100, 51 g 2,5-diphenyloxazole, and 3 g
2,2'-p-phenylenebis(4-methyl-5-phenyloxazole) can be used.
Application to Various Cell Types and Different G Proteins
In membranes of various cell types, including human neutrophils:
human platelets, 9 human leukemia cells (HL-60), ~1'°~ rat myometrium, 2~
8 .R ,reppuK .B ,dlaweD .H .K ,sbokaJ .M ,iniloiggaB .P dna ,kihcsreiG Biochem. J. ,282
924 .)2991(
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Biochem. ,7112 952 .)2991(
0l .p ,kihcsreiG .R .C ,redathgoM ,buartS .K ,hcireteiD dna .K .H ,sbokaJ Eur. J. Biochem.
,791 527 .)1991(
IIT. .IM ,srepehcS .M .E ,reirB dna .K .R ,hsieLcM J. Biol. Chem. ,762 951 .)2991(
21 .C ,nnambeiL .M ,relttinhcS .M ,htarwaN dna . K.H ,sbokaJ Eur. J. Pharmacol. 207,
76 .)1991(