- Email: [email protected]
[35] Adenylyl cyclase assay for βγ subunits of G proteins
[35]
ADENYLYL CYCLASE ASSAY
451
with a MgCI 2 linear gradient (0-0.33 M, 4.4 mM/min, flow rate 0.5 ml/ min). With this salt gradient, T/iy elutes first, in a sharp peak whose major fractions are not contaminated by Ta, which is already a minor component of the extract and elutes later as a lower and more diffuse peak (not shown). Pooled Tfly fractions from previous TaGTPvS subunit purification batches on the Na2SO4 gradient can be purified after concentration/dilution (buffer A) on a Centricon 30 microconcentrator (Amicon, Danvers, MA) up to a final dilution factor of about 1/10 (less than l0 mM Na2SO4). About 30/.~g of pure Tfly can be obtained after treatment of 1 mg rhodopsin containing membranes. Separation of Modified T3~ Subunits With some transducin extracts, particularly when the preliminary washing step in Hypo buffer, prior to the GTP addition, had been deleted, the elution profile of the final Tfly extract often appears split in two distinct peaks (Fig. 1A). Both peaks contain the same Tfl component (Fig. 1B), but the first minor peak contains a T3~ component which migrates more slowly on urea-polyacrylamide gels (Fig. 1C). Such a separation of two Tfly components with distinctive -y subunits has been described by Fukada et al.,3 who separated Tflyl plus Tfly 2 (first peak) and pure TflYz (second peak) on a DEAE-Toyopearl 650S (Toyo Soda Mfg. Co., Japan) column, then purified the TflY1 complex from TflYz onto a Mono Q HR 5/5 column (Pharmacia). They identified the slower migrating Tyl as a minor defarnesylated component and T72 as a farnesylated and partly carboxymethylated 7 subunit.4'5 4 y. Fukada, T. Takao, H. Ohguro, T. Yoshizawa, T. Akino, and Y. Shimonishi, Nature (London) 346, 658 (1990). 5 H. Ohguro, Y. Fukada, T. Takao, Y. Shimonishi, T. Yoshizawa, and T. Akino, EMBO J. 10, 3669(1991).
[35] A d e n y l y l C y c l a s e A s s a y for f13~ S u b u n i t s o f G P r o t e i n s
By JIANQIANG CHEN, DONNA J. CARTY, and RAvI IYENGAR Introduction Transmission of signals through G proteins can be achieved by the use of either a or/37 subunits.1,2 It had been generally believed that a I j. R. H e p l e r and A. G. Gilman, Trends Biochem. Sci. 17, 383 (1992). 2 L. Birnbanmer, Cell (Cambridge, Mass.) 71, I069 (1992).
METHODS IN ENZYMOLOGY, VOL. 237
Copyright © 1994by AcademicPress, Inc. All fights of reproductionin any form reserved.
452
G/3y SUBUNITS
[35]
subunits were the prime signal transmitters. Only a few systems such as certain types of K ÷ channels 3 and the effector(s) in the yeast pheromone pathway 4 were thought to be regulated by fly subunits. However, it has now been shown that f13, subunits can by themselves regulate the activity of a number of mammalian effectors such as certain types of adenylyl cyclase (adenylate cyclase) 5'6 and phospholipaseC. 7'8 Thus it has become increasingly obvious that fly subunits will play a central role in many signaling pathways. In deciphering the role of fly subunits it is useful to have simple assay systems that can be used to assess the biological activity of the purified or expressed fly subunits. Both adenylyl cyclases as well as phospholipases C offer excellent readout systems for fly subunits. Gilman and co-workers have shown that type 2 and 4 adenylyl cyclases are stimulated by /33, subunits in the presence of activated a2 subunits. 5'6 Giershick and coworkers have shown that phospholipase C from HL-60 cells and neutrophils can be stimulated by/33, subunits. 7 The use of the cytosolic HL-60 cell phospholipase C to measure/33, activity is described elsewhere. 9 In this chapter we describe a very simple adenylyl cyclase assay that can be used to assess the activity of purified/33, subunits. The assay is based on the original observations by Katada et al. 1o that/33, subunits stimulate the mouse lymphoma $49 c y c - cell membrane adenylyl cyclase. This assay works on crude $49 c y c - cell membranes and does not require the presence of any other adenylyl cyclase stimulants for the expression of /33, activity.
Preparation
of $49 cyc- Cell Membranes
The $49 c y c - cells are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated horse serum. The $49 cells grow as suspension cultures and can be grown in spinner flasks or in T-75 or T-175 bottles. Growth of $49 cells is very sensitive to the pH of the medium, and it is best to maintain a slightly acidic pH (reddish orange). We routinely do this by adding sterile 1 N HCI to the culture medium. Cells should be harvested at a density of 2-3 x 106 cells/ml. 3 D. E. Logothetis, et al., Nature (London) 325, 321 (1987). 4 C. Dietzel and J. Kurgan, Cell (Cambridge, Mass.) 50, 1001 (1987). 5 W.-J. Tang and A. G. Gilman, Science 254, 1500 (1991). 6 B. Gao and A. G. Gilman, Proc. Natl. Acad. Sci. U.S.A. 88, 10178 (1991). 7 M. Camps, H.-H. Lee, D. Park, C.-W. Lee, and K.-H. Lee, Eur. J. Biochem. 206, 821 (1992). 8 D.-K. Jhon, D. J. Yoo, and S. G. Rhee, J. Biol. Chem. 268, 6654 (1993). 9 p. Giershick and M. Camps, this series, Vol. 238 [14]. 10T. Katada, G. M. Bukoch, M. D. Smigel, M. Ui, and A. G. Gilman, J. Biol. Chem. 259, 3586 (1984).
135]
ADENYLYL CYCLASE ASSAY
453
12 A C
E
......
-i-
8
0
E e.,
T
v
o
4
12-
IYY
ll
boiled13"y
FIG. 1. Effect of bovine brain fly subunits on the $49 cyc- adenylyl cyclase. Adenylyl cyclase was assayed in the presence of 10 m M MgC12. When present the concentration of /3~/subunits was 300 mM. The final concentration of Lubrol-PX in the assay was 0.02%. Values are means -+ S.D. of triplicate determinations.
Cells are collected by low-speed centrifugation (I0 min, 900 g) and washed with ice-cold Puck's saline G without divalent cation. After this wash, all further manipulations are in the cold (4°). It is essential that all centrifuge tubes, cylinders, etc., be precooled prior to use. The cells are lysed in 25 mM sodium HEPES, 1 mM EDTA, and 120 mM NaCI, pH 8.0. Typically, cells from 1 liter of medium are suspended in 100 ml of lysis buffer. Cells are lysed by nitrogen cavitation in a Parr bomb. Cells are allowed to equilibrate for 20 min at 400 psi after which the pressure is rapidly released. The lysate is then collected and centrifuged at 900 g for 10 min. The supernatant from the first centrifugation is then centrifuged at 40,000 g for 30 min. The 40,000 g pellet is then resuspended in 10 ml of 25 mM sodium HEPES, I mM EDTA, pH 8.0, and washed by centrifugation at 40,000 g for 30 min. The wash procedure is repeated twice. The final pellet is resuspended in the wash buffer to a final concentration of 1-2 mg/ml protein, divided into aliquots, and snap-frozen in a dry ice-acetone bath. The membranes can be stored at - 7 0 ° without loss of activity for at least 6 months. The membranes can be thawed to 4 ° only once and should be assayed soon (10-15 min) after thawing. Prolonged maintainence on ice or refreezing leads to inactivation of adenylyl cyclase catalytic activity. Preparation
of fly Subunits
Protocols for the purification of/33, subunits are described elsewhere in this volume) l,]z It should be noted that most tissue sources will yield tt T. Katada, K. Kontani, A. Inanobe, I. Kobayashi, Y. Ohoka, H. Nishina, and K. Takahashi, this volume [10]. 12 j. Bigay and M. Chabre, this volume [34].
454
[35]
Gfly SUBUNITS 12
¢-
LO
O
E "O
O la.
< O
0
GDPBS [[~
+
+
GTPyS +
FIG. 2. Stimulation of $49 cyc- adenylyl cyclase by bovine brain/33, subunits in the presence of GDP/3S or GTPyS. The $49 cyc- adenylyl cyclase was assayed at either 10 m M (A) or 2 m M (B) MgC! 2 with no other additions ( - ) or in the presence of 100/~M GDPflS or GTP3,S in the presence or absence of 300 m M added/33, subunits. Values are means _+ S.D. of triplicate determinations.
heterogeneous mixtures of fly subunits. In our laboratory fly subunits are prepared from bovine brain. During elution of the heptylamine column with cholate we invariably obtain free fly subunits at higher (-3%) concentrations of cholate. These fly subunits, which are about 80-90% pure, are pooled and concentrated on a DEAE-Sephacel column. Further purification is obtained on a Mono Q column (Pharmarcia, Piscataway, N J) using conditions previously described. 13 On the salt gradient the fly subunits elute very early. At this stage very pure mixtures of fly subunits can be obtained. There do not appear to be present any measurable amounts of t3 E. Padrell, D. J. Carty, T. M. Moriarity, J. D. Hildebrandt, E. M. Landow, and R. Iyengar, J. Biol. Chem. 266, 9771 (1991).
[35]
ADENYLYL
CYCLASE
ASSAY
455
12
~E Etn ° h o.E v E v
I
I
I
100
200
300
bovine brain [~7 (nM)
FIG. 3. Effect of varying concentrations of added fly subunits on the $49 cyc- adenylyl cyclase. Varying amounts of fly subunits were added to the assay to achieve the indicated concentrations in the final assay mixture. The fly subunits were always added in a volume of 10/zl, and the final concentration of Lubrol-PX was always 0.02%. Adenylyl cyclase activity was measured in the presence of 10 mM MgCI2. Values are means of triplicate determinations. The coefficient of variance was less than 10%. a subunits as assessed by either silver staining or by ADP-ribosylation assays. The 113' subunits are stored in 25 m M sodium H E P E S , 1 m M E D T A , 0.1% Lubrol, 100 m M NaC1, and 20 m M 2-mercaptoethanol at a final concentraion of 6 0 - 1 0 0 / , g / m l . fly-Stimulated Adenylyl Cyclase Assay Standard adenylyl cyclase assay conditions are used. The assay mixture contains 0.1 m M [a-a2p]ATP [1000-2000 counts/min (cpm)/pmol], 25 m M sodium H E P E S , 1 m M E D T A , p H 8.0, indicated amounts of Mg 2÷, and an ATP-regenerating s y s t e m consisting of 20 m M creatine phosphate, 0.2 mg/ml creatine phosphokinase, and 0.02 mg/ml myokinase. The fly subunits and cyc- m e m b r a n e s are added to yield a fir al volume of 50/~1. A b o u t 5 - 1 0 / x g of cyc m e m b r a n e s are used. The 11-. subunits are diluted at least 5-fold so that the final concentration of Lubrol-PX in the assay is no m o r e than 0.02%. The assay mixture is incubated for 15 min at 32 °. At the end of the incubation the assay is terminated and the [32p]cAMP f o r m e d is quantified by the method of Salomon et al.14 Results Addition of 300 n M fly subunits results in a 2-fold stimulation of the basal adenylyl cyclase activity of cyc- m e m b r a n e s . I f the 1t7 subunits are t4 y. Salomon, C. Londos, and M. Rodbell, Anal. Biochem. 58, 541 (1974).
456
G/3-/ SUBUNITS
[35l
boiled prior to use in the assay, no stimulation is observed (Fig. 1). Although the extent of stimulation by /33, subunits is relatively modest, the stimulation is reproducibly observed. Typically we observe a 2-fold stimulation. The stimulation can be observed both at low (2 mM) and higher (I0 mM) Mg 2÷ concentrations (Fig. 2). Addition of saturating concentrations of GDP/3S or GTP3,S does not appear to affect significantly the extent of stimulation by exogenously added/33, subunits at either low or high Mg 2+ concentrations (Fig. 2). Addition of GTP3'S, however, results in lower activities, presumably owing to the activation of Giot. Thus, it appears best to assay the/33, subunits without any added guanine nucleotides. Stimulation by added/33, subunits is dependent on the concentration of/33, subunits used and is saturable (Fig. 3). The concentration required to obtain half-maximal stimulation is around 50 nM. We have not used forskolin in our assays, but essentially similar stimulations with/33, subunits are also observable when forskolin is used. l° The mechanism by which /33, subunits stimulate the $49 cyc- cell membrane adenylyl cyclase is not known. However, the stimulation by /33, subunits is observable only in the absence of Gsa. Addition of/33, subunits to wild-type $49 cell membranes results in inhibition rather than stimulation. ~° Thus, it does not appear likely that the adenylyl cyclase stimulated by/33, subunits in cyc- membranes is like type 2 or 4. Irrespective of the type of adenylyl cyclase stimulated, the $49 cyc- cell membranes offer a very simple assay system to ascertain the biological activity of/33, subunits. Acknowledgment Research was supported by National Institutesof Health Grants CA-44998 and DK-38761.
ADENYLYL CYCLASE ASSAY
451
with a MgCI 2 linear gradient (0-0.33 M, 4.4 mM/min, flow rate 0.5 ml/ min). With this salt gradient, T/iy elutes first, in a sharp peak whose major fractions are not contaminated by Ta, which is already a minor component of the extract and elutes later as a lower and more diffuse peak (not shown). Pooled Tfly fractions from previous TaGTPvS subunit purification batches on the Na2SO4 gradient can be purified after concentration/dilution (buffer A) on a Centricon 30 microconcentrator (Amicon, Danvers, MA) up to a final dilution factor of about 1/10 (less than l0 mM Na2SO4). About 30/.~g of pure Tfly can be obtained after treatment of 1 mg rhodopsin containing membranes. Separation of Modified T3~ Subunits With some transducin extracts, particularly when the preliminary washing step in Hypo buffer, prior to the GTP addition, had been deleted, the elution profile of the final Tfly extract often appears split in two distinct peaks (Fig. 1A). Both peaks contain the same Tfl component (Fig. 1B), but the first minor peak contains a T3~ component which migrates more slowly on urea-polyacrylamide gels (Fig. 1C). Such a separation of two Tfly components with distinctive -y subunits has been described by Fukada et al.,3 who separated Tflyl plus Tfly 2 (first peak) and pure TflYz (second peak) on a DEAE-Toyopearl 650S (Toyo Soda Mfg. Co., Japan) column, then purified the TflY1 complex from TflYz onto a Mono Q HR 5/5 column (Pharmacia). They identified the slower migrating Tyl as a minor defarnesylated component and T72 as a farnesylated and partly carboxymethylated 7 subunit.4'5 4 y. Fukada, T. Takao, H. Ohguro, T. Yoshizawa, T. Akino, and Y. Shimonishi, Nature (London) 346, 658 (1990). 5 H. Ohguro, Y. Fukada, T. Takao, Y. Shimonishi, T. Yoshizawa, and T. Akino, EMBO J. 10, 3669(1991).
[35] A d e n y l y l C y c l a s e A s s a y for f13~ S u b u n i t s o f G P r o t e i n s
By JIANQIANG CHEN, DONNA J. CARTY, and RAvI IYENGAR Introduction Transmission of signals through G proteins can be achieved by the use of either a or/37 subunits.1,2 It had been generally believed that a I j. R. H e p l e r and A. G. Gilman, Trends Biochem. Sci. 17, 383 (1992). 2 L. Birnbanmer, Cell (Cambridge, Mass.) 71, I069 (1992).
METHODS IN ENZYMOLOGY, VOL. 237
Copyright © 1994by AcademicPress, Inc. All fights of reproductionin any form reserved.
452
G/3y SUBUNITS
[35]
subunits were the prime signal transmitters. Only a few systems such as certain types of K ÷ channels 3 and the effector(s) in the yeast pheromone pathway 4 were thought to be regulated by fly subunits. However, it has now been shown that f13, subunits can by themselves regulate the activity of a number of mammalian effectors such as certain types of adenylyl cyclase (adenylate cyclase) 5'6 and phospholipaseC. 7'8 Thus it has become increasingly obvious that fly subunits will play a central role in many signaling pathways. In deciphering the role of fly subunits it is useful to have simple assay systems that can be used to assess the biological activity of the purified or expressed fly subunits. Both adenylyl cyclases as well as phospholipases C offer excellent readout systems for fly subunits. Gilman and co-workers have shown that type 2 and 4 adenylyl cyclases are stimulated by /33, subunits in the presence of activated a2 subunits. 5'6 Giershick and coworkers have shown that phospholipase C from HL-60 cells and neutrophils can be stimulated by/33, subunits. 7 The use of the cytosolic HL-60 cell phospholipase C to measure/33, activity is described elsewhere. 9 In this chapter we describe a very simple adenylyl cyclase assay that can be used to assess the activity of purified/33, subunits. The assay is based on the original observations by Katada et al. 1o that/33, subunits stimulate the mouse lymphoma $49 c y c - cell membrane adenylyl cyclase. This assay works on crude $49 c y c - cell membranes and does not require the presence of any other adenylyl cyclase stimulants for the expression of /33, activity.
Preparation
of $49 cyc- Cell Membranes
The $49 c y c - cells are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated horse serum. The $49 cells grow as suspension cultures and can be grown in spinner flasks or in T-75 or T-175 bottles. Growth of $49 cells is very sensitive to the pH of the medium, and it is best to maintain a slightly acidic pH (reddish orange). We routinely do this by adding sterile 1 N HCI to the culture medium. Cells should be harvested at a density of 2-3 x 106 cells/ml. 3 D. E. Logothetis, et al., Nature (London) 325, 321 (1987). 4 C. Dietzel and J. Kurgan, Cell (Cambridge, Mass.) 50, 1001 (1987). 5 W.-J. Tang and A. G. Gilman, Science 254, 1500 (1991). 6 B. Gao and A. G. Gilman, Proc. Natl. Acad. Sci. U.S.A. 88, 10178 (1991). 7 M. Camps, H.-H. Lee, D. Park, C.-W. Lee, and K.-H. Lee, Eur. J. Biochem. 206, 821 (1992). 8 D.-K. Jhon, D. J. Yoo, and S. G. Rhee, J. Biol. Chem. 268, 6654 (1993). 9 p. Giershick and M. Camps, this series, Vol. 238 [14]. 10T. Katada, G. M. Bukoch, M. D. Smigel, M. Ui, and A. G. Gilman, J. Biol. Chem. 259, 3586 (1984).
135]
ADENYLYL CYCLASE ASSAY
453
12 A C
E
......
-i-
8
0
E e.,
T
v
o
4
12-
IYY
ll
boiled13"y
FIG. 1. Effect of bovine brain fly subunits on the $49 cyc- adenylyl cyclase. Adenylyl cyclase was assayed in the presence of 10 m M MgC12. When present the concentration of /3~/subunits was 300 mM. The final concentration of Lubrol-PX in the assay was 0.02%. Values are means -+ S.D. of triplicate determinations.
Cells are collected by low-speed centrifugation (I0 min, 900 g) and washed with ice-cold Puck's saline G without divalent cation. After this wash, all further manipulations are in the cold (4°). It is essential that all centrifuge tubes, cylinders, etc., be precooled prior to use. The cells are lysed in 25 mM sodium HEPES, 1 mM EDTA, and 120 mM NaCI, pH 8.0. Typically, cells from 1 liter of medium are suspended in 100 ml of lysis buffer. Cells are lysed by nitrogen cavitation in a Parr bomb. Cells are allowed to equilibrate for 20 min at 400 psi after which the pressure is rapidly released. The lysate is then collected and centrifuged at 900 g for 10 min. The supernatant from the first centrifugation is then centrifuged at 40,000 g for 30 min. The 40,000 g pellet is then resuspended in 10 ml of 25 mM sodium HEPES, I mM EDTA, pH 8.0, and washed by centrifugation at 40,000 g for 30 min. The wash procedure is repeated twice. The final pellet is resuspended in the wash buffer to a final concentration of 1-2 mg/ml protein, divided into aliquots, and snap-frozen in a dry ice-acetone bath. The membranes can be stored at - 7 0 ° without loss of activity for at least 6 months. The membranes can be thawed to 4 ° only once and should be assayed soon (10-15 min) after thawing. Prolonged maintainence on ice or refreezing leads to inactivation of adenylyl cyclase catalytic activity. Preparation
of fly Subunits
Protocols for the purification of/33, subunits are described elsewhere in this volume) l,]z It should be noted that most tissue sources will yield tt T. Katada, K. Kontani, A. Inanobe, I. Kobayashi, Y. Ohoka, H. Nishina, and K. Takahashi, this volume [10]. 12 j. Bigay and M. Chabre, this volume [34].
454
[35]
Gfly SUBUNITS 12
¢-
LO
O
E "O
O la.
< O
0
GDPBS [[~
+
+
GTPyS +
FIG. 2. Stimulation of $49 cyc- adenylyl cyclase by bovine brain/33, subunits in the presence of GDP/3S or GTPyS. The $49 cyc- adenylyl cyclase was assayed at either 10 m M (A) or 2 m M (B) MgC! 2 with no other additions ( - ) or in the presence of 100/~M GDPflS or GTP3,S in the presence or absence of 300 m M added/33, subunits. Values are means _+ S.D. of triplicate determinations.
heterogeneous mixtures of fly subunits. In our laboratory fly subunits are prepared from bovine brain. During elution of the heptylamine column with cholate we invariably obtain free fly subunits at higher (-3%) concentrations of cholate. These fly subunits, which are about 80-90% pure, are pooled and concentrated on a DEAE-Sephacel column. Further purification is obtained on a Mono Q column (Pharmarcia, Piscataway, N J) using conditions previously described. 13 On the salt gradient the fly subunits elute very early. At this stage very pure mixtures of fly subunits can be obtained. There do not appear to be present any measurable amounts of t3 E. Padrell, D. J. Carty, T. M. Moriarity, J. D. Hildebrandt, E. M. Landow, and R. Iyengar, J. Biol. Chem. 266, 9771 (1991).
[35]
ADENYLYL
CYCLASE
ASSAY
455
12
~E Etn ° h o.E v E v
I
I
I
100
200
300
bovine brain [~7 (nM)
FIG. 3. Effect of varying concentrations of added fly subunits on the $49 cyc- adenylyl cyclase. Varying amounts of fly subunits were added to the assay to achieve the indicated concentrations in the final assay mixture. The fly subunits were always added in a volume of 10/zl, and the final concentration of Lubrol-PX was always 0.02%. Adenylyl cyclase activity was measured in the presence of 10 mM MgCI2. Values are means of triplicate determinations. The coefficient of variance was less than 10%. a subunits as assessed by either silver staining or by ADP-ribosylation assays. The 113' subunits are stored in 25 m M sodium H E P E S , 1 m M E D T A , 0.1% Lubrol, 100 m M NaC1, and 20 m M 2-mercaptoethanol at a final concentraion of 6 0 - 1 0 0 / , g / m l . fly-Stimulated Adenylyl Cyclase Assay Standard adenylyl cyclase assay conditions are used. The assay mixture contains 0.1 m M [a-a2p]ATP [1000-2000 counts/min (cpm)/pmol], 25 m M sodium H E P E S , 1 m M E D T A , p H 8.0, indicated amounts of Mg 2÷, and an ATP-regenerating s y s t e m consisting of 20 m M creatine phosphate, 0.2 mg/ml creatine phosphokinase, and 0.02 mg/ml myokinase. The fly subunits and cyc- m e m b r a n e s are added to yield a fir al volume of 50/~1. A b o u t 5 - 1 0 / x g of cyc m e m b r a n e s are used. The 11-. subunits are diluted at least 5-fold so that the final concentration of Lubrol-PX in the assay is no m o r e than 0.02%. The assay mixture is incubated for 15 min at 32 °. At the end of the incubation the assay is terminated and the [32p]cAMP f o r m e d is quantified by the method of Salomon et al.14 Results Addition of 300 n M fly subunits results in a 2-fold stimulation of the basal adenylyl cyclase activity of cyc- m e m b r a n e s . I f the 1t7 subunits are t4 y. Salomon, C. Londos, and M. Rodbell, Anal. Biochem. 58, 541 (1974).
456
G/3-/ SUBUNITS
[35l
boiled prior to use in the assay, no stimulation is observed (Fig. 1). Although the extent of stimulation by /33, subunits is relatively modest, the stimulation is reproducibly observed. Typically we observe a 2-fold stimulation. The stimulation can be observed both at low (2 mM) and higher (I0 mM) Mg 2÷ concentrations (Fig. 2). Addition of saturating concentrations of GDP/3S or GTP3,S does not appear to affect significantly the extent of stimulation by exogenously added/33, subunits at either low or high Mg 2+ concentrations (Fig. 2). Addition of GTP3'S, however, results in lower activities, presumably owing to the activation of Giot. Thus, it appears best to assay the/33, subunits without any added guanine nucleotides. Stimulation by added/33, subunits is dependent on the concentration of/33, subunits used and is saturable (Fig. 3). The concentration required to obtain half-maximal stimulation is around 50 nM. We have not used forskolin in our assays, but essentially similar stimulations with/33, subunits are also observable when forskolin is used. l° The mechanism by which /33, subunits stimulate the $49 cyc- cell membrane adenylyl cyclase is not known. However, the stimulation by /33, subunits is observable only in the absence of Gsa. Addition of/33, subunits to wild-type $49 cell membranes results in inhibition rather than stimulation. ~° Thus, it does not appear likely that the adenylyl cyclase stimulated by/33, subunits in cyc- membranes is like type 2 or 4. Irrespective of the type of adenylyl cyclase stimulated, the $49 cyc- cell membranes offer a very simple assay system to ascertain the biological activity of/33, subunits. Acknowledgment Research was supported by National Institutesof Health Grants CA-44998 and DK-38761.