- Email: [email protected]
Acth sensitive adenyl cyclase in bovine adrenal cortex membrane fractions
Life Sciences Yol . 8-, Part I, pp . 935-942, 1989 . Printed in Great Britain.
Pergamon Prees
ACTH SENSITIVE ADENYL CYCLASE IN BCNINE ADRENAL OORTEX t+D~RANE FRACTIONS 0 . Hechter, H.-P . Bhr, M. Matsuba and D. Soifer Institute for Biomedical Research American Medical Association-Education and Research Foundation Chicago, Illinois 60610 (Received 5 May 1989 ; in final form 13 June 1989)
There is considerable evidence that cyclic 3',5'-AMP is the intracellular mediator of ACTH induced steroidogenesis in the adrenal cortex . l~ 2
ACTH is be-
lieved to selectively activate an adenyl cyclase moiety associated with the plasma membrane, the 3',5'-AMP generated acting to induce the steroidogenic response .
A selective effect of ACiH to stimulate adrenal adenyl cyclase acti-
vity has been shown in particulate fractions isolated from homogenates of moose adrenal t~m~or cells . 3 ~ 4 We have undertaken a systematic investigation of adenyl cyclase in bovine adrenal cortex tissué .
The results of fractionating bovine
adrenocortical homogenates by differential centrifugation are presented here, together with same properties of the ACTH-sensitive aderryl cyclases associated with membranous fractions of this tissue . Materials and Methods Bovine adrenal cortex tissue was dissected from fresh glands (collected at a local abbatoir and stored in ice for transport to the laboratory) and homogenized with 9 volumes of MST solution, consisting of mannitol (210 mt+l), suc rose (70 ~ and Tris-HCl (30 mM), pH 7.4 in a Potter all-glass hamogenizer. Homogenization and all subsequent fractionation procedures were carried out at 1-4° .
After filtration through cheesecloth to remove connective tissue and de-
bris, the homogenate was centrifuged twice at 600 y for 10 minutes .
The super-
nates were then centrifuged successively at 8200 g for 10 minutes, and 105,000 4 for 60 minutes .
Each of the pellets obtained was washed once with 1~6T .
fied "mitochondria" were obtained by washing the 8200 q fraction four bees 935
Puri-
938
ADENYL CYCLA3E
Vol. 8, No . 17
with MST (twice at 10,000 g for 10 minutes, and twice at 14,500 g for 10 minutes) .
Electron microscopy (E .M .) revealed that the 105,000 g pellet con-
sisted predominately of typical microsanal membranes contaminated with a small number of mitochondria .
A purified 'Snicroscmal fraction", almost canpletely de-
void of mitochondria, was obtained by centrifuging the 8200 g supernate at 20,000 g for 10 minutes, prior to centrifugation at 105,000 g.
All pellets
were .sugpended in MST containing 0 .1$ bovine seem albumin (BSA) .
The fractions
obtained were assayed within 1 hour or were rapidly frozen (liquid N2), stored at -70° and subsequently assayed immediately after thawing .
Frozen suspensions
retained 70-90$ of cyclase activity for at least 1 month . Adertyl cyclase was assayed by a method developed in this laboratory . 5
The
pH maxim
The final cancentratioais of
components in the standard assay (total volume 0.05 ml) were :
45 mM Tris fiCl
(pH 8.4) ; 5 mM MgC12 ; 0 .1$ BSA (Fraction V, Armour) ; 10 mM phosphoenolpyruvate (Sigma) ; 0 .05 mg/ml pyruvate kinase (Type II, Sigma) ; 1 mM unlabeled 3',5' -~; 50-400 ug/ml protein (determined by the Lvwry method) ; 42 mM mannitol and 14 mM sucrose (resulting from addition of enzyme) ; arld 0 .1 mM ATP-a- 32P (International Che®ioal and Nuclear Corporation, 300-500 Ci/mole) .
The reaction was started
either by addition of protein or ATP and incubations were carried out at 37°, generally for 15 to 30 minutes .
Reactions were stopped by boiling for 3 minutes
after addition of 3',5'-ANIP and ATP (both at 5 mM) .
3',5'-AMP was separated by
chr®atography an precoated polyethyleneimine impregnated cellulose thinlayer sheets (Polygram PEI, Macherey Nagel/Brinkman) developed with 0 .3 M LiCl ; 3', 5'-AMP has an Rf of about 0 .4 while ATP and ADP starting line .
remain
almost entirely at the
The 3',5'-AMP and ATP spots were marked under W light, cut out
and measured by liquid scintillatia¢~ crnmting .
The $ conversion of ATP to 3',
5'-AMP was determined and absolute rates of 3',5'-AMP formation calculated .
ADENYL CYCLASE
Vol. 8, No. 17
887
ATP breakdown occurring during incubation was assessed by chromatography of an aliquot of the incubated sample an TLC-PEI in 1 M LiCl using ATP, AMP and ADP as carriers .
These nucleotides are well separated in this system and Pi (mobi-
lity similar to AMP) is crnmted together with the AMP spot . Synthetic ACTH 91-24 was a gift from Dr . W. Rittl, Ciba, Basel, Switzerland; synthetic glucagan from Dr . E . Jaeger, Max-Planck-Institut, Alfichen, Germany; synthetic aMSH (5-glutamine, 11-N formyl lysyl-a-~ from Prof . K. Hoffman, University of Pittsburgh ; ovine thyrotropin (NIH standard, TSH 1 .44 I .U ./mg) from Prof . N. Freinkel, Northwestern University .
Bovine vasapressin (56 Pressor
Units/mg) was purchased from Mann, L-epinephrine, L-and D-thyraocin and L-and Dtrüodothyranine fran Sigma. Results and Discussion Adenyl cyclase activity was consistently found to be associated with all particulate fractions .
Table 1 shows results of a typical experiment where the
aderryl cyclase activity is distributed between the 600, 8200 and 105,000 g par ticulate fractions ; the 105,000 g supernatant ("cytosol") did not contain detectable activity .
Each of the particulate fractions had higher specific acti-
vity than the total homogenate, both in the presence and absence of added NaF ; moreover the total cyclase activity recovered in the fractions was consistently found to be greater than that assayed in the unfractionated homogenates.
Addi-
tion of the 105,000 g supernate to each of the particulate fractions markedly inhibited cyclase activity (both in the absence and presence of NaF) without influencing the degree of ATP breakdown .
Following heat treatment of the cytosol
fraction (15 minutes at 100°) inhibitory activity was reduced about 501 ; the nature of these inhibitory factor is being investigated . The 8200 g pellet was consistently found to have the highest specific activity (S .A.) .
E.M . revealed that this pellet contained sütodrandria, oontaRi
nated with microsames and other membranous stnrctures .
The S.A . of the cyciase
associated with this fraction as w~eli as the sensitivity to ACIÜ and NaF xas
938
Vol . 8, No. 17
ADENYL CYCLASE
TABLE I Distribution of adenyl cyclase in bovine adrenal cortex hanogenates* Specific Activity (moles/min/mg protein)
Fractiaai
Basal
NaF (10 mM)
Total Activity (pmoles/min/total protein) Basal
NaF (10 mM)
Dole Homogenate
2 .4
9 .0
1050
3820
600 g pellet
6 .5
18 .3
970
2720
8200 g pellet
10 .8
44 .5
625
2560
8 .2
24 .6
590
2520
< 0 .1
< 0 .1
< 1.3
< 1 .3
105,000 g pellet 105,000 g supernate
"Freshly prepared fractions were incubated for 20 minutes as described in "Methods" . unchanged by four additional washes with
NLSI' .
Successive washings reduced mito-
chondrial contamination by other membranes ; however, the contaminating membranes could not be removed by various sucrose gradient centrifugations .
The defini-
tive localization of adenyl cyclase as an integral component of a mitochondrial membrane system thus remains to be established. B .M . revealed that the nuclei in the 600 g pellet were heavily contaminated with mitochondria ; fragments of the plasma membrane are also presumed to be in this fraction .
Purified nuclei, prepared by the method of Pogo et a16, were
found to have negligible cyclase activity, both in the absence and presence of NaF (10 mMJ or ACIH (10 ug/ml) ; hence the cyclase activity in the 600 g pellet cannot be due to rulclei .
Attempts to isolate purified plasma membrane fragments
by sucrose-gradient centrifugation of nuclear fractions were unsuccessful .
Mito-
choaidrial bands have been obtained fran such gradients, indicating that a large part of the cyclase activity in the 6dCl g pellet may result from mitochondria . the contribution of plasma membrane fragments to the cyclase activity of this fraction remains to be established.
Vol .
a,
No. 17
ADENYL CYCLA3E
9S9
MINUTES OF INCUBATION
FIG . 1 The rate of 3',5'-AMP foxmatia~n by purified mitochondria, purified microsomes and 105.,000 g pellets in the absence and presence of ACTH (3 x 10 -~ and NaF (10 ~ . Frozen thawed samples were assayed in a total voluDOe of 0.5 ml, ins tead of 0 .05 ml : aliquots were removed at various intervals and worked up in the standard manner . The final enzyme concentrations were 400, 300, and 300 yg/ml for mitochondria, microsomes and the 105,000 g pellet, respectively . The rate of formation of 3',5'-AMP by purified mitochondria, purified microsomes and of the 105,000 g pellet (microsomes minimally contaminated with "light" mitochondria) is shown in Figure 1 .
In the 105,000 g and purified microsomal
fractions the rates of 3',5'-AMP formation are greater with NaF than with AC1H . Both rates are approximately linear for 45 minutes, whereas basal enzyme activity falls off after 30 minutes .
With purified mitochondria, ACTH is alsoat
equivalent to NaF in stipulating the initial rate of 3',5'-AMP formation; the major differences observed later are due to the fact that with AC1H (aod the
940
ADENYL CYCLASE
Vol . 8~ No. 17
y E _x 'c .E x d Q h
.
'ri UI
0 ô
a
FIG. 2 The increase in 3',5'-AMP formation over basal rate produced by various concentrations of ACTH in mitochondria and the 105,000 g pellet fraction . Basal rates were 10 .8 and 7 .0 pmoles/min/mg for mitochondria and the 105,000 9 fraction, respectively . imstimulated enzyme) the initial rate declined after 20 minutes (although ATP levels were maintained for 60 minutes in the absence and presence of ACiIn whereas with NaF, this did not occur .
In repeated experiments with washed mito-
chondria, (20 minute incubations), ACIH and NaF increased cyclase activity 170$ (range 150-260) and 230$ (180-350), respectively ; with 105,000 g pellets and purified micros®al fractions, the comparable values were 130$ (100-160) and 285$ (210-400), respectively .
Figure 2 shows a typical dosage-response curve
with AC1H on the adenyl cyclase activity of purified mitochondria and the 105,000 g pellet .
In both preparatiaais, maximal effects were obtained with
AC1H concentrations of about 3 x 10 -6 M; half-maximal stimulation was achieved
Vol.
&,
ADENYL CYCLA3E
No . 17
941
with about 2 x 10 -8 M . Bovine vasopressin (10 ug/ml), glucagon (1 and 10 ug/ml), aM3-1 (10 and 20 ug/ml), L-and D-thyroxin (10 ug/~) and L-and D-trüodothyronine (10 ug/ml) had no effect an the adenyl cyclase activity of either washed mitochondria, 105,000 g pellets, or purified microscmes .
Epinephrine (10-20 ug/ml)
in same experi-
ments increased cyclase activity of mitochondria to a very small extent (20 30$), but in other assays had no effect .
Preincubation of mitochondria with
the monamine oxidase inhibitor, pargyline (10 -5 M, 10 minutes at 25°) did not potentiate epinephrine action upon adenyl cyclase activity . The findings presented suggest that the ACTH-sensitive adenyl cyclase in bovine adrenocortical homogenates is associated with multiple membrane systems of the cell, particularly mitochondria .
The question whether adenyl cyclase is
a fixed canpanent of the various membrane systems of the cell or is primarily associated with the plasma membrane, fragmented in different particle sizes by homogenization remains open .
The former possibility would suggest that 3',5'-
AMP, generated and locally utilized in different compartments, may regulate diverse cell functions .
The fact that the rate-limiting enzymes of the cortico-
steroidogenic sequence appear to be localized in adrenal cortex mitochandria~~ 8 association with an ACIH-sensitive cyclase, poses the question whether AC1H acts at a mitochondrial locus, as well as at the surface of the adrenocortical cell . 9 S~amiary Adenyl cyclase in bovine adrenal cortex was distributed in all particulate fractions obtained by differential centrifugation ; factors in the 105,000 q supernate markedly inhibited cyclase activity . enzyme is associated with mitochondria .
A major fraction of the total
In all membranous fractions, the en-
zyme was stimulated by NaF and IU.TH, but not by IKSH, thyrotrapin, glucaQaa or vasopressin ; epinephrine variably stimulated cyclase to a small extent relative to ACTH .
ADENYL CYCLASE
942
Vol . 8., No . 17
Aclmvwledgement - The authors thank David Ya~nkers for his skillful technical assistance . References 1.
E . W . ~, G . A . ROBISdd AND R . W . BITfC[-D;R, Circulatian 37, 279 (1968), _
2.
D . G . GRAHAM-SNIITH, R . W . B[Ti~DäR, R . L . NEY AND E . W . SUI~RLAND, Clin . Res . 15, 259 (1967) .
3.
0 . D . TAINICYd, J . BOTH AND I . PASTAN, Biochem. Biophys . Res . Camm . 29, 1 (1967) . _
4.
0 . D . TAUNTON, J . ROTH AND I . PASTAN, J . Biol . Chew . 244, 247 (1969) .
5.
H .-P . BAR AIm 0 . iBrCI-IIER, Anal . Biochem . i n press (1969) .
6.
A . 0 . POGO, V . G . ALLFREY AND A . E . MIRSKY, Proc . Nat . Acad . Sci . (U .S .) 56, 550 (1966) .
7.
0 . IB;C[f1ER AID I . D . K . HALICERSICN, in G . Pintas, K . V . Thimann and E . B . Astwood, Ed ., The Hormoaies S, 687 . Academic Press, New York (1964) .
8.
G . C . KARABOYAS AND J . B . KORITZ, Biochem . 4, 462 (1965) .
9.
0 . D . TAIMIDN, J . R(XfH AND I, PASTAN, J . Clin . Invest . 46, 1122 (1967) .
Pergamon Prees
ACTH SENSITIVE ADENYL CYCLASE IN BCNINE ADRENAL OORTEX t+D~RANE FRACTIONS 0 . Hechter, H.-P . Bhr, M. Matsuba and D. Soifer Institute for Biomedical Research American Medical Association-Education and Research Foundation Chicago, Illinois 60610 (Received 5 May 1989 ; in final form 13 June 1989)
There is considerable evidence that cyclic 3',5'-AMP is the intracellular mediator of ACTH induced steroidogenesis in the adrenal cortex . l~ 2
ACTH is be-
lieved to selectively activate an adenyl cyclase moiety associated with the plasma membrane, the 3',5'-AMP generated acting to induce the steroidogenic response .
A selective effect of ACiH to stimulate adrenal adenyl cyclase acti-
vity has been shown in particulate fractions isolated from homogenates of moose adrenal t~m~or cells . 3 ~ 4 We have undertaken a systematic investigation of adenyl cyclase in bovine adrenal cortex tissué .
The results of fractionating bovine
adrenocortical homogenates by differential centrifugation are presented here, together with same properties of the ACTH-sensitive aderryl cyclases associated with membranous fractions of this tissue . Materials and Methods Bovine adrenal cortex tissue was dissected from fresh glands (collected at a local abbatoir and stored in ice for transport to the laboratory) and homogenized with 9 volumes of MST solution, consisting of mannitol (210 mt+l), suc rose (70 ~ and Tris-HCl (30 mM), pH 7.4 in a Potter all-glass hamogenizer. Homogenization and all subsequent fractionation procedures were carried out at 1-4° .
After filtration through cheesecloth to remove connective tissue and de-
bris, the homogenate was centrifuged twice at 600 y for 10 minutes .
The super-
nates were then centrifuged successively at 8200 g for 10 minutes, and 105,000 4 for 60 minutes .
Each of the pellets obtained was washed once with 1~6T .
fied "mitochondria" were obtained by washing the 8200 q fraction four bees 935
Puri-
938
ADENYL CYCLA3E
Vol. 8, No . 17
with MST (twice at 10,000 g for 10 minutes, and twice at 14,500 g for 10 minutes) .
Electron microscopy (E .M .) revealed that the 105,000 g pellet con-
sisted predominately of typical microsanal membranes contaminated with a small number of mitochondria .
A purified 'Snicroscmal fraction", almost canpletely de-
void of mitochondria, was obtained by centrifuging the 8200 g supernate at 20,000 g for 10 minutes, prior to centrifugation at 105,000 g.
All pellets
were .sugpended in MST containing 0 .1$ bovine seem albumin (BSA) .
The fractions
obtained were assayed within 1 hour or were rapidly frozen (liquid N2), stored at -70° and subsequently assayed immediately after thawing .
Frozen suspensions
retained 70-90$ of cyclase activity for at least 1 month . Adertyl cyclase was assayed by a method developed in this laboratory . 5
The
pH maxim
The final cancentratioais of
components in the standard assay (total volume 0.05 ml) were :
45 mM Tris fiCl
(pH 8.4) ; 5 mM MgC12 ; 0 .1$ BSA (Fraction V, Armour) ; 10 mM phosphoenolpyruvate (Sigma) ; 0 .05 mg/ml pyruvate kinase (Type II, Sigma) ; 1 mM unlabeled 3',5' -~; 50-400 ug/ml protein (determined by the Lvwry method) ; 42 mM mannitol and 14 mM sucrose (resulting from addition of enzyme) ; arld 0 .1 mM ATP-a- 32P (International Che®ioal and Nuclear Corporation, 300-500 Ci/mole) .
The reaction was started
either by addition of protein or ATP and incubations were carried out at 37°, generally for 15 to 30 minutes .
Reactions were stopped by boiling for 3 minutes
after addition of 3',5'-ANIP and ATP (both at 5 mM) .
3',5'-AMP was separated by
chr®atography an precoated polyethyleneimine impregnated cellulose thinlayer sheets (Polygram PEI, Macherey Nagel/Brinkman) developed with 0 .3 M LiCl ; 3', 5'-AMP has an Rf of about 0 .4 while ATP and ADP starting line .
remain
almost entirely at the
The 3',5'-AMP and ATP spots were marked under W light, cut out
and measured by liquid scintillatia¢~ crnmting .
The $ conversion of ATP to 3',
5'-AMP was determined and absolute rates of 3',5'-AMP formation calculated .
ADENYL CYCLASE
Vol. 8, No. 17
887
ATP breakdown occurring during incubation was assessed by chromatography of an aliquot of the incubated sample an TLC-PEI in 1 M LiCl using ATP, AMP and ADP as carriers .
These nucleotides are well separated in this system and Pi (mobi-
lity similar to AMP) is crnmted together with the AMP spot . Synthetic ACTH 91-24 was a gift from Dr . W. Rittl, Ciba, Basel, Switzerland; synthetic glucagan from Dr . E . Jaeger, Max-Planck-Institut, Alfichen, Germany; synthetic aMSH (5-glutamine, 11-N formyl lysyl-a-~ from Prof . K. Hoffman, University of Pittsburgh ; ovine thyrotropin (NIH standard, TSH 1 .44 I .U ./mg) from Prof . N. Freinkel, Northwestern University .
Bovine vasapressin (56 Pressor
Units/mg) was purchased from Mann, L-epinephrine, L-and D-thyraocin and L-and Dtrüodothyranine fran Sigma. Results and Discussion Adenyl cyclase activity was consistently found to be associated with all particulate fractions .
Table 1 shows results of a typical experiment where the
aderryl cyclase activity is distributed between the 600, 8200 and 105,000 g par ticulate fractions ; the 105,000 g supernatant ("cytosol") did not contain detectable activity .
Each of the particulate fractions had higher specific acti-
vity than the total homogenate, both in the presence and absence of added NaF ; moreover the total cyclase activity recovered in the fractions was consistently found to be greater than that assayed in the unfractionated homogenates.
Addi-
tion of the 105,000 g supernate to each of the particulate fractions markedly inhibited cyclase activity (both in the absence and presence of NaF) without influencing the degree of ATP breakdown .
Following heat treatment of the cytosol
fraction (15 minutes at 100°) inhibitory activity was reduced about 501 ; the nature of these inhibitory factor is being investigated . The 8200 g pellet was consistently found to have the highest specific activity (S .A.) .
E.M . revealed that this pellet contained sütodrandria, oontaRi
nated with microsames and other membranous stnrctures .
The S.A . of the cyciase
associated with this fraction as w~eli as the sensitivity to ACIÜ and NaF xas
938
Vol . 8, No. 17
ADENYL CYCLASE
TABLE I Distribution of adenyl cyclase in bovine adrenal cortex hanogenates* Specific Activity (moles/min/mg protein)
Fractiaai
Basal
NaF (10 mM)
Total Activity (pmoles/min/total protein) Basal
NaF (10 mM)
Dole Homogenate
2 .4
9 .0
1050
3820
600 g pellet
6 .5
18 .3
970
2720
8200 g pellet
10 .8
44 .5
625
2560
8 .2
24 .6
590
2520
< 0 .1
< 0 .1
< 1.3
< 1 .3
105,000 g pellet 105,000 g supernate
"Freshly prepared fractions were incubated for 20 minutes as described in "Methods" . unchanged by four additional washes with
NLSI' .
Successive washings reduced mito-
chondrial contamination by other membranes ; however, the contaminating membranes could not be removed by various sucrose gradient centrifugations .
The defini-
tive localization of adenyl cyclase as an integral component of a mitochondrial membrane system thus remains to be established. B .M . revealed that the nuclei in the 600 g pellet were heavily contaminated with mitochondria ; fragments of the plasma membrane are also presumed to be in this fraction .
Purified nuclei, prepared by the method of Pogo et a16, were
found to have negligible cyclase activity, both in the absence and presence of NaF (10 mMJ or ACIH (10 ug/ml) ; hence the cyclase activity in the 600 g pellet cannot be due to rulclei .
Attempts to isolate purified plasma membrane fragments
by sucrose-gradient centrifugation of nuclear fractions were unsuccessful .
Mito-
choaidrial bands have been obtained fran such gradients, indicating that a large part of the cyclase activity in the 6dCl g pellet may result from mitochondria . the contribution of plasma membrane fragments to the cyclase activity of this fraction remains to be established.
Vol .
a,
No. 17
ADENYL CYCLA3E
9S9
MINUTES OF INCUBATION
FIG . 1 The rate of 3',5'-AMP foxmatia~n by purified mitochondria, purified microsomes and 105.,000 g pellets in the absence and presence of ACTH (3 x 10 -~ and NaF (10 ~ . Frozen thawed samples were assayed in a total voluDOe of 0.5 ml, ins tead of 0 .05 ml : aliquots were removed at various intervals and worked up in the standard manner . The final enzyme concentrations were 400, 300, and 300 yg/ml for mitochondria, microsomes and the 105,000 g pellet, respectively . The rate of formation of 3',5'-AMP by purified mitochondria, purified microsomes and of the 105,000 g pellet (microsomes minimally contaminated with "light" mitochondria) is shown in Figure 1 .
In the 105,000 g and purified microsomal
fractions the rates of 3',5'-AMP formation are greater with NaF than with AC1H . Both rates are approximately linear for 45 minutes, whereas basal enzyme activity falls off after 30 minutes .
With purified mitochondria, ACTH is alsoat
equivalent to NaF in stipulating the initial rate of 3',5'-AMP formation; the major differences observed later are due to the fact that with AC1H (aod the
940
ADENYL CYCLASE
Vol . 8~ No. 17
y E _x 'c .E x d Q h
.
'ri UI
0 ô
a
FIG. 2 The increase in 3',5'-AMP formation over basal rate produced by various concentrations of ACTH in mitochondria and the 105,000 g pellet fraction . Basal rates were 10 .8 and 7 .0 pmoles/min/mg for mitochondria and the 105,000 9 fraction, respectively . imstimulated enzyme) the initial rate declined after 20 minutes (although ATP levels were maintained for 60 minutes in the absence and presence of ACiIn whereas with NaF, this did not occur .
In repeated experiments with washed mito-
chondria, (20 minute incubations), ACIH and NaF increased cyclase activity 170$ (range 150-260) and 230$ (180-350), respectively ; with 105,000 g pellets and purified micros®al fractions, the comparable values were 130$ (100-160) and 285$ (210-400), respectively .
Figure 2 shows a typical dosage-response curve
with AC1H on the adenyl cyclase activity of purified mitochondria and the 105,000 g pellet .
In both preparatiaais, maximal effects were obtained with
AC1H concentrations of about 3 x 10 -6 M; half-maximal stimulation was achieved
Vol.
&,
ADENYL CYCLA3E
No . 17
941
with about 2 x 10 -8 M . Bovine vasopressin (10 ug/ml), glucagon (1 and 10 ug/ml), aM3-1 (10 and 20 ug/ml), L-and D-thyroxin (10 ug/~) and L-and D-trüodothyronine (10 ug/ml) had no effect an the adenyl cyclase activity of either washed mitochondria, 105,000 g pellets, or purified microscmes .
Epinephrine (10-20 ug/ml)
in same experi-
ments increased cyclase activity of mitochondria to a very small extent (20 30$), but in other assays had no effect .
Preincubation of mitochondria with
the monamine oxidase inhibitor, pargyline (10 -5 M, 10 minutes at 25°) did not potentiate epinephrine action upon adenyl cyclase activity . The findings presented suggest that the ACTH-sensitive adenyl cyclase in bovine adrenocortical homogenates is associated with multiple membrane systems of the cell, particularly mitochondria .
The question whether adenyl cyclase is
a fixed canpanent of the various membrane systems of the cell or is primarily associated with the plasma membrane, fragmented in different particle sizes by homogenization remains open .
The former possibility would suggest that 3',5'-
AMP, generated and locally utilized in different compartments, may regulate diverse cell functions .
The fact that the rate-limiting enzymes of the cortico-
steroidogenic sequence appear to be localized in adrenal cortex mitochandria~~ 8 association with an ACIH-sensitive cyclase, poses the question whether AC1H acts at a mitochondrial locus, as well as at the surface of the adrenocortical cell . 9 S~amiary Adenyl cyclase in bovine adrenal cortex was distributed in all particulate fractions obtained by differential centrifugation ; factors in the 105,000 q supernate markedly inhibited cyclase activity . enzyme is associated with mitochondria .
A major fraction of the total
In all membranous fractions, the en-
zyme was stimulated by NaF and IU.TH, but not by IKSH, thyrotrapin, glucaQaa or vasopressin ; epinephrine variably stimulated cyclase to a small extent relative to ACTH .
ADENYL CYCLASE
942
Vol . 8., No . 17
Aclmvwledgement - The authors thank David Ya~nkers for his skillful technical assistance . References 1.
E . W . ~, G . A . ROBISdd AND R . W . BITfC[-D;R, Circulatian 37, 279 (1968), _
2.
D . G . GRAHAM-SNIITH, R . W . B[Ti~DäR, R . L . NEY AND E . W . SUI~RLAND, Clin . Res . 15, 259 (1967) .
3.
0 . D . TAINICYd, J . BOTH AND I . PASTAN, Biochem. Biophys . Res . Camm . 29, 1 (1967) . _
4.
0 . D . TAUNTON, J . ROTH AND I . PASTAN, J . Biol . Chew . 244, 247 (1969) .
5.
H .-P . BAR AIm 0 . iBrCI-IIER, Anal . Biochem . i n press (1969) .
6.
A . 0 . POGO, V . G . ALLFREY AND A . E . MIRSKY, Proc . Nat . Acad . Sci . (U .S .) 56, 550 (1966) .
7.
0 . IB;C[f1ER AID I . D . K . HALICERSICN, in G . Pintas, K . V . Thimann and E . B . Astwood, Ed ., The Hormoaies S, 687 . Academic Press, New York (1964) .
8.
G . C . KARABOYAS AND J . B . KORITZ, Biochem . 4, 462 (1965) .
9.
0 . D . TAIMIDN, J . R(XfH AND I, PASTAN, J . Clin . Invest . 46, 1122 (1967) .