- Email: [email protected]
Characterization of [3H] corticosterone binding activity in adrenal incubation media
2925
445
OF [3Hl CORl?ICWTEXBE BINDING XTIVITY IN ADRENAL
-2ATIoN
IXUBATION MEDIA
P. G. Canpbell, J. F. Pritchett,
D. N. Marple, M. L. Till and C. H. Pahe
Departments of Animal and Dairy Science and Zoology-Hntanology; Agricultural HxperimentStation; Auburn Univ., Alabama36849 Received 2-11-82 Glucocorticoid-binding activity in adrenal incubation media was investigated with regard to characterization of a protein-like ligand. Scatchard analysis of corticosterone binding activity indicated the presence of a single non-in ratting protein with a dissociation constant (Kd) of 8.81 x 1018 M (OOC), a value which is different from that of plasma and cytoplasmic glucocorticoid binding proteins. In addition, an observed lack of affinity of the protein for dexamethasone distinguishes the protein fran Type II cytoplasmic receptor proteins. Thus our data suggest a glucocorticoid-binding protein which is distinct frcm the two krmn groups of glucocorticoid-binding proteins, corticosteroid-binding globulin (CBG)and cytoplasmic receptors.
Cmpetitive corticoids
protein bihding KPH) radioassay procedures for plasma
routinely involve an initial
extraction to remove proteins
which might otherwise compete with the assay protein for the steroid being guantitated.
Such extractions have been perform& in our
laboratory using dichloranethane as described by Murphy (1). Since
our laboratory is involved with nvmitoring invitro
adrenocortical
responsiveness to ACIHduring successive incubations and
since plasma protein concentrations were expected to be minimal in adrenal incubation media, we attempted to mdify CPHprocedures for steroid assay through elimination of the extraction step. caqarisons
of corticosteroid
were consistently
However,
values obtained fran nonextracted saqles
lower than those obtained from the sams samples
extracted with dichloranethane, an cbservation which suggested the Vohme
39,
Number 4
S
TD=OXDm
ApriZ,
2982
S
TBEOXDI
existence of a steroid-binding ligmd(s) in the media. Biological evaluationsof this bindingactivity haveindicated thatit is sensitive
to bothin X&Q andin yi&r~stimulation, thatit exhibits a circadian rhythmsimilarto thatof corticosteriod secretion in the rat,and that it is of probableadrenalorigin(2). In yifrcr adrenalreleaseof potential corticosteroid-binding proteinshas alsobeensuggested by others(3). The presentinvestigation was conducted to characterize the 1igandW so as to providea basisof coqmrisonwith otherknown corticosteroid-binding substances foundwithinthe adrenal. !WlJWXSZ4NDMGIHcoS
Male,Sprague-Dawley ratswere obtained and housedfor 1 to 3 monthsin a controlled environment maintained at 24 at1oC witha 14 hr light:10 hr darkphotoperiod. PurinaLaboratory Chowand tapwaterware consumed &J&&urn. All experimental procedures were performed on rats rangingfrom90-160daysof age maintained in groupsof 1 to 3 per cage. Animalsweremovedto singlecages1 hr priorto sacrifice and placedin a rocmseparatefrcmthe housingfacility.Animalswere sacrificed by decapitation betwe= 1900-2100 hrs sincerat adrenals exhibittheir*ak glucocorticoid production activity duringthe early trunkbloodwas collected fOK eveninghours (4,s). Upondecapit&iOn, serumand plasma,and the adrenals were remwed,quartered, and maintainedat4oCinKrebsRingerbicarbonatebuffer containing 2nq!ml glucose(KREC)and thanplacedin separate plasticincubation beakers in a Dubnoffmetabolic containing 2ml of KFBG. Tissueswere incubated shakerat 37oCand 60 oscillations/min undera 95% 02:5%Co2 atmosphere for 60 minutes.
Mediafrom7 separate adrenalincubationswere pooledtoestimate bindingproperties of therredia protein(s).The er&genous glucocorticoids were remOvedfranthe mediaby mixing500 ~1 of the pooledmedia (freshor flash-frozen in liquidnitrogen) withan equal. volumeof 2.5%dextran-coated charcoalWC) in 0.01M, pH 7.4 phosphate bufferedsalineW3S) and shakingthismixtureat 370cfor 45 min. The mediawere thencentrifuged (4W for 30 min (2000x g), and the supernatants were filtered twicethrough45 urnfilters(Millipore Corporation: B&ford, MA).To 500 ul of pooledstrippea mediawere added 25 pg of tritiated corticosterone (Specific activity = 182.1CifmnoL;
New England Nuclear; Boston, MAI in 100 ~1 of PBS containing 1% gelatin,
#I 7.4 GE’S) and 0, 50, 100, 200, 400, 800, or 1600 pg of unlabeled corticosterone in 100 ~1 of GEBS. Duplicates were prepared for each concentration of unlabeled corticosterone. Tubes were vortexed and incubated at OOCfor a minima of 6 hrs. Separation of free from bound steroid was via charcoal adsorption (500 u 1 0.25% DCC) followed by centrifugation (2000 x g for 10 min, 4W. Nine lmndred ~1 of the supernatant (representing the bound fraction) were counted in a scintillation solution of toluene-Triton X-100 (2:l) with Qmifluor (6 g/l; NewRqland Nuclear: Boston, MA). The ratio of bound to free corticosterone was plotted against the molar concentration of corticosterone bound according to the method of Scatchard (6). Rat plama was strip@ in a manner similar to that used for media. The stripped plasma was then incubated at dilutions corresponding to 0.5 and 0.25% of tile plasma and a Scatchard plot was constructed as above.
Fooled incubation media were prepared and stripped of endogenous glucocorticoids in a manner similar to the method used for the Scatchard analysis. Following stripping, tritiated corticosterone (Specific activity = 182.1 Ci/mnol; New&gland Wzlear; Boston, MA) (25 ~1 was added to 500 ul str@ped mediumin cunbination with either 0, 130, or 1000 pg of unlabeled corticosterone or dexamethasone ImX;9-fluorc-118r 17,21-trihydmxy-16a-mthyl1,4-pregnadiene-3,2O-diane) in 100 u 1 of GPBS. Tubes were incubated at OOCfor a minima of 6 hrs. Percent binding of the tritiated c%qonentJas d??te+ed and the degfee of x;zAz for a specific cortxold at a specific concentratmn . RFSULTS
Scatchard analysis of corticosterone-binding
activity
incubation media resulted in an estimated dissociation 8.81 x lo-10 M (Figure 1). class of specific protein.
in adrenal
constant U(d) of
The linear Scatchard plot suggested a single
sites for corticosterone,
The Kd of corticosterone-binding
thus a single species of globulin
mm,
usbg
nm-incubated stripped rat plasma, was estimated to be 5.00 and 2.40 x 109 M for 0.5% and 0.25% plasma respectively.
S
448
m
Representative Scatchardplot utilizingL3Hlcorticosterone in strippedadrenalmedium. Five replications utilizing differentmedia pools resultedin an estimatedKd of 8.81 + 0.64 x lo-10M.
130
E&U&L
'I?PEOIDI
1000
Corticosterone and dexamethasone competitionfor specific t3Blcorticosterone-binding sites in strippedadrenal incubationmedium. Total specificbindingin the absenceof a canpetitoris assigneda value of 100%.
s The affinity corticosterone
of media protein for DEXin the presence of
was investigated by cuqaring
unlabeled corticosterone
readily with tritiated any great extent.
the ccmpetition of
and DEXwith tritiated
are presented in Figure 2.
indicator,
449
TBEOfDI
corticosterone.
Results
Corticosterone was observed to compete
corticosterone
whereas DEXfailed
to cmpete to
Thus, using the degree of ccmpetition as an
the affinity
of the media protein was high for corticosterone
but very law for DEX. DI!XlJSSION The Kd estimate of 8.81 x lo-10 M (OW for the media protein of adrenals is compared to Kd values of glucocorticoid
cytoplasm and glucocorticoid-binding
receptors in the
proteins in the blood in Table 1.
The Kd for the protein in adrenal media is approximately one-tenth that for any other glucocorticoid Type I receptors.
binding protein cited with the exception of
However, the media protein can be readily
distinguished fran the Typa I receptor since in caqetition
studies it
has been demonstrated that the Type I receptor has a lower affinity that of aldosterone) for corticosterone
(2%
and DEX (7).
A direct canparison of Kd values to those for adrenal cytoplasmic receptors CQpe II) cannot be made since those Kd estimates exist only for DEX, but the lack of a high affinity
for DEXby the protein in the
media of adrenal incubations readily distinguishes receptors.
The observation that the media protein has a high affinity
for corticosterone affinity
it from cytoplamic
while being very low for DEXis in contrast to the
of cytosol proteins
both corticosterone
(Type II) of rat adrenals which is high for
and DEX (8).
Similar canparisons can be made to
v 0
”
2 w
0
O
::
v
0n
f v
Oo
9‘;;‘ 0
3. rr)
0
w
0
0
w5; 9s 0
0
. m
hl
v
ws ? =: “x m
1. 0
u v
vu
00
2
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W
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uv
W
00
I--*
m
0
u h
m
W
cytosol proteins higher affinity
(T&e III)
fran the bovine adrenal cortex which have a
for DEXthan cortisol
The binding affinities
or corticosterone
for corticosterone
similar to those of the media protein (10). !l?ypeII cytosol
and DEXby CBGwere The differential
binding of
receptors and CPGfor DEXis used to differentiate
two glucocorticoid affinities
(9) .
binders (11).
the
However, the similar binding
of media protein and CBGdo not necessarily
indicate that the
media protein is CBG. CDGas well as !FypeIII receptors have similar affinities
for DEXbut still
differ
with respect to other indices (12).
The present results indicate that CBGand the media protein from adrenal incubations have similar binding affinities
for corticosterone
and DEX.
However, it is doubtful that the media protein is CBGsince the principal
source of CBGis the liver.
Previous reports havs suggested
that the adrenal produces the protein in question, since media binding activity y&m
may be altered by the addition of XTH to adrenal tissue i,n
(2,3).
Furthermore, binding activity
rhythm of steroidogenesis In conclusion, glucocorticoid-binding
parallels
the circadian
(2) whereas CBGdoes not (13).
our data suggest the existence of a protein distinct
groups of glucocorticoid-binding
frm the two presently knmn
proteins,
CBGand cytoplasmic
receptors.
The secretarial assistance of Mrs. Wilrm MaddoxarCi Mrs. Charlotte Barnes is gratefully acknawledged. This manuscript is No. 4-810153 of the AlabamaAgricultural Experimant Station Journal Series.
452
S
TD&OIDS
REFERENCES 1.
Murphy,B.E.P.;J. Clin. Endocrinol.Metab.22~ 973-990 (1967). 2. Pritchett,J. F., Harper,W. L., Marple,D. N., Bradley,J. T., and Till, M. L.; Biochem.Biophys.Res. Camun. 9Q: 1355-1363(1979). 3. Goddard,C., Vinson,G. P., and Whitehouse,B. J.; J. Endocrinol. 71: lOP-1lP (1978). 4. Ret&me, K., Zimmxman, E., Schindler,W. J., Neuenschwander, J., and Lipscomb,H. S.; Acta Endocrinol.ZZ: 615-622 (1968). 5. Ran!aley, J. A.; SteroidsX: 611-630 (1972). 6. Scatchard,G.; Ann. NY Acad. Sci. a: 660-672 (1949). 7. Funder,J. W., Feldman,D. and Edelmn, I. S.; Endocrinol.92: 1005-1013(1973). 8. Loose,D. S., Do, Y. S., Chen, T. L., and Feldman,D,; Endccrinol. lQZ: 137-146 (1980) 9. Cachet,C., Job D., Dhien,A., and Chambaz,E. M.; Arch. Biochem. Biophys.l&Q: l-9 (1977). 10. Westphal,U.; J. Reprod.Fert. Suppl.l.Q:15-38 (1970). 11. Munck,A. and Leung,K. in Receptorsand Mechanismof Action of SteroidHomones, Part II (Pasqualini, J. R., Ed.), MarcelDekker, Inc.,New York (19761,pp 311-397. 12. Feldman,D., Funder,J. W. and Edelman,I. S.; Endocrinol.92: 1429-1441(1973). 13. Keller,N., SendelbeckL. R., Richardson,U. I., Moore C. and Yates, F. E.; Endocrinol.a: 884-906 (1966). 14. Funder,,J. W., Feldman,D. and Edelmn, I. S.; Endocrinol.42: 1005-1013(1973). 15. Rousseau,G. G., Baxter,J. D. and Tankins,G. M.; J. Mol. Biol.&,Z: 99-115 (1972). 16. Wagner,R. K.; Acta Endocrinol.Suppl.2l& 5-73 (1978).
J. F. Pritchett PhysiologyLaboratories Department of Zoology AuburnUniversity,AL 36849
445
OF [3Hl CORl?ICWTEXBE BINDING XTIVITY IN ADRENAL
-2ATIoN
IXUBATION MEDIA
P. G. Canpbell, J. F. Pritchett,
D. N. Marple, M. L. Till and C. H. Pahe
Departments of Animal and Dairy Science and Zoology-Hntanology; Agricultural HxperimentStation; Auburn Univ., Alabama36849 Received 2-11-82 Glucocorticoid-binding activity in adrenal incubation media was investigated with regard to characterization of a protein-like ligand. Scatchard analysis of corticosterone binding activity indicated the presence of a single non-in ratting protein with a dissociation constant (Kd) of 8.81 x 1018 M (OOC), a value which is different from that of plasma and cytoplasmic glucocorticoid binding proteins. In addition, an observed lack of affinity of the protein for dexamethasone distinguishes the protein fran Type II cytoplasmic receptor proteins. Thus our data suggest a glucocorticoid-binding protein which is distinct frcm the two krmn groups of glucocorticoid-binding proteins, corticosteroid-binding globulin (CBG)and cytoplasmic receptors.
Cmpetitive corticoids
protein bihding KPH) radioassay procedures for plasma
routinely involve an initial
extraction to remove proteins
which might otherwise compete with the assay protein for the steroid being guantitated.
Such extractions have been perform& in our
laboratory using dichloranethane as described by Murphy (1). Since
our laboratory is involved with nvmitoring invitro
adrenocortical
responsiveness to ACIHduring successive incubations and
since plasma protein concentrations were expected to be minimal in adrenal incubation media, we attempted to mdify CPHprocedures for steroid assay through elimination of the extraction step. caqarisons
of corticosteroid
were consistently
However,
values obtained fran nonextracted saqles
lower than those obtained from the sams samples
extracted with dichloranethane, an cbservation which suggested the Vohme
39,
Number 4
S
TD=OXDm
ApriZ,
2982
S
TBEOXDI
existence of a steroid-binding ligmd(s) in the media. Biological evaluationsof this bindingactivity haveindicated thatit is sensitive
to bothin X&Q andin yi&r~stimulation, thatit exhibits a circadian rhythmsimilarto thatof corticosteriod secretion in the rat,and that it is of probableadrenalorigin(2). In yifrcr adrenalreleaseof potential corticosteroid-binding proteinshas alsobeensuggested by others(3). The presentinvestigation was conducted to characterize the 1igandW so as to providea basisof coqmrisonwith otherknown corticosteroid-binding substances foundwithinthe adrenal. !WlJWXSZ4NDMGIHcoS
Male,Sprague-Dawley ratswere obtained and housedfor 1 to 3 monthsin a controlled environment maintained at 24 at1oC witha 14 hr light:10 hr darkphotoperiod. PurinaLaboratory Chowand tapwaterware consumed &J&&urn. All experimental procedures were performed on rats rangingfrom90-160daysof age maintained in groupsof 1 to 3 per cage. Animalsweremovedto singlecages1 hr priorto sacrifice and placedin a rocmseparatefrcmthe housingfacility.Animalswere sacrificed by decapitation betwe= 1900-2100 hrs sincerat adrenals exhibittheir*ak glucocorticoid production activity duringthe early trunkbloodwas collected fOK eveninghours (4,s). Upondecapit&iOn, serumand plasma,and the adrenals were remwed,quartered, and maintainedat4oCinKrebsRingerbicarbonatebuffer containing 2nq!ml glucose(KREC)and thanplacedin separate plasticincubation beakers in a Dubnoffmetabolic containing 2ml of KFBG. Tissueswere incubated shakerat 37oCand 60 oscillations/min undera 95% 02:5%Co2 atmosphere for 60 minutes.
Mediafrom7 separate adrenalincubationswere pooledtoestimate bindingproperties of therredia protein(s).The er&genous glucocorticoids were remOvedfranthe mediaby mixing500 ~1 of the pooledmedia (freshor flash-frozen in liquidnitrogen) withan equal. volumeof 2.5%dextran-coated charcoalWC) in 0.01M, pH 7.4 phosphate bufferedsalineW3S) and shakingthismixtureat 370cfor 45 min. The mediawere thencentrifuged (4W for 30 min (2000x g), and the supernatants were filtered twicethrough45 urnfilters(Millipore Corporation: B&ford, MA).To 500 ul of pooledstrippea mediawere added 25 pg of tritiated corticosterone (Specific activity = 182.1CifmnoL;
New England Nuclear; Boston, MAI in 100 ~1 of PBS containing 1% gelatin,
#I 7.4 GE’S) and 0, 50, 100, 200, 400, 800, or 1600 pg of unlabeled corticosterone in 100 ~1 of GEBS. Duplicates were prepared for each concentration of unlabeled corticosterone. Tubes were vortexed and incubated at OOCfor a minima of 6 hrs. Separation of free from bound steroid was via charcoal adsorption (500 u 1 0.25% DCC) followed by centrifugation (2000 x g for 10 min, 4W. Nine lmndred ~1 of the supernatant (representing the bound fraction) were counted in a scintillation solution of toluene-Triton X-100 (2:l) with Qmifluor (6 g/l; NewRqland Nuclear: Boston, MA). The ratio of bound to free corticosterone was plotted against the molar concentration of corticosterone bound according to the method of Scatchard (6). Rat plama was strip@ in a manner similar to that used for media. The stripped plasma was then incubated at dilutions corresponding to 0.5 and 0.25% of tile plasma and a Scatchard plot was constructed as above.
Fooled incubation media were prepared and stripped of endogenous glucocorticoids in a manner similar to the method used for the Scatchard analysis. Following stripping, tritiated corticosterone (Specific activity = 182.1 Ci/mnol; New&gland Wzlear; Boston, MA) (25 ~1 was added to 500 ul str@ped mediumin cunbination with either 0, 130, or 1000 pg of unlabeled corticosterone or dexamethasone ImX;9-fluorc-118r 17,21-trihydmxy-16a-mthyl1,4-pregnadiene-3,2O-diane) in 100 u 1 of GPBS. Tubes were incubated at OOCfor a minima of 6 hrs. Percent binding of the tritiated c%qonentJas d??te+ed and the degfee of x;zAz for a specific cortxold at a specific concentratmn . RFSULTS
Scatchard analysis of corticosterone-binding
activity
incubation media resulted in an estimated dissociation 8.81 x lo-10 M (Figure 1). class of specific protein.
in adrenal
constant U(d) of
The linear Scatchard plot suggested a single
sites for corticosterone,
The Kd of corticosterone-binding
thus a single species of globulin
mm,
usbg
nm-incubated stripped rat plasma, was estimated to be 5.00 and 2.40 x 109 M for 0.5% and 0.25% plasma respectively.
S
448
m
Representative Scatchardplot utilizingL3Hlcorticosterone in strippedadrenalmedium. Five replications utilizing differentmedia pools resultedin an estimatedKd of 8.81 + 0.64 x lo-10M.
130
E&U&L
'I?PEOIDI
1000
Corticosterone and dexamethasone competitionfor specific t3Blcorticosterone-binding sites in strippedadrenal incubationmedium. Total specificbindingin the absenceof a canpetitoris assigneda value of 100%.
s The affinity corticosterone
of media protein for DEXin the presence of
was investigated by cuqaring
unlabeled corticosterone
readily with tritiated any great extent.
the ccmpetition of
and DEXwith tritiated
are presented in Figure 2.
indicator,
449
TBEOfDI
corticosterone.
Results
Corticosterone was observed to compete
corticosterone
whereas DEXfailed
to cmpete to
Thus, using the degree of ccmpetition as an
the affinity
of the media protein was high for corticosterone
but very law for DEX. DI!XlJSSION The Kd estimate of 8.81 x lo-10 M (OW for the media protein of adrenals is compared to Kd values of glucocorticoid
cytoplasm and glucocorticoid-binding
receptors in the
proteins in the blood in Table 1.
The Kd for the protein in adrenal media is approximately one-tenth that for any other glucocorticoid Type I receptors.
binding protein cited with the exception of
However, the media protein can be readily
distinguished fran the Typa I receptor since in caqetition
studies it
has been demonstrated that the Type I receptor has a lower affinity that of aldosterone) for corticosterone
(2%
and DEX (7).
A direct canparison of Kd values to those for adrenal cytoplasmic receptors CQpe II) cannot be made since those Kd estimates exist only for DEX, but the lack of a high affinity
for DEXby the protein in the
media of adrenal incubations readily distinguishes receptors.
The observation that the media protein has a high affinity
for corticosterone affinity
it from cytoplamic
while being very low for DEXis in contrast to the
of cytosol proteins
both corticosterone
(Type II) of rat adrenals which is high for
and DEX (8).
Similar canparisons can be made to
v 0
”
2 w
0
O
::
v
0n
f v
Oo
9‘;;‘ 0
3. rr)
0
w
0
0
w5; 9s 0
0
. m
hl
v
ws ? =: “x m
1. 0
u v
vu
00
2
r-4
W
W
m
uv
W
00
I--*
m
0
u h
m
W
cytosol proteins higher affinity
(T&e III)
fran the bovine adrenal cortex which have a
for DEXthan cortisol
The binding affinities
or corticosterone
for corticosterone
similar to those of the media protein (10). !l?ypeII cytosol
and DEXby CBGwere The differential
binding of
receptors and CPGfor DEXis used to differentiate
two glucocorticoid affinities
(9) .
binders (11).
the
However, the similar binding
of media protein and CBGdo not necessarily
indicate that the
media protein is CBG. CDGas well as !FypeIII receptors have similar affinities
for DEXbut still
differ
with respect to other indices (12).
The present results indicate that CBGand the media protein from adrenal incubations have similar binding affinities
for corticosterone
and DEX.
However, it is doubtful that the media protein is CBGsince the principal
source of CBGis the liver.
Previous reports havs suggested
that the adrenal produces the protein in question, since media binding activity y&m
may be altered by the addition of XTH to adrenal tissue i,n
(2,3).
Furthermore, binding activity
rhythm of steroidogenesis In conclusion, glucocorticoid-binding
parallels
the circadian
(2) whereas CBGdoes not (13).
our data suggest the existence of a protein distinct
groups of glucocorticoid-binding
frm the two presently knmn
proteins,
CBGand cytoplasmic
receptors.
The secretarial assistance of Mrs. Wilrm MaddoxarCi Mrs. Charlotte Barnes is gratefully acknawledged. This manuscript is No. 4-810153 of the AlabamaAgricultural Experimant Station Journal Series.
452
S
TD&OIDS
REFERENCES 1.
Murphy,B.E.P.;J. Clin. Endocrinol.Metab.22~ 973-990 (1967). 2. Pritchett,J. F., Harper,W. L., Marple,D. N., Bradley,J. T., and Till, M. L.; Biochem.Biophys.Res. Camun. 9Q: 1355-1363(1979). 3. Goddard,C., Vinson,G. P., and Whitehouse,B. J.; J. Endocrinol. 71: lOP-1lP (1978). 4. Ret&me, K., Zimmxman, E., Schindler,W. J., Neuenschwander, J., and Lipscomb,H. S.; Acta Endocrinol.ZZ: 615-622 (1968). 5. Ran!aley, J. A.; SteroidsX: 611-630 (1972). 6. Scatchard,G.; Ann. NY Acad. Sci. a: 660-672 (1949). 7. Funder,J. W., Feldman,D. and Edelmn, I. S.; Endocrinol.92: 1005-1013(1973). 8. Loose,D. S., Do, Y. S., Chen, T. L., and Feldman,D,; Endccrinol. lQZ: 137-146 (1980) 9. Cachet,C., Job D., Dhien,A., and Chambaz,E. M.; Arch. Biochem. Biophys.l&Q: l-9 (1977). 10. Westphal,U.; J. Reprod.Fert. Suppl.l.Q:15-38 (1970). 11. Munck,A. and Leung,K. in Receptorsand Mechanismof Action of SteroidHomones, Part II (Pasqualini, J. R., Ed.), MarcelDekker, Inc.,New York (19761,pp 311-397. 12. Feldman,D., Funder,J. W. and Edelman,I. S.; Endocrinol.92: 1429-1441(1973). 13. Keller,N., SendelbeckL. R., Richardson,U. I., Moore C. and Yates, F. E.; Endocrinol.a: 884-906 (1966). 14. Funder,,J. W., Feldman,D. and Edelmn, I. S.; Endocrinol.42: 1005-1013(1973). 15. Rousseau,G. G., Baxter,J. D. and Tankins,G. M.; J. Mol. Biol.&,Z: 99-115 (1972). 16. Wagner,R. K.; Acta Endocrinol.Suppl.2l& 5-73 (1978).
J. F. Pritchett PhysiologyLaboratories Department of Zoology AuburnUniversity,AL 36849