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Action of actinomycin D on gastro-intestinal glucose uptake and on the glucose effect rat liver tyrosine transaminase activity
2t2
BtOCHtM]{CA E2 BIOPHYS2C~ ACZ
BBA Report BBA 21315
Action of actinomycirt D on gastro-intestinal glucose uptake and on ~he g~ucose effect upon rat liver tyrosine transamir~ase activity JACQUES HANOUNF e and ANNE-M~,R~,ECHAMBAUTe'~ Instztut de PathologTe Molkculatre ~, 24, rue deg Faubourg Saint-Jacques, Parts t4e (Fraace)
(Recewed September 2nd, 1971 )
SUMMARY
An oral load of glucose m fasted rats led to a marked and sustained decrease in the activity of laver tyrosme transammase Intraperltoneat admm:strat~on o f actlnomyc~-~ D, 1 h prior to the glucose feeding, lmhlblted tins "glucose effect", Furtbm study using a4C-labelted glucose showed that 3 h after incubation, 68% of the glucose was _trained m the stomach and intestine of rats pretreated with the drug as compaled to 28% foa she controls Concurrently, less glucose appeared an biood, musc!e and hver glycogen an the actlnomycln D-treated rats Thus, the action of actmomycm D on the glucose effect dpo~ laver tyroslne transammase as mos: ltkely ~o be secondary to lnhlblLlon of glucose absorption The so catled "glucose effect", a phenomenon appare_qtly s:mllar to the "giucose repression" m bacteria, has been shown tc affect a varaety of iwer enzymes Some enzymes revolved in glucose utlhzatlon are induced, e g g:ucokinase ~ and otheis, whach roostS? catalyst steps of the gluconeogemc pathway are represseO 2- 4 We ha',e recer,:!y reporte4 that a single, o r ~ glucose load m normal fasled rats effected a marked and sustaaqed decrease of the level of hve~ tyrosme transammase activity and we have presented data favoring an alteration an the enzyme protean synthesas at the translational level rather tha~ an mhlbitaon of its actwlty s Samflar observata3ns were made w.~:h regards to a nurnbe, c[ other enzymes and singularly to phosphoenol pyruvate carboxykmase 6 7 It was fu~hereported that simultaneous administration of either p u r o m y c m or actmomycm D was aoJe to decrease this mhmatory effect or. the later enzyme Thls was interpreted as slgmfyJng that protean synthesis was required for expression of the depresswe action of glucose o~ *Charg6 de Recherches 2t l'lnst!tut National de ta Sant6 et de la Recherche M~dlcale **Charg6e de Recherches au Centre National de la Recherche Sclentff~-que ~Instltut d'Umverslt6, Groupe UI5 de lqnstltat Nat~onat de ta SapA6et de la Recl,,ercPe M6dlcZe Laboratolre assocl6 au Centre Natmnal de la Recherche Sclev.tffNae Btochtm. Btophy~ Acta, 252 (1971) 212-2t6
BBA REPORT
213
the enzyme 6 These data and a recent report that actmomycln D could inhibit the tryptophan uptake by the gastro-lntestinal tract 8 prompted exploration of the action of actlnomycln D on the "glucose effect" upon rat liver tyrosme transamlnase Male, albino Wxstar rats (150 + 10 g body weight) were used m all experunents They were housed m screen-bottom cages and fasted for 1 day prior to use Glucose (1 g per animal) was administered in 3 ml of aqueous solution by stomach tube (1.5 mm diameter plastic tubing) For studymg glucose uptake, 2 gC of [U-14C]glucose (Amersham, 131 mC/mmole) were added to the usual glucose load for each antmal. Actmomycln D (a gift from Merck, Sharp and Dohme) and cortisone acetate (purchased from Roussel UCLAF) were rejected lntraperltoneally (5 mg and 180 gg per 100 g body weight, respectwely). Other experunental details are incorporated m the legends to figures. At the indicated tune periods, the animals were sacrificed. The livers were homogemzed m 2 vol. (v/w) of cold 0 14 M KC 1 and the homogenate centrifuged at 15 000 × g for 20 mln at 4 °. The post-mltochondnal supernatant was used for the tyrosme transammase (L-tyrosme 2-oxo-glutarate amano transferase EC 2 6 1.5) assay, accordang to the method of Dlamondstone 9 Results are expressed as/~moles of product formed per h and per g of fresh hver. All values are the average of three to nine animals + standard error of the mean. In the experiments where the uptake of labelled glucose was studied, heparmlzed blood samples were deprotelnized by adding 4 vol. of cold 5% trachloroacetlc acid Muscles from the hind-legs were cut into small pieces and heated for 5 man at 100 ° an 4 vol. of distilled water. After centrlfugatlon, ahquots of the clear supernatant were taken for measuring of radloactwlty. The stomach and small intestine were removed tn toto with their contents and dissolved an 30% KOH at 100 ° for 30 mm; 0.5-g fragments from the livers were snnllady treated and total radaoactwlty was estimated from an aliquot of these solutions The glycogen fractaon from the KOH extract of the lwer was further purified by ethanol preclpltataon according to the method of Good et al 1o and its radioactivity measured after redassolutlon in cold 5% tnchloroacetac acid. All sample counting was done an a Mark I Nuclear Chicago hqmd scintillation Counter and corrected for quenching by the addition of internal ~4C standard or by external standardization. Values are the average of four to six animals -+ standard error of the mean TABLE I INHIBITION BY ACTINOMYCIN D OF THE GLUCOSE EFFECT UPON TYROSINE TRANSAMINASE Actmomycm D (180/~g/100 g body weight) was administered 1 h prior to either corttsone admimstralion (5 mg/100 g body weight) or glucose load. Animals were sacrificed 6 h after actanomycm D. Treatment
Tyrosme transammase actlvzty (umts/g/h)
None Actinomycln D Glucose Glucose + aetmomycm D Cortisone Cortisone + actmomycm D
52 +- 3 43 _+ 8 27 + 2 41 -+ 5 302 + 25 58 -+ 7
% Change
- 17 - 48 - 21 + 480 Biochim. Btophya Acta, 252 (1971) 212-216
214
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Fig 1 Distribution of 14C eadioac~:v:ty nu actmomycm D-trea:ed (m) and :n co~troi (u) :a.s after orm mtubatlon of 114C]glucose Actmomycm D (180 t~g/t00 g body weaght) was given mtraper:tonea!iy at zero time and the glucose load containing the radloact:ve label 1 h later T1:e zats were kIiled either or 3 h after the glucose load Rad:oactav:ty is dep:cted here as the percen :age of total dose admm~stezed The percentage of total rad:oachvlty values was calculated assuming that total muscles and blood represent 45 and 5% of the body we:ght, respectively "Fae values for the stomach and small intestine include'their contents As for the hver, separate determmatmns were done for total river rad:oa~.v:ty and totN radmact:v:ty recovered as puufled hver glycogen (lower bar). Total y:eld averages 65-75% of the admm:ste~ed rad:oactlwty, Vertmal lines represent the standard dewar:on from the mean. The actual values for the 14Cradloact:vlty found m control rats aft±g,1 h were 685 000 ± 18 000 counts/ mm m the total stomada and intestine fraetmn per ammal; 1 650 ± 210 counts/ram per g muscle; 510 ± 15 counts/ram per ml blood, 11 t 0 0 ± 750 counts/ram per g h~er (of which 5 500 ± 600 were m the glycogen fractmn)
We first assessed the effect of actmomycm D on tyrosme transammase act:v:ty ~n glucose-loaded rats It ~s clear from the data m Table I that actmomyekn D affects ".he "glucose effect" upon transammase actmty m the same way prewously reported for phosphoenol pymvate carboxykmase 6. Actmomycm D led to a I7% decrease of the B~oehirn~B~ophy& Acca, 252 (1971) 2 t 2 - 2 1 6
BBA REPORT
215
enzyme actwlty over a 6-h period and completely inhibited the enhanced transammase synthesis brought about by cortisone. The glucose load depressed enzyme level by 48% after 5 h, but when both actmomycm D and glucose were given, enzyme activity fell by only 21%. We then explored the posslbdlty that actlnomycin D could act by virtue of an unpaired transport of glucose from the gastro-mtestmal tract. As shown in Fig. 1, when administered 1 h prior to the glucose load, actmomycm D effectwely prevented most of the glucose uptake In control rats, the radloactwlty remaining m the stomach and the intestine decreased between 1 and 3 h from 57 to 28% of the administered dose (Fig. la). Meanwhde, the total radioactivity m lwer and muscle rose from 7.4 and 6.2% to 22.2 and 12%, respectwely (Figs lb and c). In contrast, 3 h after glucose admlmstratlon, 68% of the initial dose remained m the stomach of the actmomycm,treated animals, while the total radaoactwlty of their liver and muscle was decreased by 70 and 61%, respectwely as compared to the control. Concurrently, the radioactwlty present m the blood was also reduced (Fig ld). That actmomycm D could diminish the hver radioactivity solely by virtue of its glycogenolytlc properties 11 is easdy dascounted since the radaoactwaty of the stomach and the intestine dad not decrease between 1 and 3 h (Fag. la). The value reported here for the total liver radloactawty at 3 h (22%) and the fractaon corresponding to purified hepatic glycogen (14.7%) are m complete agreement wath data reported prevaously 12. The present experiments show that actmomycln D is an effectwe lntubator of the glucose effect on lwer tyrosme transaminase activity but also that it can impair very strongly the glucose uptake from the gastro-mtestanal tract. It is therefore evident that the inhibitory action of actanomycin D on the glucose effect upon eather transammase or phosphoenol pyruvate carboxykmase actlvlty 6 should not be interpreted as sigmfymg an RNA protein synthesis reqmrement for the expression of the glucose effect. The data reported here are m fact a further mdacataon that the damage of the Intestinal epithehal cells brought about by the actinomycm D treatment la could prevent the glucose as well as the amino aod uptake s from the gastro-mtestinal tract, thus strongly reducing the amount of the inducer (e.g. tryptophan) or repressor (e.g. glucose)molecule reaching the lwer. In fact, we have indicated elsewheres that the glucose effect is probably located at the ribosomal level and might involve, as mtermedmry effectors, opposite modulaUons of the release of the two pancreatic hormones, glucagon and msnlm. Ttus glucose effect probably does not need any prior hepatic protein synthesis. It seems therefore relevant to stress again, after many others a4, the ambaguous meaning of all experiments done tn vtvo with inhlbators of protein synthesis. We are grateful to Drs. R. Belanger, J. Kruh and G. Schaplra for helpful and valuable dascussion of ttus study. This work was supported by the "Insatut National de la Sant~ et de la Recherche M6dacale", the "Centre National de la Recherche Sclentlfique", the D~l~gataon G~n6rale la Recherche Sclentlflque et Techmque", the "Commissariat fi l'Energle Atomlque", and the Muscular Dystrophy Assocmtxons of America.
Biochim. Btophyz Acta, 252 (1971) 212-216
2l6
BtL'< R£PGR7
REFERENCES 1 A. Sols, A SlUero and J Salas, J Ce~l Comp Phys~ol, 65 (1965) 23 2 C Perzano. C Lamar and H C Pltot,Z Bzol Chem, 241 (1966) 2944 3 H S Soling, J Kaplan, M. Erbstoeszet and H C Pl:oto m G Webe,,Ad;ances ,n Enzy,me Rpg~]a~,o~e Vol 3. Pergamon Press, New York, 1969, p 171. 4 A. Yuwfler, L Wetterberg and E. Geller, 8toch~m Btophys Acra, 208 (I970) z,28 5 J Ha~oune, A M Chambaut and Ao Joslpowmz, Bgoch!m. Blophys A cta~ 244 (197 i) 338 6 D O Foster, P D Ray and N A Lardy,Bgochemlstry, 5 (1966) 555 7 E Shrago, J W, Young ana H A Lardy, Scwnce~ 158 (1967) 1572 8 M B Yatvm and H C Pltot, Jo B~ol Chem, 245 (1970) 4673. 9 T T Dlamondstone,Anal g-9chem, 16 (1966) 395 10 C A Good, H Kramer and No SomogyL J Bgol Chem , 100 (1933) 485 1t P.D Ray, D O. Foster and H A. Lardy, J Bml Chemo, 239 (1964) 3396 12 S L Jeffcoate and A J Moody Dlabetolog~a, 5 (1969) 293. 13 H S. Schartz, S S Sterrberg and F S Phlhps. CancerRes, 23 (1963) 1125. t4 E D Bransome, Endocrinology 85 (1969) 11t4
Btochzm. Bmphy~ Acta, 252 (1971) 212--216
BtOCHtM]{CA E2 BIOPHYS2C~ ACZ
BBA Report BBA 21315
Action of actinomycirt D on gastro-intestinal glucose uptake and on ~he g~ucose effect upon rat liver tyrosine transamir~ase activity JACQUES HANOUNF e and ANNE-M~,R~,ECHAMBAUTe'~ Instztut de PathologTe Molkculatre ~, 24, rue deg Faubourg Saint-Jacques, Parts t4e (Fraace)
(Recewed September 2nd, 1971 )
SUMMARY
An oral load of glucose m fasted rats led to a marked and sustained decrease in the activity of laver tyrosme transammase Intraperltoneat admm:strat~on o f actlnomyc~-~ D, 1 h prior to the glucose feeding, lmhlblted tins "glucose effect", Furtbm study using a4C-labelted glucose showed that 3 h after incubation, 68% of the glucose was _trained m the stomach and intestine of rats pretreated with the drug as compaled to 28% foa she controls Concurrently, less glucose appeared an biood, musc!e and hver glycogen an the actlnomycln D-treated rats Thus, the action of actmomycm D on the glucose effect dpo~ laver tyroslne transammase as mos: ltkely ~o be secondary to lnhlblLlon of glucose absorption The so catled "glucose effect", a phenomenon appare_qtly s:mllar to the "giucose repression" m bacteria, has been shown tc affect a varaety of iwer enzymes Some enzymes revolved in glucose utlhzatlon are induced, e g g:ucokinase ~ and otheis, whach roostS? catalyst steps of the gluconeogemc pathway are represseO 2- 4 We ha',e recer,:!y reporte4 that a single, o r ~ glucose load m normal fasled rats effected a marked and sustaaqed decrease of the level of hve~ tyrosme transammase activity and we have presented data favoring an alteration an the enzyme protean synthesas at the translational level rather tha~ an mhlbitaon of its actwlty s Samflar observata3ns were made w.~:h regards to a nurnbe, c[ other enzymes and singularly to phosphoenol pyruvate carboxykmase 6 7 It was fu~hereported that simultaneous administration of either p u r o m y c m or actmomycm D was aoJe to decrease this mhmatory effect or. the later enzyme Thls was interpreted as slgmfyJng that protean synthesis was required for expression of the depresswe action of glucose o~ *Charg6 de Recherches 2t l'lnst!tut National de ta Sant6 et de la Recherche M~dlcale **Charg6e de Recherches au Centre National de la Recherche Sclentff~-que ~Instltut d'Umverslt6, Groupe UI5 de lqnstltat Nat~onat de ta SapA6et de la Recl,,ercPe M6dlcZe Laboratolre assocl6 au Centre Natmnal de la Recherche Sclev.tffNae Btochtm. Btophy~ Acta, 252 (1971) 212-2t6
BBA REPORT
213
the enzyme 6 These data and a recent report that actmomycln D could inhibit the tryptophan uptake by the gastro-lntestinal tract 8 prompted exploration of the action of actlnomycln D on the "glucose effect" upon rat liver tyrosme transamlnase Male, albino Wxstar rats (150 + 10 g body weight) were used m all experunents They were housed m screen-bottom cages and fasted for 1 day prior to use Glucose (1 g per animal) was administered in 3 ml of aqueous solution by stomach tube (1.5 mm diameter plastic tubing) For studymg glucose uptake, 2 gC of [U-14C]glucose (Amersham, 131 mC/mmole) were added to the usual glucose load for each antmal. Actmomycln D (a gift from Merck, Sharp and Dohme) and cortisone acetate (purchased from Roussel UCLAF) were rejected lntraperltoneally (5 mg and 180 gg per 100 g body weight, respectwely). Other experunental details are incorporated m the legends to figures. At the indicated tune periods, the animals were sacrificed. The livers were homogemzed m 2 vol. (v/w) of cold 0 14 M KC 1 and the homogenate centrifuged at 15 000 × g for 20 mln at 4 °. The post-mltochondnal supernatant was used for the tyrosme transammase (L-tyrosme 2-oxo-glutarate amano transferase EC 2 6 1.5) assay, accordang to the method of Dlamondstone 9 Results are expressed as/~moles of product formed per h and per g of fresh hver. All values are the average of three to nine animals + standard error of the mean. In the experiments where the uptake of labelled glucose was studied, heparmlzed blood samples were deprotelnized by adding 4 vol. of cold 5% trachloroacetlc acid Muscles from the hind-legs were cut into small pieces and heated for 5 man at 100 ° an 4 vol. of distilled water. After centrlfugatlon, ahquots of the clear supernatant were taken for measuring of radloactwlty. The stomach and small intestine were removed tn toto with their contents and dissolved an 30% KOH at 100 ° for 30 mm; 0.5-g fragments from the livers were snnllady treated and total radaoactwlty was estimated from an aliquot of these solutions The glycogen fractaon from the KOH extract of the lwer was further purified by ethanol preclpltataon according to the method of Good et al 1o and its radioactivity measured after redassolutlon in cold 5% tnchloroacetac acid. All sample counting was done an a Mark I Nuclear Chicago hqmd scintillation Counter and corrected for quenching by the addition of internal ~4C standard or by external standardization. Values are the average of four to six animals -+ standard error of the mean TABLE I INHIBITION BY ACTINOMYCIN D OF THE GLUCOSE EFFECT UPON TYROSINE TRANSAMINASE Actmomycm D (180/~g/100 g body weight) was administered 1 h prior to either corttsone admimstralion (5 mg/100 g body weight) or glucose load. Animals were sacrificed 6 h after actanomycm D. Treatment
Tyrosme transammase actlvzty (umts/g/h)
None Actinomycln D Glucose Glucose + aetmomycm D Cortisone Cortisone + actmomycm D
52 +- 3 43 _+ 8 27 + 2 41 -+ 5 302 + 25 58 -+ 7
% Change
- 17 - 48 - 21 + 480 Biochim. Btophya Acta, 252 (1971) 212-216
214
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Fig 1 Distribution of 14C eadioac~:v:ty nu actmomycm D-trea:ed (m) and :n co~troi (u) :a.s after orm mtubatlon of 114C]glucose Actmomycm D (180 t~g/t00 g body weaght) was given mtraper:tonea!iy at zero time and the glucose load containing the radloact:ve label 1 h later T1:e zats were kIiled either or 3 h after the glucose load Rad:oactav:ty is dep:cted here as the percen :age of total dose admm~stezed The percentage of total rad:oachvlty values was calculated assuming that total muscles and blood represent 45 and 5% of the body we:ght, respectively "Fae values for the stomach and small intestine include'their contents As for the hver, separate determmatmns were done for total river rad:oa~.v:ty and totN radmact:v:ty recovered as puufled hver glycogen (lower bar). Total y:eld averages 65-75% of the admm:ste~ed rad:oactlwty, Vertmal lines represent the standard dewar:on from the mean. The actual values for the 14Cradloact:vlty found m control rats aft±g,1 h were 685 000 ± 18 000 counts/ mm m the total stomada and intestine fraetmn per ammal; 1 650 ± 210 counts/ram per g muscle; 510 ± 15 counts/ram per ml blood, 11 t 0 0 ± 750 counts/ram per g h~er (of which 5 500 ± 600 were m the glycogen fractmn)
We first assessed the effect of actmomycm D on tyrosme transammase act:v:ty ~n glucose-loaded rats It ~s clear from the data m Table I that actmomyekn D affects ".he "glucose effect" upon transammase actmty m the same way prewously reported for phosphoenol pymvate carboxykmase 6. Actmomycm D led to a I7% decrease of the B~oehirn~B~ophy& Acca, 252 (1971) 2 t 2 - 2 1 6
BBA REPORT
215
enzyme actwlty over a 6-h period and completely inhibited the enhanced transammase synthesis brought about by cortisone. The glucose load depressed enzyme level by 48% after 5 h, but when both actmomycm D and glucose were given, enzyme activity fell by only 21%. We then explored the posslbdlty that actlnomycin D could act by virtue of an unpaired transport of glucose from the gastro-mtestmal tract. As shown in Fig. 1, when administered 1 h prior to the glucose load, actmomycm D effectwely prevented most of the glucose uptake In control rats, the radloactwlty remaining m the stomach and the intestine decreased between 1 and 3 h from 57 to 28% of the administered dose (Fig. la). Meanwhde, the total radioactivity m lwer and muscle rose from 7.4 and 6.2% to 22.2 and 12%, respectwely (Figs lb and c). In contrast, 3 h after glucose admlmstratlon, 68% of the initial dose remained m the stomach of the actmomycm,treated animals, while the total radaoactwlty of their liver and muscle was decreased by 70 and 61%, respectwely as compared to the control. Concurrently, the radioactwlty present m the blood was also reduced (Fig ld). That actmomycm D could diminish the hver radioactivity solely by virtue of its glycogenolytlc properties 11 is easdy dascounted since the radaoactwaty of the stomach and the intestine dad not decrease between 1 and 3 h (Fag. la). The value reported here for the total liver radloactawty at 3 h (22%) and the fractaon corresponding to purified hepatic glycogen (14.7%) are m complete agreement wath data reported prevaously 12. The present experiments show that actmomycln D is an effectwe lntubator of the glucose effect on lwer tyrosme transaminase activity but also that it can impair very strongly the glucose uptake from the gastro-mtestanal tract. It is therefore evident that the inhibitory action of actanomycin D on the glucose effect upon eather transammase or phosphoenol pyruvate carboxykmase actlvlty 6 should not be interpreted as sigmfymg an RNA protein synthesis reqmrement for the expression of the glucose effect. The data reported here are m fact a further mdacataon that the damage of the Intestinal epithehal cells brought about by the actinomycm D treatment la could prevent the glucose as well as the amino aod uptake s from the gastro-mtestinal tract, thus strongly reducing the amount of the inducer (e.g. tryptophan) or repressor (e.g. glucose)molecule reaching the lwer. In fact, we have indicated elsewheres that the glucose effect is probably located at the ribosomal level and might involve, as mtermedmry effectors, opposite modulaUons of the release of the two pancreatic hormones, glucagon and msnlm. Ttus glucose effect probably does not need any prior hepatic protein synthesis. It seems therefore relevant to stress again, after many others a4, the ambaguous meaning of all experiments done tn vtvo with inhlbators of protein synthesis. We are grateful to Drs. R. Belanger, J. Kruh and G. Schaplra for helpful and valuable dascussion of ttus study. This work was supported by the "Insatut National de la Sant~ et de la Recherche M6dacale", the "Centre National de la Recherche Sclentlfique", the D~l~gataon G~n6rale la Recherche Sclentlflque et Techmque", the "Commissariat fi l'Energle Atomlque", and the Muscular Dystrophy Assocmtxons of America.
Biochim. Btophyz Acta, 252 (1971) 212-216
2l6
BtL'< R£PGR7
REFERENCES 1 A. Sols, A SlUero and J Salas, J Ce~l Comp Phys~ol, 65 (1965) 23 2 C Perzano. C Lamar and H C Pltot,Z Bzol Chem, 241 (1966) 2944 3 H S Soling, J Kaplan, M. Erbstoeszet and H C Pl:oto m G Webe,,Ad;ances ,n Enzy,me Rpg~]a~,o~e Vol 3. Pergamon Press, New York, 1969, p 171. 4 A. Yuwfler, L Wetterberg and E. Geller, 8toch~m Btophys Acra, 208 (I970) z,28 5 J Ha~oune, A M Chambaut and Ao Joslpowmz, Bgoch!m. Blophys A cta~ 244 (197 i) 338 6 D O Foster, P D Ray and N A Lardy,Bgochemlstry, 5 (1966) 555 7 E Shrago, J W, Young ana H A Lardy, Scwnce~ 158 (1967) 1572 8 M B Yatvm and H C Pltot, Jo B~ol Chem, 245 (1970) 4673. 9 T T Dlamondstone,Anal g-9chem, 16 (1966) 395 10 C A Good, H Kramer and No SomogyL J Bgol Chem , 100 (1933) 485 1t P.D Ray, D O. Foster and H A. Lardy, J Bml Chemo, 239 (1964) 3396 12 S L Jeffcoate and A J Moody Dlabetolog~a, 5 (1969) 293. 13 H S. Schartz, S S Sterrberg and F S Phlhps. CancerRes, 23 (1963) 1125. t4 E D Bransome, Endocrinology 85 (1969) 11t4
Btochzm. Bmphy~ Acta, 252 (1971) 212--216