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The relationship between milk accumulation and enzyme activities in the involuting rat mammary gland
~5~
SHORT
COMMUNICATIOX
S
a n d C were o b t a i n e d from the d e n s i t o m e t e r r e a d i n g s b y m e a s u r e m e n t of t h e area u n d e r t h e c a r v e s (Fig. z). i n order to o b t a i n r e p r o d u c i b l e results it was found necessary to change tt.~e zinc a c e t a t e solution in the etectropimresis t a n k after each run as the m o l a r i t y of t h e solution alters d u r i n g a r u n due to d e p o s i t i o n of zinc on the cathode. To check t h a t t h e m e t h o d was m e a s u r i n g c h o n d r o i t i n s u l p h a t e B, a sample o~! c h o n d r o i t i n s u l p h a t e o b t a i n e d from the S e p h a d e x column was i n c u b a t e d w i t h testicw, lar h y a l u r o n i d a s e s a n d dialysed, t h e n electrophoresis was carried out on a s a m p l e in zinc a c e t a t e as above. 0 n l y one line was o b t a i n e d on s t a i n i n g t h e electrophoresis strips. T h i s result confirms t h e presence of c h o n d r o i t i n s u l p h a t e B as c h o n d r o i t i n s u l p h a t e s A a n d C a n d h y a l u r o n i c acid are d i g e s t e d b y t e s t i c n t a r hyaluronidase*~ T h e a u t h o r t h a n k s Mr. D. R o a ~ s for t e c h n i c a l assistance. R~ E. So PROem
Department of Biochemistry, University of Sheffield, She~eld (Great Britain) I 2 3 4 5 6 7 8
D. S. TRUNDLE AND G. V. ~ANX, Biochim. Bi@hys. Acta, IO~ (~965) 127. F. HAR~JKI AND J. E. KIRK, Biochim. Bi@ltys. Acta, ~36 (~967) 39x-393. G. S. BER~NSON AND E. R. DALFZRES,Biochim. Biophys. Acta, 58 (~962) 34. J- E. KIRK aND M. DYR~VX, J. Gerontol., ~2 (I957) 23. IV[.SCH~ID% Biochim. Bio~hys. Acta, 63 (~962) 346. Z. DlSCUE, J. Biol. Chem., I67 (1947) I89. T. Bl~C~gaaND H. M. MUIR, Anal. Biochem., 4 (1962) 33o. K. MEYER, E. DAVIDSON,A. LINKER AND P. HOFFMAN,Biochim. Bi@hys. Acta, 2I (~956) 506.
R e c e i v e d O c t o b e r 3~st, I968
Biochim..~?iophys. Acta, z77 (~969) I57-~58
~BA 23482
The relationship between milk accumulation and enzyme activities in the involuting rat mammary
gland
L a c t a t i o n in t h e r a t is a c c o m p a n i e d b y an increase in the a c t i v i t y of a large n u m b e r of m a m m a r y g l a n d e n z y m e s which s u b s e q u e n t l y decline r a p i d l y a f t e r weaningl,K I t is k n o w n t h a t h o r m o n e s of t h e a n t e r i o r p i t u i t a r y p l a y an l m p o r t a n z rote in t h e i n i t i a t i o n a n d m a i n t e n a n c e of l a c t a t i o n (see ref. 3 a n d h y p o p h y s e c v o m y of t h e l a c t a t i n g r a t leads to a r a p i d decline in m i l k p r o d u c t i o n a n d an a l m o s t e q u a l l y r a p i d decline in t h e activities of a n u m b e r of e n z y m e s l,~. The a c t i v i t i e s of different e n z y m e s decrease to different e x t e n t s so t h a t w i t h i n 2 4 h of o p e r a t i o n a charact e r i s t i c a l l y c h a n g e d p a t t e r n of e n z y m e a c t i v i t y has been established. These changes a f t e r h y p o p h y s e c t o m y are r e p r o d u c e d a l m o s t e x a c t l y if t h e litter is r e m o v e d from t h e l a c t a t i n g r a t a n d t h e glands allowed to b e c o m e e n g o r g e d w i t h milk a n d it is a question of some i n t e r e s t w h e t h e r this c o m m o n response is due ~o similar changes in t h e levels of c i r c u l a t i n g h o r m o n e s or w h e t h e r changes w i t h i n t h e engorged g l a n d itself in some w a y r e p r o d u c e t h e c o n d i t i o n s following h y p o p h y s e c t o m y .
~och~m. ~io~]zys. At#a, ~77 (~968) ~58-16o
159
SHORT COMMUNICATIONS
It had previously been shown that the enzymes glucose-6-phosphate dehydrogenase 6 (D-glucose-6-phosphate :NADP + oxidoreductase, EC I.I. 1.49) and ATP citrate lyase 7 (ATP:citrate oxaloacetate-lyase [CoA-acetylating and ATP-dephosphorylating~, EC 4.1.3.8) declined in activity in the milk engorged rat mammary gland when unilateral involution was produced by blocking the teats on one side with Collodion, while activities in the contralateral suckled glands remained normal. This suggested that variations in the levels of circulating hormones were probably not important in producing the changes in unsuckled animals. However, it was considered useful to investigate the decline in the activities of a wider range of enzymes with the aim of revealing any differences between the pattern of change in unsuckled rats and rats in wlhich removal of milk from the mammary glands on one side was prevented by ligation of the galactophores (unilaterally ligated rats). The enzymes assayed, chosen to include representatives of the major synthetic pathways other than protein synthesis, were: glucose-6-phosphate dehydrogenase; 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ oxidoreductase ~decarboxylating~, EC 1.1.1.44); phosphoglucomutase (~-glucose-I,6-diphosphate: D-glucose-l-phosphate phosphotransferase, EC 2.7.5.1); UDPglucose pyrophosphorylase (UTP:D-glucose-I-phosphate uridylyltransferase, EC 2.7-7.9); phosphofructokinase (ATP :D-fructose-6-phosphate I-phosphotransferase, EC 2.7.1.11); ATP citrate lyase and acetyl-CoA carboxylase (acetyl-CoA:CO~ ligase (ADP~, EC 6.4.1.2). Primiparous Norwegian hooded rats were used and killed on the Ioth or I l t h day of lactation. The weaned rats were separated from their litters 24 h before killing and the unilaterally ligated rats had the galactophores on one side tied off subcutaneously under ether anaesthesia and were returned to their litters for 24 h before killing. Details of the preparation of the particle-free fraction of the mammary glands and the assays of enzyme activity and DNA have been given previously~. The results are presented in Table I where activities are expressed on the basis of the DNA content of the homogenate. According to OTA8 no significant change in TABLE I ENZYME ACTIVITIES IN RAT MAMMARY GLANDS Activities expressed as/*moles s u b s t r a t e utilised per rain per nag h o m o g e n a t e DNA. The n u m b e r s of rats in each group are given in p a r e n t h e s e s and each value is followed b y its S.E.
Enzyme
Status of rats Control (5)
Unilaterally ligated (4) Unligated side
Acetyl-CoA carboxylase Phosphofructokinase AT1~' citrate lyase Glucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Phosphoglucomntase UDPglucose p y r o p h o s p h o r y l a s e
0.745 i.IO 2.68 17.5 2.55 1.25 16.65
4444± ± ±
o.o71 o. i i 0.44 1.2 0.45 o.18 3.14
o.791 o.91o 2.42 17.9 3.09 1.34 I2.55
4- o.213 ± o.147 4- 0.40 ± 1. 5 ~ 0.30 ± o.13 ~_ 2.07
Weaned (3)
Liga*,ed side 0.049 0.o74 o.247 6.68 0.590 0.206 6.i8
44444± ±
o.o16 O.Ol 4 0.053 o.33 o.062* o.043 o-96
0.049 o.132 o.499 8.86 1.o 5 0.297 8.13
4~2 4± 44±
* Difference significant ( P < o . o 5, t test). No other pairs of values (i.e. control vs. unligated or weaned vs. ligated) significantly different (P > o.I),
l~iochim. Biophys. Acta, I77 (1968) 158-16o
o.oi2 o.o65 o.163 1.47 o.17" 0.049 0.92
I~0
SHORT CO~{I~IU N ICATION,<~
the D N A c o n t e n t of u n s u c k l e d r a t m a m m a r y glands occurs within 2 4 h a n d ti',u,~ these results p r o b a b l y reflect changes in tots1 a c t i v i t y . I t can be seen t h a t n o t ,,;n!v do weaning a n d u n i l a t e r a l ligation b o t h p r o d u c e decreases m e n z y m e activities b u t t h a t t h e p a t t e r n of decrease is. within t h e limits of e x p e r i m e n t a l error, i d e n t i c a l ia~_ b o t h conditions w i t h the possible e x c e p t i o n of 5 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e which is r a t h e r m o r e active in t h e u n s u c k l e d rats. T h u s acetvl-CoA carboxvlase. A T P c i t r a t e l y a s e a n d p h o s p h o f r u c t o k i n a s e decrease ~o 5 - x o % of t h e c o n t r o l a c t i v i t i e s while t h e o t h e r e n z y m e s show a smaller t h o u g h stiti m a r k e d decline. On the o t h e r h a n d a c t i v i t i e s in t h e c o n t r a l a t e r a l suckled glands remain identical w i t h t h o s e in t h e c o n t r o i rats. These findings p r o v i d e s t r o n g s u p p o r t for t h e t h e o r y t h a t the e n z w n e changes in t h e m a m m a r y g l a n d s of u n s u c k l e d r a t s are a direct response to e n g o r g e m e n t w i t h milk. As activities in t h e suckled glands of t h e u n i l a t e r a l l y l i g a t e d r a t s do n o t change, t h e levels of circulating g a l a c t o p o i e t i c h o r m o n e s in this c o n d i t i o n m u s t r e m a i n withi~i n o r m a i limits a n d changes in t h e engorged glands can be a t t r i b u t e d soIely to milk a c c u m u l a t i o n . This being so t h e r e ts no n e e d to invoke a n y endocrine m e c h a n i s m ~:~ ¢xpIain t h e similar changes in t h e u n s u c k l e d rat. However, as h y p o p h y s e c t o m y p r o d u c e s c o m p a r a b l e changes, which are u n r e l a t e d to the p a r t i a l e n g o r g e m e n t of the g l a n d s which occurs in u n t r e a t e d h y p o p h y s e c t o m i z e d r a t s ~, it is possible t h a t the changes in t h e u n s u c k l e d r a t are a result of a low level of h o r m o n e s at their site of action. P r o l o n g e d e n g o r g e m e n t of t h e r a t m a m m a r y gIand w i t h m i l k leads to a coilapse of t h e alveolar c a p i l l a r y b e d s a n d t h e gland is t h e n u n r e s p o n s i v e to o x v t o c i n even a f t e r t h e occiusion is released. Such a failure of t h e circulation w o u l d also p r e v e n t t h e access of t h e g a l a c t o p o i e t ic h o r m o n e s n e c e s s a r y for t h e m a i n t e n a n c e of l a c t a t i o n . O t h e r e x p l a n a t i o n s , such as a more direct i n t r a c e l l u l a r i n h i b i t i o n of h o r m o n e action b y a c c u m u l a t i n g u n s e c r e t e d m i l k c o n s t i t u e n t s , are aIso possible. I t m a y be t h a t the s i m i l a r i t y in t h e p a t t e r n of change of e n z y m e a c t i v i t i e s in i n v o l u t i n g glands, w h a t e v e r t h e f a c t o r p r o d u c i n g t h e i n v o l u t i o n , is a reflection of t h e different rares of t u r n o v e r of t h e v a r i o u s enzymes. W h a t e v e r t h e m e c h a n i s m , this direct effect of milk a c c u m u l a t i o n on e n z y m e activities o b v i o u s l y r e p r e s e n t s a useful f e e d - b a c k s y s t e m for adi u s t i n g mi!k p r o d u c t i o n in response ro d e m a n d . I wish to t h a n k Dr. A. T. Cowie for p e r f o r m i n g t h e s u r g e r y a n d Professor So ~i. F o l I e y for his e n c o u r a g e m e n t a n d advice.
Natio~al I~stitute for Resea~'c]~ i~ Dair2i~g, Shin.~eld, Reading (Great Britai~ I 1~_. E . MUNFORD, Dai¢y ~ci. AbstY. 26 ( I 9 6 4 293. 2 R . L. BALDWIN AND L. P. ~IILLIGAN. T Biol. Chem., 24I ( I 9 6 6 2058. 3 A. T. C o w I E , i n S. ][(. K o N AND A. T. COWIE iV[ilh: The JVIam~,~a~ VoL i , A c a d e m i c P r e s s , N e w Y o r k a n d L o n d o n , 1:96I , p. I63. 4 E . A. J o N ~ s , ]3iochem. J., Io3 (I967) 4zo. 5 R . L. BALDWIN AND R . J. l~fARrrlX. J. Daf~'y Sci., 5~ 1968 748. 6 P. NIcLEAN, Biochem. ~., 9o (1964) 271. 7 A. iF. SPENCER A~¢D J. M. LOWENSTEIN. Biochem. J . , 99 ( I 9 6 6 76o. 8 IK. OTA, l~ndoc*'i~zol..[al)o~'z. ~1 I964) ix6. 9 B. A. C R o s s AND L A. SILVER. P1,oe. ~OJ]..~OC. Meal.. 49 ( r 9 5 6 978.
R e c e i v e d O c t o b e r 25th, x968 Biochim. Bio~]~ys. Acta, ~77 (~968) z 5 8 - 1 6 °
E, A. JONES
Gla¢.~d a~d i~s Secfe~io~'t
SHORT
COMMUNICATIOX
S
a n d C were o b t a i n e d from the d e n s i t o m e t e r r e a d i n g s b y m e a s u r e m e n t of t h e area u n d e r t h e c a r v e s (Fig. z). i n order to o b t a i n r e p r o d u c i b l e results it was found necessary to change tt.~e zinc a c e t a t e solution in the etectropimresis t a n k after each run as the m o l a r i t y of t h e solution alters d u r i n g a r u n due to d e p o s i t i o n of zinc on the cathode. To check t h a t t h e m e t h o d was m e a s u r i n g c h o n d r o i t i n s u l p h a t e B, a sample o~! c h o n d r o i t i n s u l p h a t e o b t a i n e d from the S e p h a d e x column was i n c u b a t e d w i t h testicw, lar h y a l u r o n i d a s e s a n d dialysed, t h e n electrophoresis was carried out on a s a m p l e in zinc a c e t a t e as above. 0 n l y one line was o b t a i n e d on s t a i n i n g t h e electrophoresis strips. T h i s result confirms t h e presence of c h o n d r o i t i n s u l p h a t e B as c h o n d r o i t i n s u l p h a t e s A a n d C a n d h y a l u r o n i c acid are d i g e s t e d b y t e s t i c n t a r hyaluronidase*~ T h e a u t h o r t h a n k s Mr. D. R o a ~ s for t e c h n i c a l assistance. R~ E. So PROem
Department of Biochemistry, University of Sheffield, She~eld (Great Britain) I 2 3 4 5 6 7 8
D. S. TRUNDLE AND G. V. ~ANX, Biochim. Bi@hys. Acta, IO~ (~965) 127. F. HAR~JKI AND J. E. KIRK, Biochim. Bi@ltys. Acta, ~36 (~967) 39x-393. G. S. BER~NSON AND E. R. DALFZRES,Biochim. Biophys. Acta, 58 (~962) 34. J- E. KIRK aND M. DYR~VX, J. Gerontol., ~2 (I957) 23. IV[.SCH~ID% Biochim. Bio~hys. Acta, 63 (~962) 346. Z. DlSCUE, J. Biol. Chem., I67 (1947) I89. T. Bl~C~gaaND H. M. MUIR, Anal. Biochem., 4 (1962) 33o. K. MEYER, E. DAVIDSON,A. LINKER AND P. HOFFMAN,Biochim. Bi@hys. Acta, 2I (~956) 506.
R e c e i v e d O c t o b e r 3~st, I968
Biochim..~?iophys. Acta, z77 (~969) I57-~58
~BA 23482
The relationship between milk accumulation and enzyme activities in the involuting rat mammary
gland
L a c t a t i o n in t h e r a t is a c c o m p a n i e d b y an increase in the a c t i v i t y of a large n u m b e r of m a m m a r y g l a n d e n z y m e s which s u b s e q u e n t l y decline r a p i d l y a f t e r weaningl,K I t is k n o w n t h a t h o r m o n e s of t h e a n t e r i o r p i t u i t a r y p l a y an l m p o r t a n z rote in t h e i n i t i a t i o n a n d m a i n t e n a n c e of l a c t a t i o n (see ref. 3 a n d h y p o p h y s e c v o m y of t h e l a c t a t i n g r a t leads to a r a p i d decline in m i l k p r o d u c t i o n a n d an a l m o s t e q u a l l y r a p i d decline in t h e activities of a n u m b e r of e n z y m e s l,~. The a c t i v i t i e s of different e n z y m e s decrease to different e x t e n t s so t h a t w i t h i n 2 4 h of o p e r a t i o n a charact e r i s t i c a l l y c h a n g e d p a t t e r n of e n z y m e a c t i v i t y has been established. These changes a f t e r h y p o p h y s e c t o m y are r e p r o d u c e d a l m o s t e x a c t l y if t h e litter is r e m o v e d from t h e l a c t a t i n g r a t a n d t h e glands allowed to b e c o m e e n g o r g e d w i t h milk a n d it is a question of some i n t e r e s t w h e t h e r this c o m m o n response is due ~o similar changes in t h e levels of c i r c u l a t i n g h o r m o n e s or w h e t h e r changes w i t h i n t h e engorged g l a n d itself in some w a y r e p r o d u c e t h e c o n d i t i o n s following h y p o p h y s e c t o m y .
~och~m. ~io~]zys. At#a, ~77 (~968) ~58-16o
159
SHORT COMMUNICATIONS
It had previously been shown that the enzymes glucose-6-phosphate dehydrogenase 6 (D-glucose-6-phosphate :NADP + oxidoreductase, EC I.I. 1.49) and ATP citrate lyase 7 (ATP:citrate oxaloacetate-lyase [CoA-acetylating and ATP-dephosphorylating~, EC 4.1.3.8) declined in activity in the milk engorged rat mammary gland when unilateral involution was produced by blocking the teats on one side with Collodion, while activities in the contralateral suckled glands remained normal. This suggested that variations in the levels of circulating hormones were probably not important in producing the changes in unsuckled animals. However, it was considered useful to investigate the decline in the activities of a wider range of enzymes with the aim of revealing any differences between the pattern of change in unsuckled rats and rats in wlhich removal of milk from the mammary glands on one side was prevented by ligation of the galactophores (unilaterally ligated rats). The enzymes assayed, chosen to include representatives of the major synthetic pathways other than protein synthesis, were: glucose-6-phosphate dehydrogenase; 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ oxidoreductase ~decarboxylating~, EC 1.1.1.44); phosphoglucomutase (~-glucose-I,6-diphosphate: D-glucose-l-phosphate phosphotransferase, EC 2.7.5.1); UDPglucose pyrophosphorylase (UTP:D-glucose-I-phosphate uridylyltransferase, EC 2.7-7.9); phosphofructokinase (ATP :D-fructose-6-phosphate I-phosphotransferase, EC 2.7.1.11); ATP citrate lyase and acetyl-CoA carboxylase (acetyl-CoA:CO~ ligase (ADP~, EC 6.4.1.2). Primiparous Norwegian hooded rats were used and killed on the Ioth or I l t h day of lactation. The weaned rats were separated from their litters 24 h before killing and the unilaterally ligated rats had the galactophores on one side tied off subcutaneously under ether anaesthesia and were returned to their litters for 24 h before killing. Details of the preparation of the particle-free fraction of the mammary glands and the assays of enzyme activity and DNA have been given previously~. The results are presented in Table I where activities are expressed on the basis of the DNA content of the homogenate. According to OTA8 no significant change in TABLE I ENZYME ACTIVITIES IN RAT MAMMARY GLANDS Activities expressed as/*moles s u b s t r a t e utilised per rain per nag h o m o g e n a t e DNA. The n u m b e r s of rats in each group are given in p a r e n t h e s e s and each value is followed b y its S.E.
Enzyme
Status of rats Control (5)
Unilaterally ligated (4) Unligated side
Acetyl-CoA carboxylase Phosphofructokinase AT1~' citrate lyase Glucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Phosphoglucomntase UDPglucose p y r o p h o s p h o r y l a s e
0.745 i.IO 2.68 17.5 2.55 1.25 16.65
4444± ± ±
o.o71 o. i i 0.44 1.2 0.45 o.18 3.14
o.791 o.91o 2.42 17.9 3.09 1.34 I2.55
4- o.213 ± o.147 4- 0.40 ± 1. 5 ~ 0.30 ± o.13 ~_ 2.07
Weaned (3)
Liga*,ed side 0.049 0.o74 o.247 6.68 0.590 0.206 6.i8
44444± ±
o.o16 O.Ol 4 0.053 o.33 o.062* o.043 o-96
0.049 o.132 o.499 8.86 1.o 5 0.297 8.13
4~2 4± 44±
* Difference significant ( P < o . o 5, t test). No other pairs of values (i.e. control vs. unligated or weaned vs. ligated) significantly different (P > o.I),
l~iochim. Biophys. Acta, I77 (1968) 158-16o
o.oi2 o.o65 o.163 1.47 o.17" 0.049 0.92
I~0
SHORT CO~{I~IU N ICATION,<~
the D N A c o n t e n t of u n s u c k l e d r a t m a m m a r y glands occurs within 2 4 h a n d ti',u,~ these results p r o b a b l y reflect changes in tots1 a c t i v i t y . I t can be seen t h a t n o t ,,;n!v do weaning a n d u n i l a t e r a l ligation b o t h p r o d u c e decreases m e n z y m e activities b u t t h a t t h e p a t t e r n of decrease is. within t h e limits of e x p e r i m e n t a l error, i d e n t i c a l ia~_ b o t h conditions w i t h the possible e x c e p t i o n of 5 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e which is r a t h e r m o r e active in t h e u n s u c k l e d rats. T h u s acetvl-CoA carboxvlase. A T P c i t r a t e l y a s e a n d p h o s p h o f r u c t o k i n a s e decrease ~o 5 - x o % of t h e c o n t r o l a c t i v i t i e s while t h e o t h e r e n z y m e s show a smaller t h o u g h stiti m a r k e d decline. On the o t h e r h a n d a c t i v i t i e s in t h e c o n t r a l a t e r a l suckled glands remain identical w i t h t h o s e in t h e c o n t r o i rats. These findings p r o v i d e s t r o n g s u p p o r t for t h e t h e o r y t h a t the e n z w n e changes in t h e m a m m a r y g l a n d s of u n s u c k l e d r a t s are a direct response to e n g o r g e m e n t w i t h milk. As activities in t h e suckled glands of t h e u n i l a t e r a l l y l i g a t e d r a t s do n o t change, t h e levels of circulating g a l a c t o p o i e t i c h o r m o n e s in this c o n d i t i o n m u s t r e m a i n withi~i n o r m a i limits a n d changes in t h e engorged glands can be a t t r i b u t e d soIely to milk a c c u m u l a t i o n . This being so t h e r e ts no n e e d to invoke a n y endocrine m e c h a n i s m ~:~ ¢xpIain t h e similar changes in t h e u n s u c k l e d rat. However, as h y p o p h y s e c t o m y p r o d u c e s c o m p a r a b l e changes, which are u n r e l a t e d to the p a r t i a l e n g o r g e m e n t of the g l a n d s which occurs in u n t r e a t e d h y p o p h y s e c t o m i z e d r a t s ~, it is possible t h a t the changes in t h e u n s u c k l e d r a t are a result of a low level of h o r m o n e s at their site of action. P r o l o n g e d e n g o r g e m e n t of t h e r a t m a m m a r y gIand w i t h m i l k leads to a coilapse of t h e alveolar c a p i l l a r y b e d s a n d t h e gland is t h e n u n r e s p o n s i v e to o x v t o c i n even a f t e r t h e occiusion is released. Such a failure of t h e circulation w o u l d also p r e v e n t t h e access of t h e g a l a c t o p o i e t ic h o r m o n e s n e c e s s a r y for t h e m a i n t e n a n c e of l a c t a t i o n . O t h e r e x p l a n a t i o n s , such as a more direct i n t r a c e l l u l a r i n h i b i t i o n of h o r m o n e action b y a c c u m u l a t i n g u n s e c r e t e d m i l k c o n s t i t u e n t s , are aIso possible. I t m a y be t h a t the s i m i l a r i t y in t h e p a t t e r n of change of e n z y m e a c t i v i t i e s in i n v o l u t i n g glands, w h a t e v e r t h e f a c t o r p r o d u c i n g t h e i n v o l u t i o n , is a reflection of t h e different rares of t u r n o v e r of t h e v a r i o u s enzymes. W h a t e v e r t h e m e c h a n i s m , this direct effect of milk a c c u m u l a t i o n on e n z y m e activities o b v i o u s l y r e p r e s e n t s a useful f e e d - b a c k s y s t e m for adi u s t i n g mi!k p r o d u c t i o n in response ro d e m a n d . I wish to t h a n k Dr. A. T. Cowie for p e r f o r m i n g t h e s u r g e r y a n d Professor So ~i. F o l I e y for his e n c o u r a g e m e n t a n d advice.
Natio~al I~stitute for Resea~'c]~ i~ Dair2i~g, Shin.~eld, Reading (Great Britai~ I 1~_. E . MUNFORD, Dai¢y ~ci. AbstY. 26 ( I 9 6 4 293. 2 R . L. BALDWIN AND L. P. ~IILLIGAN. T Biol. Chem., 24I ( I 9 6 6 2058. 3 A. T. C o w I E , i n S. ][(. K o N AND A. T. COWIE iV[ilh: The JVIam~,~a~ VoL i , A c a d e m i c P r e s s , N e w Y o r k a n d L o n d o n , 1:96I , p. I63. 4 E . A. J o N ~ s , ]3iochem. J., Io3 (I967) 4zo. 5 R . L. BALDWIN AND R . J. l~fARrrlX. J. Daf~'y Sci., 5~ 1968 748. 6 P. NIcLEAN, Biochem. ~., 9o (1964) 271. 7 A. iF. SPENCER A~¢D J. M. LOWENSTEIN. Biochem. J . , 99 ( I 9 6 6 76o. 8 IK. OTA, l~ndoc*'i~zol..[al)o~'z. ~1 I964) ix6. 9 B. A. C R o s s AND L A. SILVER. P1,oe. ~OJ]..~OC. Meal.. 49 ( r 9 5 6 978.
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