- Email: [email protected]
Catalysis of hydrogen transfer between pyridine nucleotides by 3α-hydroxysteroid dehydrogenase
VOL. ~
(I958)
PRELIMINARY NOTES
20I
T A B L E II ACTIVATION O F
SOLUBLE RIBONUCLEASE
OF MOUSE
PANCREAS
F r e s h m o u s e p a n c r e a s w a s h o m o g e n i z e d in o.25 M sucrose and, a f t e r r e m o v a l of large particles, c e n t r i f u g e d for I h a t lOO,OOO x g. T h e s u p e r n a t a n t solution was t r e a t e d as indicated. I n t h e acid t r e a t m e n t t h e sucrose solution w a s b r o u g h t to o.25 N H2SO 4, t h e n a d j u s t e d to p H 5, a n d t h e p r e c i p i t a t e r e m o v e d b y c e n t r i f u g a t i o n . I n t h e others, solid NaC1 or E D T A w a s a d d e d to b r i n g t h e solution to o.1 M, t h e solution w a s a d j u s t e d to p H 9, a n d after 3o m i n a t r o o m t e m p e r a t u r e w a s a d j u s t e d to p H 5 a n d centrifuged. All a s s a y s were carried o u t on t h e clear s u p e r n a t a n t solutions b y t h e p r o c e d u r e of DICKMAN et al. 5 a t p H 5. R i b o n u c l e a s e a c t i v i t y is reported as a b s o r b a n c e a t 26o m p / m l of original s u p e r n a t a n t solution.
Ribonudease activity
Treatment
Atoo/ml
Relative %
Untreated pH 5 pH 9--+ pH 5 o.I M NaC1, p H 9 - - + p H 5 o.I M E D T A , p H 9 - - + p H 5 o.25 N H~SO 4 - - ~ p H 5
0.30 0.23 o.31 o.85 i.i 5 1.39
ioo 77 lO3 283 383 463
(44-1o4)* (77-233) * (IO6-337) * (i 49-567) : (195-6oo)
* Figures in p a r e n t h e s e s indicate r a n g e of relative activities in t h r e e a d d i t i o n a l e x p e r i m e n t s .
Department o/Biological Chemistry, University o/ Utah College o/ Medicine, Salt Lake City, Utah (U.S.A.) 1 G. 2 C. s G. 4 D. 6 S.
SHERMAN R. DICKMAN KATHRYN M. TRUPIN
A. MORRILL AND S. R. DICEMAN, Federation Proc., 16 (1957) 223. H. W . HIRS, S. MOORE AND W. H. STEIN, J. Biol. Chem., 2oo (1953) 493. A. MORRILL AND S. R. DICKMAN, in p r e p a r a t i o n . ELSON, Biochim. Biophys. Acta, 27 (1958) 216. R. DICKMAN, J. P. AROSKAR AND R. B. KROPF, Biochim. Biophys. Acta, 21 (1956) 539. Received J u n e 9th, I958.
Catalysis of hydrogen transfer between pyridine nucleotides by 3a-hydroxysteroid dehydrogenase* I n t h e presence of low c o n c e n t r a t i o n s of estradiol-I7fl or t e s t o s t e r o n e , soluble e n z y m e s f r o m h u m a n p l a c e n t a catalyze t h e t r a n s f e r of h y d r o g e n b e t w e e n t h e oxidized a n d r e d u c e d f o r m s of dia n d t r i p h o s p h o p y r i d i n e nucleotides I :
TPNH
+ DPN + ~ DPNH
+ TPN +
The enzymic mechanism of this hydrogen transfer has been shown to involve the reversible interconversion of the hydroxy- and corresponding ketosteroids by hydroxysteroid dehydrogenases w h i c h c a n react w i t h b o t h D P N a n d T P N 1,2. M a n y of t h e well recognized b i o c h e m i c a l effects w h i c h are associated w i t h steroid h o r m o n e action m a y be a t t r i b u t e d to h y d r o g e n t r a n s f e r b e t w e e n p y r i d i n e nucleotides in specific regions of t h e cell 1. Since o t h e r h y d r o x y s t e r o i d d e h y d r o g e n a s e s of a n i m a l origin react r e a d i l y w i t h b o t h D P N a n d T P N 1, s, 4, it b e c a m e of i n t e r e s t to e x a m i n e t h e generalization t h a t s u c h e n z y m e s are c o n c e r n e d w i t h p y r i d i n e nucleotide t r a n s h y d r o g e n a t i o n . W e wish to r e p o r t t h a t r a t liver 3 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e 4 in t h e presence of m i n u t e a m o u n t s of a n d r o s t e r o n e or o t h e r 3 a - h y d r o x y s t e r o i d s c a n m e d i a t e h y d r o g e n t r a n s f e r b e t w e e n p y r i d i n e nucleotides. P r e p a r a t i o n s of soluble, r a t - l i v e r 3 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e , purified 2o-fold, c a t a l y z e d a s t e r o i d - d e p e n d e n t t r a n s f e r of h y d r o g e n f r o m T P N H to D P N . T h i s r e a c t i o n w a s d e m o n s t r a t e d * S u p p o r t e d b y g r a n t s f r o m t h e A m e r i c a n Cancer Society.
202
PRELIMINARY NOTES
VOL. 3 0 (1958)
b y m a i n t a i n i n g a low a n d c o n s t a n t c o n c e n t r a t i o n of T P N H in t h e s y s t e m b y t h e a d d i t i o n of T P N , g l u c o s e - 6 - p h o s p h a t e a n d purified y e a s t g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e . U p o n t h e f u r t h e r a d d i t i o n of purified liver 3 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e , D P N a n d a n d r o s t e r o n e , a progressive r e d u c t i o n of D P N w a s o b s e r v e d w h i c h w a s d e p e n d e n t u p o n t h e p a r t i c i p a t i o n of steroid a n d T P N H (Table I). T h e p H o p t i m u m for t h i s r e a c t i o n w a s f o u n d to lie b e t w e e n 8.0 a n d 8. 4. T h e s e o b s e r v a t i o n s are c o n s i s t e n t w i t h t h e following f o r m u l a t i o n : G l u c o s e - 6 - p h o s p h a t e + T P N + ~--- 6 - P h o s p h o g l u c o n a t e + T P N H + H + H + + TPNH
+ Androstane-3,i7-dione ~
Androsterone + TPN +
DPN + + Androsterone ~- Androstane-3,I7-dione + DPNH Sum: Glucose-6-phosphate + DPN + ~
+ H+
6-Phosphogluconate + DPNH + H + TABLE I
HYDROGEN
TRANSFER
FROM
TPNH
TO
DPN
BY 3(1-HYDROXYSTIgROID
DEHYDROGENASE
Rate ol hydrogen trans/er. ink*moles reduced pyridine nucleotide ]orraed/h
Complete system Steroid o m i t t e d TPN omitted T P N a n d steroid o m i t t e d DPN omitted D P N a n d steroid o m i t t e d
166 24 2 2 2 2
h e c o m p l e t e r e a c t i o n s y s t e m c o n t a i n e d in 3.° m l : 2oo/~moles T r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e 8. 3, i o / , m o l e s g l u c o s e - 6 - p h o s p h a t e , a n excess of purified y e a s t g l u c o s e - 6 - p h o s p h a t e d e h y d r o genase, 0.02 /~mole T P N , 1. 4 #,moles D P N , 2 # g a n d r o s t e r o n e in o.oi m l dioxane, a n d 2.5 m g purified liver 3 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e . M e a s u r e m e n t s of a b s o r b a n c e a t 34 ° m/~ were m a d e a g a i n s t a c o n t r o l c u v e t t e c o n t a i n i n g no s t e r o i d a n d no nucleotides. T e m p . 25 °.
Significant r a t e s of t r a n s h y d r o g e n a t i o n were o b s e r v e d w i t h as little as i . I o - ~ M a n d r o s t e r o n e or e t i o c h o l a n - 3 a - o l - I 7 - o n e a n d m a x i m a l r a t e s were o b t a i n e d w i t h a b o u t 1. 5. I o - S M etiocholan3a-ol-I 7-one a n d 5" lO-6 M a n d r o s t e r o n e . T h e following 3a-hydrox3;- or u n c o n j u g a t e d 3-ketosteroids p r o m o t e d t r a n s h y d r o g e n a t i o n : a n d r o s t e r o n e , a n d r o s t a n e - 3 , I 7 - d i o n e , etiocholan-3a-ol-I7one, a n d p r e g n a n e - 3 a , I 7 , 2 I - t r i o l - I I , 2o-dtone. I n c o n t r a s t , t e s t o s t e r o n e , cortisol, i 7 - e p i t e s t o s t e rone, estrone, estradiol-I7fl, a n d d i e t h y l s t i l b e s t r o l were inactive in t h i s respect, a n d t h e s e c o m p o u n d s likewise did n o t r e d u c e p y r i d i n e n u c l e o t i d e s in t h e presence of t h e e n z y m e . H y d r o g e n t r a n s f e r to D P N f r o m T P N H as such' w a s also c a t a l y z e d b y t h e liver e n z y m e in t h e presence of a n d r o s t e r o n e . T h i s r e a c t i o n could be d e t e c t e d b y following thee decrease of t h e a b s o r p t i o n of T P N H a t 34 ° m/~ in t h e presence of D P N , a n d r o s t e r o n e , liver 3 a - h y d r o x y s t e r o i d d e h y d r o genase, a c e t a l d e h y d e a n d y e a s t alcohol d e h y d r o g e n a s e . T h e acetaldehyde, a n d alcohol d e h y d r o g e n a s e s e r v e d to reoxidize t h e D P N H f o r m e d in t h e r e a c t i o n 5. T h e o x i d a t i o n of T P N H w a s d e p e n d e n t u p o n t h e p r e s e n c e of b o t h D P N a n d of steroid. 3 a - H y d r o x y s t e r o i d d e h y d r o g e n a s e in c o n j u n c t i o n w i t h a n d r o s t e r o n e c a n also c a t a l y z e h y d r o g e n t r a n s f e r f r o m D P . N H t o t h e 3 - a c e t y l p y r i d i n e a n a l o g of D P N ( A P D P N ) e. T h e r e d u c e d a n a l o g ( A P D P N H ) m a y be c o n v e n i e n t l y m e a s u r e d a t 4oo m/* w h e r e D P N H h a s negligible a b s o r p tion. A P D P N c a n s u b s t i t u t e for p y r i d i n e n u c l e o t i d e s in a n u m b e r of e n z y m i c d e h y d r o g e n a t i o n s ~ including those catalyzed by 3a-hydroxysteroid dehydrogenase. T h e e x t e n t of h y d r o g e n t r a n s f e r b e t w e e n p y r i d i n e nucleotides b y 3 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e d e p e n d s critically u p o n c o n c e n t r a t i o n s of h y d r o g e n ions, steroids, a n d t h e r a t i o s of d o n o r to a c c e p t o r nucleotides. T h e efficiency of t h i s h y d r o g e n t r a n s f e r d e p e n d s u p o n a) t h e low Michaelis c o n s t a n t s for t h e s t e r o i d s ; b) t h e m a i n t e n a n c e of s a t u r a t i n g c o n c e n t r a t i o n s of b o t h h y d r o x y - a n d k e t o s t e r o i d s in t h e r e a c t i o n s y s t e m , a n d c) t h e a p p r o p r i a t e r a t i o s of nucleotides so t h a t t h e s e do n o t displace e a c h o t h e r f r o m a single b i n d i n g site o n t h e e n z y m e surface. Evidence has been presented that rat-liver 3a-hydroxysteroid dehydrogenase can catalyze a h y d r o g e n t r a n s f e r b e t w e e n p y r i d i n e n u c l e o t i d e s in w h i c h steroids a s s u m e t h e role of h y d r o g e n carriers f o r m a l l y s i m i l a r to c o e n z y m e s . T h i s finding lends s u p p o r t to t h e view 1 t h a t all h y d r o x y -
VOL. ~
(1958)
PRELIMINARY NOTES
203
steroid dehydrogenases with dual nucleotide specificity can act as pyridine nucleotide transhydrogenases. It is hoped to publish full experimental details in the near future.
Ben May Laboratory ]or Cancer Research and Department o] Biochemistry, University o] Chicago, Chicago, Ill. (U.S.A .)
BARBARA HURLOCK PAUL TALALAY*
1 p. TALALAYAND H, G. WILLIAMN-ASHMAN,Proc. Natl. Acad. Sci., U.S., 44 (1958) 15. $ P. TALALAY, B. HURLOCKAND H. G. WILLIAMS-ASHMAN,Science, 127 (1958) io6o.
8 p. TALALAY,Physiol. Revs., 37 (1957) 362. 4 G. i . TOMKINS, J; Biol. Chem., 218 (1956) 437. 6 N. 0. KAPLAN, S. P. COLOWlCK AND E. F. ~NEUFELD,J. Biol. Chem., 195 (1952) lO7. &N. O. KAPLANAND M. M. CIOTTI, J. Biol. Chem., 221 (1956) 823. 7 ~s~.0. KAPLAN, M. M. CIOTTI AND F. E. STOLZENBACH,J. Biol. Chem., 221 (i956) 833. Received June zoth, 1958 * Aided by a faculty-level grant from the American Cancer Society.
An enzymic defect in ascites-tumor cells* Although glycogen phosphorylase is found in almost all mammalian tissues, greatly reduced phosphorylase activities recently have been ascribed to ascites tumors I and a solid tumor 2. The following hormonally-dependent sequence of reactions been proposed for phosphorylase activation in liver and adrenal cortex 3-5. ATP, Mg++ Epinephrine I Dephosphophosphoryiase Glucagon ......................... -~ (Inactive) ACTH $ I Dephosphosphophorylase Adenosine-3', if-cyclic phosphates ................~" ~kinase (ATP?) Phosphorylase (active) This communication demonstrates a complete absence of phosphorylase activity in eight different types of tumors. The cells were found to contain phosphorylase-activating enzymes but lacked specifically both active and inactive forms of phosphorylase, and therefore, they did not respond physiologically to epinephrine or glucagon. The following ascites tumors were used: Hepatoma, Ehrlich carcinoma, lymphocytic leukemia, plasma cell, mast cell, Krebs-2 carcinoma, and sarcoma-37; also the HeLa carcinoma grown in tissue culture. Freshly harvested tumors contained very low levels of glycogen, approximating 5 /zmoles glucose equivalents/g protein, as determined analytically by the method of STADm, HAUGAARD AND MARSH?. Histochemical examination with HIO4-Schiff reagent indicated that the glycogen was confined entirely to normal polymorphonuclear leukocytes which comprised 1-2 % of the ascites-cell populations. Ascites-tumor cells grown in rive and the HeLa carcinoma grown in tissue culture contained no detectable glycogen. In contrast, normal mouse-liver epithelial cells grown in tissue culture are known to contain glycogens . The phosphorylase activities of whole, undialyzed tumor and normal mouse-liver homogenates were determined by the method of SUTHERLANDAND WOSILAIT;. An active phosphorylase was found in mouse-liver homogenates (i .53/zmoles phosphate released/rag protein[Io min). Phosphatase activity was negligible. Greatly decreased phosphorylase activities were found in hepatoma homogenates ( ~ o. i/zmole phosphate/rag protein/lO min). Optimal activity was obtained between pH 6 - 7. Addition of If-AMP did not result in increased phosphorylase activity, nor did the use of a wide variety of homogenization techniques. Similar results were obtained with the previously listed tumors. A histochemical phosphorylase assay 9 was applied to the hepatoma, Ehrlich carcinoma and Krebs-2 carcinoma. An active phosphorylase was found in the polymorphonuclear leukocytes; none was present in the tumor cells. The phosphorylase reaction was then measured in the reverse direction, from glycogen to G-x-P, by following the disappearance of added glycogen * The following abbreviations are used : 5'-AMP, adenylic acid; ATP, adenosine triphosphate; G-I-P, glucose-I-phosphate; G-6-P, glucose-6-phosphate; TCA, trichloroacetic acid; TRIS, tris (hydroxymethyl) aminomethane; D P P kinase, dephosphophosphorylase kinase.
(I958)
PRELIMINARY NOTES
20I
T A B L E II ACTIVATION O F
SOLUBLE RIBONUCLEASE
OF MOUSE
PANCREAS
F r e s h m o u s e p a n c r e a s w a s h o m o g e n i z e d in o.25 M sucrose and, a f t e r r e m o v a l of large particles, c e n t r i f u g e d for I h a t lOO,OOO x g. T h e s u p e r n a t a n t solution was t r e a t e d as indicated. I n t h e acid t r e a t m e n t t h e sucrose solution w a s b r o u g h t to o.25 N H2SO 4, t h e n a d j u s t e d to p H 5, a n d t h e p r e c i p i t a t e r e m o v e d b y c e n t r i f u g a t i o n . I n t h e others, solid NaC1 or E D T A w a s a d d e d to b r i n g t h e solution to o.1 M, t h e solution w a s a d j u s t e d to p H 9, a n d after 3o m i n a t r o o m t e m p e r a t u r e w a s a d j u s t e d to p H 5 a n d centrifuged. All a s s a y s were carried o u t on t h e clear s u p e r n a t a n t solutions b y t h e p r o c e d u r e of DICKMAN et al. 5 a t p H 5. R i b o n u c l e a s e a c t i v i t y is reported as a b s o r b a n c e a t 26o m p / m l of original s u p e r n a t a n t solution.
Ribonudease activity
Treatment
Atoo/ml
Relative %
Untreated pH 5 pH 9--+ pH 5 o.I M NaC1, p H 9 - - + p H 5 o.I M E D T A , p H 9 - - + p H 5 o.25 N H~SO 4 - - ~ p H 5
0.30 0.23 o.31 o.85 i.i 5 1.39
ioo 77 lO3 283 383 463
(44-1o4)* (77-233) * (IO6-337) * (i 49-567) : (195-6oo)
* Figures in p a r e n t h e s e s indicate r a n g e of relative activities in t h r e e a d d i t i o n a l e x p e r i m e n t s .
Department o/Biological Chemistry, University o/ Utah College o/ Medicine, Salt Lake City, Utah (U.S.A.) 1 G. 2 C. s G. 4 D. 6 S.
SHERMAN R. DICKMAN KATHRYN M. TRUPIN
A. MORRILL AND S. R. DICEMAN, Federation Proc., 16 (1957) 223. H. W . HIRS, S. MOORE AND W. H. STEIN, J. Biol. Chem., 2oo (1953) 493. A. MORRILL AND S. R. DICKMAN, in p r e p a r a t i o n . ELSON, Biochim. Biophys. Acta, 27 (1958) 216. R. DICKMAN, J. P. AROSKAR AND R. B. KROPF, Biochim. Biophys. Acta, 21 (1956) 539. Received J u n e 9th, I958.
Catalysis of hydrogen transfer between pyridine nucleotides by 3a-hydroxysteroid dehydrogenase* I n t h e presence of low c o n c e n t r a t i o n s of estradiol-I7fl or t e s t o s t e r o n e , soluble e n z y m e s f r o m h u m a n p l a c e n t a catalyze t h e t r a n s f e r of h y d r o g e n b e t w e e n t h e oxidized a n d r e d u c e d f o r m s of dia n d t r i p h o s p h o p y r i d i n e nucleotides I :
TPNH
+ DPN + ~ DPNH
+ TPN +
The enzymic mechanism of this hydrogen transfer has been shown to involve the reversible interconversion of the hydroxy- and corresponding ketosteroids by hydroxysteroid dehydrogenases w h i c h c a n react w i t h b o t h D P N a n d T P N 1,2. M a n y of t h e well recognized b i o c h e m i c a l effects w h i c h are associated w i t h steroid h o r m o n e action m a y be a t t r i b u t e d to h y d r o g e n t r a n s f e r b e t w e e n p y r i d i n e nucleotides in specific regions of t h e cell 1. Since o t h e r h y d r o x y s t e r o i d d e h y d r o g e n a s e s of a n i m a l origin react r e a d i l y w i t h b o t h D P N a n d T P N 1, s, 4, it b e c a m e of i n t e r e s t to e x a m i n e t h e generalization t h a t s u c h e n z y m e s are c o n c e r n e d w i t h p y r i d i n e nucleotide t r a n s h y d r o g e n a t i o n . W e wish to r e p o r t t h a t r a t liver 3 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e 4 in t h e presence of m i n u t e a m o u n t s of a n d r o s t e r o n e or o t h e r 3 a - h y d r o x y s t e r o i d s c a n m e d i a t e h y d r o g e n t r a n s f e r b e t w e e n p y r i d i n e nucleotides. P r e p a r a t i o n s of soluble, r a t - l i v e r 3 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e , purified 2o-fold, c a t a l y z e d a s t e r o i d - d e p e n d e n t t r a n s f e r of h y d r o g e n f r o m T P N H to D P N . T h i s r e a c t i o n w a s d e m o n s t r a t e d * S u p p o r t e d b y g r a n t s f r o m t h e A m e r i c a n Cancer Society.
202
PRELIMINARY NOTES
VOL. 3 0 (1958)
b y m a i n t a i n i n g a low a n d c o n s t a n t c o n c e n t r a t i o n of T P N H in t h e s y s t e m b y t h e a d d i t i o n of T P N , g l u c o s e - 6 - p h o s p h a t e a n d purified y e a s t g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e . U p o n t h e f u r t h e r a d d i t i o n of purified liver 3 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e , D P N a n d a n d r o s t e r o n e , a progressive r e d u c t i o n of D P N w a s o b s e r v e d w h i c h w a s d e p e n d e n t u p o n t h e p a r t i c i p a t i o n of steroid a n d T P N H (Table I). T h e p H o p t i m u m for t h i s r e a c t i o n w a s f o u n d to lie b e t w e e n 8.0 a n d 8. 4. T h e s e o b s e r v a t i o n s are c o n s i s t e n t w i t h t h e following f o r m u l a t i o n : G l u c o s e - 6 - p h o s p h a t e + T P N + ~--- 6 - P h o s p h o g l u c o n a t e + T P N H + H + H + + TPNH
+ Androstane-3,i7-dione ~
Androsterone + TPN +
DPN + + Androsterone ~- Androstane-3,I7-dione + DPNH Sum: Glucose-6-phosphate + DPN + ~
+ H+
6-Phosphogluconate + DPNH + H + TABLE I
HYDROGEN
TRANSFER
FROM
TPNH
TO
DPN
BY 3(1-HYDROXYSTIgROID
DEHYDROGENASE
Rate ol hydrogen trans/er. ink*moles reduced pyridine nucleotide ]orraed/h
Complete system Steroid o m i t t e d TPN omitted T P N a n d steroid o m i t t e d DPN omitted D P N a n d steroid o m i t t e d
166 24 2 2 2 2
h e c o m p l e t e r e a c t i o n s y s t e m c o n t a i n e d in 3.° m l : 2oo/~moles T r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e 8. 3, i o / , m o l e s g l u c o s e - 6 - p h o s p h a t e , a n excess of purified y e a s t g l u c o s e - 6 - p h o s p h a t e d e h y d r o genase, 0.02 /~mole T P N , 1. 4 #,moles D P N , 2 # g a n d r o s t e r o n e in o.oi m l dioxane, a n d 2.5 m g purified liver 3 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e . M e a s u r e m e n t s of a b s o r b a n c e a t 34 ° m/~ were m a d e a g a i n s t a c o n t r o l c u v e t t e c o n t a i n i n g no s t e r o i d a n d no nucleotides. T e m p . 25 °.
Significant r a t e s of t r a n s h y d r o g e n a t i o n were o b s e r v e d w i t h as little as i . I o - ~ M a n d r o s t e r o n e or e t i o c h o l a n - 3 a - o l - I 7 - o n e a n d m a x i m a l r a t e s were o b t a i n e d w i t h a b o u t 1. 5. I o - S M etiocholan3a-ol-I 7-one a n d 5" lO-6 M a n d r o s t e r o n e . T h e following 3a-hydrox3;- or u n c o n j u g a t e d 3-ketosteroids p r o m o t e d t r a n s h y d r o g e n a t i o n : a n d r o s t e r o n e , a n d r o s t a n e - 3 , I 7 - d i o n e , etiocholan-3a-ol-I7one, a n d p r e g n a n e - 3 a , I 7 , 2 I - t r i o l - I I , 2o-dtone. I n c o n t r a s t , t e s t o s t e r o n e , cortisol, i 7 - e p i t e s t o s t e rone, estrone, estradiol-I7fl, a n d d i e t h y l s t i l b e s t r o l were inactive in t h i s respect, a n d t h e s e c o m p o u n d s likewise did n o t r e d u c e p y r i d i n e n u c l e o t i d e s in t h e presence of t h e e n z y m e . H y d r o g e n t r a n s f e r to D P N f r o m T P N H as such' w a s also c a t a l y z e d b y t h e liver e n z y m e in t h e presence of a n d r o s t e r o n e . T h i s r e a c t i o n could be d e t e c t e d b y following thee decrease of t h e a b s o r p t i o n of T P N H a t 34 ° m/~ in t h e presence of D P N , a n d r o s t e r o n e , liver 3 a - h y d r o x y s t e r o i d d e h y d r o genase, a c e t a l d e h y d e a n d y e a s t alcohol d e h y d r o g e n a s e . T h e acetaldehyde, a n d alcohol d e h y d r o g e n a s e s e r v e d to reoxidize t h e D P N H f o r m e d in t h e r e a c t i o n 5. T h e o x i d a t i o n of T P N H w a s d e p e n d e n t u p o n t h e p r e s e n c e of b o t h D P N a n d of steroid. 3 a - H y d r o x y s t e r o i d d e h y d r o g e n a s e in c o n j u n c t i o n w i t h a n d r o s t e r o n e c a n also c a t a l y z e h y d r o g e n t r a n s f e r f r o m D P . N H t o t h e 3 - a c e t y l p y r i d i n e a n a l o g of D P N ( A P D P N ) e. T h e r e d u c e d a n a l o g ( A P D P N H ) m a y be c o n v e n i e n t l y m e a s u r e d a t 4oo m/* w h e r e D P N H h a s negligible a b s o r p tion. A P D P N c a n s u b s t i t u t e for p y r i d i n e n u c l e o t i d e s in a n u m b e r of e n z y m i c d e h y d r o g e n a t i o n s ~ including those catalyzed by 3a-hydroxysteroid dehydrogenase. T h e e x t e n t of h y d r o g e n t r a n s f e r b e t w e e n p y r i d i n e nucleotides b y 3 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e d e p e n d s critically u p o n c o n c e n t r a t i o n s of h y d r o g e n ions, steroids, a n d t h e r a t i o s of d o n o r to a c c e p t o r nucleotides. T h e efficiency of t h i s h y d r o g e n t r a n s f e r d e p e n d s u p o n a) t h e low Michaelis c o n s t a n t s for t h e s t e r o i d s ; b) t h e m a i n t e n a n c e of s a t u r a t i n g c o n c e n t r a t i o n s of b o t h h y d r o x y - a n d k e t o s t e r o i d s in t h e r e a c t i o n s y s t e m , a n d c) t h e a p p r o p r i a t e r a t i o s of nucleotides so t h a t t h e s e do n o t displace e a c h o t h e r f r o m a single b i n d i n g site o n t h e e n z y m e surface. Evidence has been presented that rat-liver 3a-hydroxysteroid dehydrogenase can catalyze a h y d r o g e n t r a n s f e r b e t w e e n p y r i d i n e n u c l e o t i d e s in w h i c h steroids a s s u m e t h e role of h y d r o g e n carriers f o r m a l l y s i m i l a r to c o e n z y m e s . T h i s finding lends s u p p o r t to t h e view 1 t h a t all h y d r o x y -
VOL. ~
(1958)
PRELIMINARY NOTES
203
steroid dehydrogenases with dual nucleotide specificity can act as pyridine nucleotide transhydrogenases. It is hoped to publish full experimental details in the near future.
Ben May Laboratory ]or Cancer Research and Department o] Biochemistry, University o] Chicago, Chicago, Ill. (U.S.A .)
BARBARA HURLOCK PAUL TALALAY*
1 p. TALALAYAND H, G. WILLIAMN-ASHMAN,Proc. Natl. Acad. Sci., U.S., 44 (1958) 15. $ P. TALALAY, B. HURLOCKAND H. G. WILLIAMS-ASHMAN,Science, 127 (1958) io6o.
8 p. TALALAY,Physiol. Revs., 37 (1957) 362. 4 G. i . TOMKINS, J; Biol. Chem., 218 (1956) 437. 6 N. 0. KAPLAN, S. P. COLOWlCK AND E. F. ~NEUFELD,J. Biol. Chem., 195 (1952) lO7. &N. O. KAPLANAND M. M. CIOTTI, J. Biol. Chem., 221 (1956) 823. 7 ~s~.0. KAPLAN, M. M. CIOTTI AND F. E. STOLZENBACH,J. Biol. Chem., 221 (i956) 833. Received June zoth, 1958 * Aided by a faculty-level grant from the American Cancer Society.
An enzymic defect in ascites-tumor cells* Although glycogen phosphorylase is found in almost all mammalian tissues, greatly reduced phosphorylase activities recently have been ascribed to ascites tumors I and a solid tumor 2. The following hormonally-dependent sequence of reactions been proposed for phosphorylase activation in liver and adrenal cortex 3-5. ATP, Mg++ Epinephrine I Dephosphophosphoryiase Glucagon ......................... -~ (Inactive) ACTH $ I Dephosphosphophorylase Adenosine-3', if-cyclic phosphates ................~" ~kinase (ATP?) Phosphorylase (active) This communication demonstrates a complete absence of phosphorylase activity in eight different types of tumors. The cells were found to contain phosphorylase-activating enzymes but lacked specifically both active and inactive forms of phosphorylase, and therefore, they did not respond physiologically to epinephrine or glucagon. The following ascites tumors were used: Hepatoma, Ehrlich carcinoma, lymphocytic leukemia, plasma cell, mast cell, Krebs-2 carcinoma, and sarcoma-37; also the HeLa carcinoma grown in tissue culture. Freshly harvested tumors contained very low levels of glycogen, approximating 5 /zmoles glucose equivalents/g protein, as determined analytically by the method of STADm, HAUGAARD AND MARSH?. Histochemical examination with HIO4-Schiff reagent indicated that the glycogen was confined entirely to normal polymorphonuclear leukocytes which comprised 1-2 % of the ascites-cell populations. Ascites-tumor cells grown in rive and the HeLa carcinoma grown in tissue culture contained no detectable glycogen. In contrast, normal mouse-liver epithelial cells grown in tissue culture are known to contain glycogens . The phosphorylase activities of whole, undialyzed tumor and normal mouse-liver homogenates were determined by the method of SUTHERLANDAND WOSILAIT;. An active phosphorylase was found in mouse-liver homogenates (i .53/zmoles phosphate released/rag protein[Io min). Phosphatase activity was negligible. Greatly decreased phosphorylase activities were found in hepatoma homogenates ( ~ o. i/zmole phosphate/rag protein/lO min). Optimal activity was obtained between pH 6 - 7. Addition of If-AMP did not result in increased phosphorylase activity, nor did the use of a wide variety of homogenization techniques. Similar results were obtained with the previously listed tumors. A histochemical phosphorylase assay 9 was applied to the hepatoma, Ehrlich carcinoma and Krebs-2 carcinoma. An active phosphorylase was found in the polymorphonuclear leukocytes; none was present in the tumor cells. The phosphorylase reaction was then measured in the reverse direction, from glycogen to G-x-P, by following the disappearance of added glycogen * The following abbreviations are used : 5'-AMP, adenylic acid; ATP, adenosine triphosphate; G-I-P, glucose-I-phosphate; G-6-P, glucose-6-phosphate; TCA, trichloroacetic acid; TRIS, tris (hydroxymethyl) aminomethane; D P P kinase, dephosphophosphorylase kinase.