15.3. 1976
285
Specialia
(0.15 mmol) in a c e t o n e - d 6 (0.5 ml); m e t h a n o l - d 4 (0.2 ml) was a d d e d a n d a f t e r 24 h ( N - I t t o N - D ) t h e c u r v e s were r e c o r d e d again. T h e singlet (d 8.6-8.8) of t h e p i c r a t e a n i o n was t a k e n as p r o t o n - c o u n t i n g reference; t h e n u m ber of N - H r e s u l t e d d i r e c t l y f r o m t h e i n t e g r a t i o n c u r v e s or f r o m t h e difference w i t h t h o s e o b t a i n e d a f t e r a d d i t i o n of m e t h a n o t - d 4. T h e n u m b e r f o u n d for N-I-i w a s u s u a l l y w i t h i n 0 t o - - 1 0 % of t h e c o r r e c t one. T h e s p e c t r a do n o t s h o w s e p a r a t e d signals for E - Z isomers ~4. T h e N - H signals are g e n e r a l l y c h a r a c t e r i z e d b y : d-values in t h e r a n g e 6.7-8.2 ; h a l f - b a n d w i d t h (WII~) 5-18 H z ; t h e y d i s a p p e a r w i t h m e t h a n o l - d 4 ; &values are n o t c h a n g e d or are s l i g h t l y d e c r e a s e d (less t h a n 0.1 p p m ) b y m e t h a n o l . A n aryl s u b s t i t u e n t p r o d u c e s : a s t r o n g downfield s h i f t of t h e A r - N - H signal; a small effect on t h e p r o t o n s of t h e o t h e r N - a t o m s ; t h e N - H r e s o n a n c e s d i s a p p e a r ( ' a v e r a g e d ' w i t h M e O - H ) o r are s t r o n g l y broadened by methanol. The a r o m a t i c - H signals are d i s t i n g u i s h e d b e c a u s e t h e y are n o t r e m o v e d b y m e t h a n o l - d 4. T h e N - H signals of a m i n e s (as p i c r a t e salts) are usually v e r y b r o a d (Wl/2 > 40 Hz) a n d d i s a p p e a r b y m e t h a n o l . T h e O - H (W v 2 ca. 2 Hz) or N - H ( W ~ > 40 Hz) signals of p h e n o l s a n d c a r b o x a m i d e s are s h i f t e d downfield (0.2-0.3 p p m ) b y methanol. A f t e r t h e p r e s e n c e of t h e g u a n i d i n i u m g r o u p h a s been. a s c e r t a i n e d , its s u b s t i t u t i o n p a t t e r n r e s u l t s in m a n y e x a m p l e s f r o m t h e N - H signals in t h e s p e c t r a m e a s u r e d in p l a i n acetone. Thus, a n e x a m p l e (R ~ = R a = cycloh e x y l ; R 2 = R ~ = R 5 --- H) e x h i b i t s 4 N - H d i s t r i b u t e d in 2 signals (d 7.06 a n d 7.26; area ratio 1:3) p a r t i a l l y superposed; these data indicate non-equivalent N-H groups, a n d t h e r e f o r e t h e c o m p o u n d is a N~, Ne-disubstit u t e d g u a n i d i n i u m . T h e o b s e r v e d a r e a r a t i o is due t o s u p e r p o s i t i o n of t h e lower-field c o m p o n e n t of t h e s p l i t t e d N - H signal (doublet; t w o H - N - C - H ) w i t h t h e NHg singlet. T h e s p l i t t i n g is c o n f i r m e d b y d e c o u p l i n g w i t h ferric chloride. T h e scope is w i d e n e d , including t h e results in a c e t o n e dd, w h i c h allows t h e o b s e r v a t i o n of t h e N - C - H signals a t d 3.0-4.3; an a d j a c e n t aryl g r o u p ( N - C H - A r ) p r o d u c e s a downfield s h i f t (d 4.5-4.9). T h e m e t h o d n o w t a k e s into a c c o u n t : n u m b e r of g u a n i d i n i u m N - H ; o c c u r r e n c e of a singlet or m o r e N - H signals arising f r o m n o n - e q u i v a l e n c e or spin-spin coupling w i t h N - C - H ; n u m b e r of N - C - H m u l t i p l e t s simplified b y m e t h a n o l - d 4. One e x a m p l e is g i v e n in d e t a i l (R ~ = R ~- = e t h y l ; R ~ = m e t h y l ; R* = H ; R s = benzyl) w h i c h gives a s h a r p signal a t d 7.33 t h a t in its b a s e is p a r t i a l l y superp o s e d w i t h a b r o a d signal (total area 7 H). A f t e r a d d i t i o n
of m e t h a n o l - d 4 it r e m a i n s a 5H-singlet (aromatic-H), i n d i c a t i n g t h a t t h e c o m p o u n d is a t e t r a - s u b s t i t u t e d g u a n i d i u i u m . Besides, m e t h a n o l - d 4 simplifies t w o d o u b l e t s t o give t h e c o r r e s p o n d i n g singlets a t d 3.00 a n d 4.55; t h i s implies t h a t t w o of t h e s u b s t i t u e n t s are located on N - a t o m s b e a r i n g a p r o t o n r e s p o n s i b l e of t h e N - C - H splittings. T h e n , t h e c o m p o u n d is a N 1, N1,N~, N3-tetrasubstituted guanidinium. S o m e of t h e c o m p o u n d s were also m e a s u r e d using p l a i n a n d h e x a d e u t e r a t e d d i m e t h y l s u l p h o x i d e as s o l v e n t s ; t h e results were generally similar t o t h o s e q u o t e d above. These s o l v e n t s are m a i n l y useful for low soluble p i c r a t e s (e.g. R 1 = R 2 = m e t h y l ; R a = R ~ = R 5 = H).
1 Part XX of Studies on Plants; preceding part, R. A. CORRAL,O. O. ORAZI and I. A. BE~AGES, Tetrahedron 29, 205 (1973). 2 N. VANTHOAI and J. ROCHE,in Fortschritte der Chemic Organischer Nat~rstoffe (Ed. L. ZECHMEISTER;Springer Verlag, Wien 1960), vol. 18, p. 83. - J. NhERSCH, Biol. Rnndsch. 6, 72 (1968). - J. A. JOULE, in Annual Reports (The Chem. See., London 1969), vol. 66B, p. 485. - F. KHUO~G-Huu, J. P. LE FORESTIER and R. GOUTAREL,Tetrahedron 28, 5207 (1972). 3 See for example, J. ABERHART,R. C. JAIN, T. FEHR, P. DE MAYO and I. SZILAOYI, J. chem. See. Perkin I, 816 (1974). - T . GoTo, Y. KISHI, S. TAKAHASHI and Y. HIRATA, Tetrahedron 21, 2059 (1965). 4 I. SMITH,Chromatographic and Electrophoretic Techniques (Heinemann Medical Books, London 1960), vol. 1, p. 225. 5 T. GOTO, Y. HIRATA, S. HOSOYAand N. KOMATSU,Bull. chem. Soc., Japan 30, 729 (1957). s See for example, S. RAlVIAKRISI-n, fA and R. ADIGA, Phytoehemistry 72, 2961 (1973). 7 G. M. SFIARMAand P. R. BURKHOLDER,Chem. Conlmun. 7977, 151. s T. COTe, I{. NAKANISHIand M. OHASm, Bull. chem. See., Japan 30, 723 (1957). - E. SCHREIBER,K. PUFAHLand H. BRAEONIGER, Justus Liebigs Annln. Chem. 671, 147 (1964). 9 R. GREENHALGI~and R. A. BANNARD,Can. J. Chem. 39, 1017 (1961). - K. MATSUr~OTO and H. RAPOPOR% J. org. Chem. 33, 552 (1968). 10 R. B. CoNI~ and R. B. DAvis, Nature, Lend. 183, 1053 (1959). F. FA~RE and P. BLANQUET,Bull. Soc. Chim. biol. d3, 953 (1961). n D. WATSOIr Experientia 22, 76 (1966). - J. L6PEz-GoRoE and M. 1V~oNTEOLIVA,J. Chromat. 29, 300 (1967). 12 R. A. CORRAL,O. O. ORAZI and M. F. DE PETRUeCELLI, Rev. Iatinoam. Quire. 2, 178 (1971). 1, S. SUGIURA, S. I~OUE, Y. HAYASHI, Y. KlSm and T. GOTO, Tetrahedron 25, 5155 (1969), and references therein.
14 V. J. BAUER,W. FULMOR,G. O. M O R T O N and S. R. SAFIR, ~. Am. chem. See. 90, 6846 (1968). - H. KESSLER,Tetrahedron 30, 1861 (1974), and references therein.
Double Stranded Ribonuclease Activity in Human Lymphocyte Nuclei U. TORELLI, ST. FERRARI, G. TORELLI, R. CADOSSI a n d S. tFERRARI
Istituto di Patologia Medics dell' Universit?~, Via del Pozzo 7J, l - d / 7 0 0 Modena ( ftaly), 8 October 1975. S u m m a r y . R i b o n u c l e a s e a c t i v i t y d i r e c t e d a g a i n s t t h e s y n t h e t i c d u p l e x p o l y r C : p o l y r I was d e t e c t e d in n u c l e a r e x t r a c t s f r o m b o t h u n s t i m u l a t e d a n d P H A - s t i m u l a t e d h u m a n l y m p h o c y t e s . I n t h e l a t t e r cells, t h e a c t i v i t y was a b o u t t w i c e t h a t of small l y m p h o c y t e s . I t h a s b e e n s h o w n t h a t h u m a n small l y m p h o c y t e s synthesize mainlyunmethylated rapidly sedimenting RNA molecules w h i c h h y b r i d i z e v e r y efficiently t o D N A in v i t r o a n d are b o u n d to p o l y a d e n y l i c s e q u e n c e s in a p r o p o r t i o n u p t o 20% 1, 3. I t was t h u s c o n c l u d e d t h a t t h e m a j o r p o r t i o n of t h e R N A s y n t h e s i z e d in t h e s e cells is t h e h e t e r o g e n e o u s n u c l e a r R N A . Since it h a s b e e n s h o w n
b y several a u t h o r s t h a t a s i g n i f i c a n t p o r t i o n of n u c l e a r R N A of t h e h e t e r o g e n e o u s t y p e in a n i m a l cells is in a n R N a s e - r e s i s t a n t f o r m w i t h p r o p e r t i e s of d o u b l e - s t r a n d e d 1 U. 71"ORELLI,P. HENRY and S. WEISSMAN,J. elin. Invest. 47, 1083 (1968). 2 U. TORELLI and G, TORELLI~Acta haemat. 51, 140 (1974).
286
Specialia
EXPERIENTIA
32/3
Ribonuelease activity directed against single-stranded and double-stranded synthetic polyribonucleotides in nuclei of unstimulated and PHA-stimulated normal human lymphoeytes Donor No.
1 2 3
Substrate
8H-polyC-polyI 3H-polyrC aH-polyrC-p01yrI SH-polyrC 8H-polyrC-polyrI 9 3H-polyrC
PHA-stimulated lymphocytes
Small lymphocytes Control incubation (cpm)
Substrate radioactivity hydrolyzed (%)
Controlincubation (cpm)
Substrate radioactivity hydrolyzed (%)
6450 5100 4430 4500 5200 2800
11 12 13 11 27 18
6450 5100 4430 4500 5200 2800
25 15 27 13 56 21
After dissolution of the nuclei in 1 M NaC1 buffer, the extract was dialyzed against 0.14 M NaC1 buffer and incubations performed in buffer of this molarity.
R N A 3-v, i t was n o t u n e x p e c t e d to o b s e r v e t h a t a b o u t 2 % of t h e t o t a l l a b e l l e d cell R N A of s m a l l l y m p h o c y t e s shows d o u b l e - s t r a n d e d p r o p e r t i e s s. A t v a r i a n c e w i t h t h a t o b s e r v e d in several a n i m a l cell types, t h e t u r n o v e r r a t e of h e t e r o g e n e o u s n u c l e a r R N A i n s m a l l l y m p h o c y t e s a p p e a r s r e m a r k a b l y low, as s u g g e s t e d b y t h e a s s o c i a t i o n of t h e m a j o r i t y of a labelled p r e c u r s o r w i t h r a p i d l y s e d i m e n t i n g molecules e v e n a f t e r several h o u r s of a c t i n o m y c i n chase1, 2. T h i s c o n c l u s i o n is also s u p p o r t e d b y t h e o b s e r v a t i o n t h a t cold u r i d i n e c h a s e for several h o u r s does n o t s i g n i f i c a n t l y c h a n g e t h e p r o p o r t i o n of labelled d o u b l e - s t r a n d e d IRNA s, 9. Phytohemagglutinin ( P H A ) s t i m u l a t i o n of s m a l l l y m p h o c y t e s b r i n g s a b o u t a s h a r p c h a n g e in m e t a b o l i s m of n u c l e a r l y m p h o c y t e R N A , c h a r a c t e r i z e d b y a large increase in t h e r a t e of s y n t h e s i s a n d processing of ribosom a l p r e c u r s o r R N A , as well as b y i n c r e a s e in t u r n o v e r r a t e of t h e h e t e r o g e n e o u s n u c l e a r R N A 1 , ~. V e r y l i t t l e i n f o r m a t i o n is so f a r a v a i l a b l e a b o u t e n z y m e s y s t e m s i n v o l v e d in m e t a b o l i s m of n u c l e a r l y m p h o c y t e R N A . T h e p r e s e n c e of d o u b l e - s t r a n d e d R N A s e g m e n t s in h e t e r o g e n e o u s n u c l e a r R N A justifies t h e h y p o t h e s i s t h a t t h e c l e a v a g e of t h e large R N A molecules of t h i s R N A class i n v o l v e s t h e a c t i v i t y of a n e n z y m e w i t h a d e f i n i t e preference for d o u b l e - s t r a n d e d R N A , in a m a n n e r s i m i l a r t o w h a t h a s b e e n r e c e n t l y d e m o n s t r a t e d in b a c t e r i a l cells. I n t h e s e cells, in fact, i t w a s r e c e n t l y o b s e r v e d t h a t r i b o n u c l e a s e I I I , a n e n z y m e h i g h l y specific for d o u b l e s t r a n d e d R N A , h a s a k e y role in p r o c e s s i n g of m e s s e n g e r as well as r i b o s o m a l R N A p r e c u r s o r s 10-z3. T h i s p a p e r s h o r t l y r e p o r t s t h e r e s u l t s of a g r o u p of e x p e r i m e n t s c a r r i e d o u t in o u r l a b o r a t o r y t o a s s a y i n l y m p h o c y t e nuclei, before a n d a f t e r P H A s t i m u l a t i o n , t h e a c t i v i t y of a ribolluclease able t o digest d o u b l e s t r a n d e d regions of R N A . M a t e r i a l s a n d methods. H u m a n s m a l l l y m p h o c y t e s were o b t a i n e d f r o m t h e p e r i p h e r a l b l o o d of 3 n o r m a l donors. H e p a r i n i z e d b l o o d was s e d i m e n t e d b y g r a v i t y a n d l y m p h o c y t e suspensions, w i t h g r a n u l o c y t e c o n t a m i n a t i o n lower t h a n 0.5%, were p r e p a r e d b y f i l t r a t i o n of t h e s u p e r n a t a n t t h r o u g h c o m m e r c i a l n y l o n fibres, a c c o r d i n g to t h e t e c h n i q u e a l r e a d y d e s c r i b e d 1. C u l t u r e s were set u p in E a g l e ' s m e d i u m w i t h 2 0 % a u t o l o g o u s p l a s m a a t t h e a v e r a g e cell c o n c e n t r a t i o n of 2 • l 0 s cells p e r ml. T h e t o t a l l y m p h o c y t e yield f r o m e a c h d o n o r (2.6 t o 3 • 10 s l y m p h o c y t e s ) was d i v i d e d i n t o 2 e q u a l p a r t s , one of w h i c h was i m m e d i a t e l y h a r v e s t e d a n d t h e nuclei s e p a r a t e d . T h e o t h e r p a r t was i n c u b a t e d a t 37 ~ for 24 h a f t e r a d d i t i o n of P H A s o l u t i o n (Wellcome, R e a g e n t Grade) a t t h e c o n c e n t r a t i o n of 0.02 m l / m l , a n d t h e n h a r v e s t e d . Nuclei of h i g h p u r i t y
were o b t a i n e d w i t h o u t m e c h a n i c a I s h e a r i n g b y a m i n o r m o d i f i c a t i o n of t h e m e t h o d of TAKAKUSU e t al. 14, u s i n g a m i x t u r e of d e t e r g e n t s o l u t i o n s c o n t a i n i n g s o d i u m d e o x y c h o l a t e a n d T r i t o n W R 1339. T h e h a r v e s t e d cells were w a s h e d t w i c e w i t h 50 m l of sucrose b u f f e r (0.15 M sucrose, 0.005 M CaCI~, 0.025 M T r i s 9 HC1, p H 7.1). T h e w a s h e d cells were r e s u s p e n d e d in 15 m l of t h e s a m e buffer, a n d a n e q u a l v o l u m e of d e t e r g e n t s o l u t i o n (0.25 % s o d i u m d e o x y c h o l a t e , 0.5% T r i t o n ) was a d d e d , a n d t h e s u s p e n s i o n s h a k e n g e n t l y b y h a n d for 5 min. T h e s u s p e n s i o n w a s t h e n d i l u t e d w i t h 100 m l of sucrose b u f f e r to p r e v e n t f u r t h e r d e t e r g e n t a c t i v i t y , a n d t h e n c e n t r i f u g e d a t 1500 r p m for 10 rain, to e l i m i n a t e t h e c y t o p l a s m i c c o m p o n e n t s in t h e s u p e r n a t a n t . T h e r e s u l t i n g p e l l e t of nuclei was w a s h e d t h r i c e w i t h 20 m l of 0.25 M sucrose b u f f e r a t 4~ Microscopic e x a m i n a t i o n of G i e m s a - s t a i n e d nuclei s h o w e d t h a t t h e n u c l e a r c h r o m a t i n a n d n u c l e o l a r s t r u c t u r e were well p r e s e r v e d , a n d t h a t o n l y a v e r y few nuclei r e t a i n e d s m a l l c y t o p l a s m i c tags. T h e w a s h e d p e l l e t was t h e n easily a n d e v e n l y dissolved in 10 m l of b u f f e r (1 M NaC1, 0.02 M T r i s 9 HC1, MgAcet. 0.01 M , p H 7.6), a n d 50-200 txl a l i q u o t s were s e p a r a t e d a n d used in t h e e n z y m e assay. A p o r t i o n of t h i s s o l u t i o n was d i a l y z e d for 72 h a g a i n s t 0.14 M NaC1 buffer. T h e p r e c i p i t a t e d d e o x y r i b o n u c l e o p r o t e i n was t h e n s e p a r a t e d b y c e n t r i f u g a t i o n a n d t h e s u p e r n a t a n t a g a i n t e s t e d for d o u b l e - s t r a n d e d r i b o n u c l e a s e activity. S y n t h e t i c s u b s t r a t e for t h e e n z y m e a s s a y was p r e p a r e d f r o m e q u i m o l a r a m o u n t s of p o l y r I a n d p o l y r C c o n t a i n i n g 3 W. JELINEKand J. E. DARNELL,Proc. natn. Acad. Sci., USA 69, 2537 (1972). 4 g. H. KRONElgBERG and T. HUMPHREYS, Biochemistry 71, 2020 (1972). 5 A. P. RYSKOV, G. F. SAUI~DERS, V. ]~. FARASHYAN and G. P. GEORGIEV, Biochim. bioph. Acta 312, 152 (1973). 6 R. PATNAIKand IV[.W. TAYLOR, Biochemistry 72, 1990 (1973). 7 R. lb. ~/[ONCKTOI'rand H. NAORA, Biochim. biophys. Acta 335, 139 (1974). s U. TORELLI, G. TORELLI and R. CADOSSI, ExpI Cell Res. 88, 188 (1974). 9 H. L. COOPER, J. biol. Chem. 243, 34 (1968). 10 N. NIKOLAEV, L. SILEI~GO and D. SCHLESSINGER, J. biol. Chem. 248, 7967 (1973). 11 j'. j'. D u ~ and 1e. W. STUDIER, Proe. natn. Acad. Sci., USA 70, 1559 (1973). 12 j. j. DuNx and F. W. STUDIER,Proc. natn. Acad. Sci. USA 70, 3296 (1973). z3 3ft. ROSE'BERG, R. A. KRAMER and J. A. STEIZ, J. molec. Biol. 89, 777 (1974). z4 A. TAKA~USI~, H. LAZARUS,1Vii.LEVINE, T. A. M~cCoYand G. E. FOLEY, Expl. Cell Res. 49, 226 (1968).
15.3. 1976
Specialia
aH-polyrC. All p o l y m e r s were f r o m Miles. P o l y r I was dissolved in 0.02 M Tris b u f f e r (pH 7.8) c o n t a i n i n g 0.1 M NaC1 a n d e x t r a c t e d t w i c e w i t h b u f f e r s a t u r a t e d phenol. T h e a q u e o u s p h a s e was t h e n d i a l y z e d in t h e s a m e b u f f e r to e l i m i n a t e m i n i m a l t r a c e s of phenol. 8H-polyrC was d i l u t e d w i t h 0.02 M Tris b u f f e r a n d d i a l y z e d to e l i m i n a t e e t h a n o l f r o m t h e c o m m e r c i a l p r e p a r a t i o n . W h i l e in i n i t i a l e x p e r i m e n t s p h e n o l e x t r a c t i o n was p e r f o r m e d , t h i s was f o u n d u n n e c e s s a r y in f u r t h e r trials. T h e f o r m a t i o n of t h e bihelical h o m o p o l y m e r d u p l e x a H - p o l y r C : p o l y r I was e v a l u a t e d b y t h e h y p o c h r o m i c effect a t 233 n m . T h e t e m p e r a t u r e of t h e p r o d u c t was f o u n d b e t w e e n 60~ a n d 70~ in 0.1 M NaC1. T h e d u p l e x was also t e s t e d for s t a b i l i t y to d i g e s t i o n b y R N a s e B ( W o r t h i n g t o n ) for 30 m i n a t 37 ~ F o r c o n c e n t r a t i o n s of t h e e n z y m e u p t o 1 ~zg/ml, n o effect was o b s e r v e d o n t h e a m o u n t of acid p r e c i p i t a b l e c o u n t s of t h e d u p l e x in m o s t p r e p a r a t i o n s of ~ H - p o l y r C : p o l y r I , w h e r e a s t h e single s t r a n d e d 3H-polyrC was m a d e c o m p l e t e l y acid soluble. T h e r e a c t i o n m i x t u r e c o n t a i n e d a n a m o u n t of a H - p o l y r C : p o l y r I e q u i v a l e n t t o a b o u t 0.5 n m o l e s of P a n d v a r i a b l e a m o u n t s of n u c l e a r e x t r a c t s in a f i n a l v o l u m e of 1 m l of b u f f e r (1 M or 0.14 M NaC1, 0.01 M MgAcet. a n d 0.02 M Tris 9 HC1). T h e r e a c t i o n was s t o p p e d a f t e r 30 m i n b y a d d i t i o n of 2 m l of 10% cold TCA. 50 vg of a l b u m i n were a d d e d as carrier, a n d t h e p r e c i p i t a t e was f i l t e r e d t h r o u g h Millipore filters ( H A W P 2 5 ) . T h e filters were d r i e d a n d c o u n t e d in a P a c k a r d T r i c a r b s c i n t i l l a t i o n s p e c t r o m e t e r . A s s a y s were a l w a y s p e r f o r m e d in d u p l i c a t e , a n d a v e r a g e v a l u e s are presented. Results. All t h e n u c l e a r e x t r a c t s a s s a y e d in o u r experiments displayed a significant digesting activity on the
287
d o u b l e - s t r a n d e d s u b s t r a t e . R e s u l t s o b t a i n e d w i t h react i o n m i x t u r e s in 0.14 M NaC1 b u f f e r are s h o w n in t h e Table. T h e p r o p o r t i o n of s u b s t r a t e r a d i o a c t i v i t y d i g e s t e d in b u f f e r of t h i s m o l a r i t y was a l w a y s g r e a t e r t h a n t h a t o b s e r v e d i n 1 M NaC1 buffer, e v e n t h o u g h c o n t r o l e x p e r i m e n t s s h o w e d t h a t a s i g n i f i c a n t a m o u n t of a c t i v i t y was still b o u n d t o t h e p r e c i p i t a t e f o r m e d a f t e r dialysis. T h e r a t e of h y d r o l y s i s of t h e l a b e l l e d d u p l e x w a s i r r e g u l a r a n d p r o p o r t i o n a l i t y to t h e a m o u n t of n u c l e a r e x t r a c t a d d e d to t h e r e a c t i o n m i x t u r e could n o t b e d e m o n s t r a t e d . All t h e r e s u l t s s h o w n in t h e T a b l e were o b t a i n e d w i t h 200 tzl of e x t r a c t , c o r r e s p o n d i n g to a b o u t 3 • s nuclei. T h e a c t i v i t y in t h i s a m o u n t of nuclei f r o m u n s t i m u l a t e d l y m p h o c y t e s of d o n o r No. 2 b r o u g h t to h y d r o l y s i s of 0.08 n m o l e s of d o u b l e - s t r a n d e d s u b s t r a t e , w h e r e a s t h e a c t i v i t y in t h e s a m e n u m b e r of nuclei, a f t e r 24 h of P H A s t i m u l a t i o n , b r o u g h t t o h y d r o l y s i s of 0.17 n m o l e s of s u b s t r a t e . S i n g l e - s t r a n d e d r i b o n u c l e a s e a c t i v i t y was p r e s e n t in e a c h of t h e s a m p l e s w h e r e d o u b l e - s t r a n d e d r i b o n u c l e a s e a c t i v i t y was d e t e c t e d : H o w e v e r , in all d o n o r s e x a m i n e d , d o u b l e - s t r a n d e d r i b o n u c l e a s e a c t i v i t y in nuclei of P H A s t i m u l a t e d l y m p h o c y t e s was a b o u t t w i c e t h a t before s t i m u l a t i o n . O n t h e c o n t r a r y , o n l y a s m a l l a v e r a g e increase in single s t r a n d e d r i b o n u c l e a s e a c t i v i t y was obs e r v e d in l y m p h o c y t e nuclei a f t e r P H A s t i m u l a t i o n . F u r t h e r m o r e , h y d r o l y s i s of 3H-polyrC was m a r k e d l y stimulated by EDTA, which had, on the contrary, a slight inhibitory action on double-stranded ribonuelease a c t i v i t y . T h e s e results s u g g e s t t h a t s i n g l e - s t r a n d e d a n d d o u b l e - s t r a n d e d r i b o n u c l e a s e a c t i v i t i e s are a s s o c i a t e d w i t h d i f f e r e n t n u c l e a r p r o t e i n s . E x p e r i m e n t s are n o w in progress in t h e a t t e m p t t o s e p a r a t e t h e s e p r o t e i n s .
Dissociation-Constants of Metal-Ion-Complexes with Alkaline Phosphatase from Pig Kidney B. P. ACKERMANN1 a n d J. AHLERS ~
Institut /i~r Biochemie der Universitiit Mainz, J.-J.-Becher-Weg 28, D-65 Mainz (German Federal Republic, BRD), 76 July 7975. Summary. U s i n g m e t a l - i o n buffers i t was possible t o r e m o v e Z n 2+, Mg 2+ a n d M n 2+ ions of pig k i d n e y a l k a l i n e p h o s p h a t a s e reversibly. T h e d i s s o c i a t i o n c o n s t a n t s o b t a i n e d are K ~ g : 4 9 10 -7 M , K E ~ . : 4 9 10 -s M a n d K~zn: 8 9 10 -~a M (22~ p H : 9.6, ~z: 0.07). A l k a l i n e p h o s p h a t a s e (EC 3.1.3.1) is a m e t a l - c o n t a i n i n g e n z y m e . I t r e q u i r e s Z n 2+ ions b o t h / o r p r e s e r v a t i o n of its s t r u c t u r e a n d for its e n z y m i c a c t i v i t y (e.g. ref.~). Zinc m a y b e r e p l a c e d b y cobalt, copper, c a d m i u m , nickel or m a n g a n e s e 4-7. I n a d d i t i o n t o t h e s e m e t a l ions, t h e a l k a l i n e p h o s p h a t a s e f r o m m a m m a l i a n t i s s u e n e e d s Mg 2+ ions for a c t i v i t y S, % O t h e r d i v a l e n t cations, especially M n 2+, Co 2+, N i 2+9 a n d Ca 2+ h a v e b e e n r e p o r t e d t o b e c a p a b l e of r e p l a c i n g Mg "+ as a c t i v a t o r . M o s t of t h e s e e x p e r i m e n t s h a v e b e e n p e r f o r m e d in t h e a b s e n c e of c o m p l e x i n g agents. Therefore, d u e t o c o n t a m i n a t i o n of t h e r e a g e n t s a n d of t h e e n z y m e p r e p a r a t i o n , t h e r e are v a r i o u s m e t a l ions in t h e assay. F u r t h e r m o r e , t h e conc e n t r a t i o n of m e t a l ions were v a r i e d i n o n l y a few cases. W e d e t e r m i n e d t h e d i s s o c i a t i o n c o n s t a n t s of s e v e r a l m e t a l ion c o m p l e x e s w i t h a l k a l i n e p h o s p h a t a s e in t h e p r e s e n c e of a s u i t a b l e c o m p l e x i n g a g e n t u n d e r c o n d i t i o n s w h i c h g u a r a n t e e t h a t t h e r e m o v a l of m e t a l ions is reversible a n d t h a t a n e q u i l i b r i u m is a c h i e v e d . F u r t h e r m o r e , we solved t h e p r o b l e m s a r i s i n g f r o m a s y s t e m c o n s i s t i n g of m o r e t h a n one s o r t of m e t a l ion, c o m p l e x i n g a g e n t a n d p r o t e i n b y m e a n s of a F O R T R A N I V p r o g r a m or a n approximate formula,
Experimental. All c h e m i c a l s were o b t a i n e d f r o m E. Merck, D a r m s t a d t , G e r m a n y . P i g k i d n e y s were used as source for t h e a l k a l i n e p h o s p h a t a s e . Methods. T h e p r e p a r a t i o n of pig k i d n e y a l k a l i n e p h o s p h a t a s e a n d t h e d e t e r m i n a t i o n of e n z y m e a c t i v i t y was d e s c r i b e d r e c e n t l y s, 10. Tile t e s t m e d i u m for k i n e t i c m e a 1 Acknowledgements: The authors thank Dr. H. U. WOLF for helpful suggestions and valuable discussion and Miss H. K6T~ for techllical assistance. Present address: Dr. JA~ AHL~RS, Zentralinstitut fiir Biochemie und Biophysik, Ehrenbergstrasse 26-28, 1 Berlin 33, BRD. C. LAZDUNSKIand M. LAZDUIqSKI,]~ur. J. Bioehem. 7, 294 (1969). 4 C. LAZDUIqSKI, C. PETITCLERC and M. LAZDUNSKI, Eur. J. Biochem. 8, 510 (1969). 5 M. L. APPLEBURY and J. E. COLEmAn, J. biol. Chem. 244, 308
(1969). 6 C. PETITCLERC, C. LAZDUNSKI, D. CHAPPELET, A. I~/IOULIIqGand
M. LAZDU~SKI, Eur. J. Biochem. 74, 301 (1970). H. CsoPAx and K. E. FALK, FEBS-Lett. 7, 147 (1970). 8 j. Am]ERS, Biochem. J. 741, 257 (1974). C. BRU~EL and G. CATHALA, Biochim. biophys. Acta 3091 104 0973). 10 j . AHLERS~ Biochem. J. 7d9~535 (1975).
285
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(0.15 mmol) in a c e t o n e - d 6 (0.5 ml); m e t h a n o l - d 4 (0.2 ml) was a d d e d a n d a f t e r 24 h ( N - I t t o N - D ) t h e c u r v e s were r e c o r d e d again. T h e singlet (d 8.6-8.8) of t h e p i c r a t e a n i o n was t a k e n as p r o t o n - c o u n t i n g reference; t h e n u m ber of N - H r e s u l t e d d i r e c t l y f r o m t h e i n t e g r a t i o n c u r v e s or f r o m t h e difference w i t h t h o s e o b t a i n e d a f t e r a d d i t i o n of m e t h a n o t - d 4. T h e n u m b e r f o u n d for N-I-i w a s u s u a l l y w i t h i n 0 t o - - 1 0 % of t h e c o r r e c t one. T h e s p e c t r a do n o t s h o w s e p a r a t e d signals for E - Z isomers ~4. T h e N - H signals are g e n e r a l l y c h a r a c t e r i z e d b y : d-values in t h e r a n g e 6.7-8.2 ; h a l f - b a n d w i d t h (WII~) 5-18 H z ; t h e y d i s a p p e a r w i t h m e t h a n o l - d 4 ; &values are n o t c h a n g e d or are s l i g h t l y d e c r e a s e d (less t h a n 0.1 p p m ) b y m e t h a n o l . A n aryl s u b s t i t u e n t p r o d u c e s : a s t r o n g downfield s h i f t of t h e A r - N - H signal; a small effect on t h e p r o t o n s of t h e o t h e r N - a t o m s ; t h e N - H r e s o n a n c e s d i s a p p e a r ( ' a v e r a g e d ' w i t h M e O - H ) o r are s t r o n g l y broadened by methanol. The a r o m a t i c - H signals are d i s t i n g u i s h e d b e c a u s e t h e y are n o t r e m o v e d b y m e t h a n o l - d 4. T h e N - H signals of a m i n e s (as p i c r a t e salts) are usually v e r y b r o a d (Wl/2 > 40 Hz) a n d d i s a p p e a r b y m e t h a n o l . T h e O - H (W v 2 ca. 2 Hz) or N - H ( W ~ > 40 Hz) signals of p h e n o l s a n d c a r b o x a m i d e s are s h i f t e d downfield (0.2-0.3 p p m ) b y methanol. A f t e r t h e p r e s e n c e of t h e g u a n i d i n i u m g r o u p h a s been. a s c e r t a i n e d , its s u b s t i t u t i o n p a t t e r n r e s u l t s in m a n y e x a m p l e s f r o m t h e N - H signals in t h e s p e c t r a m e a s u r e d in p l a i n acetone. Thus, a n e x a m p l e (R ~ = R a = cycloh e x y l ; R 2 = R ~ = R 5 --- H) e x h i b i t s 4 N - H d i s t r i b u t e d in 2 signals (d 7.06 a n d 7.26; area ratio 1:3) p a r t i a l l y superposed; these data indicate non-equivalent N-H groups, a n d t h e r e f o r e t h e c o m p o u n d is a N~, Ne-disubstit u t e d g u a n i d i n i u m . T h e o b s e r v e d a r e a r a t i o is due t o s u p e r p o s i t i o n of t h e lower-field c o m p o n e n t of t h e s p l i t t e d N - H signal (doublet; t w o H - N - C - H ) w i t h t h e NHg singlet. T h e s p l i t t i n g is c o n f i r m e d b y d e c o u p l i n g w i t h ferric chloride. T h e scope is w i d e n e d , including t h e results in a c e t o n e dd, w h i c h allows t h e o b s e r v a t i o n of t h e N - C - H signals a t d 3.0-4.3; an a d j a c e n t aryl g r o u p ( N - C H - A r ) p r o d u c e s a downfield s h i f t (d 4.5-4.9). T h e m e t h o d n o w t a k e s into a c c o u n t : n u m b e r of g u a n i d i n i u m N - H ; o c c u r r e n c e of a singlet or m o r e N - H signals arising f r o m n o n - e q u i v a l e n c e or spin-spin coupling w i t h N - C - H ; n u m b e r of N - C - H m u l t i p l e t s simplified b y m e t h a n o l - d 4. One e x a m p l e is g i v e n in d e t a i l (R ~ = R ~- = e t h y l ; R ~ = m e t h y l ; R* = H ; R s = benzyl) w h i c h gives a s h a r p signal a t d 7.33 t h a t in its b a s e is p a r t i a l l y superp o s e d w i t h a b r o a d signal (total area 7 H). A f t e r a d d i t i o n
of m e t h a n o l - d 4 it r e m a i n s a 5H-singlet (aromatic-H), i n d i c a t i n g t h a t t h e c o m p o u n d is a t e t r a - s u b s t i t u t e d g u a n i d i u i u m . Besides, m e t h a n o l - d 4 simplifies t w o d o u b l e t s t o give t h e c o r r e s p o n d i n g singlets a t d 3.00 a n d 4.55; t h i s implies t h a t t w o of t h e s u b s t i t u e n t s are located on N - a t o m s b e a r i n g a p r o t o n r e s p o n s i b l e of t h e N - C - H splittings. T h e n , t h e c o m p o u n d is a N 1, N1,N~, N3-tetrasubstituted guanidinium. S o m e of t h e c o m p o u n d s were also m e a s u r e d using p l a i n a n d h e x a d e u t e r a t e d d i m e t h y l s u l p h o x i d e as s o l v e n t s ; t h e results were generally similar t o t h o s e q u o t e d above. These s o l v e n t s are m a i n l y useful for low soluble p i c r a t e s (e.g. R 1 = R 2 = m e t h y l ; R a = R ~ = R 5 = H).
1 Part XX of Studies on Plants; preceding part, R. A. CORRAL,O. O. ORAZI and I. A. BE~AGES, Tetrahedron 29, 205 (1973). 2 N. VANTHOAI and J. ROCHE,in Fortschritte der Chemic Organischer Nat~rstoffe (Ed. L. ZECHMEISTER;Springer Verlag, Wien 1960), vol. 18, p. 83. - J. NhERSCH, Biol. Rnndsch. 6, 72 (1968). - J. A. JOULE, in Annual Reports (The Chem. See., London 1969), vol. 66B, p. 485. - F. KHUO~G-Huu, J. P. LE FORESTIER and R. GOUTAREL,Tetrahedron 28, 5207 (1972). 3 See for example, J. ABERHART,R. C. JAIN, T. FEHR, P. DE MAYO and I. SZILAOYI, J. chem. See. Perkin I, 816 (1974). - T . GoTo, Y. KISHI, S. TAKAHASHI and Y. HIRATA, Tetrahedron 21, 2059 (1965). 4 I. SMITH,Chromatographic and Electrophoretic Techniques (Heinemann Medical Books, London 1960), vol. 1, p. 225. 5 T. GOTO, Y. HIRATA, S. HOSOYAand N. KOMATSU,Bull. chem. Soc., Japan 30, 729 (1957). s See for example, S. RAlVIAKRISI-n, fA and R. ADIGA, Phytoehemistry 72, 2961 (1973). 7 G. M. SFIARMAand P. R. BURKHOLDER,Chem. Conlmun. 7977, 151. s T. COTe, I{. NAKANISHIand M. OHASm, Bull. chem. See., Japan 30, 723 (1957). - E. SCHREIBER,K. PUFAHLand H. BRAEONIGER, Justus Liebigs Annln. Chem. 671, 147 (1964). 9 R. GREENHALGI~and R. A. BANNARD,Can. J. Chem. 39, 1017 (1961). - K. MATSUr~OTO and H. RAPOPOR% J. org. Chem. 33, 552 (1968). 10 R. B. CoNI~ and R. B. DAvis, Nature, Lend. 183, 1053 (1959). F. FA~RE and P. BLANQUET,Bull. Soc. Chim. biol. d3, 953 (1961). n D. WATSOIr Experientia 22, 76 (1966). - J. L6PEz-GoRoE and M. 1V~oNTEOLIVA,J. Chromat. 29, 300 (1967). 12 R. A. CORRAL,O. O. ORAZI and M. F. DE PETRUeCELLI, Rev. Iatinoam. Quire. 2, 178 (1971). 1, S. SUGIURA, S. I~OUE, Y. HAYASHI, Y. KlSm and T. GOTO, Tetrahedron 25, 5155 (1969), and references therein.
14 V. J. BAUER,W. FULMOR,G. O. M O R T O N and S. R. SAFIR, ~. Am. chem. See. 90, 6846 (1968). - H. KESSLER,Tetrahedron 30, 1861 (1974), and references therein.
Double Stranded Ribonuclease Activity in Human Lymphocyte Nuclei U. TORELLI, ST. FERRARI, G. TORELLI, R. CADOSSI a n d S. tFERRARI
Istituto di Patologia Medics dell' Universit?~, Via del Pozzo 7J, l - d / 7 0 0 Modena ( ftaly), 8 October 1975. S u m m a r y . R i b o n u c l e a s e a c t i v i t y d i r e c t e d a g a i n s t t h e s y n t h e t i c d u p l e x p o l y r C : p o l y r I was d e t e c t e d in n u c l e a r e x t r a c t s f r o m b o t h u n s t i m u l a t e d a n d P H A - s t i m u l a t e d h u m a n l y m p h o c y t e s . I n t h e l a t t e r cells, t h e a c t i v i t y was a b o u t t w i c e t h a t of small l y m p h o c y t e s . I t h a s b e e n s h o w n t h a t h u m a n small l y m p h o c y t e s synthesize mainlyunmethylated rapidly sedimenting RNA molecules w h i c h h y b r i d i z e v e r y efficiently t o D N A in v i t r o a n d are b o u n d to p o l y a d e n y l i c s e q u e n c e s in a p r o p o r t i o n u p t o 20% 1, 3. I t was t h u s c o n c l u d e d t h a t t h e m a j o r p o r t i o n of t h e R N A s y n t h e s i z e d in t h e s e cells is t h e h e t e r o g e n e o u s n u c l e a r R N A . Since it h a s b e e n s h o w n
b y several a u t h o r s t h a t a s i g n i f i c a n t p o r t i o n of n u c l e a r R N A of t h e h e t e r o g e n e o u s t y p e in a n i m a l cells is in a n R N a s e - r e s i s t a n t f o r m w i t h p r o p e r t i e s of d o u b l e - s t r a n d e d 1 U. 71"ORELLI,P. HENRY and S. WEISSMAN,J. elin. Invest. 47, 1083 (1968). 2 U. TORELLI and G, TORELLI~Acta haemat. 51, 140 (1974).
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EXPERIENTIA
32/3
Ribonuelease activity directed against single-stranded and double-stranded synthetic polyribonucleotides in nuclei of unstimulated and PHA-stimulated normal human lymphoeytes Donor No.
1 2 3
Substrate
8H-polyC-polyI 3H-polyrC aH-polyrC-p01yrI SH-polyrC 8H-polyrC-polyrI 9 3H-polyrC
PHA-stimulated lymphocytes
Small lymphocytes Control incubation (cpm)
Substrate radioactivity hydrolyzed (%)
Controlincubation (cpm)
Substrate radioactivity hydrolyzed (%)
6450 5100 4430 4500 5200 2800
11 12 13 11 27 18
6450 5100 4430 4500 5200 2800
25 15 27 13 56 21
After dissolution of the nuclei in 1 M NaC1 buffer, the extract was dialyzed against 0.14 M NaC1 buffer and incubations performed in buffer of this molarity.
R N A 3-v, i t was n o t u n e x p e c t e d to o b s e r v e t h a t a b o u t 2 % of t h e t o t a l l a b e l l e d cell R N A of s m a l l l y m p h o c y t e s shows d o u b l e - s t r a n d e d p r o p e r t i e s s. A t v a r i a n c e w i t h t h a t o b s e r v e d in several a n i m a l cell types, t h e t u r n o v e r r a t e of h e t e r o g e n e o u s n u c l e a r R N A i n s m a l l l y m p h o c y t e s a p p e a r s r e m a r k a b l y low, as s u g g e s t e d b y t h e a s s o c i a t i o n of t h e m a j o r i t y of a labelled p r e c u r s o r w i t h r a p i d l y s e d i m e n t i n g molecules e v e n a f t e r several h o u r s of a c t i n o m y c i n chase1, 2. T h i s c o n c l u s i o n is also s u p p o r t e d b y t h e o b s e r v a t i o n t h a t cold u r i d i n e c h a s e for several h o u r s does n o t s i g n i f i c a n t l y c h a n g e t h e p r o p o r t i o n of labelled d o u b l e - s t r a n d e d IRNA s, 9. Phytohemagglutinin ( P H A ) s t i m u l a t i o n of s m a l l l y m p h o c y t e s b r i n g s a b o u t a s h a r p c h a n g e in m e t a b o l i s m of n u c l e a r l y m p h o c y t e R N A , c h a r a c t e r i z e d b y a large increase in t h e r a t e of s y n t h e s i s a n d processing of ribosom a l p r e c u r s o r R N A , as well as b y i n c r e a s e in t u r n o v e r r a t e of t h e h e t e r o g e n e o u s n u c l e a r R N A 1 , ~. V e r y l i t t l e i n f o r m a t i o n is so f a r a v a i l a b l e a b o u t e n z y m e s y s t e m s i n v o l v e d in m e t a b o l i s m of n u c l e a r l y m p h o c y t e R N A . T h e p r e s e n c e of d o u b l e - s t r a n d e d R N A s e g m e n t s in h e t e r o g e n e o u s n u c l e a r R N A justifies t h e h y p o t h e s i s t h a t t h e c l e a v a g e of t h e large R N A molecules of t h i s R N A class i n v o l v e s t h e a c t i v i t y of a n e n z y m e w i t h a d e f i n i t e preference for d o u b l e - s t r a n d e d R N A , in a m a n n e r s i m i l a r t o w h a t h a s b e e n r e c e n t l y d e m o n s t r a t e d in b a c t e r i a l cells. I n t h e s e cells, in fact, i t w a s r e c e n t l y o b s e r v e d t h a t r i b o n u c l e a s e I I I , a n e n z y m e h i g h l y specific for d o u b l e s t r a n d e d R N A , h a s a k e y role in p r o c e s s i n g of m e s s e n g e r as well as r i b o s o m a l R N A p r e c u r s o r s 10-z3. T h i s p a p e r s h o r t l y r e p o r t s t h e r e s u l t s of a g r o u p of e x p e r i m e n t s c a r r i e d o u t in o u r l a b o r a t o r y t o a s s a y i n l y m p h o c y t e nuclei, before a n d a f t e r P H A s t i m u l a t i o n , t h e a c t i v i t y of a ribolluclease able t o digest d o u b l e s t r a n d e d regions of R N A . M a t e r i a l s a n d methods. H u m a n s m a l l l y m p h o c y t e s were o b t a i n e d f r o m t h e p e r i p h e r a l b l o o d of 3 n o r m a l donors. H e p a r i n i z e d b l o o d was s e d i m e n t e d b y g r a v i t y a n d l y m p h o c y t e suspensions, w i t h g r a n u l o c y t e c o n t a m i n a t i o n lower t h a n 0.5%, were p r e p a r e d b y f i l t r a t i o n of t h e s u p e r n a t a n t t h r o u g h c o m m e r c i a l n y l o n fibres, a c c o r d i n g to t h e t e c h n i q u e a l r e a d y d e s c r i b e d 1. C u l t u r e s were set u p in E a g l e ' s m e d i u m w i t h 2 0 % a u t o l o g o u s p l a s m a a t t h e a v e r a g e cell c o n c e n t r a t i o n of 2 • l 0 s cells p e r ml. T h e t o t a l l y m p h o c y t e yield f r o m e a c h d o n o r (2.6 t o 3 • 10 s l y m p h o c y t e s ) was d i v i d e d i n t o 2 e q u a l p a r t s , one of w h i c h was i m m e d i a t e l y h a r v e s t e d a n d t h e nuclei s e p a r a t e d . T h e o t h e r p a r t was i n c u b a t e d a t 37 ~ for 24 h a f t e r a d d i t i o n of P H A s o l u t i o n (Wellcome, R e a g e n t Grade) a t t h e c o n c e n t r a t i o n of 0.02 m l / m l , a n d t h e n h a r v e s t e d . Nuclei of h i g h p u r i t y
were o b t a i n e d w i t h o u t m e c h a n i c a I s h e a r i n g b y a m i n o r m o d i f i c a t i o n of t h e m e t h o d of TAKAKUSU e t al. 14, u s i n g a m i x t u r e of d e t e r g e n t s o l u t i o n s c o n t a i n i n g s o d i u m d e o x y c h o l a t e a n d T r i t o n W R 1339. T h e h a r v e s t e d cells were w a s h e d t w i c e w i t h 50 m l of sucrose b u f f e r (0.15 M sucrose, 0.005 M CaCI~, 0.025 M T r i s 9 HC1, p H 7.1). T h e w a s h e d cells were r e s u s p e n d e d in 15 m l of t h e s a m e buffer, a n d a n e q u a l v o l u m e of d e t e r g e n t s o l u t i o n (0.25 % s o d i u m d e o x y c h o l a t e , 0.5% T r i t o n ) was a d d e d , a n d t h e s u s p e n s i o n s h a k e n g e n t l y b y h a n d for 5 min. T h e s u s p e n s i o n w a s t h e n d i l u t e d w i t h 100 m l of sucrose b u f f e r to p r e v e n t f u r t h e r d e t e r g e n t a c t i v i t y , a n d t h e n c e n t r i f u g e d a t 1500 r p m for 10 rain, to e l i m i n a t e t h e c y t o p l a s m i c c o m p o n e n t s in t h e s u p e r n a t a n t . T h e r e s u l t i n g p e l l e t of nuclei was w a s h e d t h r i c e w i t h 20 m l of 0.25 M sucrose b u f f e r a t 4~ Microscopic e x a m i n a t i o n of G i e m s a - s t a i n e d nuclei s h o w e d t h a t t h e n u c l e a r c h r o m a t i n a n d n u c l e o l a r s t r u c t u r e were well p r e s e r v e d , a n d t h a t o n l y a v e r y few nuclei r e t a i n e d s m a l l c y t o p l a s m i c tags. T h e w a s h e d p e l l e t was t h e n easily a n d e v e n l y dissolved in 10 m l of b u f f e r (1 M NaC1, 0.02 M T r i s 9 HC1, MgAcet. 0.01 M , p H 7.6), a n d 50-200 txl a l i q u o t s were s e p a r a t e d a n d used in t h e e n z y m e assay. A p o r t i o n of t h i s s o l u t i o n was d i a l y z e d for 72 h a g a i n s t 0.14 M NaC1 buffer. T h e p r e c i p i t a t e d d e o x y r i b o n u c l e o p r o t e i n was t h e n s e p a r a t e d b y c e n t r i f u g a t i o n a n d t h e s u p e r n a t a n t a g a i n t e s t e d for d o u b l e - s t r a n d e d r i b o n u c l e a s e activity. S y n t h e t i c s u b s t r a t e for t h e e n z y m e a s s a y was p r e p a r e d f r o m e q u i m o l a r a m o u n t s of p o l y r I a n d p o l y r C c o n t a i n i n g 3 W. JELINEKand J. E. DARNELL,Proc. natn. Acad. Sci., USA 69, 2537 (1972). 4 g. H. KRONElgBERG and T. HUMPHREYS, Biochemistry 71, 2020 (1972). 5 A. P. RYSKOV, G. F. SAUI~DERS, V. ]~. FARASHYAN and G. P. GEORGIEV, Biochim. bioph. Acta 312, 152 (1973). 6 R. PATNAIKand IV[.W. TAYLOR, Biochemistry 72, 1990 (1973). 7 R. lb. ~/[ONCKTOI'rand H. NAORA, Biochim. biophys. Acta 335, 139 (1974). s U. TORELLI, G. TORELLI and R. CADOSSI, ExpI Cell Res. 88, 188 (1974). 9 H. L. COOPER, J. biol. Chem. 243, 34 (1968). 10 N. NIKOLAEV, L. SILEI~GO and D. SCHLESSINGER, J. biol. Chem. 248, 7967 (1973). 11 j'. j'. D u ~ and 1e. W. STUDIER, Proe. natn. Acad. Sci., USA 70, 1559 (1973). 12 j. j. DuNx and F. W. STUDIER,Proc. natn. Acad. Sci. USA 70, 3296 (1973). z3 3ft. ROSE'BERG, R. A. KRAMER and J. A. STEIZ, J. molec. Biol. 89, 777 (1974). z4 A. TAKA~USI~, H. LAZARUS,1Vii.LEVINE, T. A. M~cCoYand G. E. FOLEY, Expl. Cell Res. 49, 226 (1968).
15.3. 1976
Specialia
aH-polyrC. All p o l y m e r s were f r o m Miles. P o l y r I was dissolved in 0.02 M Tris b u f f e r (pH 7.8) c o n t a i n i n g 0.1 M NaC1 a n d e x t r a c t e d t w i c e w i t h b u f f e r s a t u r a t e d phenol. T h e a q u e o u s p h a s e was t h e n d i a l y z e d in t h e s a m e b u f f e r to e l i m i n a t e m i n i m a l t r a c e s of phenol. 8H-polyrC was d i l u t e d w i t h 0.02 M Tris b u f f e r a n d d i a l y z e d to e l i m i n a t e e t h a n o l f r o m t h e c o m m e r c i a l p r e p a r a t i o n . W h i l e in i n i t i a l e x p e r i m e n t s p h e n o l e x t r a c t i o n was p e r f o r m e d , t h i s was f o u n d u n n e c e s s a r y in f u r t h e r trials. T h e f o r m a t i o n of t h e bihelical h o m o p o l y m e r d u p l e x a H - p o l y r C : p o l y r I was e v a l u a t e d b y t h e h y p o c h r o m i c effect a t 233 n m . T h e t e m p e r a t u r e of t h e p r o d u c t was f o u n d b e t w e e n 60~ a n d 70~ in 0.1 M NaC1. T h e d u p l e x was also t e s t e d for s t a b i l i t y to d i g e s t i o n b y R N a s e B ( W o r t h i n g t o n ) for 30 m i n a t 37 ~ F o r c o n c e n t r a t i o n s of t h e e n z y m e u p t o 1 ~zg/ml, n o effect was o b s e r v e d o n t h e a m o u n t of acid p r e c i p i t a b l e c o u n t s of t h e d u p l e x in m o s t p r e p a r a t i o n s of ~ H - p o l y r C : p o l y r I , w h e r e a s t h e single s t r a n d e d 3H-polyrC was m a d e c o m p l e t e l y acid soluble. T h e r e a c t i o n m i x t u r e c o n t a i n e d a n a m o u n t of a H - p o l y r C : p o l y r I e q u i v a l e n t t o a b o u t 0.5 n m o l e s of P a n d v a r i a b l e a m o u n t s of n u c l e a r e x t r a c t s in a f i n a l v o l u m e of 1 m l of b u f f e r (1 M or 0.14 M NaC1, 0.01 M MgAcet. a n d 0.02 M Tris 9 HC1). T h e r e a c t i o n was s t o p p e d a f t e r 30 m i n b y a d d i t i o n of 2 m l of 10% cold TCA. 50 vg of a l b u m i n were a d d e d as carrier, a n d t h e p r e c i p i t a t e was f i l t e r e d t h r o u g h Millipore filters ( H A W P 2 5 ) . T h e filters were d r i e d a n d c o u n t e d in a P a c k a r d T r i c a r b s c i n t i l l a t i o n s p e c t r o m e t e r . A s s a y s were a l w a y s p e r f o r m e d in d u p l i c a t e , a n d a v e r a g e v a l u e s are presented. Results. All t h e n u c l e a r e x t r a c t s a s s a y e d in o u r experiments displayed a significant digesting activity on the
287
d o u b l e - s t r a n d e d s u b s t r a t e . R e s u l t s o b t a i n e d w i t h react i o n m i x t u r e s in 0.14 M NaC1 b u f f e r are s h o w n in t h e Table. T h e p r o p o r t i o n of s u b s t r a t e r a d i o a c t i v i t y d i g e s t e d in b u f f e r of t h i s m o l a r i t y was a l w a y s g r e a t e r t h a n t h a t o b s e r v e d i n 1 M NaC1 buffer, e v e n t h o u g h c o n t r o l e x p e r i m e n t s s h o w e d t h a t a s i g n i f i c a n t a m o u n t of a c t i v i t y was still b o u n d t o t h e p r e c i p i t a t e f o r m e d a f t e r dialysis. T h e r a t e of h y d r o l y s i s of t h e l a b e l l e d d u p l e x w a s i r r e g u l a r a n d p r o p o r t i o n a l i t y to t h e a m o u n t of n u c l e a r e x t r a c t a d d e d to t h e r e a c t i o n m i x t u r e could n o t b e d e m o n s t r a t e d . All t h e r e s u l t s s h o w n in t h e T a b l e were o b t a i n e d w i t h 200 tzl of e x t r a c t , c o r r e s p o n d i n g to a b o u t 3 • s nuclei. T h e a c t i v i t y in t h i s a m o u n t of nuclei f r o m u n s t i m u l a t e d l y m p h o c y t e s of d o n o r No. 2 b r o u g h t to h y d r o l y s i s of 0.08 n m o l e s of d o u b l e - s t r a n d e d s u b s t r a t e , w h e r e a s t h e a c t i v i t y in t h e s a m e n u m b e r of nuclei, a f t e r 24 h of P H A s t i m u l a t i o n , b r o u g h t t o h y d r o l y s i s of 0.17 n m o l e s of s u b s t r a t e . S i n g l e - s t r a n d e d r i b o n u c l e a s e a c t i v i t y was p r e s e n t in e a c h of t h e s a m p l e s w h e r e d o u b l e - s t r a n d e d r i b o n u c l e a s e a c t i v i t y was d e t e c t e d : H o w e v e r , in all d o n o r s e x a m i n e d , d o u b l e - s t r a n d e d r i b o n u c l e a s e a c t i v i t y in nuclei of P H A s t i m u l a t e d l y m p h o c y t e s was a b o u t t w i c e t h a t before s t i m u l a t i o n . O n t h e c o n t r a r y , o n l y a s m a l l a v e r a g e increase in single s t r a n d e d r i b o n u c l e a s e a c t i v i t y was obs e r v e d in l y m p h o c y t e nuclei a f t e r P H A s t i m u l a t i o n . F u r t h e r m o r e , h y d r o l y s i s of 3H-polyrC was m a r k e d l y stimulated by EDTA, which had, on the contrary, a slight inhibitory action on double-stranded ribonuelease a c t i v i t y . T h e s e results s u g g e s t t h a t s i n g l e - s t r a n d e d a n d d o u b l e - s t r a n d e d r i b o n u c l e a s e a c t i v i t i e s are a s s o c i a t e d w i t h d i f f e r e n t n u c l e a r p r o t e i n s . E x p e r i m e n t s are n o w in progress in t h e a t t e m p t t o s e p a r a t e t h e s e p r o t e i n s .
Dissociation-Constants of Metal-Ion-Complexes with Alkaline Phosphatase from Pig Kidney B. P. ACKERMANN1 a n d J. AHLERS ~
Institut /i~r Biochemie der Universitiit Mainz, J.-J.-Becher-Weg 28, D-65 Mainz (German Federal Republic, BRD), 76 July 7975. Summary. U s i n g m e t a l - i o n buffers i t was possible t o r e m o v e Z n 2+, Mg 2+ a n d M n 2+ ions of pig k i d n e y a l k a l i n e p h o s p h a t a s e reversibly. T h e d i s s o c i a t i o n c o n s t a n t s o b t a i n e d are K ~ g : 4 9 10 -7 M , K E ~ . : 4 9 10 -s M a n d K~zn: 8 9 10 -~a M (22~ p H : 9.6, ~z: 0.07). A l k a l i n e p h o s p h a t a s e (EC 3.1.3.1) is a m e t a l - c o n t a i n i n g e n z y m e . I t r e q u i r e s Z n 2+ ions b o t h / o r p r e s e r v a t i o n of its s t r u c t u r e a n d for its e n z y m i c a c t i v i t y (e.g. ref.~). Zinc m a y b e r e p l a c e d b y cobalt, copper, c a d m i u m , nickel or m a n g a n e s e 4-7. I n a d d i t i o n t o t h e s e m e t a l ions, t h e a l k a l i n e p h o s p h a t a s e f r o m m a m m a l i a n t i s s u e n e e d s Mg 2+ ions for a c t i v i t y S, % O t h e r d i v a l e n t cations, especially M n 2+, Co 2+, N i 2+9 a n d Ca 2+ h a v e b e e n r e p o r t e d t o b e c a p a b l e of r e p l a c i n g Mg "+ as a c t i v a t o r . M o s t of t h e s e e x p e r i m e n t s h a v e b e e n p e r f o r m e d in t h e a b s e n c e of c o m p l e x i n g agents. Therefore, d u e t o c o n t a m i n a t i o n of t h e r e a g e n t s a n d of t h e e n z y m e p r e p a r a t i o n , t h e r e are v a r i o u s m e t a l ions in t h e assay. F u r t h e r m o r e , t h e conc e n t r a t i o n of m e t a l ions were v a r i e d i n o n l y a few cases. W e d e t e r m i n e d t h e d i s s o c i a t i o n c o n s t a n t s of s e v e r a l m e t a l ion c o m p l e x e s w i t h a l k a l i n e p h o s p h a t a s e in t h e p r e s e n c e of a s u i t a b l e c o m p l e x i n g a g e n t u n d e r c o n d i t i o n s w h i c h g u a r a n t e e t h a t t h e r e m o v a l of m e t a l ions is reversible a n d t h a t a n e q u i l i b r i u m is a c h i e v e d . F u r t h e r m o r e , we solved t h e p r o b l e m s a r i s i n g f r o m a s y s t e m c o n s i s t i n g of m o r e t h a n one s o r t of m e t a l ion, c o m p l e x i n g a g e n t a n d p r o t e i n b y m e a n s of a F O R T R A N I V p r o g r a m or a n approximate formula,
Experimental. All c h e m i c a l s were o b t a i n e d f r o m E. Merck, D a r m s t a d t , G e r m a n y . P i g k i d n e y s were used as source for t h e a l k a l i n e p h o s p h a t a s e . Methods. T h e p r e p a r a t i o n of pig k i d n e y a l k a l i n e p h o s p h a t a s e a n d t h e d e t e r m i n a t i o n of e n z y m e a c t i v i t y was d e s c r i b e d r e c e n t l y s, 10. Tile t e s t m e d i u m for k i n e t i c m e a 1 Acknowledgements: The authors thank Dr. H. U. WOLF for helpful suggestions and valuable discussion and Miss H. K6T~ for techllical assistance. Present address: Dr. JA~ AHL~RS, Zentralinstitut fiir Biochemie und Biophysik, Ehrenbergstrasse 26-28, 1 Berlin 33, BRD. C. LAZDUNSKIand M. LAZDUIqSKI,]~ur. J. Bioehem. 7, 294 (1969). 4 C. LAZDUIqSKI, C. PETITCLERC and M. LAZDUNSKI, Eur. J. Biochem. 8, 510 (1969). 5 M. L. APPLEBURY and J. E. COLEmAn, J. biol. Chem. 244, 308
(1969). 6 C. PETITCLERC, C. LAZDUNSKI, D. CHAPPELET, A. I~/IOULIIqGand
M. LAZDU~SKI, Eur. J. Biochem. 74, 301 (1970). H. CsoPAx and K. E. FALK, FEBS-Lett. 7, 147 (1970). 8 j. Am]ERS, Biochem. J. 741, 257 (1974). C. BRU~EL and G. CATHALA, Biochim. biophys. Acta 3091 104 0973). 10 j . AHLERS~ Biochem. J. 7d9~535 (1975).
