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Nuclei from frozen calf thymus suspended in buffer were analyzed for metal content prior to and after repeated washing. After three such extractions about 0.1 micrograms Zn/mg DNA and 0.025 micrograms Cu/mg DNA remained tightly associated with chromatin. This level of metal was essentially unchanged with subsequent washings. Digestion of extracted nuclei with micrococcal nuclease yielded soluble nucleoprotein containing zinc and copper. Metal enriched regions of chromatin appeared to be preferentially solubilized by digestion, and the solubilized metal was only partially dializable either with or without EDTA. Metal profiles generated from gel (A-5m) chromatography analysis of chelated and non-chelated solubilized chromatin were distinctive in that copper was undetectable (by flame AA) while zinc was associated only with low molecular weight products when EDTA was used. In contrast, both metals were detected with higher molecular weight oligonucleosomes in the absence of chelating agents. Additionally, the two metals localized within nucleoprotein peaks and these metal-containing regions were only resolved by gel chromatography when EDTA was omitted throughout the procedure. A discrete Cu-rich species in a region of the profile suggests a subset of Cu-rich nucleoprotein complexes.

The r e n i n -a n g i o t e n s i n system is c u s t o m a r i l y thought o f as a h o m e o s t a t i c feedback l o o p i n v o l v e d i n t h e r e g u l a t i o n o f i n t r av a s c u l a r volume and p r e s s u r e . However, o v e r t h e l a s t t h r e e d e c a d e s , r e n i n h a s been i d e n t i f i e d i n a wide v a r i e t y o f n o n r e n a l t i s s u e s and indeed o u r l a b o r a t o r y h a s confirmed s y n t h e s i s o f r e n i n i n a t l e a s t one e x t r a r e n a l s i t e , namely, t h e a r t e r i a l smooth muscle c e l l ( 1 ) . Although an i m p o r t a n t n e u r o r e g u l a t o r y r o l e f o r angioprovided e v i d e n c e s u g g e s t i n g a d i r e c t effect o f a n g i o t e n s i n I1 on RNA polymerase I1 a c t i v i t y ( 4 ) a s well a s on c o n f o r m a t i o n a l changes i n chromatin o f t h e s o r t a s s o c i a t e d w i t h enhanced t r a n s c r i p t i o n ( 5 , 6 1. A l l t h e s e o b s e r v a t i o n s s u g g e s t t h a t t h e i n t r a c e l l u l a r s y n t h e s i s o f r e n i n may b e d i r e c t e d toward t h e i n t r a c e l l u l a r s y n t h e s i s o f a n g i o t e n s i n I1 and subsequent e f f e c t s on i n t r a c e l l u l a r homeostasis. W e now e x t e n d t h e s e o b s e r v a t i o n s . MATERIALS AND METHODS I s o l a t i o n o f N u c l e i , Nuclease D i g e s t i o n and F r a c t i o n a t i o n o f Chromatin. P r o c e d u r e s have been p r e v i o u s l y d e s c r i b e d ( 5 , 6 ) . N u c l e i were o b t a i n e d from e i t h e r rat l i v e r o r bovine thymus as i n d i c a t e d under r e s u l t s . Hormone I n c u b a t i o n : Washed n u c l e a r p e l l e t o b t a i n e d from r a t l i v e r o r bovine thymus w a s suspended i n hormone i n c u b a t i o n b u f f e r (150 mM N a C 1 , 10 mM T r i s , 5 mM DTT, 1 mM PMSF, pH 7.5) @ tvgj d e s i r e d conc e n t r a t i o n p r i o r t o t h e a d d i t i o n o f e i t h e r

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