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The intracellular localization of exonuclear and endonuclear “phosphodiesterase” activity by histochemical methods
BIOCHIMICA ET BIOPHYSICA ACTA
212
PRELIMINARY
NOTES
BBA 61163
The intracellular localization of exonuclear and endonuclear "phosphodiesterase" activity by histochemical methods The present state of knowledge concerning the intracellular locahzatlon of the polynucleotidases, ,.e., those enzymes that attack ohgo- and polynucleotldes, is due largely to cellular fractionatlon techniques The difficulties encountered in obtaining comparable evidence by standard hlsto- and cytochemlcal means which, incidentally, apply as well to cellular fractionatlon techniques, were outlined by SIERAKOWSKAAND SHUCAR1,2 Moreover, a procedure for the hlstochemlcal demonstration of kidney orthophosphoric dlester phosphohydrolase (EC 3 i 4_i, phosphodiesterase I) was reported by these authors a who utilized a specific synthetic substrate, naphthyl-pT The method is based on a standard azo dye couphng of enzynllcally liberated anaphthol On the other hand, attempts to localize spleen phosphodiesterase I I with Tp-naphthyl, proved to be unsuccessful The resistance of the substrate to enzymic attack stands in contrast to the reasonably facile hydrolysis of Tp-nltrophenyl by phosphodlesterase II4, 5 This difference has been ascribed to an increased steric requirement of the a-naphthyl moiety 2. Recent studies m this laboratory have led to the histochemlcal demonstration of both acid and alkaline phosphomonoesterase" (orthophosphoric monoester phosphohydrolase, EC 3 I 3 2, and EC 3.1 3 I, respectively) through the use of the synthetic substrate p-toluidlnlum 5-bromo-4-chloro-3-Indolyl phosphate 7 Enzymic hydrolysis of this substrate liberates 5-bromo-4-chlorolndol-3-ol, which is rapidly and irreversibly oxidized to 5,5'-dlbromo-4,4'-dlchl°r°lndlg° at the sites of activity On the basis of these findings, efforts were directed to the application of the lndlgogenlc principle to the hlstochemlcal locahzatlon of phosphodlesterases I and I I The syntheses, which were modeled after those described by RAZZELLAND KHORANAs for nltrophenyl-pT and Tp-nltrophenyl, proceeded from I-acetyl-5-bromo-4-chloromdol-3-ol (ret 9) to give 5-bromo-4-chloro-3-indolyl-pT and Tp-5-bromo-4-chloro3-Indolyl in moderate yields_ Phosphodiesterase I activity was observed at pH 9 2 (Trls buffer) with 5-bromo4-chloro-3-1ndolyl-pT which affords a granular cytoplasmic deposition of the indigo in the proximal and distal convoluted tubules of rat kidney, liver, gastrointestinal tract and epldldymus (Fig I) Cytoplasmic activity was heavy in the brush border zone of kidney tubules with no evidence of nuclear activity. These observations are m essential agreement with earher hlstochemmal findings a_ By contrast, Tp-5-bromo-4-chloro-3-indolyl produced a marked histochemlcal reaction at pH 4.8 5 2 (acetate buffer) In tissues of rat and mouse that was locahzed chiefly in cell nuclei and was particularly prominent In the spleen (Fig. 2A). At pH 5 9 (acetate buffer), nuclear staining was markedly reduced, instead the reaction was observed mainly in cytoplasmic granules of retlculum cells in hver, spleen, gastroAbbreviations reference is made in the t e x t to several a r y l - p T and Tp-aryl derivatives where p T and Tp are t h y m l d m e ~'-phosphate and t h y m z d m e 3"-phosphate, respectively
Bwchzm Bwphys dcta, 159 (i968) 212-214
PRELIMINARY NOTES
213
2A .4
,#
Fig I A_ An unfixed frozen section of rat small Intestine demonstratmg granular cytoplasmic deposition of indigo in the cytoplasm of the mucosal cells and the reticulum cells The indigo is at the site of phosphodlesterase I activity at pH 9 o utlllzmg 5-bromo-4-chloro-3-mdolyl-pT × 4o0 B Higher magnification ( X 12oo) of A emphaslzmg the granular nature of mdlgogenlc stamlng Fig 2 A An unfixed frozen section of rat spleen demonstrating marked deposition of indigo in nuclei of retlculum cells at periphery of follicle There is minimal cytoplasmic staining The substrate utilized was Tp-5-bromo-4-chloro-3-1ndolyl at pH 5 2 X 12oo B An unfixed frozen section of rat small mte~tme demonstratmg marked cytoplasmic and granular droplet deposition of indigo in mucosal cells and retlculum cells at pH 5 9 The nuclei are negative The substrate utlhzed was Tp-5-bromo-4-chloro-3-1ndoly] × 12oo
i n t e s t i n a l t r a c t a n d l u n g ( F i g 2B). T h e a d d i t i o n of t h e r e d o x s y s t e m p o t a s s i u m f e r r o a n d f e r r l c y a n l d e s , w h i c h is f r e q u e n t l y i n c l u d e d in t h e m c u b a t l o n m e d i u m t o effect t h e o x i d a t i o n of t h e i n t e r m e d i a t e l n d o x y l t o m d l g o 1°, l e a d s t o a s i g n i f i c a n t m h l b l t i o n of s t a i n i n g of t h e c y t o p l a s m R e c e n t l y , BERNARDI a n d c o - w o r k e r s i l , 12 h a v e s h o w n t h a t a h i g h l y p u r i f i e d f o r m
B~och,rn B2ophys .4cta, 159 (1968) 212 214
214
PRELIMINARY NOTES
of h o g s p l e e n a c i d d e o x y r l b o n u c l e a s e ( d e o x y r i b o n u c l e a t e 3 ' - o h g o n u c l e o t l d o h y d r o l a s e , E C 3 I 4_6, d e o x y r l b o n u c l e a s e II) s h o w s " p h o s p h o d l e s t e r a s e " a c t i v i t y t o w a r d a series of p - n l t r o p h e n y l p h o s p h o d i e s t e r s These findings, together with the histochemical d e m o n s t r a t i o n of b o t h e x o - a n d e n d o p o l y n u c l e o t l d a s e ( s ) i n t h e s p l e e n as well as o t h e r t i s s u e s of t h e r a t a n d m o u s e w i t h T p - 5 - b r o m o - 4 - c h l o r o - 3 - 1 n d o l y l , c a s t d o u b t o n t h e c u r r e n t l y a c c e p t e d p r o c e d u r e for t h e specific a s s a y of p h o s p h o d i e s t e r a s e I I w i t h T p - n l t r o p h e n y l la T h i s i n v e s t i g a t i o n w a s s u p p o r t e d In p a r t b y P u b l i c H e a l t h S e r v i c e R e s e a r c h G r a n t No. C A - o 2 6 2 4 f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e a n d In p a r t b y a n i n s t i t u t i o n a l g r a n t t o t h e D e t r o i t I n s t i t u t e of C a n c e r R e s e a r c h D i v i s i o n of t h e M i c h i g a n C a n c e r F o u n d a t i o n f r o m t h e U n i t e d F o u n d a t i o n of G r e a t e r D e t r o i t .
Detroit Instztute of Cancer Research, Dzvzs, on of the M,ehzgan Cancer Foundation, Departments of Pathology and Oncology, Wayne State Unwers,ty School of Medzeme, Detroit, Mzch ( U . S A . ) i 2 3 4 5 6 7 8 9 IO II 12 13
PAUL L WOLF JEROME P HORWlTZ JOSEF V FREISLER JANICE VAZQUEZ ELISABETH VON DER MUEHLL
SIERAKOWSKA AND D SHUGAR,Ac~ct Bzoch~m Polon , 7 (196°) 475 SIERAKOWSKA AND D SHUGAR,Acta B~ochzm Polon , 8 (1961) 427 SIERAKOV¢SKAAND D SHUGAR,Acta Bzochzm Polon , IO (1963) 399 E RAZZELL, J Bzol Chem , 236 (1961) 3028 E RAZZELL, J Bzol Chem, 236 (1961) 3031 L WOLF, J P HORWlTZ, J VAZQUEZ, J CHUA, M S Y PAK AND E VON DER MUEHLL, Exper,entza, 23 (1967) 183 J P HORWlTZ, J CnUA, M NOEL, J T DONATTI AND J FREISLER, J ~'VIed Chem, 9 (1966) 447 ~V E RAZZELL AND H G KHORANA, J Btol Chem , 234 (1959) 21o5 S J HOLT, A E KELLIE, D G O'SULLIVAN AND P W SADLER, J Chem Soc, (1958) 1217 A G E PEARSE, Htstochemzstry, Little Brown, Boston, Mass, 196o, p 888 G BERNARDI AND M GRIFFE, Bzoehemzstry, 3 (1964) 1419 G BERNARDI, A BERNARDI AND A CHERSI, Bzoch*m Btophys Aeta, 129 (1966) I W E RAZZELL, in S P COLOWlCK AND N O KAPLAN, Methods ~n Enzymologv, Vo] 6, Academic Press, New York, 1963, p 245 H H H W W P
R e c e i v e d F e b r u a r y 5 t h , 1968
Bzoch~m B~ophvs Acta, 159 (1968) 212-214
212
PRELIMINARY
NOTES
BBA 61163
The intracellular localization of exonuclear and endonuclear "phosphodiesterase" activity by histochemical methods The present state of knowledge concerning the intracellular locahzatlon of the polynucleotidases, ,.e., those enzymes that attack ohgo- and polynucleotldes, is due largely to cellular fractionatlon techniques The difficulties encountered in obtaining comparable evidence by standard hlsto- and cytochemlcal means which, incidentally, apply as well to cellular fractionatlon techniques, were outlined by SIERAKOWSKAAND SHUCAR1,2 Moreover, a procedure for the hlstochemlcal demonstration of kidney orthophosphoric dlester phosphohydrolase (EC 3 i 4_i, phosphodiesterase I) was reported by these authors a who utilized a specific synthetic substrate, naphthyl-pT The method is based on a standard azo dye couphng of enzynllcally liberated anaphthol On the other hand, attempts to localize spleen phosphodiesterase I I with Tp-naphthyl, proved to be unsuccessful The resistance of the substrate to enzymic attack stands in contrast to the reasonably facile hydrolysis of Tp-nltrophenyl by phosphodlesterase II4, 5 This difference has been ascribed to an increased steric requirement of the a-naphthyl moiety 2. Recent studies m this laboratory have led to the histochemlcal demonstration of both acid and alkaline phosphomonoesterase" (orthophosphoric monoester phosphohydrolase, EC 3 I 3 2, and EC 3.1 3 I, respectively) through the use of the synthetic substrate p-toluidlnlum 5-bromo-4-chloro-3-Indolyl phosphate 7 Enzymic hydrolysis of this substrate liberates 5-bromo-4-chlorolndol-3-ol, which is rapidly and irreversibly oxidized to 5,5'-dlbromo-4,4'-dlchl°r°lndlg° at the sites of activity On the basis of these findings, efforts were directed to the application of the lndlgogenlc principle to the hlstochemlcal locahzatlon of phosphodlesterases I and I I The syntheses, which were modeled after those described by RAZZELLAND KHORANAs for nltrophenyl-pT and Tp-nltrophenyl, proceeded from I-acetyl-5-bromo-4-chloromdol-3-ol (ret 9) to give 5-bromo-4-chloro-3-indolyl-pT and Tp-5-bromo-4-chloro3-Indolyl in moderate yields_ Phosphodiesterase I activity was observed at pH 9 2 (Trls buffer) with 5-bromo4-chloro-3-1ndolyl-pT which affords a granular cytoplasmic deposition of the indigo in the proximal and distal convoluted tubules of rat kidney, liver, gastrointestinal tract and epldldymus (Fig I) Cytoplasmic activity was heavy in the brush border zone of kidney tubules with no evidence of nuclear activity. These observations are m essential agreement with earher hlstochemmal findings a_ By contrast, Tp-5-bromo-4-chloro-3-indolyl produced a marked histochemlcal reaction at pH 4.8 5 2 (acetate buffer) In tissues of rat and mouse that was locahzed chiefly in cell nuclei and was particularly prominent In the spleen (Fig. 2A). At pH 5 9 (acetate buffer), nuclear staining was markedly reduced, instead the reaction was observed mainly in cytoplasmic granules of retlculum cells in hver, spleen, gastroAbbreviations reference is made in the t e x t to several a r y l - p T and Tp-aryl derivatives where p T and Tp are t h y m l d m e ~'-phosphate and t h y m z d m e 3"-phosphate, respectively
Bwchzm Bwphys dcta, 159 (i968) 212-214
PRELIMINARY NOTES
213
2A .4
,#
Fig I A_ An unfixed frozen section of rat small Intestine demonstratmg granular cytoplasmic deposition of indigo in the cytoplasm of the mucosal cells and the reticulum cells The indigo is at the site of phosphodlesterase I activity at pH 9 o utlllzmg 5-bromo-4-chloro-3-mdolyl-pT × 4o0 B Higher magnification ( X 12oo) of A emphaslzmg the granular nature of mdlgogenlc stamlng Fig 2 A An unfixed frozen section of rat spleen demonstrating marked deposition of indigo in nuclei of retlculum cells at periphery of follicle There is minimal cytoplasmic staining The substrate utilized was Tp-5-bromo-4-chloro-3-1ndolyl at pH 5 2 X 12oo B An unfixed frozen section of rat small mte~tme demonstratmg marked cytoplasmic and granular droplet deposition of indigo in mucosal cells and retlculum cells at pH 5 9 The nuclei are negative The substrate utlhzed was Tp-5-bromo-4-chloro-3-1ndoly] × 12oo
i n t e s t i n a l t r a c t a n d l u n g ( F i g 2B). T h e a d d i t i o n of t h e r e d o x s y s t e m p o t a s s i u m f e r r o a n d f e r r l c y a n l d e s , w h i c h is f r e q u e n t l y i n c l u d e d in t h e m c u b a t l o n m e d i u m t o effect t h e o x i d a t i o n of t h e i n t e r m e d i a t e l n d o x y l t o m d l g o 1°, l e a d s t o a s i g n i f i c a n t m h l b l t i o n of s t a i n i n g of t h e c y t o p l a s m R e c e n t l y , BERNARDI a n d c o - w o r k e r s i l , 12 h a v e s h o w n t h a t a h i g h l y p u r i f i e d f o r m
B~och,rn B2ophys .4cta, 159 (1968) 212 214
214
PRELIMINARY NOTES
of h o g s p l e e n a c i d d e o x y r l b o n u c l e a s e ( d e o x y r i b o n u c l e a t e 3 ' - o h g o n u c l e o t l d o h y d r o l a s e , E C 3 I 4_6, d e o x y r l b o n u c l e a s e II) s h o w s " p h o s p h o d l e s t e r a s e " a c t i v i t y t o w a r d a series of p - n l t r o p h e n y l p h o s p h o d i e s t e r s These findings, together with the histochemical d e m o n s t r a t i o n of b o t h e x o - a n d e n d o p o l y n u c l e o t l d a s e ( s ) i n t h e s p l e e n as well as o t h e r t i s s u e s of t h e r a t a n d m o u s e w i t h T p - 5 - b r o m o - 4 - c h l o r o - 3 - 1 n d o l y l , c a s t d o u b t o n t h e c u r r e n t l y a c c e p t e d p r o c e d u r e for t h e specific a s s a y of p h o s p h o d i e s t e r a s e I I w i t h T p - n l t r o p h e n y l la T h i s i n v e s t i g a t i o n w a s s u p p o r t e d In p a r t b y P u b l i c H e a l t h S e r v i c e R e s e a r c h G r a n t No. C A - o 2 6 2 4 f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e a n d In p a r t b y a n i n s t i t u t i o n a l g r a n t t o t h e D e t r o i t I n s t i t u t e of C a n c e r R e s e a r c h D i v i s i o n of t h e M i c h i g a n C a n c e r F o u n d a t i o n f r o m t h e U n i t e d F o u n d a t i o n of G r e a t e r D e t r o i t .
Detroit Instztute of Cancer Research, Dzvzs, on of the M,ehzgan Cancer Foundation, Departments of Pathology and Oncology, Wayne State Unwers,ty School of Medzeme, Detroit, Mzch ( U . S A . ) i 2 3 4 5 6 7 8 9 IO II 12 13
PAUL L WOLF JEROME P HORWlTZ JOSEF V FREISLER JANICE VAZQUEZ ELISABETH VON DER MUEHLL
SIERAKOWSKA AND D SHUGAR,Ac~ct Bzoch~m Polon , 7 (196°) 475 SIERAKOWSKA AND D SHUGAR,Acta B~ochzm Polon , 8 (1961) 427 SIERAKOV¢SKAAND D SHUGAR,Acta Bzochzm Polon , IO (1963) 399 E RAZZELL, J Bzol Chem , 236 (1961) 3028 E RAZZELL, J Bzol Chem, 236 (1961) 3031 L WOLF, J P HORWlTZ, J VAZQUEZ, J CHUA, M S Y PAK AND E VON DER MUEHLL, Exper,entza, 23 (1967) 183 J P HORWlTZ, J CnUA, M NOEL, J T DONATTI AND J FREISLER, J ~'VIed Chem, 9 (1966) 447 ~V E RAZZELL AND H G KHORANA, J Btol Chem , 234 (1959) 21o5 S J HOLT, A E KELLIE, D G O'SULLIVAN AND P W SADLER, J Chem Soc, (1958) 1217 A G E PEARSE, Htstochemzstry, Little Brown, Boston, Mass, 196o, p 888 G BERNARDI AND M GRIFFE, Bzoehemzstry, 3 (1964) 1419 G BERNARDI, A BERNARDI AND A CHERSI, Bzoch*m Btophys Aeta, 129 (1966) I W E RAZZELL, in S P COLOWlCK AND N O KAPLAN, Methods ~n Enzymologv, Vo] 6, Academic Press, New York, 1963, p 245 H H H W W P
R e c e i v e d F e b r u a r y 5 t h , 1968
Bzoch~m B~ophvs Acta, 159 (1968) 212-214