V o l u m e ~W, number 2.21t1~21t4 FEBS 09126 ~, Ill11 Fider~tllon or i~uropc~n Ittoihcmk'ltl S~l,tttli lXlt4S'ttt)#liil/$).~lg
IFebrunry 1991
A DONI,f 0014~79~ I OCillos&
Inhibitory effect of calcium-binding protein regucalcin on Ca z *-activated D N A fragmentation in rat liver nuclei Masayoshi Yamaguchi and Takashi Sakurai Deparm;¢nt nf Environmental Rtaehcmi~tO,. Sckaot of Pharmaceutical Sciences, Univer.~tty af Shl'.uoka, 39J Yada. Shhuoka.City 422, Japan Received 13 December 1990 Incubation o1' isolated rnl liver nuclei with ATP, N A D ' , and micromolar Ca=" concentrations o1"various metal ions resulted in extensive D N A hydrolysis. Halt'.maxim~t activity o¢~ttrred with 1,0 pM Ca=" added, and saturattion of the process w.s observed with 10 p M Ca=*, The Ca=" (10 tsM).~clivaled DNA frallmentalion wzts inhibited by the presence of Ca ="-bindintt protein reltucalctn tsolated from rat liver cytosol. The inhibitory effect of r¢llUcal¢lnwa~ complete at 0, S pM. At 25 p M Ca! ° added, such an effect of relluealcin (1,0 pM ) w~= not seen, Rcgucalci n also inhibited Ca=*..cliv~ted DNA fragment~ttion in the presence or calmodulin (10 and 20 pg). The results thow that relluc=lcin can =nhibit the Ca='.activated D N A t'rallmeniation due to binding the met,d, suigeslin@ a role in rellulution of liver nuclear functions.
Calcium: R¢gucalcin; DNA.fragmentation; Rat liver nucleus
1. I N T R O D U C T I O N C a a÷ p l a y s an i m p o r t a n t r o l e in t h e r e g u l a t i o n o f m a n y cell f u n c t i o n s [1]. T h e r o l e o f C a 2÷ in liver m e t a b o l i s m has b e e n d e m o n s t r a t e d in recent i n v e s t i g a t i o n s [2,3]. Liver m e t a b o l i s m is r e g u l a t e d b y t h e inc r e a s e o f Ca 2 + in t h e c y t o s o l o f tiver cells d u e to h o r m o n a l s t i m u l a t i o n , R e c e n t a c c u m u l a t i n g e v i d e n c e sugg e s t s t h a t Ca 2+ p l a y s a role in liver n u c l e a r f u n c t i o n [4-9]. Calmodulin, a calcium-binding protein which c a n a m p l i f y C a 2+ e f f e c t [10], exists in rat liver nuclei [4], T h e existence o f a n A T P - s t i m u t a t e d C a 2÷ seq u e s t r a t i o n system in rat liver n u c l e i t h a t r e q u i r e s c a l m o d u l i n a n d g e n e r a t e s a n e t i n c r e a s e in n u c l e a r m a t r i x free C a 2 + c o n c e n t r a t i o n has b e e n r e p o r t e d [5]. C a l m o d u l i n s t i m u l a t e s D N A s y n t h e s i s b y liver cells [6]° a n d the c a l m o d u l i n e f f e c t m a y be m e d i a t e d t h r o u g h ~ a d r e n e r g i c s t i m u l a t i o n [7,8]. O n t h e o t h e r h a n d , a novel c a l c i u m - b i n d i n g p r o t e i n ( r c g u c a l c i n ) , w h i c h d i f f e r s f r o m c a l m o d u l i n [10] a n d other calcium.binding proteins (caligulin [11], c a l r e g u l i n [I 2] a n d c a l r e t i c u l i n [13]) is d i s t r i b u t e d in r a t liver c y t o s o l [14-16]. R e g u c a l c i n m a y p l a y a cell p h y s i o l o g i c a l role d i f f e r e n t f r o m t h o s e o f o t h e r c a l c i u m - b i n d i n g p r o t e i n s in t h e r e g u l a t i o n o f l i v e r cell f u n c t i o n s ; r e g u c a l c i n c a n reverse the e f f e c t o f C a 2 * o n m a n y e n z y m e s in liver cells [17-20]. R e g u c a l c i n m a y p l a y a r o l e a s a r e g u l a t o r y p r o t e i n f o r Ca z ÷ e f f e c t s in liver cells;
Correspondence address: M, Yamaguchi, Department of En. vtronmental Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 395 Yada, Shizuoka.City 422, Japan
Published by Elsevier Science Publishers B, V.
M o r e r e c e n t l y , it h a s b e e n r e p o r t e d t h a t i s o l a t e d r a t liver nuclei c o n t a i n a D N A ¢ n d o n u c l e a s e a c t i v i t y d e p e n d e n t upon C a 1 ÷ in t h e s u b m i c r o m o l a r r a n g e , a n d t h a t Cai+ r e s u l t s in e x t e n s i v e D N A h y d r o l y s i s [21], T h e r e f o r e , t h e present i n v e s t i g a t i o n w a s u n d e r t a k e n to clarify whether regucalcin has an effect on C a 2 + - a c t i v a t e d D N A f r a g m e n t a t i o n in i s o l a t e d r a t liver nuclei. It w a s found that regucalcin inhibits C a 2 + - a c t i v a t e d D N A f r a g m e n t a t i o n in t h e nuclei, 2. M A T E R I A L S
AND METHODS
2, |, Chemicals Adenosine S'.triphosphate (ATP), nlcotinamide adenine dinucleotide (NAD * ), et hyleneglycol-bis.taminoethyl ether)N,N ~.tetraacetie aid (EGTA), and calmodulin (52 000 units/rag protein from bovine brain) were obtained from the Sigma Chemical Co, (St, Louis, MO. USA), CaCl~ 2H~O and other reagents were purchased from Wake Pure Chemical Co. (Osaka, Japan), The reagents were dissolved in distilled water and tllen passed through ionexchange resin to remove metal ions, 2,2, Isolation of reguealein Male Wistar rats, weighing 100-120 g, purchased from the Japan Inc. (Hamamatsu, Japan), were fed commercial laboratory chow (solid) containing 57.5°7o carbohydrate. 1,1070Ca. and 1.1°70 P, and distilled water, freely, After one week on this diet animals were killed by bleeding. The livers were perfused with Tris-HC1 buffer (pH 7.4, containing 100 mM Tris, 120 mM NaCI, 4 mM KCI, cooled to 4"C), The livers were removed, cut into small pieces, suspended 1:4 in TrisHCI buffer (pH 7.4) and the homogenate was spun at 5500 × g in a refrigerated centrifuge for |0 rain and the supernatant was spun at 105 000 x g for 60 min. Regucatcin in the 105 000 × g supernatant (cytosol) was purified to electrophoretic homogeneity by gel filtration on Sephadex G-75 and 0.$0 followed by ion exchange chromatography on diethylaminoethyl(DEAE)-cellulose, as reported previously [14], Protein concentration was determined by the method of Lowry et al. [22] using albumin as a standard.
281
V o l u m e 2?% number 2
FEB$ LEr'TERS
2,3, Ixolmlm~ of nuclei Liver nuclei were isolaled by th¢ procedure or J~n¢~ qt =1, [211 with a minor modification, R~t~ ~ere killed by cardiac puncture, and the liver w~s perfused with ~ppro~lmately 10 ml or i,~e.cold TKM solution (~0 rr~M Trls.HCl, pH 7,~, 2~ mM KCI, S mM MIICI~) to remove blood. Livers were then removed, cut into ~mnll pieees, ~ud homogenized in = Po, t~r,~lvehjern homogenizer with a Teflon pestle in 40 ml of the same solution containing 0,25 M ~u
IFebrunry 1991
A DONI,f 0014~79~ I OCillos&
Inhibitory effect of calcium-binding protein regucalcin on Ca z *-activated D N A fragmentation in rat liver nuclei Masayoshi Yamaguchi and Takashi Sakurai Deparm;¢nt nf Environmental Rtaehcmi~tO,. Sckaot of Pharmaceutical Sciences, Univer.~tty af Shl'.uoka, 39J Yada. Shhuoka.City 422, Japan Received 13 December 1990 Incubation o1' isolated rnl liver nuclei with ATP, N A D ' , and micromolar Ca=" concentrations o1"various metal ions resulted in extensive D N A hydrolysis. Halt'.maxim~t activity o¢~ttrred with 1,0 pM Ca=" added, and saturattion of the process w.s observed with 10 p M Ca=*, The Ca=" (10 tsM).~clivaled DNA frallmentalion wzts inhibited by the presence of Ca ="-bindintt protein reltucalctn tsolated from rat liver cytosol. The inhibitory effect of r¢llUcal¢lnwa~ complete at 0, S pM. At 25 p M Ca! ° added, such an effect of relluealcin (1,0 pM ) w~= not seen, Rcgucalci n also inhibited Ca=*..cliv~ted DNA fragment~ttion in the presence or calmodulin (10 and 20 pg). The results thow that relluc=lcin can =nhibit the Ca='.activated D N A t'rallmeniation due to binding the met,d, suigeslin@ a role in rellulution of liver nuclear functions.
Calcium: R¢gucalcin; DNA.fragmentation; Rat liver nucleus
1. I N T R O D U C T I O N C a a÷ p l a y s an i m p o r t a n t r o l e in t h e r e g u l a t i o n o f m a n y cell f u n c t i o n s [1]. T h e r o l e o f C a 2÷ in liver m e t a b o l i s m has b e e n d e m o n s t r a t e d in recent i n v e s t i g a t i o n s [2,3]. Liver m e t a b o l i s m is r e g u l a t e d b y t h e inc r e a s e o f Ca 2 + in t h e c y t o s o l o f tiver cells d u e to h o r m o n a l s t i m u l a t i o n , R e c e n t a c c u m u l a t i n g e v i d e n c e sugg e s t s t h a t Ca 2+ p l a y s a role in liver n u c l e a r f u n c t i o n [4-9]. Calmodulin, a calcium-binding protein which c a n a m p l i f y C a 2+ e f f e c t [10], exists in rat liver nuclei [4], T h e existence o f a n A T P - s t i m u t a t e d C a 2÷ seq u e s t r a t i o n system in rat liver n u c l e i t h a t r e q u i r e s c a l m o d u l i n a n d g e n e r a t e s a n e t i n c r e a s e in n u c l e a r m a t r i x free C a 2 + c o n c e n t r a t i o n has b e e n r e p o r t e d [5]. C a l m o d u l i n s t i m u l a t e s D N A s y n t h e s i s b y liver cells [6]° a n d the c a l m o d u l i n e f f e c t m a y be m e d i a t e d t h r o u g h ~ a d r e n e r g i c s t i m u l a t i o n [7,8]. O n t h e o t h e r h a n d , a novel c a l c i u m - b i n d i n g p r o t e i n ( r c g u c a l c i n ) , w h i c h d i f f e r s f r o m c a l m o d u l i n [10] a n d other calcium.binding proteins (caligulin [11], c a l r e g u l i n [I 2] a n d c a l r e t i c u l i n [13]) is d i s t r i b u t e d in r a t liver c y t o s o l [14-16]. R e g u c a l c i n m a y p l a y a cell p h y s i o l o g i c a l role d i f f e r e n t f r o m t h o s e o f o t h e r c a l c i u m - b i n d i n g p r o t e i n s in t h e r e g u l a t i o n o f l i v e r cell f u n c t i o n s ; r e g u c a l c i n c a n reverse the e f f e c t o f C a 2 * o n m a n y e n z y m e s in liver cells [17-20]. R e g u c a l c i n m a y p l a y a r o l e a s a r e g u l a t o r y p r o t e i n f o r Ca z ÷ e f f e c t s in liver cells;
Correspondence address: M, Yamaguchi, Department of En. vtronmental Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 395 Yada, Shizuoka.City 422, Japan
Published by Elsevier Science Publishers B, V.
M o r e r e c e n t l y , it h a s b e e n r e p o r t e d t h a t i s o l a t e d r a t liver nuclei c o n t a i n a D N A ¢ n d o n u c l e a s e a c t i v i t y d e p e n d e n t upon C a 1 ÷ in t h e s u b m i c r o m o l a r r a n g e , a n d t h a t Cai+ r e s u l t s in e x t e n s i v e D N A h y d r o l y s i s [21], T h e r e f o r e , t h e present i n v e s t i g a t i o n w a s u n d e r t a k e n to clarify whether regucalcin has an effect on C a 2 + - a c t i v a t e d D N A f r a g m e n t a t i o n in i s o l a t e d r a t liver nuclei. It w a s found that regucalcin inhibits C a 2 + - a c t i v a t e d D N A f r a g m e n t a t i o n in t h e nuclei, 2. M A T E R I A L S
AND METHODS
2, |, Chemicals Adenosine S'.triphosphate (ATP), nlcotinamide adenine dinucleotide (NAD * ), et hyleneglycol-bis.taminoethyl ether)N,N ~.tetraacetie aid (EGTA), and calmodulin (52 000 units/rag protein from bovine brain) were obtained from the Sigma Chemical Co, (St, Louis, MO. USA), CaCl~ 2H~O and other reagents were purchased from Wake Pure Chemical Co. (Osaka, Japan), The reagents were dissolved in distilled water and tllen passed through ionexchange resin to remove metal ions, 2,2, Isolation of reguealein Male Wistar rats, weighing 100-120 g, purchased from the Japan Inc. (Hamamatsu, Japan), were fed commercial laboratory chow (solid) containing 57.5°7o carbohydrate. 1,1070Ca. and 1.1°70 P, and distilled water, freely, After one week on this diet animals were killed by bleeding. The livers were perfused with Tris-HC1 buffer (pH 7.4, containing 100 mM Tris, 120 mM NaCI, 4 mM KCI, cooled to 4"C), The livers were removed, cut into small pieces, suspended 1:4 in TrisHCI buffer (pH 7.4) and the homogenate was spun at 5500 × g in a refrigerated centrifuge for |0 rain and the supernatant was spun at 105 000 x g for 60 min. Regucatcin in the 105 000 × g supernatant (cytosol) was purified to electrophoretic homogeneity by gel filtration on Sephadex G-75 and 0.$0 followed by ion exchange chromatography on diethylaminoethyl(DEAE)-cellulose, as reported previously [14], Protein concentration was determined by the method of Lowry et al. [22] using albumin as a standard.
281
V o l u m e 2?% number 2
FEB$ LEr'TERS
2,3, Ixolmlm~ of nuclei Liver nuclei were isolaled by th¢ procedure or J~n¢~ qt =1, [211 with a minor modification, R~t~ ~ere killed by cardiac puncture, and the liver w~s perfused with ~ppro~lmately 10 ml or i,~e.cold TKM solution (~0 rr~M Trls.HCl, pH 7,~, 2~ mM KCI, S mM MIICI~) to remove blood. Livers were then removed, cut into ~mnll pieees, ~ud homogenized in = Po, t~r,~lvehjern homogenizer with a Teflon pestle in 40 ml of the same solution containing 0,25 M ~u
