Increased activity of deoxyribonuclease inhibitor in rat serum after partial hepatectomy

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BBA 93498

Increased activity of deoxyribonuclease inhibitor in rat serum after partial

hepatectomy The inhibitor of deoxyribonuclease in tissues of animal origin have been described b y several investigators 1-s. Recently, LINDBERG4 has succeeded in crystallizing two closely related inhibitors from calf spleen to act on pancreatic deoxyribonuclease (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.5). The presence of deoxyribonuclease inhibitor in rat serum was first described by OKADA et a/.~,and BERGER AND MAYs have recently purified the inhibitor from rat serum and shown it to have close similarity to calf spleen inhibitor I I extracted by LINDBERG4. This paper describes an investigation of the finding that partial hepatectomy caused an increase in the activity of deoxyribonuclease inhibitor in serum. Surgical removal under ether anesthesia was carried out as described b y HIGGINS AND ANDERSON7 on male Wistar rats weighing 8O-lOO g. Blood samples were taken b y heart puncture under ether anesthesisa, and the serum was separated b y centrifugation. In assaying deoxyribonuclease inhibitor, the serum was dialyzed against o.oi M Tris buffer (pH 7-5) at 4 ° overnight. Inhibitor was assayed b y estimating the inhibition of pancreatic deoxyribonuclease b y the dialyzed rat serum. A unit of inhibitor activity was defined as that causing 50 % inhibition ot 0.02 #g crystalline pancreatic deoxyribonuclease under assay conditions. As shown in Fig. I, partial hepatectomy caused an increase in the activity of deoxyribonuclease inhibitor in serum. Increased activity remained the same at 24 h after the operation.

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Fig. i. I n h i b i t i o n of d e o x y r i b o n u c l e a s e a c t i v i t y in s e r u m f r o m n o r m a l a n d p a r t i a l l y h e p a t e c t o m i z e d rats. I n h i b i t o r a c t i v i t y w a s m e a s u r e d in t h e dialyzed s e r u m f r o m n o r m a l a n d p a r t i a l l y h e p a t e c t o m i z e d r a t s a t 18 h after t h e operation. A s s a y s were carried o u t in a t o t a l v o l u m e s of 0.3 m l c o n t a i n i n g : 3 0 / , m o l e s Tris-HC1 buffer (pH 7-5), 2.5 /~moles MgC1 v 300/~g of n a t i v e calf t h y m u s DNA, 0.02/zg of crystalline p a n c r e a t i c d e o x y r i b o n u c l e a s e , a n d dialyzed s e r u m . T h e reaction w a s s t o p p e d w i t h 3 m l of 5 % HC104. T h e a b s o r b a n c e of t h e acid s u p e r n a t a n t w a s d e t e r m i n e d a t 260 m/~. 0 , s e r u m f r o m n o r m a l r a t s ; O , s e r u m f r o m paxtially h e p a t e e t o m i z e d rats. Fig, 2. T h e r m a l s t a b i l i t y of d e o x y r i b o n u c l e a s e i n h i b i t o r in s e r u m f r o m n o r m a l a n d p a r t i a l l y h e p a t e e t o m i z e d rats. T h e a c t i v i t y of p a n c r e a t i c d e o x y r i b o n u c l e a s e w a s a s s a y e d b y a d d i n g t h e i n h i b i t o r p r e p a r a t i o n (7° / z g of protein) after h e a t i n g for 5 m i n in o.oi M Tris-HC1 b u f f e r (pH 7.5) a t t h e i n d i c a t e d t e m p e r a t u r e . A s s a y c o n d i t i o n s are described in Fig. i. O , s e r u m f r o m n o r m a l r a t s ; O , s e r u m f r o m p a r t i a l l y h e p a t e c t o m i z e d rats.

Biochim. Biophys. Acta, 204 (197 o) 255-256

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The thermal stability of the inhibitor was determined by assaying the activity remaining after incubation for 5 mid at various temperatures between 45 and 9 °0 in o.oi M Tris buffer (pH 7-5). The results shown in Fig. 2 indicate that the activity ot the inhibitor protein was almost completely destroyed at 60 °. The inhibitor activities in normal and partially hepatectomized animals were indistinguishably affected by heat. Similar observations have been reported for ribonuclease inhibitor, that the activity of ribonuclease inhibitor was found to be higher in serum after partial hepatectomy than in serum from normal rats s. Whether increased activity in deoxyribonuclease inhibitor is due to de novo synthesis or to activation is not known, though the former seems more likely in view of the results that increased activity m a y be responsible for the increased protein synthesis 8,9. I t is possible that this inhibitor m a y actually control the activity of deoxyribonucleases. We are now attempting to find the function of deoxyribonuclease inhibitor in the overall control of DNA synthesis.

Department o/ Biochemistry, Drug Research Institute, Faculty o/Pharmaceutical Science, Toyama University, Go/uku, Toyama (Japan)

NOBUKO HAYASAKI KINJI TSUKADA

I G. SIEBERT, R. LANG, S. LUCINS-LANG, Z. HERBERT, G. STARK, G. ROSSMULLER AND H. JOCKEL, Z. Physiol. Chem., 295 (1953) 229. 2 W. FRISCH-NIGGEMEYER AND O. HOFFMANN-OSTENHOF, Monatsh., 81 (I95 o) 607. 3 L. M. GILBERT, W. G. OVEREND AND M. WEBB, Exptl. Cell Res., 2 / I 9 5 I ) 349. 4 M. L. LINDBERG, dr. Biol. Chem., 241 (1966) 1246. 5 S. OKADA, E. 1~. GORDON, R. KING AND L. H. HEMPELMANN, Arch. Biochem. Biophys. 7° (I957) 469 . 6 G. BERGER AND P. MAY, Biochim. Biophys. Acra, I39 (1967) 148. 7 C. M. HIGGINS AND R. M. ANDERSON, Arch. Pathol., i2 (1931) 186. 8 K. TSUKADA, Biochim. Biophys. Acta, 186 (1969) 2I. 9 ]~. TSUKADA, T. MORIYAMA, O. DOI AND I. LIEBERMAN, J. Biol. Chem., 243 (1968) 1152.

Received December 29th , 1969 Biochim. Biophys. Acta, 204 (197 o) 255-256