Dominant Role of the Liver in Biosynthesis of the Plasma Proteins with

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to the Plasma Mucoproteins (Seromucoid), Ceruloplasmin, and Fibrinogen LEON L. MILLER, HELEN R. H A N A V A N , N A N T H A TITTHASIRI, AND ANUBHA

CHOWDHURY

Department of Radiation Biology, School of Medicine and Dentistry, University of Rochester, Rochester, Ν. Y. The techniques of isolated rat liver perfu sion, preparative starch-block electropho resis, and the use of isotopically labeled metabolites (lysine-6-C , leucine-1-C , sulfur -labeled sulfate, and copper ) have been combined to examine more critically previously published conclusions that the liver is the site of biosynthesis of plasma albumin, and of the plasma α-globulins and ß-globulins, including fibrinogen. The data reaffirm the view that all plasma albumin, virtually all of the normally occurring perchloric acid nonprecipitable proteins (seromucoid), ceruloplasmin, the plasma lipoproteins, and all fibrinogen are synthesized by the liver. Data support the view that nonhepatic tissues are primarily concerned with producing γ-globulins and ß-globulins generically related to the γ-globulins. Also described are net biosynthesis of fibrino gen and its inhibition by puromycin, mito mycin, and ethionine in the isolated per fused liver. 14

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More than ten years ago we devised a direct approach toward a defi nition of the role of the liver in plasma protein biosynthesis by com bining the techniques of isolated rat liver perfusion, starch-block elec trophoresis, and the use of carbon-14-labeled aminoacids (23,24). The 17

Stekol; Amino Acids and Serum Proteins Advances in Chemistry; American Chemical Society: Washington, DC, 1964.

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ADVANCES IN CHEMISTRY SERIES

c l a s s i c a l p r o c e d u r e of l i v e r p e r f u s i o n was g r e a t l y s i m p l i f i e d and adapted to a study of the s m a l l m a m m a l i a n l i v e r of the r a t (25). It was d e m o n s t r a t e d that the i s o l a t e d r a t l i v e r perfused w i t h homologous hepa r i n i z e d r a t blood s i m u l a t e s p h y s i o l o g i c a l l y the q u a l i t a t i v e and q u a n t i tative p e r f o r m a n c e of the l i v e r i n the i n t a c t r a t f o r 6 to 8 h o u r s . It w a s q u i c k l y r e c o g n i z e d that t h i s t i m e i n t e r v a l w a s m o r e than sufficient to p e r m i t d e t a i l e d o b s e r v a t i o n s of the m e t a b o l i s m of i s o t o p i c a l l y l a b e l e d a m i n o a c i d s u s e d , s u c h as l y s i n e and h i s t i d i n e , and to o b s e r v e t h e i r i n c o r p o r a t i o n i n t o the p r o t e i n s d i s c h a r g e d by the l i v e r i n t o the c i r c u l a t i n g p l a s m a . W i t h the a p p l i c a t i o n of p r e p a r a t i v e zone e l e c t r o p h o r e s i s to the s e p a r a t i o n of the p l a s m a p r o t e i n s l a b e l e d w i t h C - a m i n o a c i d s i n the c o u r s e of l i v e r p e r f u s i o n , i t was r e v e a l e d that the i s o l a t e d perfused l i v e r i n c o r p o r a t e d l a b e l e d a m i n o a c i d s into a l l of the p l a s m a p r o t e i n f r a c t i o n s , w i t h one notable exception, the y - g l o b u l i n s . T h i s r e p o r t d e s c r i b e s further studies w i t h the i s o l a t e d perfused l i v e r , f r o m w h i c h i t w i l l be c l e a r that the p l a s m a s e r o m u c o i d — i . e . , p r o teins s o l u b l e i n 0 . 6 4 N p e r c h l o r i c a c i d (34, 35)—plasma f i b r i n o g e n , and c e r u l o p l a s m i n a r e s y n t h e s i z e d e x c l u s i v e l y by the l i v e r . In the c a s e of f i b r i n o g e n by u t i l i z i n g d e f i b r i n a t e d r a t blood f o r l i v e r p e r f u s i o n , i t has been p o s s i b l e to d e m o n s t r a t e i m p r e s s i v e net b i o s y n t h e s i s of f i b r i n o g e n p r o t e i n to an extent depending upon the d e g r e e of h y p o f i b r i n o g e n e m i a i n the c i r c u l a t i n g p e r f u s i n g b l o o d . B y way of supplementing and c o n t r o l l i n g the o b s e r v a t i o n s made w i t h i s o l a t e d l i v e r p e r f u s i o n , studies have been c a r r i e d out on the e v i s c e r a t e d s u r v i v i n g r a t ( a l l a b d o m i n a l v i s c e r a excepting k i d n e y s r e moved) and the hepatectomized r a t , and t h e i r p l a s m a s subjected to s i m i l a r f r a c t i o n a t i o n techniques. In a n u m b e r of e x p e r i m e n t s the e v i s c e r ated s u r v i v i n g r a t w a s p r e p a r e d i n s u c h a manner as to leave the l i v e r hepatic a r t e r i a l c i r c u l a t i o n i n t a c t . Such a r a t had a l l a b d o m i n a l v i s c e r a except the k i d n e y s and l i v e r e x c i s e d . In k e e p i n g w i t h the c o n c l u s i o n s d e r i v a b l e f r o m the l i v e r p e r f u s i o n s t u d i e s , the studies i n the e v i s c e r ated s u r v i v i n g r a t and the h e p a t e c t o m i z e d r a t d e m o n s t r a t e that the n o n hepatic t i s s u e s a r e p r i m a r i l y c o n c e r n e d w i t h the b i o s y n t h e s i s of the y g l o b u l i n s and s o m e β - g l o b u l i n s w h i c h m a y be r e l a t e d to the y - g l o b u l i n s . 1 4

Methods and

Experimental

A d u l t m a l e S p r a g u e - D a w l e y r a t s w e i g h i n g 250 to 450 g r a m s w e r e u s e d i n m o s t of these s t u d i e s . T h e y w e r e m a i n t a i n e d on c o m m e r c i a l r a t food and w e r e a l l o w e d a c c e s s to w a t e r at a l l t i m e s . The techniques of i s o l a t e d l i v e r p e r f u s i o n and the p r e p a r a t i o n of e v i s c e r a t e d s u r v i v i n g r a t have been d e s c r i b e d i n d e t a i l (24, 25). The e v i s c e r a t e d s u r v i v i n g r a t w a s p r e p a r e d a c c o r d i n g to the p r o c e d u r e of Ingle (13); the t o t a l l y h e p a t e c t o m i z e d r a t w a s p r e p a r e d a c c o r d i n g to the two-stage p r o c e d u r e of M e e h a n (21). D L - l y s i n e - 6 - C ' H C l and a c e tate-l-C w e r e p r e p a r e d i n this l a b o r a t o r y by accepted methods and w e r e c h r o m a t o g r a p h i c a l l y and r a d i o c h e m i c a l l y homogeneous. C a r r i e r f r e e s u l f u r - 3 5 - l a b e l e d sulfate w a s obtained f r o m the Oak R i d g e N a t i o n a l L a b o r a t o r i e s and u s e d without f u r t h e r p u r i f i c a t i o n . C u acetate w a s obtained as a s t e r i l e s o l u t i o n f r o m Abbott L a b o r a t o r i e s . T h e m a n u f a c 1 4

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h i r e r ' s a s s a y of the S 0 and C u acetate w a s u s e d i n d i l u t i o n s of the dose. In a l l c a s e s the r a d i o a c t i v e dose w a s i n t r o d u c e d into the p e r f u s i o n o r injected i n t r a v e n o u s l y i n s m a l l v o l u m e s (1 to 4 m l . ) of R i n g e r ' s s o l u t i o n . In the c e r u l o p l a s m i n study C u acetate was found v e r y t o x i c if g i v e n i n t r a v e n o u s l y ; i t was t h e r e f o r e injected i n t r a m u s c u l a r l y i n the eviscerated surviving rat. In l i v e r p e r f u s i o n s s a m p l e s of b l o o d w e r e g e n e r a l l y r e m o v e d at h o u r l y i n t e r v a l s after a z e r o t i m e s a m p l e at the outset. S a m p l e s of blood obtained f r o m the p e r f u s i o n s o r at the t e r m i n a t i o n (5 to 6 hours) of e v i s c e r a t e d s u r v i v i n g r a t o r h e p a t e c t o m i z e d r a t e x p e r i m e n t s w e r e centrifuged and the p l a s m a w a s h a r v e s t e d and m i x e d w i t h 1 m l . of 0.9% s o d i u m c h l o r i d e s o l u t i o n c o n t a i n i n g c a r r i e r l y s i n e , c a r r i e r acetate, o r c a r r i e r sulfate. The p l a s m a s w e r e then d i a l y z e d at 4 ° C . against t h r e e o r four changes of p h y s i o l o g i c a l s a l i n e , the l a t t e r b e i n g changed at l e a s t t h r e e t i m e s i n the c o u r s e of 24 h o u r s . A t the c l o s e of the d i a l y s i s the p l a s m a was centrifuged to r e m o v e s m a l l amounts of p r e c i p i t a t e and the c l e a r p l a s m a r e s u l t i n g w a s s u b j e c t e d to s t a r c h - b l o c k p r e p a r a t i v e zone e l e c t r o p h o r e s i s b y the t e c h nique of K u n k e l and S l a t e r (15). A t the c l o s e of the e l e c t r o p h o r e s i s 1 - c m . segments of the s t a r c h b l o c k w e r e cut and t r a n s f e r r e d to s i n t e r e d g l a s s funnels, and the p r o t e i n s w e r e quantitatively eluted w i t h five s u c c e s s i v e 2 - m l . aliquots of 0.9% s o d i u m c h l o r i d e . The f i l t r a t e s c o n t a i n i n g the p r o t e i n f r a c t i o n s w e r e then made up to a constant v o l u m e and m i x e d , and s m a l l a l i q u o t s w e r e a n a l y z e d f o r t o t a l p r o t e i n content by the method of L o w r y et a l . (18). T o e a c h tube a sufficient quantity of concentrated p e r c h l o r i c a c i d (12.IN) was added w i t h a s e r o l o g i c a l pipet to g i v e a f i n a l c o n c e n t r a t i o n of 0 . 6 4 N p e r c h l o r i c a c i d . The tubes w e r e m i x e d r a p i d l y after the a d d i tion of p e r c h l o r i c a c i d , a l l o w e d to stand at r o o m t e m p e r a t u r e f o r 10 m i n u t e s , and centrifuged r a p i d l y f o r 10 m i n u t e s at 3000 r . p . m . i n a T y p e 3 I n t e r n a t i o n a l centrifuge. The c l e a r o r s l i g h t l y opalescent s u p e r natant f l u i d was quantitatively t r a n s f e r r e d to a c o r r e s p o n d i n g s e r i e s of tubes and a l i q u o t s w e r e again r e m o v e d f o r p r o t e i n a n a l y s i s . P r o t e i n s s o l u b l e i n 0.64 M p e r c h l o r i c w e r e a r b i t r a r i l y designated s e r o m u c o i d s . The s e r o m u c o i d s w e r e quantitatively p r e c i p i t a t e d by the f u r t h e r a d d i t i o n of 5% phosphotungstate i n 2 N H C l and the r e s u l t a n t p r e c i p i t a t e s w e r e centrifuged off. P r e c i p i t a t e s w e r e then quantitatively t r a n s f e r r e d to c o m b u s t i o n f l a s k s and c o n v e r t e d to c a r b o n d i o x i d e w i t h the d i g e s t i o n m i x t u r e of V a n S l y k e , P l a z i n , and W e i s i g e r (33), and t h e i r C content was d e t e r m i n e d i n an i o n i z a t i o n c h a m b e r w i t h the v i b r a t i n g r e e d e l e c t r o m e t e r . R e s u l t s of a l l C a s s a y s a r e a r b i t r a r i l y e x p r e s s e d i n t e r m s of v o l t s p e r m i n u t e , w h e r e 1 v o l t p e r minute i s equivalent to 2.1 χ 1 0 d i s i n t e g r a t i o n s p e r minute. In e x p e r i m e n t s i n w h i c h s u l f u r - 3 5 - l a b e l e d sulfate w a s u s e d , the supernatant s o l u t i o n s c o n t a i n i n g the p l a s m a s e r o m u c o i d s w e r e m i x e d w i t h 1.0 m l . of 1% s o d i u m sulfate and d i g e s t e d on a s t e a m bath w i t h 10 m l . of 20% H C l f o r about 2 h o u r s , a t i m e sufficient to c o n v e r t a l l e s t e r sulfate to i n o r g a n i c sulfate. A f t e r a n e x c e s s of 5% b a r i u m c h l o r i d e w a s added, p r e c i p i t a t e s w e r e digested f o r another 20 o r 30 minutes on a s t e a m bath and centrifuged. Supernatant c l e a r f l u i d w a s r e m o v e d , 4

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and the p r e c i p i t a t e s w e r e washed once w i t h d i l u t e H C l , then w i t h 95% e t h y l a l c o h o l , and t r a n s f e r r e d q u a n t i t a t i v e l y to planchette cups as a s l u r r y w i t h the a i d of s m a l l v o l u m e s of a l c o h o l . T h e a l c o h o l w a s a l lowed to evaporate, l e a v i n g a u n i f o r m deposit of b a r i u m sulfate w h i c h w a s a s s a y e d f o r r a d i o a c t i v i t y i n a w i n d o w l e s s flow c o u n t e r . T h e amount of c a r r i e r sulfate added was s u c h that the p r e c i p i t a t e s w e r e a s s a y e d e s s e n t i a l l y at i n f i n i t e t h i c k n e s s . In a n u m b e r of e x p e r i m e n t s s t u d y i n g the i n c o r p o r a t i o n of S 0 into s e r o m u c o i d f r a c t i o n s 1 0 - μ 1 . s a m p l e s of p l a s m a w e r e a p p l i e d to paper s t r i p s and subjected to e l e c t r o p h o r e s i s i n the B e c k m a n - S p i n c o paper e l e c t r o p h o r e s i s apparatus. T h e r e s u l t a n t o v e n - d r i e d paper s t r i p s w e r e attached to an 8 χ 10 sheet of p a p e r and a p p l i e d d i r e c t l y to a sheet of E a s t m a n N o - s c r e e n x - r a y f i l m . E x p o s u r e extended o v e r a p e r i o d of 1 month. A t the end of this t i m e the f i l m was developed i n the u s u a l m a n n e r (see F i g u r e 2). T h e paper s t r i p s w e r e then s t a i n e d w i t h b r o m o p h e n o l blue to d e m o n s t r a t e the m a j o r p r o t e i n f r a c t i o n s . In the c e r u l o p l a s m i n e x p e r i m e n t s a p p r o p r i a t e a l i q u o t s of s p e c i m e n s w e r e p l a c e d i n s m a l l test tubes and a s s a y e d f o r C u a c t i v i t y i n a w e l l - t y p e s c i n t i l l a t i o n c o u n t e r . A n a l y s e s w e r e made w i t h r e f e r e n c e to a C u standard p r e p a r e d and a s s a y e d at the outset and whenever other samples were assayed. This permitted c o r r e c t i o n for radioactivity de cay of t h i s 12.8-hour h a l f - l i f e i s o t o p e . 3 5

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Experimental

Results

F i g u r e 1 p r e s e n t s r e s u l t s of the e l e c t r o p h o r e t i c s e p a r a t i o n of p l a s m a p r o t e i n s f r o m an i s o l a t e d p e r f u s e d r a t l i v e r e x p e r i m e n t to w h i c h both D L - l y s i n e - 6 - C and S 0 w e r e added. It i s c l e a r that the s e r o m u c o i d f r a c t i o n s a r e p r i m a r i l y r e s t r i c t e d to the a l b u m i n , a - , and β - g l o b u l i n r e g i o n s , and c o r r e s p o n d to the M P and M P f r a c t i o n s of M e h l , H u m p h r e y , and W i n z l e r (22). T h e quantitative v a r i a t i o n s i n the i n c o r p o r a t i o n of C and S 0> a r e best i n t e r p r e t e d i n t e r m s of the s e r o m u c o i d f r a c t i o n s b e i n g g r o s s l y heterogeneous. A l l of the p l a s m a s e r o m u c o i d f r a c t i o n s contained s i g n i f i c a n t amounts of c a r b o n - 1 4 and s u l f u r - 3 5 sulfate. The amounts of l y s i n e - C p r e s e n t a r e r o u g h l y p r o p o r t i o n a l to the amounts of p r o t e i n i n the s e r o m u c o i d f r a c t i o n s . The l a r g e s t amount of sulfate-35 a c t i v i t y i s a s s o c i a t e d w i t h the f a s t - m o v i n g l e a d i n g edge of the s e r o m u c o i d f r a c t i o n . F i g u r e 2 p r e s e n t s the r e s u l t s of a u t o r a d i o g r a p h y of the e l e c t r o p h o r e t i c a l l y s e p a r a t e d p l a s m a p r o t e i n f r a c t i o n s taken at z e r o t i m e and at h o u r l y i n t e r v a l s d u r i n g the c o u r s e of an i s o l a t e d l i v e r p e r f u s i o n to w h i c h only s u l f u r - 3 5 - l a b e l e d sulfate had been added. The z e r o t i m e c o n t r o l s t r i p r e v e a l s no s i g n i f i c a n t a c t i v i t y a s s o c i a t e d w i t h any of the p l a s m a p r o t e i n f r a c t i o n s . R a d i o a c t i v i t y of the p r o t e i n f r a c t i o n s i s seen as e a r l y as 1 hour and a p p e a r s w i t h i n c r e a s i n g i n t e n s i t y i n a v a r i e t y of f r a c t i o n s c o m p a r a b l e w i t h s e r o m u c o i d f r a c t i o n s noted i n the s t a r c h b l o c k e l e c t r o p h o r e s i s . Of s o m e i n t e r e s t i s the appearance of two r a d i o a c t i v e f r a c t i o n s m o v i n g w e l l ahead of the a l b u m i n . T h e i r i d e n t i t y r e m a i n s to be e s t a b l i s h e d . I n F i g u r e 2 the a r a b i c n u m b e r s r e f e r only to 1 4

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-9-6-7-6-5-4-3-e-l I 2 34 5 67891011 13 15 17 19 0ISTANCE OF MIGRATION FROM ORIGIN IN CM.

Figure 1. Electrophoretic from isolated perfused

separation of plasma rat liver with S 0l and 35

mucoproteins lysine-6-C

z

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Liver and blood donors, 18 hours fasted. Liver weight, 6.25 grams. Perfusion blood volume, 181 ml. containing 500 mg. glucose. 1000 mg. glucose total infused continuously in 17.5 ml. Ringer's solution through 6 hours' perfusion. Dose, 12.0 mg. DL-lysine-Q-C · HCl (600 volts/min. as L-lysine-6-C ). S 0 as carrier-free sulfate (total activity, 400 microcuries). 4.0 ml. dialyzed plasma (total C activity, 1.752 volts/min.) separated by electrophoresis. After electrophoresis, total C in nonmucoprotein fractions was 0.928 volt/min.; in mucoprotein fractions, 0.812 volt/min^ M

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the g r o s s o r d e r of i n t e n s i t y of the v a r i o u s a u t o r a d i o g r a p h i c a l l y d e t e c t able f r a c t i o n s . F i g u r e 3 shows the r e s u l t s of the e l e c t r o p h o r e t i c s e p a r a t i o n of a s p e c i m e n of p l a s m a taken f r o m an e v i s c e r a t e d s u r v i v i n g r a t 4 hours after an infusion of 320 m i c r o c i i r i e s of S 0 g i v e n as c a r r i e r - f r e e i n o r g a n i c sulfate and analogous to the dose u s e d i n the i s o l a t e d l i v e r p e r fusion of F i g u r e 1. T h e l o w e r half of F i g u r e 3 r e v e a l s that the amounts of S p r e s e n t i n the s e r o m u c o i d f r a c t i o n s of the p l a s m a f r o m the e v i s 3 5

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Figure

2. Autoradiograph of plasma proteins labeled with S lated perfused rat liver

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0 by iso 4

Blood and liver donors not fasted. Liver donor weight, 396 grams. Liver weight, 11.6 grams. Perfusion blood volume, 106 ml. Dose of 6.0 millicuries carrier-free S CU mixed into perfusion blood at zero time. Samples of blood, 2.2 to 3.5 ml. taken hourly, experiment ter minated at 7 hours. Perfusion rate, 13 to 15ml./min. throughout. Total bile volume, 2.9 ml. Samples of plasma, ΙΟμΙ. each, placed on paper strips and electrophoresed with Veronal bufferV ionic strength, 0.5, pH 8.5. Strips oven-dried 10 min. at 105°before 1-month exposure to No-screen x-ray film (Eastman Kodak Co.). Strips stained with bromophenol blue according to Beckman-Spinco manual. Location of stained protein fractions shown by brackets in diagram on right 5

c e r a t e d r a t a r e detectable but u n i m p r e s s i v e l y s m a l l and only a tiny f r a c t i o n of the a c t i v i t y i n c o r p o r a t e d into the p l a s m a s e r o m u c o i d b y the i s o l a t e d r a t l i v e r . T h e c u r v e f o r the l a t t e r c o m p a r i s o n i s the s a m e as that i n F i g u r e 1. If t h i s c o m p a r i s o n i s to be meaningful, i t i s n e c e s s a r y to a s s u m e that the s p e c i f i c a c t i v i t i e s of p r e c u r s o r sulfate i n the i s o l a t e d r a t l i v e r p e r f u s i o n and i n the e v i s c e r a t e d s u r v i v i n g r a t w e r e e s s e n t i a l l y s i m i l a r . A l t h o u g h s p e c i f i c a c t i v i t i e s of the sulfate w e r e not a c t u a l l y m e a s u r e d , i t s e e m s r e a s o n a b l e to a s s u m e that, i f anything, the s p e c i f i c a c t i v i t y of the sulfate i n the l i v e r p e r f u s i o n w a s l o w e r than that i n the e v i s c e r a t e d s u r v i v i n g r a t . The b l o o d v o l u m e i n the f o r m e r i s r o u g h l y f i v e to s i x t i m e s the i n t r a - and e x t r a v a s c u l a r f l u i d v o l u m e of the e v i s c e r a t e d r a t . T h e f a i l u r e to i n c o r p o r a t e s i g n i f i c a n t amounts of sulfate into the p l a s m a s e r o m u c o i d by the e v i s c e r a t e d s u r v i v i n g r a t i s not s u r p r i s i n g , i n v i e w of the fact that l y s i n e - C w a s not i n c o r p o r a t e d into the p l a s m a s e r o 1 4


2.

MILLER ET AL.

Role of the Liver In Protein Biosynthesis

•9-β -7-6-5-4-3-2 -I I 2 3 4 5 6 7 8 9 (Oil

23

13 15 17 19

- 9-8-7-6-5-4-3-2H I 2 3 4 5 6 78 9 Κ) 11 13 15 17 19 DISTANCE OF MIGRATION FROM ORIGIN IN CM.

Figure 3. Electrophoretic separation of plasma mucoproteins from surviving eviscerated rat with S 0 " (upper) and comparison of S 0 incorporation by evis cerated rat and isolated liver (lower) 3 5

2

4

3 5

4

Rat wt. 400 grams. After total evisceration, dose 320 microcuries carrier-free S 0 given intraven ously at zero time. Continuous infusion 10% glucose in Ringer's solution; 1.35 ml. plus 0.16 unit insulin per hour for 4 hours of experiment. 2 ml. dialyzed plasma separated by electrophoresis and resulting fractions treated with per chloric acid to obtain mucoproteins. Measured S 0 activity of eviscerated rat plasma mucoproteinfractions multiplied by 2 X 1.5 to permit indicated comparison with data from 4 ml. of plasma of 6-hour experiment of isolated rat liver perfusion of Figure 1 t

3 5

4

3 5

4

m u c o i d f r a c t i o n s of the e v i s c e r a t e d s u r v i v i n g r a t o r the h e p a t e c t o m i z e d rat. F i g u r e 4 p r e s e n t s the r e s u l t s of e l e c t r o p h o r e t i c s e p a r a t i o n of p l a s m a p r o t e i n f r a c t i o n s obtained after p e r f u s i o n of an i s o l a t e d r a t l i v e r w i t h a c e t a t e - 1 - C . F o r the p u r p o s e s of t h i s p r e s e n t a t i o n , i t i s noteworthy that v i r t u a l l y a l l of the a c t i v i t y a s s o c i a t e d w i t h p l a s m a p r o t e i n f r a c t i o n s 8 through 13 w a s p r e s e n t i n the s e r o m u c o i d f r a c t i o n s and 14



24

O-o-o TOTAL PROTEIN · - # - · TOTAL MUCOPROTEIN

ALB.

-β-9-4-8'2 "I I 2 3 4 5 6 7 · · 10 12 o-o-o

0.200 TOTAL «14 C

16 18

1 4

0.150

ACTIVITY

l

14

1

PLASMA PROTEIN C " MUCOPROTEIN C

Λ

1

Λ

Λ

0.100

N

ARBITRARY UNITS 0.050 -6-5*4 It'l

12345678910

12 14 16

18

DISTANCE OF MIGRATION FROM ORIGIN IN CM.

Figure protein

4. Electrophoretic separation of plasma from isolated rat liver perfusion with acetate-1-C 14

Liver donor fed, blood donors fasted. Liver weight, 10.2 grams. Perfusion blood volume, 202 ml. Solution of35mg. sodium acetate-1-C plus 500mg. glucose in 16 ml. of Ringer's solu tion given by continuous infusion for first 2 1/2 hours of 6-hour perfusion. Six-hour sample of dialyzed plasma, 4.0 ml. separated by electro phoresis 14

1 4

that the highest C - a c t i v i t y d e r i v a b l e f r o m acetate i n the s e r o m u c o i d s i s seen i n the l e a d i n g edge of the s e r o m u c o i d f r a c t i o n s , m a i n l y 13, 14, and 15. It i s t h i s p o r t i o n of the s e r o m u c o i d f r a c t i o n s w h i c h w e p r e v i o u s l y noted i n F i g u r e 1 had the highest s p e c i f i c sulfate a c t i v i t y . A s i g n i f i c a n t p o r t i o n of the s e r o m u c o i d r a d i o a c t i v i t y w a s p r e s u m ably associated with plasma lipides. A f t e r the s e r o m u c o i d s w e r e p r e c i p i t a t e d w i t h phosphotungstateH C 1 , they w e r e e x h a u s t i v e l y e x t r a c t e d w i t h hot a l c o h o l - e t h e r (3 to 1 by v o l u m e ) , f o l l o w e d b y two hot e x t r a c t i o n s w i t h c h l o r o f o r m - m e t h a n o l (1 to 1). The c o m b i n e d s o l v e n t e x t r a c t s w e r e evaporated on the s t e a m bath under n i t r o g e n and the l i p i d e s taken up w i t h p e t r o l e u m e t h e r . T h e p e t r o l e u m ether e x t r a c t s w e r e w a s h e d s e v e r a l t i m e s w i t h w a t e r c o n t a i n i n g acetate and β - h y d r o x y b u t y r a t e and acetoacetate (10). T h e c o m b i n e d washed p e t r o l e u m ether e x t r a c t s of the s e r o m u c o i d f r a c t i o n s contained 41% of the t o t a l s e r o m u c o i d - C - a c t i v i t y ; the c o m bined p e t r o l e u m ether e x t r a c t s of the n o n s e r o m u c o i d p r o t e i n f r a c t i o n s contained 37% of the t o t a l C - a c t i v i t y p r e s e n t i n those f r a c t i o n s before e x t r a c t i o n . Such an e x p e r i m e n t w i t h a c e t a t e - l - C l e a v e s open q u e s tions c o n c e r n i n g the n a t u r e of the n o n l i p i d e a c t i v i t y . On the b a s i s of 1 4

1 4

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2.

MILLER ET AL.

Role of the Liver in Protein Biosynthesis 14

25

known m e t a b o l i c c o n v e r s i o n s of a c e t a t e - 1 - C , g l u t a m i c and a s p a r t i c a c i d s as w e l l as g l u c o s e , mannose, and h e x o s a m i n e s c o u l d c o n c e i v a b l y be l a b e l e d . B e c a u s e the p l a s m a s e r o m u c o i d s appear i n i n c r e a s e d c o n c e n t r a tions i n the b l o o d of a n i m a l s w i t h i n f l a m m a t o r y p r o c e s s e s o r malignant t u m o r s (34), we studied the p a t t e r n s of C - i n c o r p o r a t i o n into the p l a s m a p r o t e i n s of a n u m b e r of r a t s b e a r i n g subcutaneously i m p l a n t e d W a l k e r c a r c i n o m a 256. F i g u r e 5 p r e s e n t s the r e s u l t s of e l e c t r o p h o r e t i c f r a c t i o n a t i o n of p l a s m a p r o t e i n s obtained f r o m an e v i s c e r a t e d s u r v i v i n g t u m o r - b e a r i n g 1 4

DISTANCE OF MIGRATION FROM ORIGIN IN CM. 1 4

Figure 5. Electrophoretic separation of C labeled proteins from eviscerated surviving rat (with liver) Walker

tumor

present

Rat wt., 470 grams. Eviscerated by usual procedure, except liver left with functioning hepatic arterial blood supply. Tumor wt., 37 grams; dose given at zero time, 6.0 mg. DL-lysine-6C · HCl (340 volts/min. as L-lysine-6-C ). Experiment terminated at 6 hours. 2.0 ml. dialyzed plasma (total C * activity 3.152 volts/min.) separated by electrophoresis 1 4

lA

1



26

r a t i n w h i c h the l i v e r w a s left f u n c t i o n a l l y i n t a c t s u p p l i e d w i t h the h e p a t i c a r t e r y . H e r e the l i v e r i s s u p p l i e d w i t h b l o o d i n s p i t e of the a b sence of the g a s t r o i n t e s t i n a l t r a c t , s p l e e n , p a n c r e a s , and other m e s e n t e r i c s t r u c t u r e s . F i g u r e 5 r e v e a l s that i n spite of the absence of these nonhepatic v i s c e r a , the p a t t e r n of C - i n c o r p o r a t i o n i n t o the p l a s m a p r o t e i n s i s not q u a l i t a t i v e l y different f r o m that noted i n the n o r m a l n o n t u m o r - b e a r i n g , unoperated r a t [for c o m p a r i s o n see (23)]. H e r e i t i s obvious that the a - g l o b u l i n s , w h i c h we know i n c l u d e many of the s e r o m u c o i d s , have the highest s p e c i f i c a c t i v i t y , f o l l o w e d by the a l b u m i n , the β - g l o b u l i n , and the y - g l o b u l i n f r a c t i o n s . T h i s i s r o u g h l y the o r d e r of i n c o r p o r a t i o n p r e v i o u s l y noted i n the n o r m a l a n i m a l and i n the i s o l a t e d perfused l i v e r (23, 24). F i g u r e 6 p r e s e n t s the r e s u l t s of a p a r a l l e l study i n w h i c h the e v i s c e r a t e d s u r v i v i n g t u m o r - b e a r i n g r a t w a s a l s o s u r g i c a l l y d e p r i v e d of i t s l i v e r . T h e r e a r e a n u m b e r of s t r i k i n g quantitative and q u a l i t a t i v e d i f f e r e n c e s between the p a t t e r n of i n c o r p o r a t i o n noted i n t h i s a n i m a l w i t h out a l i v e r and that found i n the e x p e r i m e n t of F i g u r e 5 i n w h i c h the e v i s c e r a t e d r a t r e t a i n e d i t s l i v e r . Q u a l i t a t i v e l y one notes i n F i g u r e 6 the dominant i n c o r p o r a t i o n i n t o the y - g l o b u l i n s w i t h highest s p e c i f i c a c t i v i t y , and i n t o the β - g l o b u l i n s . S i m i l a r l y to the p a t t e r n we have d e s c r i b e d , f o r the n o r m a l e v i s c e r a t e d s u r v i v i n g r a t , t h e r e a r e h e r e s m a l l q u a n t i t a t i v e l y i n s i g n i f i c a n t amounts of r a d i o a c t i v i t y i n the ot - g l o b u l i n and a l b u m i n f r a c t i o n s . It i s i m p o r t a n t to r e c o g n i z e that w i t h a g i v e n dose of l y s i n e - C , the total C a c t i v i t y i n c o r p o r a t e d i n t o the p l a s m a p r o t e i n s of the e v i s c e r a t e d s u r v i v i n g r a t without a l i v e r i n t h i s and s i m i l a r e x p e r i m e n t s i s about 10% of the t o t a l a c t i v i t y i n c o r p o r a t e d i n t o the p l a s m a p r o t e i n s by an i n t a c t a n i m a l , o r by an e v i s c e r a t e d s u r v i v i n g r a t w i t h the l i v e r left f u n c t i o n a l l y i n t a c t s u p p l i e d w i t h the hepatic a r t e r y , o r by the i s o l a t e d p e r f u s e d l i v e r . T h i s c o m p a r i s o n i s q u a n t i t a t i v e l y the m o r e i m p r e s s i v e i f one r e c a l l s that i n the p r e s e n c e of the n o r m a l l y functioning l i v e r a m a j o r f r a c t i o n (15 to 50% of an L - l y s i n e - C dose) i s o x i d i z e d to c a r b o n d i o x i d e (26). In the a b s e n c e of the l i v e r , o x i d a t i o n of l a b e l e d l y s i n e to C Q î i s a p p r o x i m a t e l y 1 / 2 0 that seen i n the p r e s e n c e of the l i v e r (23, 2 4 , 2 5 ) . W h e n the s e r o m u c o i d f r a c t i o n s (those p r o t e i n s s o l u b l e i n 0 . 6 4 N p e r c h l o r i c a c i d and i n s o l u b l e i n phosphotungstate H C l ) f r o m the t o t a l l y e v i s c e r a t e d r a t e x p e r i m e n t s — e . g . , that of F i g u r e 6—were a s s a y e d f o r C , they contained no s i g n i f i c a n t a c t i v i t y . Such r e s u l t s add to the e v i dence i n d i c a t i n g that the nonhepatic t i s s u e s do not p r o d u c e p l a s m a s e r o m u c o i d s , even i n r a t s h a r b o r i n g a n e x p e r i m e n t a l t u m o r p r e s u m a b l y known to enhance the s y n t h e s i s of p l a s m a s e r o m u c o i d s . F i g u r e 7 p r e s e n t s the r e s u l t s of a study i n w h i c h a t o t a l l y h e p a t e c t o m i z e d r a t m a i n t a i n e d a l i v e w i t h a continuous i n f u s i o n of g l u c o s e and R i n g e r ' s solution was given l y s i n e - 6 - C i n t r a v e n o u s l y . A l t h o u g h the s e p a r a t e i n d i v i d u a l s m a l l f r a c t i o n s f r o m the e l e c t r o p h o r e s i s w e r e not a s s a y e d i n d i v i d u a l l y , they w e r e p o o l e d a c c o r d i n g to the g r o s s l y p e r c e p t i b l e e l e c t r o p h o r e t i c p a t t e r n as d e t e r m i n e d by a n a l y s i s of p r o t e i n and i n d i c a t e d b y the dotted l i n e s . A p p r o p r i a t e a l i q u o t s of these f r a c t i o n pools were analyzed for C and the r e s u l t s a r e i n d i c a t e d i n the table a s s o c i a t e d w i t h F i g u r e 7. H e r e a g a i n i t i s apparent that t h e r e a r e 1 4

1 4

1 4

1 4

1 4

1 4

1 4

1 4


2.

MILLER ET AL.

Role of the Liver in Protein Biosynthesis

10.0 TOTAL PROTEIN IN MGS.

27

Alb.

_ 5

0 Globs,

0

r Globs.

Dominant Role of the Liver in Biosynthesis of the Plasma Proteins with

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