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Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes
BIOCHIMIE, 1975, 57, 10~65-1071.
Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes. C h r i s t i a n e Guau~N, Claudine GnEGom and F a n n y SCHAPInA~. * I n s t i t u t d e P a t h o l o g i e M o l ~ c u l a i r e , 24, r u e d u F a u b o u r g S a i n t - J a c ? u e s , 75014 P a r i s ( F r a n c e ) . (27.6-1975).
S u m m a r y . - - Pyruvate k,inase isozymic changes were stud~ied in the adu]t he.patoeyte cultures, by eleetropl~o~etic, kinetic and immum)togical methods. VCe were .able to li~aintain par~enchymal cells from normal adult rat 1.leer in' no.n-proliferating mono.layer cuKures up t,o 10 days. Hep.atocytes appeared to contain a dominant ~K I type up to 4-5 days of culture. After day 5, PK III type wa~ ~eg.ul,arl,y pl~esent with PK I and after 7 days PK III type was always the .only isozyme detected in culture. It must b e p o i n t e d out that, by the Ouchterlony method and sometimes by electropl~oresi~, coneen.trated ~xtracts from fves~lT isol~ated h,elaa~0ocytes or sta~ing hep~atoeyte cultures did' also contain" Pyruvate kinase PK III type. These results suggest that Pyr~vate kina,se III is present but pavtl.y repressed in the adult parench.ymal eel,l.s and becomes dcrepressed in cu:lt'ure.
T w o types of p y r u v a t e - k i n a s e (PK) in adult rat tissues h a v e been d e s c r i b e d b y T a n a k a et al. [1]. Later, Susor a n d Rutter [2] h a v e s h o w n that the p r e d o m i n a n t f o r m in adult l i v e r w a s PK I type w h i c h was f o u n d to h a v e kinetic, e l e c t r o p h o r e t i c and i n u n u n o l o g i c a l p.roperties different f r o m PK II Muscle-type. T h e y h a v e also s h o w n that the m i n o r f o r m of PK in l i v e r w a s d i f f e r e n t f r o m the muscle form. S c h a p i r a and Gregori [3] h a v e r e p o r t e d that this PK III type w a s also f o u n d in fetal liver, in p l a c e n t a a n d in fast g r o w i n g hepatomas. I m a m u r a a n d T a n a k a [4] h a v e found this PK n I type in m a n y tissues, p r i n c i p a l l y in kidney. Several authors h a v e suggested that PK I type was f o u n d only in the p a r e n c h y m a l cells an.d that the PK [II type w a s p r e s e n t in the non p a r e n c h y m a l f r a c t i o n s [5, 6, 7, 8]. One fact was p a r t i c u l a r l y int e r e s t i n g : a l t h o u g h n o r m a l l y p r e s e n t as a m i n o r c o m p o n e n t in liver, i n r i v e , this PK III f o r m i s the only one s y n t h e s i z e d in rat l i v e r cells in v i t r o [9, 10]. W a l k e r et al. [9] i n d i c a t e d that cell selection o c c u r r e d in p r i m a r y cultures in cells w h i c h a l r e a d y c o n t a i n e d PK HL A n o t h e r h y p o t h e s i s w o u l d be that c o n d i t i o n s of c u l t u r e t h e m s e l v e s r e p r e s s the synthesis of some specific proteins. But little is k n o w n about the e n z y m i c c h a n g e s w h i c h o c c u r in fully d i f f e r e n t i a t e d h epatocytes m a i n t a i n e d in m o n o l a y e r cultures. (*) Gro.upe U 129 4e l'Institut Nation,a.1 d.e la S,ant6 et de l,a l~echerehe M~d.iea.le, Laboratoire associ6 au Gentre National d,e la Re~heveh.e Scientifique. T.o wh,om a~ll eorrespon&ence should b~e addres.sed.
In a p r e v i o u s paper, one of us has s h o w n that hepatoeytes retain ultrastructural characteristics up to 8-10 days in n o n - p r o l i f e r a t i n g cultures in the p r e s e n c e of h y d r o e o r t i s o n e h e m i s u c c i n a t e [11]. The aim of this w o r k is to study the isozymes of PK p r e s e n t in these s u r v i v i n g h e p a t o c y t e s .
MATERIAL AND METHODS. W i s t a r rats w e i g h t i n g 200 to 300 g w e r e used. T h e m e t h o d for the p r e p a r a t i o n of isolated h e p a tocytes has been d e s c r i b e d e l s e w h e r e [117 ; it is a m o d i f i c a t i o n of the m e t h o d of Seglen [12]. The h e p a t o c y t e s w e r e w a s h e d six times and i n c u b a t e d in E12F12 m e d i u m . Cultures w e r e p e r f o r m e d in this m e d i u m c o n t a i n i n g 0,.1 p e r c e n t glucose and s u p p l e m e n t e d w i t h 10 p e r c e n t fetal calf s e r u m a n d 0.5 p e r c e n t c h i c k e m b r y o e x t r a c t e q u i l i b r a t e d a f t e r w a r d s w i t h a 90 p e r c e n t a i r - 10 p e r c e n t CO 2 m i x t u r e . In some e x p e r i m e n t s 1.8 mM h e m i s u c c i nate h y d r o c o r t i s o n e w a s a d d e d ~ h e n the m e d i u m w a s c h a n g e d 5 h r s after s e e d i n g and then e v e r y t w o or t h r e e days. E v e r y day, ceils w e r e h a r v e s t e d : the m e d i u m w a s d i s c a r d e d and the plates w a s h e d t w i c e w i t h 154 mM N a t l . The cells w e r e s c r a p e d off the plates and c o l l e c t e d in a c o n i c a l c e n t r i f u g e tube w i t h a buffer c o m p o s e d of 10 mM Tris HC1 p H 8.0 20 mM MgC12 and 200 mM KC1. The cell suspension w a s frozen and tha-wed twice.
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C. G u g u e n , C. Gregori a n d F. S c h a p i r a .
Electrophoresis : it was c a r r i e d out on starch gel c o n t a i n i n g Fructose Diphosphate (Fru-l-0-P) 3 mM w i t h a phosphate citric acid buffer pH 7.0 for 18 hrs at 4°C w i t h a potential gradient of 3 V / c m as described by Schapira and Gregori !31. After niigration, the starch-gel was sliced and covered w i i h the reactive m i x t u r e of Susor and Rutter for specific s t a i n i n g of PK [21. I n some exp e r i m e n t s , isozymes w e r e located by a modification of the m e t h o d of I m a l n u r a a n d T a n a k a [4~. Kinetic studies : the cell homogenate was centrifuged at 2000 g for 20 m i n at 0°C in order to s e d i m e n t cell debris. The r e s u l t i n g s u p e r n a l a n t was used for the enzyme assays a n d for soluble p r o t e i n d e t e r m i n a t i o n s by the m e t h o d of Lowry et al [13] u s i n g b o v i n e plasma a l b u m i n as standard. Measurement of PK was c a r r i e d out as d e s c r i b e d by Biicher a n d P f l e i d e r e r [14] with variable Concentrations of p h o s p h o e n o l p y r u v a t e as substrate (PEP) 0.05 mM to 1.5 mM with or w i t h o u t F r u - l - 6 - P 0.1 raM. Adenosine diphosphate was used at the n o n i n h i b i t i n g c o n c e n t r a t i o n of 0.3 mM as described by Cartier et al [15].
vant. The specific anti PK II a n t i s e r u m did not cross-react w i t h PK I type, but cross-reacted with PK III type [16]. I m m u n o l o g i c a l e x p e r i m e n t s were p e r f o r m e d a c c o r d i n g to the double diffnsion method of Ouehterlony with 2 p e r c e n t NaC1 added to 1 percent agar in 50 mM veronal buffer. Radioautographic studies : the nlethod of Pelc and H o w a r d [17] modified by Maciera~Coelho et al [18] was used for the localization of 3H-thymidine i n c o r p o r a t i o n . Monolayers were i n c u b a t e d for 15 hrs witll 1 ~Ci/ml 3H-thymidine (27 m C i / raM) and then fixed in Bouin solution.
tk~ESULTS. 1.
--
MORPHOLOGICAL OBSERVATIONS.
Tile results obtained were s i m i l a r to those described in the previous papers of one of us [11, 19]. Isolated hepatocytes became attached to the s u b s t r a t u m w i t h i n 4 hrs and formed m o n o l a y e r s
Fro. 1. - - Adult liver parenchymal cells after 5 days of culture. Phase-contrast photograph of living cell9 (X 320).
Immunological methods : the isozymic modifications were f o l l o w e d by the study of the action of a specific a n t i s e r u m . A n t i s e r u m was p r e p a r e d by i n j e c t i n g crystalline PK II from r a b b i t muscle (Boehringer) into chicken, w i t h F r e u n d ' s adjuBIOCHIMIE, 1975, 57, n ° 9.
w i t h i n 24 hrs (fig. 1) ; this figure shows also the p u r i t y of the cultures w h i c h c o n t a i n only hepatocytes w i t h o u t n o n p a r e n c h y m a l cells. The use of E12FI2 m e d i u m i n c r e a s e d the c o n s e r v a t i o n o.f hepatocytes up to 8 days. With addition of h y d r o -
P!lruvate-kinase
in hepatocyle
cortisone h e m i s u c c i n a t e , the cells were m a i n t a i ned up to 10 days. Uptake of 3H-thymidine was located in culture after 3 days by radioautogra-
1067
cultures.
5th day, but mitotic figures were scarcely seen (less t h a n I percent). A s i m i l a r p e r c e n t a g e was o b t a i n e d i n h o r m o n e - t r e a t e d cultures. 2. - - ELECTROlaHORETIC MODIFICATIONS. Figure 2 compares e l e c t r o p h o r e t i c c h a r a c t e r i s tics of PK i n adult liver, muscle, k i d n e y a n d i n fetal liver (15-16.th day old). The m a i n b a n d of the liver PK I h a d the highest m o b i l i t y t o w a r d s the a n o d e ; the muscle PK II m i g r a t e d more slowly a n d differed from the k i d n e y PK III w h i c h h a d the lowest mobility. The fetal liver PK was resolved into 3 b a n d s : PK I a n d PK HI types, a n d an i n t e r m e d i a r y type ( p r o b a b l y due to erythrocytes).
FIo. 2. - - Electrophoresis of p y r u u a i e k i n a s e isozyraes. Isozyrnes vcerc .1,ocat.ed by a modification, of the
method .of Imamura and' Tan.aka E47. a : ad'ult liver. b : fetal Hver (16th d~y). c : kidnvy. d : muscle.
I
Figure 3 shows the p a t t e r n o b t a i n e d w i t h cultured hepatocytes. They a p p e a r e d to c o n t a i n a dol n i n a n t PK I type up to 4-5 days of culture. After day 5, PK III type was r e g u l a r l y p r e s e n t w i t h PK I (fig. 4). On day 7, PK III b a n d was the strongest. In some experiments, an e l e c t r o p h o r e t i c p a t t e r n s h o w i n g i n t e r m e d i a r y b a n d s b e t w e e n PK I a n d PK III, was observed after 6 days of culture. PK III was always the only isozyme seen after the
-
+
Culture day 2
FIG. 3. - - Electrophoresis o f p y r u v a t e kinase i$oz y m e s in cultured hepatocytes. Isozymes~ were located
with the rea,ctiva mixture of Susor and Rutter [2].
phy. The m a x i m u m of labelled nuclei (about 20 p e r c e n t of total nuclei) was r e a c h e d on the BIOCHIMIE, 1975, 57, n ° 9.
7th day i n cultures main,rained w i t h or w i t h o u t hydroeortisone hemisuceinate.
C. G u g u e n , C. G r e g o r i a n d F. S c h a p i r a .
1068 3.
---
KINETIC
STUDIES.
I n f i g u r e 5 a r e p l o t t e d t i m a c t i v i t i e s of h o m o g c n a t e s of m u s c l e a n d i s o l a t e d p a r e n c h y m a l cells. T h e P K II M u s c l e t y p e s h o ' w e d M i c h a e l i a n b e h a -
s h o w n i n t a b l e I w h i c h s u m m a r i z e s t h e e f f e c t of F r u - l - 6 - P at a n o n - i n h i b i t i n g c o n c e n t r a t i o n o f P E P , o n t h e P K a c t i v i t y of m u s c l e , l i v e r a n d p a r e n c h y m a l cells at d i f f e r e n t t i m e s o f c u l t u r e . TABLE I.
Action oi' Fru-l-6-P on P y r u v a t e kinase activity at a non saturating concentration o[ substrate P E P (0.0625 mM).
÷
Without Fru-t-6-P
With Fra-t-6-P
Adult muscle . . . . . . . . . . . . A d u l t liver . . . . . . . . . . . . . . I Fetal liver (15 th-16 t h day)
40 4 18
55 42 46
Hepatocyte culture 4 days . . . . . . . . . . . . . . . 7 days ~. . . . . . . . . . . . . . .
8 13
51 53
Tissues
.voeo
. v o ~ o (--1
(+)
i I
! L
Results a~re, expressed in percentage of the activity detected in the presence of PEP l mM (mean of 3 determinations).
m
FIG. 4. Electrophoresis of pyruoate kinase isoz!Imes in hepatoctltes ma, i n t a i n e d w i t h or w i t h o u t
4.
-l~6ro~eoriison~ h e m i s u c e i n a t e ; a f t e r 6 days of culture.
v i o u r a n d w a s n o t i n f l u e n c e d b y a d d i t i o n of F r u 1-6-P. I n c o n t r a s t t h e P K I t y p e a c t i v i t y s h o w e d a sigmoid curve when plotted against an increased c o n c e n t . r a t i o n of P E P w h i c h c o u l d b e c o n v e r t e d
--
IMMUNOLOGICAL
RESULTS.
Figure 7 shows the precipitation line obtained with the Ouchterlony method, using antiserum a n t i P K II, b e t w e e n e x t r a c t s of d i f f e r e n t r a t t i s s u e s o r cells w h i c h w e r e d i l u t e d i n o r d e r to have the same final concentration into the wells. A slight precipitation line was obtained with the
200
80
Y_ E
c E
0 o <3
(o)
0
!
0.5
! 1
[PEP] m M
4o (b)
0 ~
0.5 ~PEP~ m M
1
FIG. 5. - - Pyruvate kinase activity of muscle (a) and liver (b) h o m o g e n a t e s versus t h e P E P con,centration, a t a concentration of ADP 0.3 mM, in the presence (× ..... x) o r absence ( o - - - o ) of F r u - l - 6 - P .
into a hyperbolic curve by adding Fru-l-6;P 0.1 m M . F i g u r e 6 s h o w s t h e P K I a c t i v i t y of t h e h o m o g e n a t e of c u l t u r e d h e p a t o c y t e s u p to t h e 4th day. The curve was partly converted into a h y p e r b o l i c o n e ~)n t h e 7 t h o r t h e 8 t h d a y , b u t t h e a c t i v i t i e s w e r e still i n f l u e n c e d b y F r u - l - 6 - P , as
BIOCHIMIE, 1975, 57, n ° 9.
adult liver extracts, a strong line with the plac e n t a a n d m u s c l e e x t r a c t s . E x t r a c t s of h e p a t o c y t e s freshly isolated reacted against antiserum. The precipitation lines obtained with the hepatocytes m a i n t a i n e d in vitro w e r e s t r e n g h t e n e d a l o n g t h e c u l t u r e : s l i g h t o n t h e 2rid d a y , m o d e r a t e o n t h e
Pyruvate-kinase
in h e p a t o c y t e
4th day a n d strongest on the 6th day of culture. In other experiments, we have f o u n d that the pre-
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cultures.
layers in vitro. These results also agree w i t h previous reports of B o n n e y et al [20, 21].
.# ¢./
g
E
E
~2 o
(a)
"
o
2
(b] l
0.5
1
[PE P] m M
,
o.s 1 [PEP]mM
Fro. 6. - - Pyruvate kinase activity of hepatocyte homogenates after 4 days (a) and 7 days of culture (b), versus the PEP concentration in the presence (× ........ ~,) or absenve ( • - - • ) of Fru-I=6-P (0.1 ml~).
c i p i t a t i o n line was still stronger on the 8th or 9th day of culture. But a p a t t e r n of p a r t i a l i d e n t i t y w i t h a spur was always seen w i t h the p l a c e n t a line.
I n the presence of E12F12 m e d i u m w i t h embryonic chick extracts and fetal calf serum, they m a i n t a i n e d their m o r p h o l o g i c a l a n d f u n c t i o n a l characteristics for more t h a n 7 d a y s a n d took up tritiated t h y m i d i n e (~5 to 20 p e r c e n t nuclei on the 5th day). W h e n h y d r o c o r t i s o n e was added, the culture p e r i o d was i n c r e a s e d up to 10 days without i m p o r t a n t m o d i f i c a t i o n s of the s n b c e l l u l a r elements of the hepatocytes [11]. The s c a r c i t y of mitotic figures suggests a p r o l o n g e d <> phase, p e r h a p s as i n other cellular cultures [22!. The k i n e t i c and electrophoretic results i n d i c a t e that we were able to m a i n t a i n the p r e s e n c e of the PK I isozyme i n adult rat p a r e n c h y m a l cells in vitro for up to 5 days, in c o n t r a s t w i t h p r e v i o u s results of W a l k e r et al [9]. Very recently, Lin a n d Snodgrass [23] have suc.ceeded i n m a i n t a i n i n g urea cycle enzymes i n n o r m a l adult rat liver cells for 4 days. T h e i r results m a y he c o m p a r e d to ours on p y r n v a t e kinase PK I. The p r e s e n c e of PK I isozyme, d o m i n a n t i n these m o n o l a y e r ce'lls, toge. ther wi,th t h e i r m o r p h o l o g i c a l c h a r a c t e r i s t i c s suggests that all the cultured cells were, i n fact, hepatocytes.
FI6. 7 , - Precipitation lines in double immunodiffusion of placenta extracts (P), freshly isolated cells (IC) and hepatocytes after 2 Says (G2) 4 days (C4), 7 days (C7~, of culture, ag,air~st antisel~um .am.tipyruvate kinase II: ~al~lex,trac~s we~-e at the. same activity). DISCUSSION. We c o n f i r m that adu'll rat h e p a t o c y t e s isolated a c c o r d i n g - t o the method of Seglen, formed m o n o BIOCHIMIE, 1975, 57, n ° 9.
This result seems to exclude the o v e r g r o w t h of the hepatocytes by n o n - p a r e n c h y m a l cells i n i t i a l l y p r e s e n t w h e n s t a r t i n g the culture. Isozymic changes o c c u r r e d after 5 days of culture w i t h the a p p e a r a n c e of the PK III type. The possibility can he ruled out that cell selection o c c u r r e d in a p r i m a r y c u l t u r e from n e w cells w h i c h have just divided, because of the scarcity
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C. G u g u e n ,
C. G r e g o r i a n d F . S c h a p i r a .
of m i t o t i c figures a n d because the 3H-thymidine i n c o r p o r a t i o n g r a d u a l l y d e c r e a s e d after 5 days of culture, c o n t r a s t i n g w i t h the a p p e a r a n c e of PK III. The p o s s i b i l i t y can be also r u l e d out that c u l t u r e c o n d i t i o n s affected the nature of the kinet i c p r o p e r t i e s of the p y r u v a t e isozymes in these cells as d e s c r i b e d by W a l k e r el al. [24] because the e n z y m e s w e r e always e x t r a c t e d f r o m cells g r o w n in the p r e s e n c e of the same c o n c e n t r a t i o n of glucose. This i s o z y m i c change i n d i c a t e s that h e p a t o c y t e s p r o b a b l y u n d e r g o an arrest of synthesis of an adult e n z y m e and a shift t o w a r d s the synthesis of the fetal type. Moreover, by the O u c h t e r l o n y m e t h o d and s o m e t i m e s by electrophoresis, we w e r e able to s h o w that c o n c e n t r a t e d extracts f r o m f r e s h l y isolated h e p a i o c y t e s or f r o m starting cultures, do also c o n t a i n PK III. This result suggests that the PK III type is p a r t l y r e p r e s s e d in the cells at the b e g i n n i n g of the culture a n d bec o m e s d e r e p r e s s e d d u r i n g ageing. The same phen u m e n o n w a s a l r e a d y o b s e r v e d w i t h Muscle-type of L a c t a t e d e h y d r o g e n a s e w h i c h is d e r e p r e s s e d in c u l t u r e [ ~ ] . We plan to c h e c k that synthesis is really r e p r e s s e d or d e r e p r e s s e d in our system.
same h e p a t i c cell. This p h e n o m e n o n m a y c o n t r i bute to the u n d e r s t a n d i n g of the m e c h a n i s m s of r e p r e s s i o n and d e r e p r e s s i o n of specific p r o t e i n s in culture. I~SUM~. Les modifications des isozymes de l,a Pyruvate Kinase ,ont ~t~ ~tudi~es sur des eul,tures d'h~patocytes d,e rat ad~u.Re pay des m~thod.es ~lectrop,hor~tiques, cin~tiques et immun,ologiques. Des mon
W e h a v e also no~ed the p r e s e n c e o~ a spur b e t w e e n the cell extracts and the p l a c e n t a e x t r a c t ('which is of PK III type), w h i l e the cell extracts d u r i n g the w h o l e cell e v o l u t i o n display a p a t t e r n of total identity. It m i g h t be postulated that the t y p e of PK in cell cu~lure is not p u r e PK III type, but an h y b r i d . H y d r o c o r t i s o n e does not influ,ence this synthesis although the i n d u c t i o n effect of glucocortic o l d s in v i v o and in vitro on t y r o s i n e t r a n s a m i nase [20, 23, 26] and also on aldolase B [27] is w e l l - k n o w n . But in these e x p e r i m e n t s , dexamet h a s o n e or h y d r ~ c o r t i s o n e w e r e g e n e r a l l y a d d e d after 4.8 hrs of culture and f o r a short time, whereas o u r assays w e r e p e r f o r m e d in cultures always m a i n t a i n e d w R h h y d r o c o r f i s o n e . This possible ind u c t i o n effect of h y d r o c o r t i s o n e tends p e r h a p s to d i s a p p e a r after some days of treatment. In c o n c l u s i o n , w e w e r e abte to m a i n t a i n in vitro p r i m a r y cul.tures af adult rat h e p a t o c y t e s w h i c h kept not only t h e i r m a i n m o r p h o l o g i c a l p a r e n c h y m a l c h a r a c t e r i s t i c s but also, up to the 5th day, t h e i r i s o z y m i c adult c o m p l e m e n t . We w e r e able also to d e m o n s t r a t e that p u r e h e p a t o c y t e s m a y s y n t h e s i z e PK I I I a l t h o u g h in slight amount, even at t h e b e g i n n i n g of thee culture. L a t e r these hepatocytes stops s y n t h e s i z i n g adult PK I type w h i l e the s y n t h e s i s of fetal PK III type increases, s h o w i n g that the t w o PK types m a y be s y n t h e s i z e d by the BIOCHIMIE, 1975, 57, n ° 9.
1. T~anaka, T., ~arano, Y., M,o.rimarra, M. & Mort, R. (1965'), Biochem. Biophys. Res. Commttn., 21, 5560. 2. Susor, W. A. a Rutter, W. J. (1968), Biochem. Biophys. Res. Commun., 30, 14-20. 3. Setmpira, F. a Greffori, C. (1971~), C. B. Acad. Set., Sdrie D, 272, li10~-1,172. 4. In~am.ura, K. ,¢ Ta,naka, T. (1972). J. Biochem., 71, 1043-1051. 5. Van Berkel, J. C., Koster, J. F. & Hiil~smann; W. C. (1972), Biochim. Biophys. Aeta, 276, 425-429. 6. Garnett, M. E., Dyson, R. D. & Dost, F. N. (1974), J. Biol. Chem., 249, 522~L5226. 7. Bonney, R. J., Walker, P. R. & Potter, V. R. (1~973), Biochem. J., 136, 947"954. 8. L.enlz, P. E. & Diluzio, N. R. (1971), Exptl. Cell Res., 67, 1'7-26. 9. W.al,ker, P. R. ~ Potter, V. R. (1973), J. Biol. Chem., 248, 4610-4616. 1~0. Lan~biotfe, M., Susur, W. A. & Cohn, R. D. (1972~, Bioehimie, 54, 1479-11,87. 11. Guguen, C., GuiHouzo, A., Boisn~r4, M., Le Cam, A. Bourel, M. (1,975) Biol. Gastroenterol., 8, 223-231. 12. Seglen, P. O. (1E973)~ Exptl. Cell. Res., 76, 25-30. 13. L(>wry, O. H., R~rsebr~ugh, N. I., F.arr, A. L. a R.anda11, R. J. (19519, J. Biol. Chem., 193, 265-275. 14. Biieher, T. a Pfleiderer, G. (1955) In : Methods in Enzymology. S. P. CArlowick and N. O. Kaphm (eds) Academic Press, 1, 435-440. 15. Cartier, P., Najman, A., Leroux, J. P. a Temkine, H. (~968'), Clin. Chim. Aeta, ~2, 1-65-181. 16. Imam~ra, K., Taniuehi, K. a Tan.aka, T. (1972~ J. Biochem., 72, 10~1-1015. 17. Pelc, S. R. a Howard, A. (1952) Brit. Med. Bull., 8, 13~-135. 18. ~aeiera-Coelho~ A., Ponten, J. a Philipson, L. (1966) Exptl. Cell Res., 42, 673-084. 1,9. Guguen, C., ~uil,l,ou~o, A., Boisnard, Yi. ~ Le Cam, A. (1974) J. Microscopic (Paris), 20, .~,59.
Pyruvale-kinase
in h e p a t o c y t e
20. Bonney, R. J., Becker, J. E. , Walker, P. R. a Potter, V. R. (19.7'4:) In Vitro, 9, 399-41~3. 21. B~on.ney, R. J. (19~74) In Vitro, 10, 139-142. 22. M,aeiera-Coelho, A. a Berumen, L. (1973) Proc. Soc. Exp. Biol. Med., 144, 43-47. ~3. Lin, B. C. a Snodgrass, P. J. (19q5~) Biochem. Biophys. Res. Commun., 64, 725-734.
BIOCHIMIE,
1 9 7 5 , 57, n ° 9.
cultures.
1071
24. Walker, P. R., Bonney, R. J., P~ecker, J. E. & Potter, V. R. (197% In Vitro, 8, 107-114. 25. Phi.lip, J. & V~es~l, E. S. (1962) Proc. Soc. Exp. Biol. Med., 110, 582-585. 26. Gers,chen.son, L. E., Anderson, M., N,ols,on, J. & Okigaki, T. (1L9.7~ff)Science, 170, 85~9,-86.1. 27. Schapira, F., Delain, D. & Lacroix, Y. (197I) Enzyme, 12, 545-552.
Modification of pyruvate kinase isozymes in prolonged primary cultures of adult rat hepatocytes. C h r i s t i a n e Guau~N, Claudine GnEGom and F a n n y SCHAPInA~. * I n s t i t u t d e P a t h o l o g i e M o l ~ c u l a i r e , 24, r u e d u F a u b o u r g S a i n t - J a c ? u e s , 75014 P a r i s ( F r a n c e ) . (27.6-1975).
S u m m a r y . - - Pyruvate k,inase isozymic changes were stud~ied in the adu]t he.patoeyte cultures, by eleetropl~o~etic, kinetic and immum)togical methods. VCe were .able to li~aintain par~enchymal cells from normal adult rat 1.leer in' no.n-proliferating mono.layer cuKures up t,o 10 days. Hep.atocytes appeared to contain a dominant ~K I type up to 4-5 days of culture. After day 5, PK III type wa~ ~eg.ul,arl,y pl~esent with PK I and after 7 days PK III type was always the .only isozyme detected in culture. It must b e p o i n t e d out that, by the Ouchterlony method and sometimes by electropl~oresi~, coneen.trated ~xtracts from fves~lT isol~ated h,elaa~0ocytes or sta~ing hep~atoeyte cultures did' also contain" Pyruvate kinase PK III type. These results suggest that Pyr~vate kina,se III is present but pavtl.y repressed in the adult parench.ymal eel,l.s and becomes dcrepressed in cu:lt'ure.
T w o types of p y r u v a t e - k i n a s e (PK) in adult rat tissues h a v e been d e s c r i b e d b y T a n a k a et al. [1]. Later, Susor a n d Rutter [2] h a v e s h o w n that the p r e d o m i n a n t f o r m in adult l i v e r w a s PK I type w h i c h was f o u n d to h a v e kinetic, e l e c t r o p h o r e t i c and i n u n u n o l o g i c a l p.roperties different f r o m PK II Muscle-type. T h e y h a v e also s h o w n that the m i n o r f o r m of PK in l i v e r w a s d i f f e r e n t f r o m the muscle form. S c h a p i r a and Gregori [3] h a v e r e p o r t e d that this PK III type w a s also f o u n d in fetal liver, in p l a c e n t a a n d in fast g r o w i n g hepatomas. I m a m u r a a n d T a n a k a [4] h a v e found this PK n I type in m a n y tissues, p r i n c i p a l l y in kidney. Several authors h a v e suggested that PK I type was f o u n d only in the p a r e n c h y m a l cells an.d that the PK [II type w a s p r e s e n t in the non p a r e n c h y m a l f r a c t i o n s [5, 6, 7, 8]. One fact was p a r t i c u l a r l y int e r e s t i n g : a l t h o u g h n o r m a l l y p r e s e n t as a m i n o r c o m p o n e n t in liver, i n r i v e , this PK III f o r m i s the only one s y n t h e s i z e d in rat l i v e r cells in v i t r o [9, 10]. W a l k e r et al. [9] i n d i c a t e d that cell selection o c c u r r e d in p r i m a r y cultures in cells w h i c h a l r e a d y c o n t a i n e d PK HL A n o t h e r h y p o t h e s i s w o u l d be that c o n d i t i o n s of c u l t u r e t h e m s e l v e s r e p r e s s the synthesis of some specific proteins. But little is k n o w n about the e n z y m i c c h a n g e s w h i c h o c c u r in fully d i f f e r e n t i a t e d h epatocytes m a i n t a i n e d in m o n o l a y e r cultures. (*) Gro.upe U 129 4e l'Institut Nation,a.1 d.e la S,ant6 et de l,a l~echerehe M~d.iea.le, Laboratoire associ6 au Gentre National d,e la Re~heveh.e Scientifique. T.o wh,om a~ll eorrespon&ence should b~e addres.sed.
In a p r e v i o u s paper, one of us has s h o w n that hepatoeytes retain ultrastructural characteristics up to 8-10 days in n o n - p r o l i f e r a t i n g cultures in the p r e s e n c e of h y d r o e o r t i s o n e h e m i s u c c i n a t e [11]. The aim of this w o r k is to study the isozymes of PK p r e s e n t in these s u r v i v i n g h e p a t o c y t e s .
MATERIAL AND METHODS. W i s t a r rats w e i g h t i n g 200 to 300 g w e r e used. T h e m e t h o d for the p r e p a r a t i o n of isolated h e p a tocytes has been d e s c r i b e d e l s e w h e r e [117 ; it is a m o d i f i c a t i o n of the m e t h o d of Seglen [12]. The h e p a t o c y t e s w e r e w a s h e d six times and i n c u b a t e d in E12F12 m e d i u m . Cultures w e r e p e r f o r m e d in this m e d i u m c o n t a i n i n g 0,.1 p e r c e n t glucose and s u p p l e m e n t e d w i t h 10 p e r c e n t fetal calf s e r u m a n d 0.5 p e r c e n t c h i c k e m b r y o e x t r a c t e q u i l i b r a t e d a f t e r w a r d s w i t h a 90 p e r c e n t a i r - 10 p e r c e n t CO 2 m i x t u r e . In some e x p e r i m e n t s 1.8 mM h e m i s u c c i nate h y d r o c o r t i s o n e w a s a d d e d ~ h e n the m e d i u m w a s c h a n g e d 5 h r s after s e e d i n g and then e v e r y t w o or t h r e e days. E v e r y day, ceils w e r e h a r v e s t e d : the m e d i u m w a s d i s c a r d e d and the plates w a s h e d t w i c e w i t h 154 mM N a t l . The cells w e r e s c r a p e d off the plates and c o l l e c t e d in a c o n i c a l c e n t r i f u g e tube w i t h a buffer c o m p o s e d of 10 mM Tris HC1 p H 8.0 20 mM MgC12 and 200 mM KC1. The cell suspension w a s frozen and tha-wed twice.
10'66
C. G u g u e n , C. Gregori a n d F. S c h a p i r a .
Electrophoresis : it was c a r r i e d out on starch gel c o n t a i n i n g Fructose Diphosphate (Fru-l-0-P) 3 mM w i t h a phosphate citric acid buffer pH 7.0 for 18 hrs at 4°C w i t h a potential gradient of 3 V / c m as described by Schapira and Gregori !31. After niigration, the starch-gel was sliced and covered w i i h the reactive m i x t u r e of Susor and Rutter for specific s t a i n i n g of PK [21. I n some exp e r i m e n t s , isozymes w e r e located by a modification of the m e t h o d of I m a l n u r a a n d T a n a k a [4~. Kinetic studies : the cell homogenate was centrifuged at 2000 g for 20 m i n at 0°C in order to s e d i m e n t cell debris. The r e s u l t i n g s u p e r n a l a n t was used for the enzyme assays a n d for soluble p r o t e i n d e t e r m i n a t i o n s by the m e t h o d of Lowry et al [13] u s i n g b o v i n e plasma a l b u m i n as standard. Measurement of PK was c a r r i e d out as d e s c r i b e d by Biicher a n d P f l e i d e r e r [14] with variable Concentrations of p h o s p h o e n o l p y r u v a t e as substrate (PEP) 0.05 mM to 1.5 mM with or w i t h o u t F r u - l - 6 - P 0.1 raM. Adenosine diphosphate was used at the n o n i n h i b i t i n g c o n c e n t r a t i o n of 0.3 mM as described by Cartier et al [15].
vant. The specific anti PK II a n t i s e r u m did not cross-react w i t h PK I type, but cross-reacted with PK III type [16]. I m m u n o l o g i c a l e x p e r i m e n t s were p e r f o r m e d a c c o r d i n g to the double diffnsion method of Ouehterlony with 2 p e r c e n t NaC1 added to 1 percent agar in 50 mM veronal buffer. Radioautographic studies : the nlethod of Pelc and H o w a r d [17] modified by Maciera~Coelho et al [18] was used for the localization of 3H-thymidine i n c o r p o r a t i o n . Monolayers were i n c u b a t e d for 15 hrs witll 1 ~Ci/ml 3H-thymidine (27 m C i / raM) and then fixed in Bouin solution.
tk~ESULTS. 1.
--
MORPHOLOGICAL OBSERVATIONS.
Tile results obtained were s i m i l a r to those described in the previous papers of one of us [11, 19]. Isolated hepatocytes became attached to the s u b s t r a t u m w i t h i n 4 hrs and formed m o n o l a y e r s
Fro. 1. - - Adult liver parenchymal cells after 5 days of culture. Phase-contrast photograph of living cell9 (X 320).
Immunological methods : the isozymic modifications were f o l l o w e d by the study of the action of a specific a n t i s e r u m . A n t i s e r u m was p r e p a r e d by i n j e c t i n g crystalline PK II from r a b b i t muscle (Boehringer) into chicken, w i t h F r e u n d ' s adjuBIOCHIMIE, 1975, 57, n ° 9.
w i t h i n 24 hrs (fig. 1) ; this figure shows also the p u r i t y of the cultures w h i c h c o n t a i n only hepatocytes w i t h o u t n o n p a r e n c h y m a l cells. The use of E12FI2 m e d i u m i n c r e a s e d the c o n s e r v a t i o n o.f hepatocytes up to 8 days. With addition of h y d r o -
P!lruvate-kinase
in hepatocyle
cortisone h e m i s u c c i n a t e , the cells were m a i n t a i ned up to 10 days. Uptake of 3H-thymidine was located in culture after 3 days by radioautogra-
1067
cultures.
5th day, but mitotic figures were scarcely seen (less t h a n I percent). A s i m i l a r p e r c e n t a g e was o b t a i n e d i n h o r m o n e - t r e a t e d cultures. 2. - - ELECTROlaHORETIC MODIFICATIONS. Figure 2 compares e l e c t r o p h o r e t i c c h a r a c t e r i s tics of PK i n adult liver, muscle, k i d n e y a n d i n fetal liver (15-16.th day old). The m a i n b a n d of the liver PK I h a d the highest m o b i l i t y t o w a r d s the a n o d e ; the muscle PK II m i g r a t e d more slowly a n d differed from the k i d n e y PK III w h i c h h a d the lowest mobility. The fetal liver PK was resolved into 3 b a n d s : PK I a n d PK HI types, a n d an i n t e r m e d i a r y type ( p r o b a b l y due to erythrocytes).
FIo. 2. - - Electrophoresis of p y r u u a i e k i n a s e isozyraes. Isozyrnes vcerc .1,ocat.ed by a modification, of the
method .of Imamura and' Tan.aka E47. a : ad'ult liver. b : fetal Hver (16th d~y). c : kidnvy. d : muscle.
I
Figure 3 shows the p a t t e r n o b t a i n e d w i t h cultured hepatocytes. They a p p e a r e d to c o n t a i n a dol n i n a n t PK I type up to 4-5 days of culture. After day 5, PK III type was r e g u l a r l y p r e s e n t w i t h PK I (fig. 4). On day 7, PK III b a n d was the strongest. In some experiments, an e l e c t r o p h o r e t i c p a t t e r n s h o w i n g i n t e r m e d i a r y b a n d s b e t w e e n PK I a n d PK III, was observed after 6 days of culture. PK III was always the only isozyme seen after the
-
+
Culture day 2
FIG. 3. - - Electrophoresis o f p y r u v a t e kinase i$oz y m e s in cultured hepatocytes. Isozymes~ were located
with the rea,ctiva mixture of Susor and Rutter [2].
phy. The m a x i m u m of labelled nuclei (about 20 p e r c e n t of total nuclei) was r e a c h e d on the BIOCHIMIE, 1975, 57, n ° 9.
7th day i n cultures main,rained w i t h or w i t h o u t hydroeortisone hemisuceinate.
C. G u g u e n , C. G r e g o r i a n d F. S c h a p i r a .
1068 3.
---
KINETIC
STUDIES.
I n f i g u r e 5 a r e p l o t t e d t i m a c t i v i t i e s of h o m o g c n a t e s of m u s c l e a n d i s o l a t e d p a r e n c h y m a l cells. T h e P K II M u s c l e t y p e s h o ' w e d M i c h a e l i a n b e h a -
s h o w n i n t a b l e I w h i c h s u m m a r i z e s t h e e f f e c t of F r u - l - 6 - P at a n o n - i n h i b i t i n g c o n c e n t r a t i o n o f P E P , o n t h e P K a c t i v i t y of m u s c l e , l i v e r a n d p a r e n c h y m a l cells at d i f f e r e n t t i m e s o f c u l t u r e . TABLE I.
Action oi' Fru-l-6-P on P y r u v a t e kinase activity at a non saturating concentration o[ substrate P E P (0.0625 mM).
÷
Without Fru-t-6-P
With Fra-t-6-P
Adult muscle . . . . . . . . . . . . A d u l t liver . . . . . . . . . . . . . . I Fetal liver (15 th-16 t h day)
40 4 18
55 42 46
Hepatocyte culture 4 days . . . . . . . . . . . . . . . 7 days ~. . . . . . . . . . . . . . .
8 13
51 53
Tissues
.voeo
. v o ~ o (--1
(+)
i I
! L
Results a~re, expressed in percentage of the activity detected in the presence of PEP l mM (mean of 3 determinations).
m
FIG. 4. Electrophoresis of pyruoate kinase isoz!Imes in hepatoctltes ma, i n t a i n e d w i t h or w i t h o u t
4.
-l~6ro~eoriison~ h e m i s u c e i n a t e ; a f t e r 6 days of culture.
v i o u r a n d w a s n o t i n f l u e n c e d b y a d d i t i o n of F r u 1-6-P. I n c o n t r a s t t h e P K I t y p e a c t i v i t y s h o w e d a sigmoid curve when plotted against an increased c o n c e n t . r a t i o n of P E P w h i c h c o u l d b e c o n v e r t e d
--
IMMUNOLOGICAL
RESULTS.
Figure 7 shows the precipitation line obtained with the Ouchterlony method, using antiserum a n t i P K II, b e t w e e n e x t r a c t s of d i f f e r e n t r a t t i s s u e s o r cells w h i c h w e r e d i l u t e d i n o r d e r to have the same final concentration into the wells. A slight precipitation line was obtained with the
200
80
Y_ E
c E
0 o <3
(o)
0
!
0.5
! 1
[PEP] m M
4o (b)
0 ~
0.5 ~PEP~ m M
1
FIG. 5. - - Pyruvate kinase activity of muscle (a) and liver (b) h o m o g e n a t e s versus t h e P E P con,centration, a t a concentration of ADP 0.3 mM, in the presence (× ..... x) o r absence ( o - - - o ) of F r u - l - 6 - P .
into a hyperbolic curve by adding Fru-l-6;P 0.1 m M . F i g u r e 6 s h o w s t h e P K I a c t i v i t y of t h e h o m o g e n a t e of c u l t u r e d h e p a t o c y t e s u p to t h e 4th day. The curve was partly converted into a h y p e r b o l i c o n e ~)n t h e 7 t h o r t h e 8 t h d a y , b u t t h e a c t i v i t i e s w e r e still i n f l u e n c e d b y F r u - l - 6 - P , as
BIOCHIMIE, 1975, 57, n ° 9.
adult liver extracts, a strong line with the plac e n t a a n d m u s c l e e x t r a c t s . E x t r a c t s of h e p a t o c y t e s freshly isolated reacted against antiserum. The precipitation lines obtained with the hepatocytes m a i n t a i n e d in vitro w e r e s t r e n g h t e n e d a l o n g t h e c u l t u r e : s l i g h t o n t h e 2rid d a y , m o d e r a t e o n t h e
Pyruvate-kinase
in h e p a t o c y t e
4th day a n d strongest on the 6th day of culture. In other experiments, we have f o u n d that the pre-
1069
cultures.
layers in vitro. These results also agree w i t h previous reports of B o n n e y et al [20, 21].
.# ¢./
g
E
E
~2 o
(a)
"
o
2
(b] l
0.5
1
[PE P] m M
,
o.s 1 [PEP]mM
Fro. 6. - - Pyruvate kinase activity of hepatocyte homogenates after 4 days (a) and 7 days of culture (b), versus the PEP concentration in the presence (× ........ ~,) or absenve ( • - - • ) of Fru-I=6-P (0.1 ml~).
c i p i t a t i o n line was still stronger on the 8th or 9th day of culture. But a p a t t e r n of p a r t i a l i d e n t i t y w i t h a spur was always seen w i t h the p l a c e n t a line.
I n the presence of E12F12 m e d i u m w i t h embryonic chick extracts and fetal calf serum, they m a i n t a i n e d their m o r p h o l o g i c a l a n d f u n c t i o n a l characteristics for more t h a n 7 d a y s a n d took up tritiated t h y m i d i n e (~5 to 20 p e r c e n t nuclei on the 5th day). W h e n h y d r o c o r t i s o n e was added, the culture p e r i o d was i n c r e a s e d up to 10 days without i m p o r t a n t m o d i f i c a t i o n s of the s n b c e l l u l a r elements of the hepatocytes [11]. The s c a r c i t y of mitotic figures suggests a p r o l o n g e d <
FI6. 7 , - Precipitation lines in double immunodiffusion of placenta extracts (P), freshly isolated cells (IC) and hepatocytes after 2 Says (G2) 4 days (C4), 7 days (C7~, of culture, ag,air~st antisel~um .am.tipyruvate kinase II: ~al~lex,trac~s we~-e at the. same activity). DISCUSSION. We c o n f i r m that adu'll rat h e p a t o c y t e s isolated a c c o r d i n g - t o the method of Seglen, formed m o n o BIOCHIMIE, 1975, 57, n ° 9.
This result seems to exclude the o v e r g r o w t h of the hepatocytes by n o n - p a r e n c h y m a l cells i n i t i a l l y p r e s e n t w h e n s t a r t i n g the culture. Isozymic changes o c c u r r e d after 5 days of culture w i t h the a p p e a r a n c e of the PK III type. The possibility can he ruled out that cell selection o c c u r r e d in a p r i m a r y c u l t u r e from n e w cells w h i c h have just divided, because of the scarcity
10'70
C. G u g u e n ,
C. G r e g o r i a n d F . S c h a p i r a .
of m i t o t i c figures a n d because the 3H-thymidine i n c o r p o r a t i o n g r a d u a l l y d e c r e a s e d after 5 days of culture, c o n t r a s t i n g w i t h the a p p e a r a n c e of PK III. The p o s s i b i l i t y can be also r u l e d out that c u l t u r e c o n d i t i o n s affected the nature of the kinet i c p r o p e r t i e s of the p y r u v a t e isozymes in these cells as d e s c r i b e d by W a l k e r el al. [24] because the e n z y m e s w e r e always e x t r a c t e d f r o m cells g r o w n in the p r e s e n c e of the same c o n c e n t r a t i o n of glucose. This i s o z y m i c change i n d i c a t e s that h e p a t o c y t e s p r o b a b l y u n d e r g o an arrest of synthesis of an adult e n z y m e and a shift t o w a r d s the synthesis of the fetal type. Moreover, by the O u c h t e r l o n y m e t h o d and s o m e t i m e s by electrophoresis, we w e r e able to s h o w that c o n c e n t r a t e d extracts f r o m f r e s h l y isolated h e p a i o c y t e s or f r o m starting cultures, do also c o n t a i n PK III. This result suggests that the PK III type is p a r t l y r e p r e s s e d in the cells at the b e g i n n i n g of the culture a n d bec o m e s d e r e p r e s s e d d u r i n g ageing. The same phen u m e n o n w a s a l r e a d y o b s e r v e d w i t h Muscle-type of L a c t a t e d e h y d r o g e n a s e w h i c h is d e r e p r e s s e d in c u l t u r e [ ~ ] . We plan to c h e c k that synthesis is really r e p r e s s e d or d e r e p r e s s e d in our system.
same h e p a t i c cell. This p h e n o m e n o n m a y c o n t r i bute to the u n d e r s t a n d i n g of the m e c h a n i s m s of r e p r e s s i o n and d e r e p r e s s i o n of specific p r o t e i n s in culture. I~SUM~. Les modifications des isozymes de l,a Pyruvate Kinase ,ont ~t~ ~tudi~es sur des eul,tures d'h~patocytes d,e rat ad~u.Re pay des m~thod.es ~lectrop,hor~tiques, cin~tiques et immun,ologiques. Des mon
W e h a v e also no~ed the p r e s e n c e o~ a spur b e t w e e n the cell extracts and the p l a c e n t a e x t r a c t ('which is of PK III type), w h i l e the cell extracts d u r i n g the w h o l e cell e v o l u t i o n display a p a t t e r n of total identity. It m i g h t be postulated that the t y p e of PK in cell cu~lure is not p u r e PK III type, but an h y b r i d . H y d r o c o r t i s o n e does not influ,ence this synthesis although the i n d u c t i o n effect of glucocortic o l d s in v i v o and in vitro on t y r o s i n e t r a n s a m i nase [20, 23, 26] and also on aldolase B [27] is w e l l - k n o w n . But in these e x p e r i m e n t s , dexamet h a s o n e or h y d r ~ c o r t i s o n e w e r e g e n e r a l l y a d d e d after 4.8 hrs of culture and f o r a short time, whereas o u r assays w e r e p e r f o r m e d in cultures always m a i n t a i n e d w R h h y d r o c o r f i s o n e . This possible ind u c t i o n effect of h y d r o c o r t i s o n e tends p e r h a p s to d i s a p p e a r after some days of treatment. In c o n c l u s i o n , w e w e r e abte to m a i n t a i n in vitro p r i m a r y cul.tures af adult rat h e p a t o c y t e s w h i c h kept not only t h e i r m a i n m o r p h o l o g i c a l p a r e n c h y m a l c h a r a c t e r i s t i c s but also, up to the 5th day, t h e i r i s o z y m i c adult c o m p l e m e n t . We w e r e able also to d e m o n s t r a t e that p u r e h e p a t o c y t e s m a y s y n t h e s i z e PK I I I a l t h o u g h in slight amount, even at t h e b e g i n n i n g of thee culture. L a t e r these hepatocytes stops s y n t h e s i z i n g adult PK I type w h i l e the s y n t h e s i s of fetal PK III type increases, s h o w i n g that the t w o PK types m a y be s y n t h e s i z e d by the BIOCHIMIE, 1975, 57, n ° 9.
1. T~anaka, T., ~arano, Y., M,o.rimarra, M. & Mort, R. (1965'), Biochem. Biophys. Res. Commttn., 21, 5560. 2. Susor, W. A. a Rutter, W. J. (1968), Biochem. Biophys. Res. Commun., 30, 14-20. 3. Setmpira, F. a Greffori, C. (1971~), C. B. Acad. Set., Sdrie D, 272, li10~-1,172. 4. In~am.ura, K. ,¢ Ta,naka, T. (1972). J. Biochem., 71, 1043-1051. 5. Van Berkel, J. C., Koster, J. F. & Hiil~smann; W. C. (1972), Biochim. Biophys. Aeta, 276, 425-429. 6. Garnett, M. E., Dyson, R. D. & Dost, F. N. (1974), J. Biol. Chem., 249, 522~L5226. 7. Bonney, R. J., Walker, P. R. & Potter, V. R. (1~973), Biochem. J., 136, 947"954. 8. L.enlz, P. E. & Diluzio, N. R. (1971), Exptl. Cell Res., 67, 1'7-26. 9. W.al,ker, P. R. ~ Potter, V. R. (1973), J. Biol. Chem., 248, 4610-4616. 1~0. Lan~biotfe, M., Susur, W. A. & Cohn, R. D. (1972~, Bioehimie, 54, 1479-11,87. 11. Guguen, C., GuiHouzo, A., Boisn~r4, M., Le Cam, A. Bourel, M. (1,975) Biol. Gastroenterol., 8, 223-231. 12. Seglen, P. O. (1E973)~ Exptl. Cell. Res., 76, 25-30. 13. L(>wry, O. H., R~rsebr~ugh, N. I., F.arr, A. L. a R.anda11, R. J. (19519, J. Biol. Chem., 193, 265-275. 14. Biieher, T. a Pfleiderer, G. (1955) In : Methods in Enzymology. S. P. CArlowick and N. O. Kaphm (eds) Academic Press, 1, 435-440. 15. Cartier, P., Najman, A., Leroux, J. P. a Temkine, H. (~968'), Clin. Chim. Aeta, ~2, 1-65-181. 16. Imam~ra, K., Taniuehi, K. a Tan.aka, T. (1972~ J. Biochem., 72, 10~1-1015. 17. Pelc, S. R. a Howard, A. (1952) Brit. Med. Bull., 8, 13~-135. 18. ~aeiera-Coelho~ A., Ponten, J. a Philipson, L. (1966) Exptl. Cell Res., 42, 673-084. 1,9. Guguen, C., ~uil,l,ou~o, A., Boisnard, Yi. ~ Le Cam, A. (1974) J. Microscopic (Paris), 20, .~,59.
Pyruvale-kinase
in h e p a t o c y t e
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