Immunochemical titration of liver glucokinase from normal, fasted, and diabetic rats

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i n t h e l i v e r .of r o d e n t s n n d o ~ h e r r n a n m m l i : a n s p e c i e s [ 1.]. ln-fact~ g ! u c o k i n a s e w h i c h is o n e o f f o u r i s o e n zYmes"catalyzing.this reaction 12], decreases ~nalloxan d i a b e t i c . r a t s , a n d i n - n o r m a l I a t s w:]fich h a v e 'been de;prived. o f f o o d o I iia-ve b e e n f e d - ~ ' t h .a . g l u c o s e , f r e e " " d i e I [ 3 - - . 6 ] . N o r m a l Jeve.'ls-ot~1he e n z y . m e are r e c o v e r e d foll0~ngadministrafion o f g l u c o s e a n d / o / i n s u l i n ~[3-6 ] , I~ ~pRe o f t h e •,time e l a p s e d ~ipce t h e s e o b ~ e r v a itions w e r e firs* m a d e •t h e r e is still n o g o o d e v i d e n c e ".that chma.ges i n g l ~ c o M n a s e , a c f i v i W p a r a l l e l c h a n g e s i n the amount of the ,enzyme protein, The fact that - a c t i n o m y c J n D, p u r o m y c i n .or c y c ] o h e x i m i d e , as w.ell as a m i n o .acid a n d . n u c t e o l i d e an.al~gs p a ' e v e n t ~ u c ~ kLuase in,crease af,te~ d e p l e t i o n [ 4 , 5, 7 - , 9 ] giyes .suppor,t t o ',the i d e a t h a t d e n o v o s y n t h e s i s .of 1he onz y m e is i m p l i c a l e d in i h a t i n c r e a s e . T h u s , "the p h e n o m o r t o n i s - r e f e r r e d t o as . e n z y m e i n d u c t i o n . . T h e W.ef e r e n t i N / a b e ! i n . g wi~h a4 G ] a b e l ] e d a m i n o a c i d s o f a

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T]TRATION.OF.LiVER GLucOK]NASE . FASTED," AND DIABETt C RATS .-

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. ~tions w e r e p r e p a r e d : i) A b s e n c e o f l:6wrKin: h e x 0 k i .nases.6,phosp}iogluconate,dehydi0gehase and sugar-:dehyi~lr0gen~ses~- all-~0f w h i c l u - ~ m e r f e r e w~h*he-Nueo-,." " . . ;. . " • '~, . - ; - -. - . . . . . " . . . : - . . . . . " .. . xmaseassay;

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2.3. Immun.oehemice] ~itralion The li'¢er extracts we.z,e ~]neubated with the antise,,vam ~n.a meclharn eon..tNn.~ng (final concemrations):_ ,D.t M T f i s - H C 1 , p H 7,5; 0.0D1 M E D T A ; 0.]5 M K C I ; 0.1 M ~ u c o s e ; 0 . 0 0 3 M D T I . The m i x t u r e s containing a FLx,ed a,reount o f A G S ant: ~'afiab]e a m o u n t s " a f ~ u c o M n a s e u n i t s were ~ncubaled ~aI 3 0 rain a-f 37 C and 1hen 1,ef~ o v e l m g h t in ~ e ] ~ n g Joe. A£t.ei c o n tfifugalion; conv,en~-ent a]iquots f r o m the supelnat~_ut fluid, were taken ~or d u p l i c a t e ~ucc, Mnsse assays. :Glueokinase activity Was meas.uz~ed in a Gi~ford spot.

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:References

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•... .of]3iolo:gical ~kcti~5~y.{Gaede~ K.,.}-lozec:kex, :B..aaad " - . :.7 ' : . ~ • " • " '. " • . _ "" . " " W h e l z n , W . a . , . e d s . ) , p . 2 2 1 , A ~ a d e m a c P , ess, Ne'~, F i g . ] . l m m u n o e h e m ~ e a l fiim't3on o f x ~ I ~v.er extr.ael'so Y o r k . . I~creasiaag '.v.g!~m.es ,of li.,az~.~ exl~ac~s .C.gl~aeokim~e aiad,ed') . [2 i] ,G~azfitez~ ,C., g x e l a ; T . , B~bxal., J.,- R a b z j i l t % E. ma.d 'w.e~e:jncuba'ted w i t h a ~oaastap.l z ~ : o u n t (1.92 m y ) of N i d m c y e l , H.. ( 1 9 6 7 ) B i ~ c l a e m i s l r y ,6, 4.60. . ~ m ' m } e p*o.l ein ,IAGS).ms d e s c l ~ e d i n :the rex1...Gl~co-, t 3 ] N i e m e y e 2, H., Claxk-.Tnrr], L: 'and ,Itab~jille; E . 0 9 6 3 ) .kmase z e c o ,ere.d coa-respondS t0 t h e e n z y m : e acfiid~]es N . a l m e . 1 9 8 , 109.6. - " a s s a y e d :after i n c u b a t i o n a n d ,centfifuga-d,0n. T h e ext~a,e~s 1.4 ]. Zalas, M., V i f i ~ e l a , E. a n d 8~]s, A. ( 1 9 6 3 ) 3. B~ol. ~eexe:prepa~ed f.x,~m *.a~-~alm-tiCti~l ~t,~:v,a r i ~ m ~xperim:enlwl C h b m . 2318, 3 5 3 5 . : : "" c o n d i t i o n s '(see k , e y , 0 ~ " Sy/TJl~'Ol~"10n t a b l e 1 ). T h e c o n I r o ] ~5 ] 8 h a r m a , C . ; M a n j e s h ~ a r , R . a n d .WeJnhome, 8. { 1 9 6 3 ) .line (--51GS) c o , x e s p o n d s l o llae theox.etical . v o m p l e t e .. - J . BIN.. C h e m . . 2 3 8 , 3 8 4 0 . . a n d C o d o c e o . K. { 1 9 6 7 ) :recovery o f , e n z y m e ~ctivities ~ f t e r i~cul~afima i n ¢.':he . . ~,6] Naean:eyer, " " " H . , Pexez,.N. " 3. 'Biol. C h e m . 2 4 2 , ,8:60. ~ a b s e n c e o f a n t i s e r u m . T h e e x , p e r i m e n l a l. .l.i.n e ( + A G S ) was drawa~ p~rall.e.1 .tO *he ,co.n~t~,~l a n d -i¥1fi~g ~ t a e p o i n t s [7 ] N i e a n ~ v e , , ,H., P e r e z , N . , ~3arc~z, E . a n d V e r g a l a , F . E : e 6 r z e s p o n d i n g 1o n e r m a l ~nim--_ls. T h e ,equi~a]enee poin~ {19.fi2) Bi.oetdna. Bi.ophys. Ae,ta 6 2 , 4 1 1 . ~,as 0 b ~ a i n e d b y ~X.t.,rapolalion. " " [B] N i e r n e y e r , I-£, Clalk-Taarri, L,~ ?6xez, N.. a n d R a ~ a j i l I e , E. (19.55) A z c h . B i o e h e n x N o p h y s . 1 0 9 , 634. [ 9] C h a m o n o , G, a n d S c N l k . m I , R. {1 9 6 9 ) Cin~fi,ea ,de .lndxacci~Sny d e Degra.daeign d e G ] u e o q a ~ S s ~ ,eng~gaa,o d e R~'ta. A]glmosI2a¢~tOres q u e infl~ay,en en el p_*.oeeso. Tesi~. F a t a l t a d ,de Me.dicina. U. d e ~.~." e. i l 0 ] Pjlkis, $: J.. { 1 9 7 0 ) B i o c h i m . B i o p h y s . A c l a 2 1 5 , 4 8 1 . t l 1 ] K e n d a l l ; F . ~ : ( 1 9 3 S ) . t o l d S p r i n g / - I ~ z b ~ z Syan:p. . Qua.nt, Bie]..'6, ~ 7 ~ . . . . . 1112] PJlkis, S. 3., H a p s e n , R . ,J. a n d Krah], h~I. E. ,{19,68) : C.omp. Bioe~hem. PhNNS~]. 2 5 , 9 0 3 o : .

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,gicai Support t o t h e . ¢ o m : e n t i o n t h a t ,cllanges in ] i v e I ,,'glucokinase activities obse.wed aft,er die¢,ary . a n d h o t ' • r n c n a l I n a n i p u I a I ] o n s ~ r e 'file i e s u l ~ o f amount of.enzyme pxo*eln.

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