Multiple forms of phosvitin kinase from rat liver cytosol

346 BBA

BIOCHIMICA ET BIOPHYSICA ACTA

66437

M U L T I P L E FORMS OF P H O S V I T I N K I N A S E FROM RAT L I V E R CYTOSOL B B A G G I O AND \'

MORE'I

Instztute of Bzolog~cal Chemistry of the ly mver~ try of Padova and Centro per lo qtudw della F~swlog~a dez Mztocondrz del Conszgho Naz,onale delle R,cerche, Padova (Italy) ( Recel~ed M a y 4th, 1971)

SUMMARY

Rat liver cytosol phosvltm kinase can exist In more than one active form, dlstmgulshable by phosphorylated cellulose chromatography and Sephadex filtration It is probable that they consist of different types of aggregates dlssoclable in solutions of high ionic strength

INTRODUCTION

In a previous paper a simple procedure was described/or the purification from rat liver cytosol of phosvItln klnase which is completely free of protamine kmase i The present paper shows that phosvltm kmase from rat liver cytosol can exist in more than one active form, distinguishable by means of P-cellulose chromatography and Sephadex filtration MATERIALS AND METHODS

Pur,ficatzon of phoswt,n k,nase free of protamme k,nase The crude cytosol was prepared by centrifuging for 60 rain at lO5 ooo x g the 25 ooo × g post-mitochondrlal supernatant of a o 25 M sucrose homogenate of rat hver cells 2 The cytosol preparation (about 500 ml), containing about 6 50 g protein, was dialyzed against several changes of o 05 M TrIs-HC1 buffer (pH 7 5) It was then sublected to chromatography on a phosphorylated cellulose (P-cellulose) column (4 5 cm × 15 cm) previously equlhbrated with the same buffer used for the dialysis The column was eluted with the same buffer contamlng increasing concentrations of NaC1, as indicated in the figures (continuous or stepwlse gradient) The flow rate was I 5 ml/mln and 9-ml fractions ~ere collected Phosvitin klnase activity was assa~ ed in the smgle tubes as described later

sephade ~Jdtratwn Each P-cellulose fraction, concentrated by Dlaflo-membrane ultrafiltration, was dialyzed overnight against o 05 M TrIs-HC1 buffer (pH 7 5) containing the required NaC1 concentration (o I or o 5 M NaC1) as indicated in the single experiment It was then submitted to molecular filtration on a Sephadex G-2oo column (I 8 cm × 13o cm) previously equilibrated with the same buffered solution used in the previous dlalybi~ Flow rates were about 4 5 ml/h, and I 8-ml fractions were collected Bzoch~m Bzophys dcta, 250 (1971) 346-35 °

347

MULTIPLE FORMS OF PHOSVITIN KINASE

Phosv,tzn k,nase determznatwn Phosvitm kmase was determined by mcubating the enzyme sample in I ml of a medium containing I mg of p h o s w t m , 6 #moles MgCI~, 200/~moles of Tns buffer (pH 7 5) and o 25/,mole of ATP contamlng 1-2/~C as [7-32P]ATP The reaction was started by addition of the enzyme sample After IO rain, at 37 °, the incubation was stopped by adding o 3 ml 50% trlchloroacetlc acid and the precipitate washed 4 times with 5 ml of lO% trichloroacetlc acid Finally the precipitate was transferred to a stainless steel planchet and counted with a t h i n - w i d o w Gelgel counter ~7-a2P]ATP was prepared by the method of GLYNN AND CHAPPEL3 Phosvltin was prepared from fresh egg yolk according the procedure of MECHAM AND OLCOTT4 RESULTS AND DISCUSSION

Fig I shows the profile obtained when crude rat hver cytosol was submitted to chromatography on a P-cellulose column, equilibrated with o 05 M Tns-HC1 buffer (pH 7 5), and eluted with a continuous linear gradient of NaC1

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Fig I C h r o m a t o g r a p h y profile for cytosol r a t liver p h o s v l t l n kinase on P-cellulose c o l u m n e q u l h b r a t e d w i t h o 05 M T n s - H C 1 buffer (pH 7 5) and eluted w i t h 15o ml of the same buffer containing o 25 M NaC1 followed b y a linear g r a d m n t r a n g i n g from o 25 to o 75 M NaC1 (mlxmg c h a m b e r o 25 M NaC1 and reservoir o 75 M NaC1, 250 ml each) 0 - - 0 , a b s o r b a n c e at 28o n m , C A - - - O , phosvatln k m a s e activity, expressed as 8~p transferred from [3*P]ATP to p h o s v l t m

I t can be seen that while the bulk of the cytosol proteins was not retained by the column, the phosvltln kmase activity was eluted at comparatively high lomc strength and resolved into two peaks The peaks were better separated when the lomc strength of the elutmg buffer was increased in a step~lse manner (Fag 2)* The two P-cellulose fractions, when submitted separately to molecular filtration on Sephadex G-2oo at low ionic strength (o I M NaC1), exhibited different elution patterns (Fig 3a) " T w o peaks of p h o s v l t l n kinase activity were o b t a i n e d also w h e n the cytosol ~ a s chrom a t o g r a p h e d on D E A E cellulose column e q m h b r a t e d with o 0 5 M Trm buffer (pH 9 o) and elnted b v mcreaslng stepwlse the c o n c e n t r a t i o n of the same buffer

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Fig 2 C h r o m a t o g r a p h y prohle for c2¢tosol r a t h~er p h o s v l t m klnase on P-cellulose c o l u m n e q m h b r a t e d w i t h o o 5 M T n s - H C 1 buffer (pH 7 5) and eluted stepwlse w i t h the same buffer cont a m m g lncreasmg c o n c e n t r a t i o n s of NaC1, as Indicated b y the arrows 1~---O, a b s o r b a n c e at 28o n m This a b s o r b a n c e was n o t detectable m the single t u b e s of the p e a k A a n d t3 because of too low p r o t e m c o n c e n t r a t i o n The a b s o r b a n c e ~ a s detectable only w h e n the t w o protein fractions ere separately collected and c o n c e n t r a t e d bTy Dlaflo-membrane ultrafiltratlon, @ - - - @, phos~ l t m klnase acti~lt), expressed as 8~p t r a n s f e r r e d from [a2P]ATP to p h o s v l t m The specific a c t i v i t y of the f o r m A (5 9 mg protein) and 13 (i 5 m g protein) was a b o u t 927 and 31oo c o u n t s / r a m p e r / t g protein, r e s p e c t w e l v

These different patterns Indicate that the two enzyme fractions eluted from P-cellulose are not homogeneous However, when the two P-cellulose fractions were submitted separately or together to molecular filtration on Sephadex G-2oo at high ionic strength (o 5 M NaCI), a single enzyme peak (Fig 3b), more retarded than those obtained at low ionic strength (Fig 3a), was obtained The enzyme fraction of this peak exhibited a specific activity much higher than that of the form A and B of Fag 2, thus Indicating that during the Sephadex filtration at high lomc strength some other protein fractions devoid of phosvltin kinase activity were removed These results suggest that the enzyme m a y exist an different forms depending on the ionic strength conditions The two enzyme forms separated b y P-ceillulose chromatography (Fig 2) m a y consist of two different types of aggregates of an enzyme protein (Fag 3b) with some other cytosol component (possibly proteins) The aggregates differ in electrical charge and molecular weight and are dis~oclated by high ionic strength, thus giving rise to the enzyme form eluted as a single peak from Sephadex G-2oo (Fig 3b) The enzyme form present in this Sephadex peak, when submitted to P-cellulose chromatography under the same conditions as in Fig 2, can be eiluted with o 7 M NaC1 but not with lower NaC1 concentrations as those used an the experiment of Fig 2 Moreover when the enzyme form eiluted as a single peak from Sephadex with o 5 M NaC1 (see Fig 3b) is submitted agam to molecular filtration on the same Sephadex column at low ionic strength (o I M NaC1), it is converted into some aggregate forms showing an elutlon pattern (Fig 3c), different from each one of those obtained by submitting directly the P-cellulose fractions (Fig 2) to Sephadex filtration at the same low ionic strength (compare Fig 3c with Fig 3a) As appears from a comparison of the areas of peaks of Fig 3b and Fag 3e, Bzoch,m B w p h y s Acta, 250 (1971) 346-35 °

349

M U L T I P L E FORMS OF P H O S V I T I N K I N A S E a

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Fig 3 Molecular filtration on S e p h a d e x G-zoo column (I 8 cm × 13o cm) a Profiles obtained at low ionic s t r e n g t h (o i M NaC1) from the two P-cellulose fractions, p r e c i o u s l y dialyzed as d e s c r i b e d in MATERIALS AND METHODS (Curve A from o 4 M NaC1 peak, Curve B from o 5 M NaCI peak) b profile o b t a i n e d at high ionic s t r e n g t h (o 5 M NaC1) from a m i x t u r e of the t~vo P-cellulose fractions (4 6 m g protein) The eluted e n z v m e form (I i m g of protein) exhibited a s p e c l h c activity m u c h higher t h a n t h a t of the initial m i x t u r e subjected to S e p h a d e x filtration O - - - O , p h o s v l t m k m a s e activity, / ~ - - A , a b s o r b a n c e a t 220 n m c Profile obtained f r o m refiltratlon at low ionic s t r e n g t h (o I M NaC1) of the p e a k eluted from Sephadex at high lomc s t r e n g t h in the abo~ e e x p e r i m e n t

aggregahon 1s a c c o m p a n i e d b y a decrease of e n z y m e activity The loss of activity is dependent on the lowernlg of the l o m c strength of the m e d m m and not on the loss of s o m e e n z y m e c o m p o n e n t during the last Sephadex filtration at low ionic strength Indeed the loss of activity does take place also when the e n z y m e is merely diluted with water to o I M NaC1, while it does not take place ]f the d]luhon is performed at constant lomc strength, , e by using o 5 M NaC1 or o 5 M p o t a s s m m acetate Once the reactivation has occurred, it cannot be reversed b y increasing the ionic strength of the m e d m m The crude e n z y m e preparation (original cytosol or the e n z y m e only partially B~och,m B,ophys Acta, 25o (1971) 346-35o

350

B BAGGI0, V MORET

purified b y (NH4)2SO 4 fractlonatlon) once dissolved in o 5 M NaC1 does n o t undergo a n y r e a c t i v a t i o n b y dilution to o I M NaC1 This s t r e n g t h e n s t h e i d e a t h a t in the crude s t a t e the e n z y m e is b o u n d to borne o t h e r c y t o p l a s m c o m p o n e n t (likely proteins) whmh could p r e v e n t such r e a c t i v a t i o n of the e n z y m e occurring ~ h e n it is in a purified s t a t e RLFERENCES I B BAGGIO, L A PIN~*, V MORET AND iN SILIPRANDI, Bzochzm Bzophys Acta, 212 (197 o) 515 2 ~A" W UMBREIT, R H BURRIS AND J F bTAUFFER, Manomet~zc Ttchmques, Burgess, M m n e a p o h s , 1957, P 194 3 J M GLYNN AND J B CHAPPEL, B*ochem J , 90 (1964) 147 4 D K MECH~M AND H S OLCOTT, J A m Chem Soc, 71 (1949) 367 °

Bzochtm Bzophys Acta, 250 (1971 ) 316-35o