Adenosine triphosphatase activity of adenosine triphosphate-creatine phosphotransferase

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  • 270 BIOCHIMICA ET BIOPHYSICA ACTA

    BBA 12318

    ADENOSINE TR IPHOSPHATASE ACTIVITY OF ADENOSINE TR IPHOSPHATE-CREATINE PHOSPHOTRANSFERASE

    TSUTOMU SASA* AND LAFAYETTE NODA

    Department of Biochemistry, Dartmouth Medical School, Hanover, N.H. ( U.S.A .)

    (Received June 4th, 1963)

    SUMMARY

    Creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) 1 has been found to have a low but constant amount of ATPase activity 2. The ratio of ATPase activity to creatine kinase activity remains the same during repeated crystallization and dnring repeated precipitations. Loss of kinase activity by a variety of treatments using phys- ical and chemical agents has been found to result in a proportionate loss of ATPase activity. Twice-crystallized preparations show no evidence of protein or activity heterogeneity on DEAE-cellulose column chromatography.

    Kinetic studies like those carried out for the kinase activity 3 lead to the identical conclusion that MgATP 2- is also the true substrate for ATPase activity. It may be postulated that the trace ATPase activity is due to a water molecule acting as a nucle- ophilic agent instead of the creatine molecule.

    INTRODUCTION

    It was first suggested to us by Dr. M. COHN* that the crystallized creatine kinase (ATP- creatine phosphotransferase, EC 2.7.3.2) we had provided for her experiments contain- ed a trace of ATPase activity. Assay of other samples of enzyme preparations showed this to be generally true. This hydrolyzing activity is of such a low order of magnitude that by comparison with the creatine kinase activity, the ATPase activity may be considered absent as has been previously reported 5. It is only under conditions de- signed to detect very low catalytic activity that hydrolytic activity is apparent. The reversible kinase reaction is shown in Eqn. I and the ATPase activity in Eqn. 2.

    MgATP 2- + creatine ~- MgADP 1~ + creat ine-P 2- + H + (i)

    MgATP 2- + H20 ---> MgAD Px- + Pl*- + H + (2)

    This trace secondary hydrolyzing activity of a phosphate transferring enzyme recalls the very similar ATPase activity of hexokinase (EC 2.7.1.1 ) reported by

    Abbreviat ions: PCMB, p-chloromercuribenzoate; NBS, N-bromosuccinimide; NEM, N- ethylmaleimide.

    * Present address: The Tokugawa Inst i tute for Biological Research, and the Depar tment of Biochemistry, Univers i ty of Tokyo, Tokyo (Japan).

    Biochim. Biophys. Acta, 81 (1964) 27o-279

  • ATPAsE ACTIVITY OF CREATINE KINASE 271

    TRAYSER AND COLOWICK 6, and KAj I , TRAYSER AND COLOWICK 7. These authors found the ratio of ATPase to hexokinase activities to be about 5-8" io -8 and found no evidence on the basis of further experiments for the formation of an enzyme-phosphate inter- mediate. Similar to hexokinase, evidence on creatine kinase does not support the post- ulate of the formation of a phosphoryl-enzyme intermediate s. For creatine kinase, kinetic and other studies3, 8 support the postulate that there are two adjacent sites on the enzyme, one for binding the adenine nucleotide and the other for binding creatine or creatine phosphate. In terms of enzyme mechanism it appears that in ATPase activity, water merely substitutes for the creatine molecule.

    Evidence is presented below that ATPase activity of creatine kinase is not a contaminant of the enzyme preparations but is a property of the enzyme molecule. Studies of some characteristics of the ATPase activity show close similarity to the properties of the kinase activity.

    MATERIALS AND METHODS

    ATP-creatine phosphotransferase

    The enzyme was purified from rabbit skeletal muscle3,9,1 and crystallized at least twice. Specific activities under the standard conditions in the presence of MgSO 4 and the method of activity calculation using the second order rate constant were generally in the range 65-72 units/mg. The enzyme was stored for use as a concentrated solution (50-60 mg/ml) after thorough dialysis against o.ooi M glycine buffer (pH 9). Protein was determined by the biuret procedure of GORNALL, BARDAWlLL AND DAVID 11.

    Reagents

    Salts used were reagent grade.Glycine,gtycyigiycme,hlstidine and creatine hydrate were A grade (California Corporation for Biochemical Research). Nucleotides were the highest grade available from Sigma Chemical Company. Other chemicals and their sources included NEM (Schwarz Bio Research, Inc.), NBS (Matheson, Coleman and Bell), Tris (Sigma Chemical Co,).

    Enzymic assay

    Creatine kinase activity was me3:sured by determining the creatine phosphate formed under the standard conditions 9.

    ATPase activity was measured by the inorganic phosphate liberated from ATP at 3 0 , correcting for the blank mixture without enzyme incubated for the same time intervals. The io-ml final volume reaction mixture was composed of o.I M glycine buffer (pH 9.o), 6 mM magnesium acetate, i mM ATP added at zero time, and an en- zyme concentration of about 6 mg/ml. At o, 20, 30 and 4 min, 2-ml aliquots of the incubation mixture and the blank were each pipetted and rapidly mixed with i.o ml cold 24% trichloroacetic acid contained in separate tubes in an ice bath. Sample and blank were treated in the same way. The denatured protein was rapidly removed by using Whatman No. 40 filter paper on pre-cooled glass funnels and the filtrate was

    Biochim. Biopkys. Acta, 81 (1964) 27o-279

  • 272 T. SASA, L. NODA

    collected in tubes in an ice bath. Inorganic phosphate in 2 ml filtrate was determined by the KING procedure 1~ (added 6 ml 8% perchloric acid, I ml 5 % ammonium molyb- date, o.4 ml I-amino-2-naphthol-4-sulfonic acid solution and water to a total volume of IO ml in graduated test tubes). After standing at room temperature exactly IO min from the time of addition of the last reagent, the absorbancy at 66o m/, was read in a Beckman DU spectrophotometer and compared with the absorbancy of a standard containing o.5oo #mole phosphate.

    Care was taken during trichloroacetic acid treatment to keep the solutions cold in order to minimize ATP hydrolysis. The initial rate of hydrolysis was determined by plotting the P1 values against time and it was found to be linear up to lO% hydrol- ysis of the total ATP.

    RESULTS

    The stoichiometry of the ATPase activity indicated in Eqn. 2 was shown by incubating for periods up to 3 h at 3 o 0.07 mM enzyme with I mM ATP, 6 mM magnesium acetate, and o.I M glycine buffer (pH 9), followed by paper chromatography is. In- creasing amounts of Pl were shown by the inorganic phosphate determinations. In- cidentally, it was observed that ADPase activity of an enzyme preparation was less than 5 % of the ATPase activity, both being measured at 3 in glycine buffer (pH 9).

    Activity ratio during purification

    Creatine kinase and ATPase activities determined for the various fractions of the purification procedure ~ are shown in Table I. With increasing purity of the fractions and increasing creatine kinase activity the ATPase activity decreases. Early fractions compared to later purer fractions are very high in the hydrolyzing activity, possibly because of the presence of myosin. After crystallization the ratios of ATPase to kinase activities are constant with repeated crystallization. Table II shows the constancy of the activity ratio during repeated precipitations of the enzyme from a solution containing 3 mM MgSO, and 60 vol.% of 95% ethyl alcohol.

    TABLE I

    CREATINE KINASE AND ATPAsE ACTIVITIES DURING ENZYME PURIFICATION*

    Incubat ion cond i t ions fo r k inase act iv i ty : i m~~ ATP , 6 mM MgSO, , o.o2 4 M c reat ine , o . I M g lyc ine , a t pH 9 and 3 o. Cond i t ions fo r ATPase act iv i ty : I mM ATP , 6 mM magnes ium acetate , o . I M g lyc ine , a t pH 9 and 3 o. The amount o f enzyme taken was ad jus ted fo r the par t i cu la r

    assay . Speci f ic ac t iv i t ies a re expressed in / *moles P i p roduced] ra in / rag .

    Crea3ine kinase A TPase A TPase (units/rag) (unitslmg) Crealine kinase

    Fract ion I 5.7 o . i 1 .8. lO -2 F ract ion I I 29 0.02 7 . 1o -5 F ract ion I I I 45 o .o i 22 . lO -5 F ract ion IV 54 i lO -3 1.9" lO -5 is* c rys ta l l i za t ion 65 0 .84" IO -8 1.3" IO-5 2nd c rys ta l l i za t ion 66 o .86 . io -s I . 3 IO -s

    * See ref. 9.

    Bioch im. B iophys . Acta, 8 i (1964) 27o-279

  • ATPASE ACTIVITY OF CREATINE KINASE 273

    TABLE II

    CONSTANCY OF ATPAsE/CREAT INE KINASE RATIO DURING REPEATED PRECIPITATION AND DIALYSIS

    The enzyme was precipitated at 3 mM MgSO 4 and 60 vol. % of 95 % ethanol at -- i o and dialyzed against cold o.ooi M glycine (pH 9.o), equal in vo lume to about 3ooo volume of enzyme

    solution. Assay as in Table I.

    A TPase A TPase Precipitation Crea$ine kinase (units/rag x xo ~ and dialysis (units/rag) x zo* ) Creatine kinase

    Original sample 63 o,81 1. 3 Once 65 o.83 t.3 Twice 63 0.86 1. 4 Thrice 63 0.80 i. 3

    I I I. I I = l

    .1o

    ~ 3.o 7 / / / , / 7 7 0.03 ~ ~ o

    2O Tube number

    Fig. I. Chromatography of enzyme on 2 I9-cm colurr~ of DEAE-cel lulose. Freshly prepared and twice crystal l ized enzyme. 17 mg was eluted by O.Ol-O.O 5 M Tr is -acetate buffer (pH 8.05) (total vo lume 30o ml) wi th l inear gradient. Flow rate was about io ml /h and collector was set for 5-ml fractions. E luates were concentrated by precipitat ion as in Table II. -- , absorbancy at 280 m/~ (left-hand ordinate) ; . . . . . , concentrat ion of Tris--acetate buffer ( f ight-hand ordinate).

    Tube Creatine A TPase A TPase kinase (units~rag x zo ~

    No. (units/mg) x zo s) Creatine kinase

    37-38 58 0.77 1.3 39 63 o.74 1.2 4 6o o.75 1.3 41 64 o.86 1. 3 42 6o o.75 1.3

    43-45 58 o.7o 1.2

    Bioch im. B iophys . Acta , 81 (i964) 27o-279

  • 274 T. SASA, L. NODA

    DE AE-cellulose chromatography

    In further attempts to separate ATPase activity from creatine kinase activity, DEAE-cellulose chromatography was run in a cold room using linear gradient elution, o.oi to 0.05 M Tris buffer (pH 8.05). A sample of twice crystallized enzyme containing 17o mg protein was placed on a 2 29-cm column which had been thoroughly washed and equilibrated. Absorbancy at 28o m# was measured on each 5-ml sample from the fraction collector. Since a comparatively large quantity of enzyme is required for the ATPase activity measurements, when it was deemed necessary, several fractions were combined and the protein was precipitated in 3 mM MgSO4 and 60 vol.% of 95% ethyl alcohol at --lO% After centrifugation the precipitate was dissolved in o.ooi M glycine buffer (pH 9.0) and dialyzed overnight against the same buffer. Conditions for creatine kinase and ATPase assay are given in Table I. In preliminary trials it was found that enzyme solutions which had been stored near o for long periods as a con- centrated solution in o.ooi M glycine (pH 9.0) showed small additional peaks having ATPase/kinase ratios greater than that of the main peak. Even in these cases, however, the ATPase/kinase ratios ~ of the main peak did not change on re-running the sample through a second column.

    Fig. I shows the elution pattern of a flesh enzyme preparation. A single protein peak is found. Enzymic activities of various cuts are indicated in the legend of Fig. I. The ratio of ATPase to kinase activity is essentially the same for all fractions, thus supporting the proposition that the ATPase activity is a secondary property of the creatine kinase molecule and not a contaminant of the enzyme preparation.

    Effect of various treatments on enzymic activities

    In the following experiments unless otherwise specified enzyme concentration was adjusted to a final concentration of 2o mg/ml in o.o5 M glycine buffer (pH 9.o) and temperature was maintained at o with an ice bath. I f other buffers than glycine or a chemical reagent had been used, the protein solution was dialyzed overnight against o.ooi M glycine buffer (pH 9.o). I f insoluble protein was present at the time of assay, it was removed by centrifugation at o . Protein was determined by the biuret procedure 11 and enzymic activities measured under conditions given in Table I. Table I I I summarizes the data for inactivation by a varie~r of agents. The agents include those known to cause denaturation of proteins (heat, acid and urea) and group specific agents (NBS reacting with tryptophan and other grbups14, ~5 and mercuric chloride, PCMB and NEM reacting with the sulfhydryl group). In the various treatments it is seen in Table I I I that the ratio of the ATPase activity to creatine kinase activity is essentially constant with variation of the condition of treatment and furthermore, that the ratio is very nearly the same for all the different kinds of inactivation proce- dures.

    Some experiments were done using PCMB as the inactivating agent. At pH 7.4, o.oi M glycylglycine buffer, ATPase as well as kinase activity was decreased in the presence of PCMB, but on treating the enzyme at pH 9.o, glycine buffer, only the ATPase activity was inhibited. This is the only exception found to the constancy of the ratios of the two enzymic activities by treatment of the enzyme.

    These experiments clearly show that treatment leading to the decrease of one of

    Biochim. Biophys. Acta, 8i (I964) 270-279

  • ATPAsE ACTIVITY OF CREATINE KINASE 275

    TABLE II I

    THE EFFECT OF VARIOUS TREATMENTS ON ENZYMIC ACTIVITIES

    Enzyme samples were treated for 3 min except as indicated, while at a concentration of zo mg/ml in o.o 5 M glycine buffer (pH 9) unless otherwise indicated and at o (except 5 o heat treatment), followed by exhaustive dialysis against o.ooi M glycine buffer (pH 9) and centrifugation at o c

    if precipitation occurred. Assays were run under conditions indicated in Table I.

    Condition Creatine kinase A TPase A TPase Treatment varied (units/rag) (units/rag x xo s

    zo s) Creatine kinase

    Heat (5 o) Acid (pH 2.I)** Urea

    bIBS***

    NEM

    Mercuric chloride

    PCMB

    o.o5 M glycyl-glycine

    (pH 7.4)

    PCMB

    5 miD* 20 O.23 1.2

    60 in.in* 35 0.45 1.3 7.o M* 21 0.27 L3 NBS

    io* 19 o.26 1. 4 enzyme NEM

    8* lO o.13 1.3 enzyme HgC1,

    3" 20 0.30 1.5 enzyme PCMB

    o 56 0.67 1.2 enzyme PCMB

    I.O 33 0.43 1.3 enzyme PCMB

    1. 5 25 0.28 I.I enzyme PCMB

    2.0 14 o.17 1.2 enzyme PCMB

    o 62 o.8o 1.3 enzyme PCMB

    I.O 60 0.60 I.O enzyme PCMB

    1.5 65 0.44 0.67 enzyme PCMB

    2.0 61 0.26 o.4z enzyme

    * Four or five lower values including control, not reported here, gave similar values of decreased creatine kinase and decreased ATPase activities compared to the control but with essentially the same ATPase/creatine kinase values,

    ** o.0 5 M glycine-HC1. *** o.o 5 M acetate (pH 5.2).

    the cata lyt ic p roper t ies of the enzyme s imul taneous ly results in a cor respond ing loss

    of the o ther cata ly t ic p roper ty , thus suggest ing the over lap, i f not the ident i ty of the enzymic s ite for the two act iv i t ies.

    Kinetic properties of ATPase compared with creatine kinase

    App ly ing the analys is prev ious ly used for the creat ine k inase fo rward react ion 3 to the AT...

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