z86 P R E L I M I N A R Y NOTES

On the activation of myosin ATPase by EDTA PN 61007

The activation of myosin ATPase (ATP phosphohydrolase, EC 3.6.1.3) by EDTA at high ionic strength, first described by FRIESS 1 and studied by many workers si.nce, is still unexplained. The assumption of removal of some inhibiting metal ions by chelation was excluded in the experiments of FRIESS z by the fact that after treatment with EDTA and removal of the reagent, the activity of myosin remained unchanged and the enzyme was still activated by the chelating agent. The other plausible possi- bility, i.e. a binding of EDTA to the protein, was excluded by ultrafiltration experi- ments *. The fact that EGTA, a chelating agent which binds.Ca (but not Mg) does not enhance ATPase activity 3, plus the finding that the enzymic activity is enhanced by EDTA only for those nucleotidetriphosphates, the hydrolysis of which is inhibited by Mg (ref. 4), made it worthwile to reconsider the possibility that EDTA activates by depressing the concentration of ionized Mg present.

We determined the ATPase activity of myosin using systematically varied ad- ditions of Mg and EDTA. We then evaluated the results in terms of the free Mg, taking into account the equilibrium of Mg with EDTA and ATP. (For the method of calculation see ref. 5-) The results, ATPase vs. log Mg, showed a dependence o11 the concentration of free EDTA. However, it was clearly seen, that concentrations of Mg as low as IO -e M can event an influence. We estimated the Mg in the ultrafiltrate of our standard test solution including all additions except Mg and EDTA, by flame spectrophotometry, using the Unicam SP 900 instrument, and we found the concen- tration of Mg to be several times io -s M. If we take this initial concentration of Mg into account ill the calculation of the concentration of free Mg in the different samples, the dependence on free EDTA practically disappears as shown in Fig. I, the activity depending only on the concentration of free Mg. The curve of ATPase vs. log Mg which was obtained, resembles a simple dissociation diagram. The half maximum activity was found to be at about 7" IO-~ M Mg.

According to our considerations if activation by EDTA is caused by the de- pression of the concentration of free Mg, in a system free of Mg there should be an enhanced activity and a diminished activation by the chelating agent. We found that this was the case after the purification of the components of the system. We purified the salt solutions and myosin by passing them through a column of Dowex AI. ATP and distilled water were passed through a column of Dowex 50. (The chief source of Mg contamination was ATP, our commercial ATP, Reanal, Budapest contained one atom of Mg per IOO mole of ATP.) We found that, in the purified systems in all experiments, the activity of myosin was enhanced 200-300% relative to the unpurified system; in the case of seven preparations the specific activities were found between 0.095 and o.261 #M P per min/mg. Accordingly, the activation by means of EDTA was much depressed in all experiments. A typical example is given in Table I.

Our results are in line with a great number of published data concerning the different "modifiers" which activate myosin. These activations are generally thought to be caused by the supression of an inhibition resulting from the binding of the

Abbreviations: EGTA, ethylene glycol bis (~-aminoethyl)-N,N'-tetraacetic acid; PCMB, p-chloromercuribenzoate.

Biochim. Biophys. Acta, 89 (1964) 186-188

PRELIMINARY NOTES 187

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F i g . i . ATPase activity of myosin in relation to the concentration of free Mg in the presence of different concentrations of EDTA. I mg/ml myosin, prepared according to WEBERS; 4 ° mM Tris-HC1 buffer (pH 7.o) ; 4 mM ATP; 4o0 mM KC1; temperature 2o°; time of hydrolysis varied to obtain measurable amounts of phosphorus, but remaining below 2 5 ~o of the hydrolysable phosphorus present. Phosphorus determined according to FISKE AND SUBBAROW 7. The ultra- filtrate of the complete reaction mixture with all additions except EDTA and Mg contained 4.8"IO -s M Mg. Free EDTA concentrations (as calculated): O, none; ×, 5.o. Io-5; o, 5.o. Io-4;

A, 5. o" xo-a. Abscissa log Mg, ordinate / ,a tom P hydrolyzed/miu/mg of myosin.

nucleotidetriphosphates containing NH 2 in position 6 of the ring and involving also Mg ~+ (refs. 8-1o). An experiment in line with this hypothesis is shown in Table II, comparing the inhibition by Mg of untreated myosin with that treated by PCMB. PCMB treatment, by eliminating the -SH groups of the active centre involved in the formation of the unfavourable complex, diminishes the sensitivity of the enzyme towards Mg. At lower concentrations of Mg, there is a depressed inhibition and, at higher concentrations, a substantial activation. If Mg inhibits by taking part in the

TABLE I

A C T I V A T I O N O F M Y O S I N B Y EDTA IN U N P U R I F I E D S Y S T E M S

A N D I N T H O S E P U R I F I E D F R ] g E F R O M M g

Details of ATPase measurement as in the legend of Fig. i. EDTA added as indicated in the table. Purified system: all ingredients, including myosin, purified as outlined in the text ; Mg in the ultrafiltrate of the complete system: less than 2.5.!o -5 M. Unpurified system: common analytical grade reagents, myosin not treated by Dowex AI; Mg concentration of the ultra-

filtrate: 7. i5. IO -5 M.

A TPase activity

EDTA added As lw2om Plmin]mg As ~e'rc~age of adivity

(M × xo'} withou* EDTA

u ~ r ( ~ #uri~l un~rif~ed puri~

none 0.084 o.179 ioo Ioo 2.0 o .193 0 .266 228 149 4.0 o.22o o.27i 260 i52 6.o o .222 o .242 262 135

B i o c h i m . B i o p h y s . A c t a , 89 (x964) i86-i88

1 8 8 P R E L I M I N A R Y N O T E S

T A B L E I I

INFLUENCE OF P C M B TREATMENT ON THE INHIBITION OF A T P A s E OF MYOSIN BY M g

C o n d i t i o n s o f A T P a s e m e a s u r e m e n t a s i n t h e l e g e n d o f F ig . i M g i n t h e u l t r a f i l t r a t e o f t h e c o m p l e t e t e s t s o l u t i o n ( e x c e p t Mg) a s i n t h e e x p e r i m e n t o f F ig . I . I n t h e e x p e r i m e n t s w i t h P C M B , t h e r e a g e n t w a s a d d e d t o t h e s a m p l e s a t b e t w e e n I a n d 2 m i n b e f o r e t h e a d d i t i o n o f A T P , a t a

c o n c e n t r a t i o n o f 4 M / I o 5 g m y o s i n .

Activity with A TPase activity PCMB as

Calculated percentage of Added Mg free Mg PCMB PCMB activity

not added added without PCMB

o 1 .15" IO-S 0 . 0 9 5 ° 0 -035 ° 37 4 .09 • IO -7 I . 17 • IO - s 0 . 0 7 9 0 0 . 0 4 8 0 61 2 .o 4 • lO -6 1.2 • IO - s o . 0 6 8 o 0 . 0 4 8 0 71 4 .09 . IO-e 1 .25" IO-e o . o 4 8 5 0 . 0 3 8 o 79 2 .o 4 • IO - s 1.7 " I ° - S ° . ° 2 4 2 ° , ° 3 3 ° 135 4 .06 • IO - s 2 . I 4 • IO -6 o . o i 2 1 o . o i 2 I i o o 1.95 ' lO -4 6 . 0 0 . IO - s 0 . 0 0 4 5 o . o 1 2 3 274 3 .74 " 1 ° - 4 1.o2 • lO -5 0 .003 ° 0 . 0 0 8 0 267

formation of the unfavourable complex, it is to be expected that its inhibitory action will be lost when the blocking of -SH groups renders the formation of this complex impossible. Similar depressed sensitivity towards Mg was found also with myosin which had been activated by dinitropheno111 and by S- (2-aminoethyl)isothiuronium 1~.

Biochemistry Group of the Department of Phylogenetics and Genetics,

E6tv6s Lordnd University, Budapest (Hungary)

A. Mf3HLRAD F. FABIAN N. A. BIRd

1 E . T. la'RIESS, Arch. Bioahem. Biophys. , 51 (1954) 17. 2 W . J . BOWEN AND T. D. KERWlN, J . Biol. Chem., 211 (1954) 237. s S. EBASHI, F . EBASttI AND Y. FUJIE, J . Biochem. Tokyo, 47 (196o) 54- 4 W . I-IASSELBACH, Biochim. Biophys. Acta, 25 (1957) 365 • s A. MOHLRAD, G. FEKETE AND IN'. A . BIRd, Acta Physiol. Acad. Sci. Hung. , i n t h e p r e s s . e H . H . WEBI~tR, Biochim. Biophys. Acta, 4 (195 °) I2 .

C. H . FIsKl~ ~ND Y. SUBBAROW, J . Biol. Chem., 66 (1925) 375. s D . GILMOOR, Nature, 186 (196o) 295. 9 H . M. LEVY, N . SHARON, E . M . RYAN AND D. E . KOSHLAND, Biochim. Biophys. Acta 5 6

(1962) 118. x0 j . j . BLUM, Arch. Biochem. Biophys. , 87 (196o) lO 4. 11 H . M. LEVY AND E. M. RYAN, Biochim. Biophys. Acta, 4 6 (1961) 193. a3 M. F . MORALES AND t{. HOTTA, J . Biol. Chem., 235 (196o) 1979.

Received March ioth, 1964

Biochim. Biophys. Acta, 89 (1964) 1 3 6 - 1 8 8