1097
Septenibcr 196s cm column of Dowex 2 resin (XS, 200-400 mesh, C1-) a t 4'. The entire sample was passed, at 15 ml/min, through the column, which was then washed with H20 (1 1.) followed by 1 1. of 0.003 .V HC1. The fractionation of the products was achieved by elution, at the same rate, with successive 1-1. portions of 0.003 A' HCl containing increasing concentrations of LiCl, the first portion being 0.0055 -1.I in LiCl, the second 0.011 M , the third 0.0163 M , etc. One-liter fractions were collected and stored in the cold room. Total and acid-labile (10 min at 100" in 1 Ar H2S04)P were determined on 0.3 ml of each fraction by the procedure of Bartlett.8 The acid-stable P (total minus acid labile) was found in three well-separated peaks. Peak I (1.8 mg of acid-stable phosphorus) appeared in fraction 4, eluted with 0.022 i1.I LiC1. It. was probably a mixture of methyl phosphate and inorganic phosphate. Peak I1 (fractions 13-15) contained 23.7 mg of acid-st,able P but very little acid-labile P. It is believed to consist primarily of P1,P2-dimethyl pyrophosphate. Peak 111 (fractions 27-31) contained approximately 60 mg of acid-stable P (59% of the starting methyl phosphat'e). All fractions in this peak had a ratio of total P/acid-labile P of close to 1.3. Inorganic polyphosphates were eluted shortly after this peak 111. Isolation of the Lithium Salt of MTP.-The fractions of peak I11 were pooled and brought to p H 7.3 with 1 iV LiOH. The solution was concentrated at 40" in a flash evaporator t o about' 50 ml, then 64 ml of MeOH was added. The solution was filtered through sintered glass and the flask and the filter were rinsed with hIeOH (30 ml). To the filtrate was added acetone (1880 ml). The precipitate, which formed immediately, was collerted, after 18 hr in the cold, by decantation and centrifugation and was washed six times with ;\leOH-Me&O (1:20) and three times with dry ether. The product was dried overnight under vacuum at room temperature over P2Oj and weighed 644 mg. A n a l . Calcd for CH3Li4O10P3.H20: P, 29.6. Found: P , 29.1. The ratio of total/acid-labile P was 1.48. Less than 1% of the P was present as inorganic phosphate. S o C1- was detect,ed. Titration between p H 8.5 and 4 gave one acid group per three P atoms. Hydrolysis of M T P by Myosin.-A sample of myosin from rabbit skeletal muscle was kindly provided by Dr. G. Richards of the Department of Sutrition, University of California, Berkeley. I t had been prepared by the method of Tonomura and coworkerslo and preserved a t - 18" in 50y0 glycerol. The protein concentration was determined by Lolvry procedure11 with standardization by Kjrldahl. Possible changes in the color yield of the Lowry proredure were checked using albumin as a standard. h solution in 0.5 111 KCI was obtained after precipitation of the myosin by dilut,ing with water (IO: l), centrifugation, and redissolution in and dialysis against 0.3 M KCl (pH 8.1 with 1 m J l Tris-chloride buffer). Glass-distilled water was used in all procedures. T h e was kept in a plastic myosin solution so obtained (200 pg,"l) (cellulose nitrate) tube and in ice. Conditions for the AITPase and ATPase assays were as follows: O..? X KCI, 5 mJ1 CaC12,0.5 m J l substrate, p H 8.2 (Tris-chloride buffer 0.02 M),temperature 2,j". The substrates were used as K + salts, obtained by passing their solutions through a column of Dowex 50, K+. The enzymic reaction was started by adding, with magnetic stirring, 0.5 ml of the 3 m M substrate to 2.5 ml of a solution containing the other components, including myosin (100 pg). The reaction was stopped by adding 1 ml of a 207" trichloroacetic acid solution. After filtration, Pi was determined in 3 ml of the filtrate by the phosphomolybdate extraction method of Dreisbach,lZ 4 ml of the xylene-i-BuOH solvent being used for the extraction. The enzymic activity of the myosin, obtained from the amount of Pi released after a given time and expressed as micromoles of Pi per minute per gram, was found to be 390 with ATP and 48 with MTP. Azuma and c.oworkers4ahave reported that ribose triphosphate, another simple ATP analog, is hydrolyzed, also in the presence of Cat+, at one-twentieth the rate of ATP. However, we rioted that the loss of activity during storage in ice was not parallel for the two substrates, the ATPase being better preserved than the MTPase. Afkr 35 days the ATPase (9) G. R. Bartlett, J . B i d . Chem.. 234, 466 (1959). (10) f a ) T.Tonomura, P. Appel, a n d hl. Norales, Biochemintry, I , 515 f1966); ( h ) E. G. Richards, C . 4 . Chung, D. B. XIensel, and H. S. Olcott, i h t d . , 6 , 528 ( l 9 6 i ) . (11) R . .J. Henry, "Clinical Chemistry, Principles and Technics." Hoeber Medical Division of Harper and Row, Ne\\- Tork. N . T..lY6-i. p 190. (12) R . H. Dreisbacli. A n d . Bioehem., 10, 169 (lYti5).
i
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OUR.
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.,
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Figure l.--Ca2+ dependence of myosin methy triphoephalase Experimental conditioiis: 0.3 M KC1, 0.3 mdl AITP, pH 8.2, 2.3' activity was still at 70% of the original value while the MTPabe had declined to 27%. The loss of AITPase activity with time appears to be a first-order reaction with a half-life period of about 19 days in our conditions of storage of the myosin. The dependence of XTPase activity on Ca2 concentration is shown in Figure 1. Xaximum activity was obtained when Ca*+was about 5 mdl. So activity m-as observed in the absence of Ca2+. Green and lIommaerts,13 working in somewhat ditferent conditions (0.13 M KC1, p H 9.0), have reported an optimal Ca2+ concentration of approximately 1 mM in the case of ATP. +
Acknowledgment.-I am indebted t o Dr. J. Eiler and to Dr. 11. Alorales who sponsored this ~ o r and k to Dr. G. Richards for gifts of myosin arid much helpful advice. (13) I. Green and I\ E'. f i . A1 XIolnmaert6. J . Btoi (1954).
C l i e m , a l 0 , bY5
2-Hydroxy-3-naphthoic Acid Anilide Phosphate as a Fluorescent Histochemical Substrate for Phosphatase' K. C. T s o ~A N D
si0.10A 1 1 ~ s u ~ ~ i i . i
Harrison Depart?tient of Surgical Research, School of d l e d u n e , Cniversity of Pennsylvunia, Philadelphia, Pennsylvania 19104 Received Sugust 24, 1967 Revised J l anuscript Received March
4,1568
The development of fluorogenic substrates for histochemistry of normal and cancer cells is of importance because of the inherent sensitivity of fluorescence measurement over that of the absorbance measurement. Several years ago, Rutenberg, et d ,reported2 the use of 2-hydroxy-3-naphthoic acid anilide phosphate for histochemical demonstration of phosphatase. Because of the presence of a small amount of naphtholAS, a simultaneous coupling procedure was recommended and fluorescent study was not possible. A more detailed study was therefore undertaken in order (1) This work was supported by Research Grant AT-30-1-3784 from t h e
U. S. Atomic Energy Commission and U. S. Public Health Grant C.1 07339. (2) A. AI. Rutenberg. R. J. Barrnett. K. C. Tsou, B. Alonis, and R. Teague, J . Hislochem. Cytuchem., 6 , 90 (1998).
