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 myosin solution so obtained (200 pg,"l) was kept in a plastic (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
>YIO.'
,*IO-J
'a++
3x
\
OUR.
1
.,
L--
0'
Y
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).
I
Hk C,H la
cwritiibut(+ t o t the iiaphthol to itig this compound with I’OCl, or 1’C tiori of the corresponding plioiphatc, it is thus iiere-b:ir\ to overcome the hydrogen bonding iii order to fnvilitatc the reaction. Favorable corictitiorih for the prcpuratioti of this substrate can he fouiicl i n the esperimerital pal t . Two of’ the by-produc*t.;of tliih rea~tioliare ideiitifietl. .I diester of isoprop? 1 ?-iiaphth> 1-3-c-arboxjiitiilid(~ phosphate (111) \vas i5ol:itccl. when isopropj 1 :11(~0hol cwlicetitrattioti
aiitl
atid substantivit~of
0
O[;CH(CH /I
t IIC‘
J-
t IN,
CONHC,H,
I1I
u w l a- it component oi the c.hromatographic4 wlem. This c*ompouiitl \\ ti- i i o t h j drol>zed h \ intestinal alkaline phosphatase, hut m u l d be I i \ drol>zed by liver and kidric.> e i i q inch in tissue experiments. Xlqo isolated wa- the caorrehponding p j yophosphate (IT)whivh w : ~ i iiot unanticipated iinc(b :I ith
Experimental Section ,111 ~ - l i ~ ~ i l i ~ ~ ~ ~ ~ - ~ ~ -t i i( ,ii i :l ~ :~iiiIidi~ ~ i l i ~ J ~ i ~ ii ii ~ ~ i , ~ ~ l: tii 1 ~ 11 l i ~ ~ l - . ~ derivative> arc>piii’ified fi,om c~ornniercialranipleh (Pfihtcr (’tivniitallimtioii f i u m 31eC>ONl\le2n i i d 1IeOIi. Preparation of 2-Hydroxy-3-naphthoicAcid Anilide Phosphate. A. POCIa.--~-~I?.(li,o~~-~~.naphthoic. avid anilide (napht,hol-AS) (.;.% g, 0.01 mole) 5 .lispended in anhydrous THF (130 ml) : I [ 60’. POC&(3.68 g, slight excess) tdded with stirring. Aftei, 20 min, pyridine (1.9 g ) was added and the mixture was warmed for 1.J min. ,ifter the yeacation was allowed to stand at room temperature overnight the pyridine hydrochloride ( 2 - 3 g) wa5 removed by filtration. This precipitat>e contained a small amount. of iiaphthol-Ad. The solvent,was removed under redurrd p i ’ w s i i y e xith 3111 efFic.it,lit i ~ i i a r yevaporator, and an oily yellow hillid reniaiiieci. ?‘hi,< h o l i t l W:L> dirsolved iri a small ~ I I I C I ( I I I ( i f : i < v t o i I ( - : c i i d q i : i i , : i l c t l h y d(,(wit:ttioii froni the tiaphthol-AS, \ v l i i i * l i i h *p:iriiigly -i~liiiiii~ ill : i i v t i i i i v , Tliv i + w t i i i i v s o l i i l i o i i \v:I:
September 1965
NOTES
then concentrated to give R crude pruduct which showed three spots upon hydrolysis and paper chromatography: Rr 0.78 (AS), Rt 0.90 (cyclic dimer), Rt 0.22 (AS - PO4). Thus, V could be isolated in a very low yield by paper chromatography, as seen later. B. With PCls.-Naphthol-AS (1.3 g, 0.005 mole) was SI& pended in 50 ml of dioxane and PCL (2.6 g, 0.0125 mole) was added with stirring. The reaction was exothermic and a clear yellow solution was obtained after continuous stirring. Pyridine (0.09 g, 0.0125 mole) was then added after 10 min, and the mixt,ure was $lowed to stand overnight. Pyridine hydrochloride (0.4 g ) w a ~removed by filtration. From the precipitate, 33 mg of naphthol-AS was recovered. The dioxane solution was poured into ice water with stirring. A light yellow crystalline precipitate was obtained which was collected and treated with 1 N Na2C08. Napht,hol-AS (0.7 g) was %gainrecovered as an insoluble product. T h e alkaline solution was now treated with Amberlite-IR-120 and lyophilized to obtain 225 mg of the crude napht.hol-AS phosphate. An additional 339 mg (yield 35%) of the AS phosphate was recovered from the dioxane rolution. This criide acid is adequnte for most histochemical p u r p o s q hut is not pnre enough for Huorescence study as its paper chromatogram shows three spots, Rr 0.21 (AS phosphate), Rr 0.80 (AS), and R r 0.!)5 (cyclic dimer), even though the latter two spots are weak. Purification of Naphthol-AS Phosphate by Paper Chromatagraphy.-Since the analytical sample and bhe ammoniiim Halt are obtained irom extensive purification and commercial ssmple' of this and related phosphates is now available irom various SUIIICBS, it is necessary for 11s to include the purification of this compound here. Ascending paper chromatography was used in testing. When BIOH was used, the Rr values were very close. Only CPrOHNHO€l-H20 (7:1:2) wss iound to he useful for paper strip separation (Rr 0.20 (AS phosphate), Rt 0.78 (AS), Rr 0.80 (cyclic dimer)). Preparstive paper chromatography employed 20 mg uf compuund dissolved in 0.5 ml oi MenCO, spotted on four sheets of Whatman No. 1 paper of 2Scm widt,h, and ehromatographed, using the i-PrOH-NHs-H20 system. The low Rr and high Rr value strips were cut out and exbracted with warm H 2 0or IllelCO. A low Rr compound (14 mg) was analyaed and found to he the iaopropyl ester of 111. 4nd. C . I ~ H * ~ N O ~ PNHJ 9HsO: C, 42.55; H, 7.26; N, 4.96; P, 5.50. Found: C , 42.76; H, 7.31; N, 5.05; P, 5.40. After extensive drying on PzOr, a trihydrate could be obtained (P: calcd, 6.80; found, 6.80). B e cause of the low P content, this compound cannot be a triphosphat,e. Titrat,ion with 0.1 N NsOH on the iree acid ( p w pared imm Amberlite Ill-120) shows basic monoacid and has the same equivalent, M expected. I r spectroscopy shows an i-Pr hand a t 7.60 p (1370 em-'). The high R r compound agrees with the expected IVa as a monoammonium salt and as x dihydrate. A1~al.Ca.H.,N,O,P, NHa 2H.O: C, 56.68; H, 4.57; N, 5.82. Found: C, 55.83; H, 4.08; N, 5.82. The ir spectrum supports the cyclic structure based on the presence of II band st 1660 cm-' (-C=N-), the absence of a hand at 1650 c m P (amide I1 hand), and a strong band at 1375 em? in the -P-O-C deformation region. Ascending Paper Column Preparatory Chromatography.-The paper cnlomn was packed with Whatman cellulose powder CF-1 I in R tapered column to ahoot 12 cm (1,Fmm diameter). Five uf these were ,wed for 101) mg of the sample. After the compound was applied to the bol,tom of the t,nhe, chromatography was done with the us~tsli-PrOH-NHs-H,O (7:1:2) in B large graduated cylinder. Solvent may be added if n e w s m y to maintain the reservoir at, the bottom of 1,hegraduated cylinder. It took 1-1.5 hr tu complete this development and the column was pushed out while we1 and c u t out under light into five portions and extracted (HtO). From the top, 28 mg was recovered as the pyrophosphate. The next, layer contained a violet fraction, Rt 0.23 (0.8 mg), which ix AS phosphate; the third fraction oontrtined 69 mg, Rr 022. The ir spectrum showed t h s t they are the same monoammonium salt. This salt was converted into the free acid by treating with IR-120 and then lyophilized. The reulting acid showed a weak 1 6 6 0 - ~ m -hand, ~ sintered st 110-120", and melted a t 240-243'. A n d . ClrH14NOsP~2HzO:C, 53.78; H, 4.74; N, 3.70; P,8.17. Found: G54.10; H,4.41; N,3.99; P, 8.34. Dilute NH,OH was used now to convert this free acid to the
moiioe.mmwiium ti& which is readily soluble in HIO aiid is useful for fluorescence atudy. Fluorescence Assay.-Fluorescence measurementr were carried out on an Amineo fluorometer and recorded with an X-Y recorder. Naphthol-AS was dissolved in 0.1 N NaOH and was found to be excited at 240 mw to give fluorescence maxims at 520 w, whereas the phosphate has no 520-mp emission. Enzymatic hydrolysis uf the phosphate was therefore carried out in glycine buffer and its product could be detected as low as 0.01 wg/ml. Histochemical Study.-Fresh frozen sections of tiaaue were cut on a Pearse cryost,at and incubated immediately for alkaline phosphatase with the lollowing substrate d u t i o n , in a small coupling jar: nsphthol-AS phosphate smmonium salt (0.1 mg), 3 ml of glycine buffer (pH 9.8),3 ml oi 2 M NaCI, and 1 ml of 1% I\iIgCL. Alter 30 mi", the sections were rinsed quickly in distilled Ha0 and mounted on glycerol for examination. They can also be dehydrated in the usual manner and mormted in Histoelad. T h e bluish green Huoreseence of the dye can be seen vividly along thevilli (Figure 1). I n the case of the intestine, leucocytea and macrophaga are also stained.
+
+
1099
stlalod by lhe Hnurcsveru.c mcl hod.
+
+
For acid phosphatase, ~alciom-furmalin-fixed sections were wed and citrate buffer (pH 5.0) was used in place of the glycine buffer. There was conspicuously more shining in the peribiliary region of liver when compared with the 5-iodoindoxyl method.6 At least part of this activit,y is attributed to phosphodiesterase activity. An applieat,ion of this method to glioma. ahowed brilliant yellowish fluorescent tumor cells. I n Leydig cell tumor of the rat (Figure 2) this method reveals ditiwentid stitin around tumor cells and some clearly in the nucleus.
Figtire 2.-Acid
p h ~ ~ s p l u ~inl ~rill ~ ~ 1,eydir c rell tumor with fliarrrceirce phase coutvsst.
Acknowledgment.-We wish to acknodedge the generous cooperation of Dr. Gerald Peskin through the use of his Aminco spectrophotometer in part of this work, and Miss Diana Weiss and Miss Patty Lu Kaliher for the fluorometric study.
