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Chemical modification of tyrosine residues in heavy meromyosin ATPase
3io
BIOCHIMICAET BIOPHYSICAACTA
m~A 35737 CHEMICAL MODIFICATION OF T Y R O S I N E R E S I D U E S IN HEAVY MEROMYOSIN ATPase
K. UCHll)A AND 3". HIRATSUKA Department of Chemistry, Fac*dty of Science, Hokkaido University, Sapporo (Japan)
(Received June 26th, 197o) (Revised manuscript received October i2th, i97 o)
SUMMARY
When heavy meromyosin was treated with a 2oo-fold molar excess of cystamine for 72 h in an ice-bath, most of the free SH groups disappeared, resulting in complete inactivation of the ATPase. The content of free SH groups, ATPase activity and the --b 0 value were restored completely to those of the original heavy meromyosin by further treatment with a 3ooo-fold molar excess of thioglycollate. The effect of diazo-IH-tetrazole and tetranitromethane on heavy meromyosin, whose SH groups were protected, has been studied. Treatment of the enzyme with diazo-IH-tetrazole caused a stoichiometric loss of ATPase activity accompanying the formation of two monoazotyrosine residues. The --b 0 value and the content of free amino groups of the diazotized enzyme were also reduced as the extent of diazotization increased. In contrast, nitration of 2 tyrosine residues with tetranitromethane merely reduced the activity to 60°/'0 of that of the control without causing a change in the --b o value. A possible explanation for the decrease in ATPase activity induced by modification with the two reagents is discussed.
INTRODUCTION The functional importance of cysteine, histidine and lysine residues in myosin and heavy meromyosin ATPase has already been suggested as a consequence of chemical modification and of the study of enzymic properties 1-9. Recently, from experiments on the difference spectrum of heavy meromyosin induced by substrate, some tyrosine and tryptophan chromophores were found to be buried in the protein moiety as a secondary effect of the binding of ATP to the active site of heavy meromyosin 1°-12. However, no proof was given that tyrosine residues actually play a role in the active center. An account is given here on the participation of tyrosine residues in the reaction Abbreviations: I)HT, diazo-tH-tetrazole; monoazo-Z-tyrosine, monoazocarbobenzoxytyrosine. Biochim. Biophys. Acta. 22q (I97 l) 31o-321
MODIFICATION OF TYROSINE RESIDUES
311
of heavy meromyosin ATPase. Diazo-IH-tetrazole (DHT) was proposed as a coupling reagent for histidine residues in protein 13. However, this reagent was found to be unspecific, and in addition to histidine residues it also reacts with tyrosine, cysteine and lysine residuesl4,15. Diazotization of heavy meromyosin, the SH groups of which were protected in advance through the disulfide-SH exhcange reaction using cystamine, resulted in significant changes in ATPase activity and in the conformation of heavy meromyosin. The loss of ATPase activity was correlated with the formation of two monoazotyrosine residues per mole of heavy meromyosin. In addition, nitration of the cystamine-treated heavy meromyosin with tetranitromethanel~, 17 was also examined to get further information on the functional and conformational changes of the enzyme resulting from specific modification of tyrosine residues. The complete loss of ATPase caused by diazotization seems to be due both to modification of tyrosine residues and to structural alteration induced by modification of a large number of lysine residues. MATERIALS AND METHODS Enzyme Heavy meromyosin was prepared by the procedure described by YAGI AND YAZAWAis. The protein was lyophilized in the presence of sucrose and stored at 4 °. Before use, it was dissolved in 2 mM borate buffer (pH 8.o), and dialyzed exhaustively against the same buffer. The molecular weight of heavy meromyosin was taken as 3.5" lO5 (ref. 19). Model compounds N-Carbobenzoxymonoazotetrazole tyrosine was prepared according to the method of SOKOLOVSKY AND VALLEE 14. For preparation of bisazo-derivatives, N-acetyltyrosine ethyl ester and bacitracin were treated with a 3oo-fold molar excess of D H T in 0.67 M KHCO 3 buffer (pH 9.0) for 60 min at room temperature. Treatment with cystamine and thioglycollate To heavy meromyosin solution (20 mg/ml, in 50 mM borate buffer, pH 8.0, and i M KC1) a 2oo-fold molar excess of cystamine over heavy meromyosin was added at 4 °. After an appropriate period of incubation, the mixture was passed through a Sephadex G-5o column (2.5 c m x 30 cm) equilibrated with 2 mM borate buffer (pH 7.o), and IO mlV[ KC1. The fraction eluted without retardation is referred to as cystamine-treated heavy meromyosin. The cystamine-treated heavy meromyosin was then incubated with a 3ooo-fold molar excess of thioglycollate in 50 mM borate buffer (pH 8.0) and 0. 5 M KC1 at 4 °. The excess thioglycollate was eliminated by gel filtration through a Sephadex column. Diazotization with D H T The cystamine-treated heavy meromyosin was diazotized with D H T according to the method of HORINISHI et al. x3 with a slight modification. D H T was prepared by diazotization of 5-amino-IH-tetrazole with NaNO2 at the molar ratio of 1:0. 9. The unreacted NO 2- were not detectable in the D H T solution. The concentration of D H T was not directly determined, but it was assumed from the concentration of Biochim. Biophys. Acta, 229 (I97I) 3to-32I
312
K. UCHII)A, T. HIRATSUKA
5-amino-IH-tetrazole originally used. Diazotization of the cystamine-treated h e a v y meromyosin was carried out at 4 ° for 5 min in o.67 M KHCO3 buffer (pH 9.o). The reaction was terminated b y addition of a large excess of thioglycollate. The mixture was passed through a Sephadex G-5o column. The protein eluted was again treated with thioglycollate to restore the SH groups under the conditions described above. Alternatively, the diazotized protein was precipitated with (NH4)2SO 4 (6o% saturation) and dissolved in a small volume of 5 ° mM borate buffer (pH 8.0) e()ntaining o.5 M KC1 and a 3ooo-fold molar excess of thioglycollate. The mixture was allowed to stand overnight at 4 °, and then dialyzed against 2 hiM borate buffer (pH 8.o) at 4 °. A control sample was treated in the same m a n n e r but without addition of D H T . Nitration with tetranitromethane
Nitration of the eystamine-treated heavy meromyosin was carried out according to the method of SOKOLOVSKY et al) 6 with a slight modification. Tetranitromethane was saturated in 5 ° mM Tris buffer (pH 8.0) and 0.5 M KC1 at 25 °. The concentratkm of tetranitromethane in the solution was determined from the absorbance of nitroformate anions ~ formed by the addition of a large excess of thioglycollate. A calculated volume of tetranitromethane solution was added to the cystamine-treated h e a v y meromvosin (IO mg/ml) under two different conditions: in 50 mM Tris buffer (pH 8.0) and o. 5 M KC1 at 15 °, and in 50 mM borate buffer (pH 7.o) and o.5 M KC1 at 4 °. The reaction mixtures were allowed to stand for 20 min and I5 h, respectively. The nitrated protein was precipitated with (NH4),,SO 4 (60% saturation) and dissolved in a small volume of 5o mM borate buffer (pH 8.0) containing o.5 M KCI and a 3ooo-fold molar excess of thioglycollate. After the incubation, the mixture was dialyzed in the same manner as described in the section on diazotization. The extent of nitration was estimated by measuring the absorbance at 428 nm and at p H 8.o, assuming a molar extinction coefficient of 4zoo for 3-nitrotyrosine residueUL Protein concentration
The protein concentration was determined b y measuring the absorbance at 28o n m using a factor o.63 per mg per ml, and also by the biuret method or the m e t h o d of LOWRy et al. "°.
E~tz3~rne assay The ATPase activity of h e a v y meromyosin and the modified heavy meromyosin was measured in a solution containing, unless otherwise stated, 0.08 mg of protein per ml, 2 mM ATP, 5 mM CaCI~, 0. 5 M KC1 and 2o mM Tris-maleate buffer (pH 7.o). The measurements were carried out at 25 ° in tile I-ml mixture. I n c u b a t i o n was terminated b y addition of I ml of IO °:/O triehloroacetic acid. Pi liberated was determined b y the m e t h o d of Fiske-SubbaRow or by the method of MARTIN AND DOTY21. Specific ATPase activity is expressed in units given as #moles Pi liberated per min per m g of protein. Determination of S H groups
SH groups were determined according to tile method of EIA.MAN22 in which Biochim. Biophys. Acta, 229 (197 l) 31o 32I
313
MODIFICATION OF TYROSINE RESIDUES
excess 5,5'-dithiobis-(2-nitrobenzoic acid) was added to 0.2-0.3 mg of protein per ml of o.I M borate buffer (pH 8.0) containing 6 M urea. Titration of the protein with p-chloromercuribenzoate was also carried out according to the method of BOYER~s.
Determination of amino groups The free amino groups were determined by means of the ninhydrin reaction 24 using leucine as a standard.
optical rotatory dispersion measurement Optical rotatory dispersion was measured with a Jasco automatic recording spectropolarimeter, model ORD/UV-5. The measurements were performed in I - c m ceils at 20 ° in the range from 2800 to 5000 A. The instrument was calibrated to give zero rotation for the buffer blank. The solvent used was 0. 5 M KC1-5o mM borate buffer (pH 8.0). The usual equation of MOFFITT AND YANG25 was used to calculate the parameter, --bo, assuming a ~0 value of 212o A. RESULTS
Disulfide-SH exchange and recovery of the free SH groups The enzyme, at a concentration of about 2o mg per ml in i M KC1-5o mM borate buffer (pH 8.o), was treated with a 2oo-fold molar excess of cystamine over heavy meromyosin at 4 ° before the ATPase activity and the content of free SH groups were determined. As shown in Fig. I, an initial increase of activity was observed during I h by which time the activation had reached its maximum. Concomitantly, about one-third of the free SH groups disappeared at a rapid rate. On prolonged treatment, the decrease in activity was accompanied by a more gradual disappearance of the other free SH groups. After 17 h of treatment, 6- 7 moles of
f
6
z 4
q ~ 0.~
5 2 v---
o
c*
"o
,= ~
,~"~ ~5
t i m e o hours
Fig. I. Effect of c y s t a m i n e on t h e A T P a s e a c t i v i t y of h e a v y m e r o m y o s i n (HMM) a n d reversal o f t h e effect c a u s e d b y t r e a t m e n t w i t h thioglycollate. H e a v y m e r o m y o s i n , 20 m g / m l in I M KC1 a n d 5o m M b o r a t e buffer (pH 8.0) a t 4 °, was t r e a t e d w i t h a 2oo-fold m o l a r excess of c y s t a m i n e over h e a v y m e r o m y o s i n . A f t e r 72 h o f t r e a t m e n t , t h e c y s t a m i n e - t r e a t e d h e a v y m e r o m y o s i n , 5 m g / m l in I M KC1 a n d 5 ° m M b o r a t e buffer (pH 8.o) a t 4 °, w a s f u r t h e r t r e a t e d w i t h a 3ooo-fold m o l a r excess o f thioglycollate. T h e A T P a s e a c t i v i t y ( O ) a n d t h e c o n t e n t o f free S H g r o u p s ( × ) were d e t e r m i n e d i m m e d i a t e l y a f t e r gel filtration t h r o u g h a S e p h a d e x G-5o c o l u m n (1.5 c m × 3 ° cm).
Biochim. Biophys. Acta, 229 (1971) 31o-321
314
K.
UCHIDA,
T. HIRATSUKA
SH groups o u t of 8.o--8. 5 moles per IoS g of h e a v y m e r o m y o s i n were modified, resulting in a c o m p l e t e loss of a c t i v i t y . A f t e r 72 h of t r e a t m e n t , a b o u t one S H group per IoS g of h e a v y m e r o m y o s i n r e m a i n e d unreacted. These changes in A T P a s c a c t i v i t y a n d in the c o n t e n t of free S H groups were similar to those r e p o r t e d by GAETJENS el al. 2~ a n d SEKIYA ~.!t al. 27 who used the d i m e t h y l e s t e r of dithioglycollic acid as the disulfide reagent. The c y s t a m i n e - t r e a t e d h e a v y m e r o m y o s i n , which was i n c u b a t e d tk)r 72 h under the conditions described a n d passed t h r o u g h a S e p h a d e x column, was then t r e a t e d with a 3ooo-fold m o l a r excess of thioglycollate in I M KC1 a n d 50 mM b o r a t e lmffer (pH 8.o). Complete r e v e r s i b i l i t y of the inhibition of the A T P a s e a c t i v i t y was o b t a i n e d when the c y s t a m i n e - t r e a t e d h e a v y m e r o m y o s i n was i n c u b a t e d at 4 ' for IO h or more. F r e e S H groups were also c o m p l e t e l y regenerated. Fig. 2 shows M o f f i t t - Y a n g plots of the optical r o t a t o r y d i s p e r s i o n - d a t a of the original a n d the c y s t a m i n e - a n d t h i o g l y c o l l a t e - t r e a t e d h e a v y m e r o m y o s i n . The b0 values of b o t h p r e p a r a t i o n s were the same, 312. However, when h e a v v m e r o m y o s i n was t r e a t e d with c y s t a m i n e at p H 9.o i n s t e a d of p H 8.o a n d for more than ()o h, no c o m p l e t e recoveries of A T P a s e a c t i v i t y and the - b0 value were observed even after t r e a t m e n t with thioglycollate. These o b s e r v a t i o n s i n d i c a t e t h a t t r e a t m e n t of the c y s t a m i n e - t r e a t e d h e a v y m e r o m y o s i n with thioglycollate u n d e r the present conditions resulted in disulfide interchange a c c o m p a n i e d b y the r e c o v e r y of A T P a s e a c t i v i t y , the c o n t e n t of free S H groups a n d the - b o value of the original h e a v y meromyosin. The e y s t a m i n e t r e a t e d h e a v y m e r o m y o s i n was therefore used for further studies to e l i m i n a t e the c o m p l e x i t y due to modification of SH groups. Diazotization of hea W meromvosin
As r e p o r t e d b y SOKOLOVSKY AND VALLEE14, the visible a b s o r p t i o n s p e c t r m n of m o n o a z o - Z - t y r o s i n e was m a r k e d l y d e p e n d e n t on p H (Fig. 3a). Monoazo-Z-tyrosine in I M b i c a r b o n a t e buffer (pH 9.o) showed a m a x i m u m at 480 m#. The m o l a r extim> 1.£
DHIOO
"1(1
% x
,
-20
I
"5¢ i
i
i
I0
Z0
30
s~o nrtu
mu
Fig. 2. Moffitt-Yang plots of optical rotatory dispersion data of the original and cystaminc- and thioglycollate-treated heavy meromyosin. 0 , original heavy meromyosin; :~, heavy meromyosin treated with cystamine for 72 h and followed by treatment with thioglycollate for 15 h under the same conditions as given in Fig. [. Fig. 3. Absorption spectra of monoazo-N-carbobenzoxytyrosine, 1.2 • to- 't M (a), and the diazotized heavy meromyosin, 5.1 mg/ml (b), at the pH indicated. The following buffers were employed: pI2I 9.o and io.o, i M potassium bicarbonate; pH 8.o and 8.2, 0.2 M potassium phosphate. Biochim. Biophys. Acta, 229 (~97 I) 31o-32t
MODIFICATION OF TYROSINE RESIDUES
315
tion coefficient at this wavelength calculated from the concentration of this model compound was 5.2 "1o3. When the pH was lowered to 8.2, the molar extinction coefficient at 480 nm decreased; the isosbestic point was at 416 m#, as shown by SOKOLOVSKYAND VALLEE14. The difference molar extinction coefficient was obtained as 3.5.1o 8. The absorption spectrum of the diazotized heavy meromyosin also showed a maximum at 480 m/~ in I M bicarbonate buffer (pH 9.o), and was dependent on pH (Fig. 3b). The isosbestic point was at 412 m#, which is close to the 416 m/z observed with monoazo-Z-tyrosine. This would strongly suggest that the absorbance of the diazotized heavy meromyosin at 480 m# is mainly due to monoazotyrosine residues formed. The possibility that the reaction of D H T with tyrosine and histidine residues in protein would yield a mixture of monoazo- and bisazo-derivatives of these residues has been reported. Bisazotyrosine residues could be determined directly by measuring the absorbance at 550 m#, at pH 9.0. The molar extinction coefficient was 1.4 • lO4, as determined from the absorbance of bisazoacetyltyrosine ethyl ester. If the contribution of monoazotyrosine residues to the absorbance of the diazotized heavy meromyosin at 480 m/~ can be calculated and subtracted, bisazohistidine residues could also be determined by measuring the absorbance at this wavelength and pH 9.0. The molar extinction coefficient of bisazohistidine was 2.1-IO4, which was ascertained by using bacitracin as a model compound. The values of the molar extinction coefficient for bisazotyrosine and bisazohistidine are in agreement with those reported by SOKOLOVSKY AND V A L L E E 14. Using these values, the concentrations of monoazotyrosine, bisazotyrosine and bisazohistidine residues in the diazotized heavy meromyosin solution were determined as follows: monoazotyrosine, (ApI~9, 480 ma--ApI-I 8.2,480ma)/3.5"103; bisazotyrosine, A p t t 9,550 m u / I ' 4 " 1 ° 4 ; bisazohistidine, { ( A p i t v 480 m u - - A p I ' I v 550 mu/2) --5.2" lO3 mono azotyrosine) }/2. i . lO4. The change in the absorbance of bisazohistidine residues at 480 m/z when the pH was lowered from 9.0 to 8.2 was not taken into account. Similar spectral analysis of these modified residues in protein was proposed by TAKENAKAet al. 15 who accounted for the change in absorbance of bisazohistidine residues. The contents of monoazotyrosine, bisazotyrosine and bisazohistidine residues in the diazotized heavy meromyosin estimated according to the present procedure and the equations of TAKENAKA et al. 15 are shown in Table I. The values for monoazotyrosine residues obtained from TABLE I NUMBER OF DIAZOTIZED RESIDUES PER MOLE OF HEAVY MEROMYOSIN PRODUCED BY TREATMENT WITH A I70-FOLD MOLAR EXCESS OF D H T The v a l u e s were o b t a i n e d a c c o r d i n g to t h e e q u a t i o n s p r e s e n t e d in t h i s p a p e r (a), a n d b y TAKENAKA
et al. 15 (b). Diazotized residue
Monoazotyrosine Bisazotyrosine Bisazohistidine
Moles per mole of heavy meromyosin
(a)
(b)
2.67 o. 34 o. 62
2.7 ° o. 48 o. 23
Biochim. Biophys. Acta, 229 (1971) 31o-321
3I(/
K. U C H I D A , T. H I R A T S U K A he
ioo
i
a
monoazotyrosine formed/mole of HMM
F i g . 4- C o r r e l a t i o n o f A T P a s e a c t i v i t y ( O ) a n d t h e c o n t e n t o f f r e e S H g r o u p s ( / ) o f t h e d i a z o t i z e d heavy meromyosin (HMM) with number of monoazotyrosine residues. A molar excess of DHT ranging from 2o to I4 o was employed to obtain the various degrees of diazotization. The activity is e x p r e s s e d r e l a t i v e t o t h a t o f t h e c o n t r o l .
both equations are in good agreement, but those fl)r bisazohistidine residue are considerably different. The values for monoazotyrosine residues obtained from both equations were always in good agreement when the coupling reaction of the cystaminetreated heavy meromyosin with D H T was carried out under the conditions described in MATERIALS AND METHODS. On the other hand, when the coupling reaction was carried out at room temperature and for 6o nfin, the values for bisazotyrosine and bisazohistidine residues were relatively high and those for monoazotyrosine residues obtained from both equations were not in agreement. The effect of diazotization on ATPase activity is shown in Fig. 4- Increasing the molar excess of D H T over heavy meromyosin progressively increased the degree of the formation of monoazotyrosine residues and abolished the ATPase. At pH 9.o. and 4 °, a I4o-fold molar excess of D H T completely inactivated the ATPase for 5 min. Analysis for monoazotyrosine residues showed a close correlation between the fraction, 2 moles of which had formed, and the loss of activity. The content of free SH groups was nearly the same as the original content, even in the preparations that showed low activity (Fig. 4)- Recently, SHIMADA28 reported that the formation of I monoazotyrosine residue per mole of myosin correlated with a complete loss of ATPase activity, The difference between the stoichiometry of heavy meromyosin and myosin could not be explained. TABLE
II
KINETIC
PARAMFTERS
FOR HEAVY
MEROMYOSIN
AND THF DIAZOTIZED
IIEAVY MEROMYOSIN
ATPAsI,:
A T P a s e a c t i v i t i e s w e r e m e a s u r e d in o . o o 2 m g e n z y m e p e r nil o f t h e r e a c t i o n m i x t u r e . ( ) t h o r c o n d i t i o n s w e r e t h e s a m e a s t h o s e d e s c r i b e d i n MATERIALS AND METHODS. P i w a s d e t e r m i n e d b y t h e MARTIN--DoTY
method
~1.
Heavy meromyosin Heavy meronlyosin Modified heavy meromyosin Modified heavy meromyosin
,'~Jonoazo-
]x m
Umax
tyrosine residues per mole of heavy
(:1/I)
(l*moh'/mg per rain)
i. i - i o 5 ~.2 • Io 5 1-3 " I o 5 i. 1 • I o -'~
0.54 o.56 o.36 o.o8
o.61 I. 7
Biochim. Biophys. ,4eta, 2 2 9 ( I 9 7 I ) 3Io--321
317
MODIFICATION OF TYROSINE RESIDUES TABLE III AN OPTICAL ROTATORY
DISPERSION
PARAMETER,
--bo,
A N D F R E E A M I N O G R O U P S OF T H E D I A Z O T I Z E D
HEAVY MEROMYOSIN The c o n t e n t of m o n o a z o t y r o s i n e residues was based on the e q u a t i o n as described in the t e x t . Specific A T P a s e activity of the control h e a v y m e r o m y o s i n was 0.57 unit.
Monoazotyrosine residues per mole of heavy meromyosin
-- bo (degrees)
Decrease of free amino groups (%)
None None 0.86 o.95 i .53 1.7o 2.45
322 312 297 238 246 240 252
None None 12 N o t measured 20 38 41
Kinetic constants for heavy meromyosin and the diazotized heavy meromyosin derived from initial rate data are shown in Table II. Diazotization of heavy meromyosin had no effect on the Kin. Only Vmax for the diaotized heavy meromyosin was markedly reduced. When IO mM ATP and IO mM Mg 2÷ were added to the reaction mixture in which the cystamine-treated heavy meromyosin was treated with DHT, no protective effect was observed. It seems likely that modification would occur without interference with the substrate binding. It is of interest to know whether the loss of ATPase activity is a direct result of modification of particular tyrosine residues or the indirect result of unspecific changes in the secondary or tertiary structure of heavy meromyosin. It was reported by SHIMADA2s that the --b 0 value of the diazotized myosin was lower than that of the native myosin. Optical rotatory dispersion of the diazotized heavy meromyosin was also measured in 0. 5 M KC1 and 5 ° mM borate buffer (pH 8.0) at 20 °. As shown in Table I I I , the --b o value of the preparations that contained monoazotyrosine residues less than 2 moles/mole of heavy meromyosin was reduced by lO-25% compared with that of the control. However, it is not clear whether the alteration of secondary structure is a result of modification of tyrosine residues or of modification of other residues attacked by DHT. As shown in Table I I I , the changes in the --b 0 values after diazotization were accompanied by a decrease in free amino groups. These values were obtained after determination of the amino groups with ninhydrin and of course are only rough estimates.
Nitration of tyrosine residues Tetranitromethane nitrates tyrosine and oxidizes cysteine residues in protein le. Since the cystamine-treated heavy meromyosin does not contain the reactive SH groups, attention was directed to tyrosine residues. Nitration is markedly dependent on p H and temperature. Increasing amounts of tetranitromethane were added to the cystamine-treated heavy meromyosin in 0.5 M KC1 and 50 mM Tris buffer (pH 8.o). The reaction was allowed to proceed for 20 rain at 15 °. In a different Biochim. Biophys. Acta, 229 (1971) 31o-321
JI~
K. U C H l l ) A , T. H I R A T S U K A
! ?
, ! 6 _e
I00
--..
L; 4
a 'i
_ _ _ o- ~
i2
o
e
Tetronitrornelhone odded ( m o l e s / m o l e of
HMM)
Fig. 5. N i t r a t i o n o f h e a v y m e r o m y o s i n ( H M M ) w i t h t e t r a n i t r o m e t h a n e . T h e f o r n I a t i o n o f n i t r o t y r o s i n e r e s i d u e , a n d c h a n g e s in t h e A T P a s e a c t i v i t y w i t h i n c r e a s i n g a m o u n t s o f t e t r a n i t r o m e t h a n e u n d e r t h e t w o d i f f e r e n t c o n d i t i o n s . A t p H 8, 15 ° for 2o m i n ( ~ ) a n d a t p H 7, 4" f o r / 15 h (@). -. . . . . . . , r e l a t i v e A T P a s e a c t i v i t y , ; . . . . . , n u m b e r o f n i t r o t y r o s i n e s f o r m e d per m o l e of heavv meromyosin; , n u m b e r o f free S H g r o u p s p e r l o 5 g h e a v y n i e r o m y 0 s i n .
experiment, the cystamine-treated heavy meromyosin in 0. 5 M KC1 and 5o mM borate buffer (pH 7.o), with increasing amounts of tetranitromethane, was allowed to stand in an ice-bath overnight. The dependence of nitrotyrosine residue formation on amount of tetranitromethane is shown in Fig. 5. With the same amount of tetranitromethane more nitrotyrosine residues were formed at pH 8.o and I5 ° for 2o rain than at pH 7.o and 4 ° for I5 h. The difference spectrum of nitrated minus control heavy meromyosin in o. 5 M KC1 and 5 o m M Tris buffer (pH 8.o) was virtually identical with that of 3-nitrotyrosine minus tyrosine which was reported by RIORDAN et al. 17, and provides evidence for the existence of nitrotyrosine residues in the modified heavy meromyosin. About 85% of the decrease in ATPase activity observed on nitration of heavy meromyosin with a 75-fold molar excess of tetranitromethane at pH 7.o and TABLE AN
IV
OPTICAL
ROTATORY
THE DIFFERENT
DISPERSION
PARAMETER
,-b0,
Specific ATPasc tively.
Conditions
tyrosine
. . . . . . . . .
residues per mole of
pH
Temp.
Time
-- b o
8 8 8 7 7 7
15' 15' t5 ' 4:' 4' 4':
20 20 20 15 15 15
(degrees)
heavy meromyosin
2.1 7.i None 2.2 4.3
PREPARATIONS
NITRATED
tINDER
a c t i v i t i e s o f t h e c o n t r o l s t r e a t e d a t p H 8.o a n d 7.o w e r e o . 6 I a n d o.5o, r e s p e c -
Nitro-
None
OF THE
CONDITIONS
t~iochim. Biophys..4 cta, 229-
rain rain rain tl h h
( 1 9 7 I) 3 i ° - 3 2 1
312 312 312 302 302 296
MODIFICATION OF TYROSINE RESIDUES
319
4 ° correlated with the nitration of 6.5 tyrosine residues. At p H 8.0 and IO °, o n the other hand, only 60% of the decrease in activity observed on nitration with a 9-fold molar excess of tetranitromethane correlated with the nitration of 7.1 tyrosine residues. In each instance, on nitration of up to 2 tyrosine residues, the activity fell to about 60% of the control. The content of free SH groups in each preparation recovered to the same level as that of the control, but was lower than that of the original heavy meromyosin. The content of free SH groups in the original heavy meromyosin was 8-8.5 moles per lOs g of heavy meromyosin. Diazotization of two tyrosine residues with D H T caused complete inactivation of ATPase accompanied by a decrease in the --b o value. In marked contrast, the decrease in ATPase activity induced by nitration in both experiments was not accompanied by detectable changes in the --b o value (Table IV). On prolonged treatment of the cystamine-treated heavy meromyosin in the reaction mixture, the --b o value of all preparations decreased very slightly, from 312 to 302, but this does not seem to be significant. DISCUSSION As reported b y SOKOLOVSKY AND VALLEE 14, diazo-derivatives of amino acid residues are destroyed under standard conditions for protein hydrolysis. Thus, the number of diazotized residues in the protein can be determined b y measuring their loss on acid hydrolysis as compared with the native protein. From the results of amino acid analysis of the diazotized enzymes with a relatively small molecular weight29, ~°, it is known that cysteine, tyrosine, lysine and histidine residues couple with DHT. When protein is allowed to react with a large excess of DHT, the methionine content is also decreased. As shown in Fig. 4, Table I and Table I I I , it is clear that two tyrosine residues and about 40 % of the total amino groups, corresponding to 115 lysine residues, coupled with DHT, and heavy meromyosin was completely inactivated. In addition, the formation of less than one residue of bisazohistidine per mole heavy meromyosin was observed. SuzuKI et al. 31 have indicated, in their study of insulin modified by DHT, that monoazohistidine residues also formed before the formation of bisazohistidine residues. It is possible to determine spectroscopically the amount of monoazohistidine residue, which has a spectrum with a m a x i m u m at 359-360 m# and a shoulder with a midpoint at 375 m/~ at p H 8.8 (ref. 14). The product of the reaction with only a large excess of D H T showed a spectrum with a m a x i m u m at 320 m# in this range. No stoichiometric relation was observed between the changes in ATPase activity of the diazotized heavy meromyosin and in its absorbance in the range from 300 to 400 m#. Thus, the complete loss of activity which occurred concomitantly with the formation of two monoazotyrosine residues should not be due to the formation of monoazohistidine residue, if any. B~.R2{NY AND B2~R~NY8~ modified the lysine residues of myosin using Ncarboxy-L-cysteine anhydride and found that ATPase activity was lost. KUBO et al. ~ found that the binding of two moles of trinitrobenzene sulfonate led to a loss of the ATPase activity of myosin. FABIAN AND MOHLRAD33 modified the lysine residues of myosin with trinitrobenzene sulfonate in the presence and absence of ATP, and found Biochim. Biophys. Acta, 229 (1971) 31o-321
];2o
K. UCHIDA, T. HIRATSUKA
a decrease in the K+-activated and the Ca2+-activated ATPase activities. These results suggest that the lysine residues are somehow involved in the functioning of myosin ATPase. When heavy meromyosin was modified with DHT, about 115 lysine residues coupled with D H T and heavy meromyosin was completely inactivated concomitantly with a change in the secondary structure of protein. Nitration of 7 tyrosine residues in heavy meromyosin resulted in a change in activity but n~,t in conformation detectable by optical rotatory dispersion. It appears that the lvsine residues in heavy meromyosin are important for activity. However, the possibility that the lysine residues in heavy meromvosin are the binding site for the substrate seems unlikely. In the present experiments, it is impossible to refer to a role ot7 methionine residues because of the lack of its estimation. STRACHER9 found, in a study' of the photooxidation of myosin, that tile methionine residues were oxidized at a rate too slow to account for the loss in enzymic activity. The important criteria of active site labeling are stoichiometric inactivation and specific protection against inactivation a4. Although treatnlent of heavy meromyosin with D H T caused complete inactivation upon the formation of two moles of monoazotyrosine residue per mole of protein, diazotization in the presence of the substrate did not result in a significantly smaller degree of inactivation than occurred in the absence of substrate. Nitration of tyrosine residues with tetranitromethane under milder conditions resulted in the loss of 85°4, of the activity, and 6 tyrosine residues were nitrated. 2 tyrosine residues per mole of protein were nitrated with tetranitromethane, though about 4 o % of the activity remained. There was no stoichiometric loss of activity for every mole of label covalently attached per mole of protein. It is evident from these results that the tyrosine residue m a y not be involved in the binding of the substrate. From the above discussion, it is concluded that the complete inactivation of heavy meromyosin ATPase by reaction with D H T is due to modification of both tyrosine and lysine residues in heavv meromyosin accompanied by a change in the conformation of the protein molecule. ACKNOWLEI)GMENT
The authors wish to thank Dr. K. Yagi for his discussion and support. This work was supported by a grant from the Muscular Dystrophy Association of America. I~EFERENCES I J. J. BLUM, Arch. Biochem. Biophys., 97 (1962) 309. 2 T. SEKINE AND V~r. V~. KIELLEY, Biochim. Biophys. Acta, 81 (1964) 336. 3 W. YAMASHITA, Y. SOMA, S. KOBAYASHI, W. SEKINE, K. TITANI AND K. NARITA, ,]. Biochem. Tokyo, 55 (1964) 576. 4 M. F. MORALES AND ]c~. HOTTA, J. Biol. Chem., 235 (196o) 1979. 5 A. STRACHER, in W. A. PAUL, E. E. DANIEL, C. M. KAY AND G. MONKTON, Muscle, P e r g a m o n , Oxford, 1965, p. 85. 6 S. KUBO, S. TOKURA AND Y. TONOMURA, J. Biol. Chem., 235 (r96o) 28.35. 7 S. I{UBO, H. TOKUYAMA AND Y. TONOMURA, Biochim. Biophys. Acla, i o o (1965) 4598 K. I-[OTTA, J. Biochem. Tokyo, 5 ° (1961) 218. 9 A. STRACHER, .]. Biol. Chem., 240 (1965) 958 PC. lO F. MORITA ANt) K. YAGI, Biochem. Biophys. Res. Commun., 22 (1966) 297.
Biochim. Biophys. Acta, 229 (1971) 31o-321
MODIFICATION OF TYROSINE RESIDUES
321
i i F. MORITA,J. Biol. Chem., 242 (1967) 45Ol. 12 K. SEKIYA AND Y. TONOMURA, J. Biochem. Tokyo, 61 (1967) 787 . 13 H. HORINISm, Y. HACHIMORI, K. KURIHARA AND K. SHIBATA, Biochim. Biophys. Acta, 86 (1964) 477. 14 M. SOKOLOVSK¥ AND B. L. VALLEE, Biochemistry, 5 (1966) 3574. 15 A. TAKENAKA, T. SUZUKI, O. TAKENAKA, H. HORINISHI AND K. SHIBATA, Biochim. Biophys. Acta, 194 (1969) 293. 16 M. SOKOLOVSKY,J. F. RIORDAN AND B. L. VALLEE, Biochemistry, 5 (1966) 3582. 17 J. F. RIORDAN, M. SOKOLOVSKY AND B. L. VALLEE, Biochemistry, 6 (1967) 3609. 18 K. YAGI AND Y. YAZAWA, J. Biochem. Tokyo, 60 (1964) 450. 19 H. MUELLER, J. Biol. Chem., 239 (1964) 797. 20 O. H. L o w R y , N. J. ROSEBROUGH, A . L . FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951 ) 265. 21 J. B. MARTIN AND D. M. DUTY, Anal. Chem., 21 (1949) 965. 22 G. L. ELLMAN, Arch. Biochem. Biophys., 82 (1959) 7 o. 23 P. D. BUYER, J. Am. Chem. Soc., 76 (1954) 4331. 24 S. MOORE AND W. H. STEIN, J. Biol. Chem., 211 (1954) 907. 25 W. MOFFITT AND J. T. WANG, Proc. Natl. Acad. Sci. U.S., 42 (1956) 596. 26 E. GAETJENS, T. THERATTIL-AUTOMY AND M. BARAN¥, Biochim. Biophys. Acta, 86 (1964) 554. 27 K. SEKIYA, S. MII AND Y. TONOMURA, J. Biochem. Tokyo, 57 (1965) 192. 28 T. SHIMADA, J. Biochem. Tokyo, 67 (197 o) 185. 29 M. ~V[URAMATUAND S. FUJII, J. Biochem. Tokyo, 66 (1969) 455. 3 ° 1R. H. SCHIRMER, I. SCHIRMER AND L. NODA, Bioehim. Biophys. Acta, 207 (197 o) 165. 31 T. SUZUKI, O. TAKENAKA AND K. SHIBATA, .]. Biochem. Tokyo, 66 (1969) 815. 32 M. BkR,~NY AND K. BkRANV, Bioehim. Biophys. Acta, 35 (1959) 293. 33 F. FABIAN AND A. MOI-ILRAD, Biochim. Biophys. Acta, 162 (1968) 596. 34 S. J. SINGER, Advan. Protein Chem., 22 (1967) I.
Biochim. Biophys. Acta, 229 (1971) 31o-321
BIOCHIMICAET BIOPHYSICAACTA
m~A 35737 CHEMICAL MODIFICATION OF T Y R O S I N E R E S I D U E S IN HEAVY MEROMYOSIN ATPase
K. UCHll)A AND 3". HIRATSUKA Department of Chemistry, Fac*dty of Science, Hokkaido University, Sapporo (Japan)
(Received June 26th, 197o) (Revised manuscript received October i2th, i97 o)
SUMMARY
When heavy meromyosin was treated with a 2oo-fold molar excess of cystamine for 72 h in an ice-bath, most of the free SH groups disappeared, resulting in complete inactivation of the ATPase. The content of free SH groups, ATPase activity and the --b 0 value were restored completely to those of the original heavy meromyosin by further treatment with a 3ooo-fold molar excess of thioglycollate. The effect of diazo-IH-tetrazole and tetranitromethane on heavy meromyosin, whose SH groups were protected, has been studied. Treatment of the enzyme with diazo-IH-tetrazole caused a stoichiometric loss of ATPase activity accompanying the formation of two monoazotyrosine residues. The --b 0 value and the content of free amino groups of the diazotized enzyme were also reduced as the extent of diazotization increased. In contrast, nitration of 2 tyrosine residues with tetranitromethane merely reduced the activity to 60°/'0 of that of the control without causing a change in the --b o value. A possible explanation for the decrease in ATPase activity induced by modification with the two reagents is discussed.
INTRODUCTION The functional importance of cysteine, histidine and lysine residues in myosin and heavy meromyosin ATPase has already been suggested as a consequence of chemical modification and of the study of enzymic properties 1-9. Recently, from experiments on the difference spectrum of heavy meromyosin induced by substrate, some tyrosine and tryptophan chromophores were found to be buried in the protein moiety as a secondary effect of the binding of ATP to the active site of heavy meromyosin 1°-12. However, no proof was given that tyrosine residues actually play a role in the active center. An account is given here on the participation of tyrosine residues in the reaction Abbreviations: I)HT, diazo-tH-tetrazole; monoazo-Z-tyrosine, monoazocarbobenzoxytyrosine. Biochim. Biophys. Acta. 22q (I97 l) 31o-321
MODIFICATION OF TYROSINE RESIDUES
311
of heavy meromyosin ATPase. Diazo-IH-tetrazole (DHT) was proposed as a coupling reagent for histidine residues in protein 13. However, this reagent was found to be unspecific, and in addition to histidine residues it also reacts with tyrosine, cysteine and lysine residuesl4,15. Diazotization of heavy meromyosin, the SH groups of which were protected in advance through the disulfide-SH exhcange reaction using cystamine, resulted in significant changes in ATPase activity and in the conformation of heavy meromyosin. The loss of ATPase activity was correlated with the formation of two monoazotyrosine residues per mole of heavy meromyosin. In addition, nitration of the cystamine-treated heavy meromyosin with tetranitromethanel~, 17 was also examined to get further information on the functional and conformational changes of the enzyme resulting from specific modification of tyrosine residues. The complete loss of ATPase caused by diazotization seems to be due both to modification of tyrosine residues and to structural alteration induced by modification of a large number of lysine residues. MATERIALS AND METHODS Enzyme Heavy meromyosin was prepared by the procedure described by YAGI AND YAZAWAis. The protein was lyophilized in the presence of sucrose and stored at 4 °. Before use, it was dissolved in 2 mM borate buffer (pH 8.o), and dialyzed exhaustively against the same buffer. The molecular weight of heavy meromyosin was taken as 3.5" lO5 (ref. 19). Model compounds N-Carbobenzoxymonoazotetrazole tyrosine was prepared according to the method of SOKOLOVSKY AND VALLEE 14. For preparation of bisazo-derivatives, N-acetyltyrosine ethyl ester and bacitracin were treated with a 3oo-fold molar excess of D H T in 0.67 M KHCO 3 buffer (pH 9.0) for 60 min at room temperature. Treatment with cystamine and thioglycollate To heavy meromyosin solution (20 mg/ml, in 50 mM borate buffer, pH 8.0, and i M KC1) a 2oo-fold molar excess of cystamine over heavy meromyosin was added at 4 °. After an appropriate period of incubation, the mixture was passed through a Sephadex G-5o column (2.5 c m x 30 cm) equilibrated with 2 mM borate buffer (pH 7.o), and IO mlV[ KC1. The fraction eluted without retardation is referred to as cystamine-treated heavy meromyosin. The cystamine-treated heavy meromyosin was then incubated with a 3ooo-fold molar excess of thioglycollate in 50 mM borate buffer (pH 8.0) and 0. 5 M KC1 at 4 °. The excess thioglycollate was eliminated by gel filtration through a Sephadex column. Diazotization with D H T The cystamine-treated heavy meromyosin was diazotized with D H T according to the method of HORINISHI et al. x3 with a slight modification. D H T was prepared by diazotization of 5-amino-IH-tetrazole with NaNO2 at the molar ratio of 1:0. 9. The unreacted NO 2- were not detectable in the D H T solution. The concentration of D H T was not directly determined, but it was assumed from the concentration of Biochim. Biophys. Acta, 229 (I97I) 3to-32I
312
K. UCHII)A, T. HIRATSUKA
5-amino-IH-tetrazole originally used. Diazotization of the cystamine-treated h e a v y meromyosin was carried out at 4 ° for 5 min in o.67 M KHCO3 buffer (pH 9.o). The reaction was terminated b y addition of a large excess of thioglycollate. The mixture was passed through a Sephadex G-5o column. The protein eluted was again treated with thioglycollate to restore the SH groups under the conditions described above. Alternatively, the diazotized protein was precipitated with (NH4)2SO 4 (6o% saturation) and dissolved in a small volume of 5 ° mM borate buffer (pH 8.0) e()ntaining o.5 M KC1 and a 3ooo-fold molar excess of thioglycollate. The mixture was allowed to stand overnight at 4 °, and then dialyzed against 2 hiM borate buffer (pH 8.o) at 4 °. A control sample was treated in the same m a n n e r but without addition of D H T . Nitration with tetranitromethane
Nitration of the eystamine-treated heavy meromyosin was carried out according to the method of SOKOLOVSKY et al) 6 with a slight modification. Tetranitromethane was saturated in 5 ° mM Tris buffer (pH 8.0) and 0.5 M KC1 at 25 °. The concentratkm of tetranitromethane in the solution was determined from the absorbance of nitroformate anions ~ formed by the addition of a large excess of thioglycollate. A calculated volume of tetranitromethane solution was added to the cystamine-treated h e a v y meromvosin (IO mg/ml) under two different conditions: in 50 mM Tris buffer (pH 8.0) and o. 5 M KC1 at 15 °, and in 50 mM borate buffer (pH 7.o) and o.5 M KC1 at 4 °. The reaction mixtures were allowed to stand for 20 min and I5 h, respectively. The nitrated protein was precipitated with (NH4),,SO 4 (60% saturation) and dissolved in a small volume of 5o mM borate buffer (pH 8.0) containing o.5 M KCI and a 3ooo-fold molar excess of thioglycollate. After the incubation, the mixture was dialyzed in the same manner as described in the section on diazotization. The extent of nitration was estimated by measuring the absorbance at 428 nm and at p H 8.o, assuming a molar extinction coefficient of 4zoo for 3-nitrotyrosine residueUL Protein concentration
The protein concentration was determined b y measuring the absorbance at 28o n m using a factor o.63 per mg per ml, and also by the biuret method or the m e t h o d of LOWRy et al. "°.
E~tz3~rne assay The ATPase activity of h e a v y meromyosin and the modified heavy meromyosin was measured in a solution containing, unless otherwise stated, 0.08 mg of protein per ml, 2 mM ATP, 5 mM CaCI~, 0. 5 M KC1 and 2o mM Tris-maleate buffer (pH 7.o). The measurements were carried out at 25 ° in tile I-ml mixture. I n c u b a t i o n was terminated b y addition of I ml of IO °:/O triehloroacetic acid. Pi liberated was determined b y the m e t h o d of Fiske-SubbaRow or by the method of MARTIN AND DOTY21. Specific ATPase activity is expressed in units given as #moles Pi liberated per min per m g of protein. Determination of S H groups
SH groups were determined according to tile method of EIA.MAN22 in which Biochim. Biophys. Acta, 229 (197 l) 31o 32I
313
MODIFICATION OF TYROSINE RESIDUES
excess 5,5'-dithiobis-(2-nitrobenzoic acid) was added to 0.2-0.3 mg of protein per ml of o.I M borate buffer (pH 8.0) containing 6 M urea. Titration of the protein with p-chloromercuribenzoate was also carried out according to the method of BOYER~s.
Determination of amino groups The free amino groups were determined by means of the ninhydrin reaction 24 using leucine as a standard.
optical rotatory dispersion measurement Optical rotatory dispersion was measured with a Jasco automatic recording spectropolarimeter, model ORD/UV-5. The measurements were performed in I - c m ceils at 20 ° in the range from 2800 to 5000 A. The instrument was calibrated to give zero rotation for the buffer blank. The solvent used was 0. 5 M KC1-5o mM borate buffer (pH 8.0). The usual equation of MOFFITT AND YANG25 was used to calculate the parameter, --bo, assuming a ~0 value of 212o A. RESULTS
Disulfide-SH exchange and recovery of the free SH groups The enzyme, at a concentration of about 2o mg per ml in i M KC1-5o mM borate buffer (pH 8.o), was treated with a 2oo-fold molar excess of cystamine over heavy meromyosin at 4 ° before the ATPase activity and the content of free SH groups were determined. As shown in Fig. I, an initial increase of activity was observed during I h by which time the activation had reached its maximum. Concomitantly, about one-third of the free SH groups disappeared at a rapid rate. On prolonged treatment, the decrease in activity was accompanied by a more gradual disappearance of the other free SH groups. After 17 h of treatment, 6- 7 moles of
f
6
z 4
q ~ 0.~
5 2 v---
o
c*
"o
,= ~
,~"~ ~5
t i m e o hours
Fig. I. Effect of c y s t a m i n e on t h e A T P a s e a c t i v i t y of h e a v y m e r o m y o s i n (HMM) a n d reversal o f t h e effect c a u s e d b y t r e a t m e n t w i t h thioglycollate. H e a v y m e r o m y o s i n , 20 m g / m l in I M KC1 a n d 5o m M b o r a t e buffer (pH 8.0) a t 4 °, was t r e a t e d w i t h a 2oo-fold m o l a r excess of c y s t a m i n e over h e a v y m e r o m y o s i n . A f t e r 72 h o f t r e a t m e n t , t h e c y s t a m i n e - t r e a t e d h e a v y m e r o m y o s i n , 5 m g / m l in I M KC1 a n d 5 ° m M b o r a t e buffer (pH 8.o) a t 4 °, w a s f u r t h e r t r e a t e d w i t h a 3ooo-fold m o l a r excess o f thioglycollate. T h e A T P a s e a c t i v i t y ( O ) a n d t h e c o n t e n t o f free S H g r o u p s ( × ) were d e t e r m i n e d i m m e d i a t e l y a f t e r gel filtration t h r o u g h a S e p h a d e x G-5o c o l u m n (1.5 c m × 3 ° cm).
Biochim. Biophys. Acta, 229 (1971) 31o-321
314
K.
UCHIDA,
T. HIRATSUKA
SH groups o u t of 8.o--8. 5 moles per IoS g of h e a v y m e r o m y o s i n were modified, resulting in a c o m p l e t e loss of a c t i v i t y . A f t e r 72 h of t r e a t m e n t , a b o u t one S H group per IoS g of h e a v y m e r o m y o s i n r e m a i n e d unreacted. These changes in A T P a s c a c t i v i t y a n d in the c o n t e n t of free S H groups were similar to those r e p o r t e d by GAETJENS el al. 2~ a n d SEKIYA ~.!t al. 27 who used the d i m e t h y l e s t e r of dithioglycollic acid as the disulfide reagent. The c y s t a m i n e - t r e a t e d h e a v y m e r o m y o s i n , which was i n c u b a t e d tk)r 72 h under the conditions described a n d passed t h r o u g h a S e p h a d e x column, was then t r e a t e d with a 3ooo-fold m o l a r excess of thioglycollate in I M KC1 a n d 50 mM b o r a t e lmffer (pH 8.o). Complete r e v e r s i b i l i t y of the inhibition of the A T P a s e a c t i v i t y was o b t a i n e d when the c y s t a m i n e - t r e a t e d h e a v y m e r o m y o s i n was i n c u b a t e d at 4 ' for IO h or more. F r e e S H groups were also c o m p l e t e l y regenerated. Fig. 2 shows M o f f i t t - Y a n g plots of the optical r o t a t o r y d i s p e r s i o n - d a t a of the original a n d the c y s t a m i n e - a n d t h i o g l y c o l l a t e - t r e a t e d h e a v y m e r o m y o s i n . The b0 values of b o t h p r e p a r a t i o n s were the same, 312. However, when h e a v v m e r o m y o s i n was t r e a t e d with c y s t a m i n e at p H 9.o i n s t e a d of p H 8.o a n d for more than ()o h, no c o m p l e t e recoveries of A T P a s e a c t i v i t y and the - b0 value were observed even after t r e a t m e n t with thioglycollate. These o b s e r v a t i o n s i n d i c a t e t h a t t r e a t m e n t of the c y s t a m i n e - t r e a t e d h e a v y m e r o m y o s i n with thioglycollate u n d e r the present conditions resulted in disulfide interchange a c c o m p a n i e d b y the r e c o v e r y of A T P a s e a c t i v i t y , the c o n t e n t of free S H groups a n d the - b o value of the original h e a v y meromyosin. The e y s t a m i n e t r e a t e d h e a v y m e r o m y o s i n was therefore used for further studies to e l i m i n a t e the c o m p l e x i t y due to modification of SH groups. Diazotization of hea W meromvosin
As r e p o r t e d b y SOKOLOVSKY AND VALLEE14, the visible a b s o r p t i o n s p e c t r m n of m o n o a z o - Z - t y r o s i n e was m a r k e d l y d e p e n d e n t on p H (Fig. 3a). Monoazo-Z-tyrosine in I M b i c a r b o n a t e buffer (pH 9.o) showed a m a x i m u m at 480 m#. The m o l a r extim> 1.£
DHIOO
"1(1
% x
,
-20
I
"5¢ i
i
i
I0
Z0
30
s~o nrtu
mu
Fig. 2. Moffitt-Yang plots of optical rotatory dispersion data of the original and cystaminc- and thioglycollate-treated heavy meromyosin. 0 , original heavy meromyosin; :~, heavy meromyosin treated with cystamine for 72 h and followed by treatment with thioglycollate for 15 h under the same conditions as given in Fig. [. Fig. 3. Absorption spectra of monoazo-N-carbobenzoxytyrosine, 1.2 • to- 't M (a), and the diazotized heavy meromyosin, 5.1 mg/ml (b), at the pH indicated. The following buffers were employed: pI2I 9.o and io.o, i M potassium bicarbonate; pH 8.o and 8.2, 0.2 M potassium phosphate. Biochim. Biophys. Acta, 229 (~97 I) 31o-32t
MODIFICATION OF TYROSINE RESIDUES
315
tion coefficient at this wavelength calculated from the concentration of this model compound was 5.2 "1o3. When the pH was lowered to 8.2, the molar extinction coefficient at 480 nm decreased; the isosbestic point was at 416 m#, as shown by SOKOLOVSKYAND VALLEE14. The difference molar extinction coefficient was obtained as 3.5.1o 8. The absorption spectrum of the diazotized heavy meromyosin also showed a maximum at 480 m/~ in I M bicarbonate buffer (pH 9.o), and was dependent on pH (Fig. 3b). The isosbestic point was at 412 m#, which is close to the 416 m/z observed with monoazo-Z-tyrosine. This would strongly suggest that the absorbance of the diazotized heavy meromyosin at 480 m# is mainly due to monoazotyrosine residues formed. The possibility that the reaction of D H T with tyrosine and histidine residues in protein would yield a mixture of monoazo- and bisazo-derivatives of these residues has been reported. Bisazotyrosine residues could be determined directly by measuring the absorbance at 550 m#, at pH 9.0. The molar extinction coefficient was 1.4 • lO4, as determined from the absorbance of bisazoacetyltyrosine ethyl ester. If the contribution of monoazotyrosine residues to the absorbance of the diazotized heavy meromyosin at 480 m/~ can be calculated and subtracted, bisazohistidine residues could also be determined by measuring the absorbance at this wavelength and pH 9.0. The molar extinction coefficient of bisazohistidine was 2.1-IO4, which was ascertained by using bacitracin as a model compound. The values of the molar extinction coefficient for bisazotyrosine and bisazohistidine are in agreement with those reported by SOKOLOVSKY AND V A L L E E 14. Using these values, the concentrations of monoazotyrosine, bisazotyrosine and bisazohistidine residues in the diazotized heavy meromyosin solution were determined as follows: monoazotyrosine, (ApI~9, 480 ma--ApI-I 8.2,480ma)/3.5"103; bisazotyrosine, A p t t 9,550 m u / I ' 4 " 1 ° 4 ; bisazohistidine, { ( A p i t v 480 m u - - A p I ' I v 550 mu/2) --5.2" lO3 mono azotyrosine) }/2. i . lO4. The change in the absorbance of bisazohistidine residues at 480 m/z when the pH was lowered from 9.0 to 8.2 was not taken into account. Similar spectral analysis of these modified residues in protein was proposed by TAKENAKAet al. 15 who accounted for the change in absorbance of bisazohistidine residues. The contents of monoazotyrosine, bisazotyrosine and bisazohistidine residues in the diazotized heavy meromyosin estimated according to the present procedure and the equations of TAKENAKA et al. 15 are shown in Table I. The values for monoazotyrosine residues obtained from TABLE I NUMBER OF DIAZOTIZED RESIDUES PER MOLE OF HEAVY MEROMYOSIN PRODUCED BY TREATMENT WITH A I70-FOLD MOLAR EXCESS OF D H T The v a l u e s were o b t a i n e d a c c o r d i n g to t h e e q u a t i o n s p r e s e n t e d in t h i s p a p e r (a), a n d b y TAKENAKA
et al. 15 (b). Diazotized residue
Monoazotyrosine Bisazotyrosine Bisazohistidine
Moles per mole of heavy meromyosin
(a)
(b)
2.67 o. 34 o. 62
2.7 ° o. 48 o. 23
Biochim. Biophys. Acta, 229 (1971) 31o-321
3I(/
K. U C H I D A , T. H I R A T S U K A he
ioo
i
a
monoazotyrosine formed/mole of HMM
F i g . 4- C o r r e l a t i o n o f A T P a s e a c t i v i t y ( O ) a n d t h e c o n t e n t o f f r e e S H g r o u p s ( / ) o f t h e d i a z o t i z e d heavy meromyosin (HMM) with number of monoazotyrosine residues. A molar excess of DHT ranging from 2o to I4 o was employed to obtain the various degrees of diazotization. The activity is e x p r e s s e d r e l a t i v e t o t h a t o f t h e c o n t r o l .
both equations are in good agreement, but those fl)r bisazohistidine residue are considerably different. The values for monoazotyrosine residues obtained from both equations were always in good agreement when the coupling reaction of the cystaminetreated heavy meromyosin with D H T was carried out under the conditions described in MATERIALS AND METHODS. On the other hand, when the coupling reaction was carried out at room temperature and for 6o nfin, the values for bisazotyrosine and bisazohistidine residues were relatively high and those for monoazotyrosine residues obtained from both equations were not in agreement. The effect of diazotization on ATPase activity is shown in Fig. 4- Increasing the molar excess of D H T over heavy meromyosin progressively increased the degree of the formation of monoazotyrosine residues and abolished the ATPase. At pH 9.o. and 4 °, a I4o-fold molar excess of D H T completely inactivated the ATPase for 5 min. Analysis for monoazotyrosine residues showed a close correlation between the fraction, 2 moles of which had formed, and the loss of activity. The content of free SH groups was nearly the same as the original content, even in the preparations that showed low activity (Fig. 4)- Recently, SHIMADA28 reported that the formation of I monoazotyrosine residue per mole of myosin correlated with a complete loss of ATPase activity, The difference between the stoichiometry of heavy meromyosin and myosin could not be explained. TABLE
II
KINETIC
PARAMFTERS
FOR HEAVY
MEROMYOSIN
AND THF DIAZOTIZED
IIEAVY MEROMYOSIN
ATPAsI,:
A T P a s e a c t i v i t i e s w e r e m e a s u r e d in o . o o 2 m g e n z y m e p e r nil o f t h e r e a c t i o n m i x t u r e . ( ) t h o r c o n d i t i o n s w e r e t h e s a m e a s t h o s e d e s c r i b e d i n MATERIALS AND METHODS. P i w a s d e t e r m i n e d b y t h e MARTIN--DoTY
method
~1.
Heavy meromyosin Heavy meronlyosin Modified heavy meromyosin Modified heavy meromyosin
,'~Jonoazo-
]x m
Umax
tyrosine residues per mole of heavy
(:1/I)
(l*moh'/mg per rain)
i. i - i o 5 ~.2 • Io 5 1-3 " I o 5 i. 1 • I o -'~
0.54 o.56 o.36 o.o8
o.61 I. 7
Biochim. Biophys. ,4eta, 2 2 9 ( I 9 7 I ) 3Io--321
317
MODIFICATION OF TYROSINE RESIDUES TABLE III AN OPTICAL ROTATORY
DISPERSION
PARAMETER,
--bo,
A N D F R E E A M I N O G R O U P S OF T H E D I A Z O T I Z E D
HEAVY MEROMYOSIN The c o n t e n t of m o n o a z o t y r o s i n e residues was based on the e q u a t i o n as described in the t e x t . Specific A T P a s e activity of the control h e a v y m e r o m y o s i n was 0.57 unit.
Monoazotyrosine residues per mole of heavy meromyosin
-- bo (degrees)
Decrease of free amino groups (%)
None None 0.86 o.95 i .53 1.7o 2.45
322 312 297 238 246 240 252
None None 12 N o t measured 20 38 41
Kinetic constants for heavy meromyosin and the diazotized heavy meromyosin derived from initial rate data are shown in Table II. Diazotization of heavy meromyosin had no effect on the Kin. Only Vmax for the diaotized heavy meromyosin was markedly reduced. When IO mM ATP and IO mM Mg 2÷ were added to the reaction mixture in which the cystamine-treated heavy meromyosin was treated with DHT, no protective effect was observed. It seems likely that modification would occur without interference with the substrate binding. It is of interest to know whether the loss of ATPase activity is a direct result of modification of particular tyrosine residues or the indirect result of unspecific changes in the secondary or tertiary structure of heavy meromyosin. It was reported by SHIMADA2s that the --b 0 value of the diazotized myosin was lower than that of the native myosin. Optical rotatory dispersion of the diazotized heavy meromyosin was also measured in 0. 5 M KC1 and 5 ° mM borate buffer (pH 8.0) at 20 °. As shown in Table I I I , the --b o value of the preparations that contained monoazotyrosine residues less than 2 moles/mole of heavy meromyosin was reduced by lO-25% compared with that of the control. However, it is not clear whether the alteration of secondary structure is a result of modification of tyrosine residues or of modification of other residues attacked by DHT. As shown in Table I I I , the changes in the --b 0 values after diazotization were accompanied by a decrease in free amino groups. These values were obtained after determination of the amino groups with ninhydrin and of course are only rough estimates.
Nitration of tyrosine residues Tetranitromethane nitrates tyrosine and oxidizes cysteine residues in protein le. Since the cystamine-treated heavy meromyosin does not contain the reactive SH groups, attention was directed to tyrosine residues. Nitration is markedly dependent on p H and temperature. Increasing amounts of tetranitromethane were added to the cystamine-treated heavy meromyosin in 0.5 M KC1 and 50 mM Tris buffer (pH 8.o). The reaction was allowed to proceed for 20 rain at 15 °. In a different Biochim. Biophys. Acta, 229 (1971) 31o-321
JI~
K. U C H l l ) A , T. H I R A T S U K A
! ?
, ! 6 _e
I00
--..
L; 4
a 'i
_ _ _ o- ~
i2
o
e
Tetronitrornelhone odded ( m o l e s / m o l e of
HMM)
Fig. 5. N i t r a t i o n o f h e a v y m e r o m y o s i n ( H M M ) w i t h t e t r a n i t r o m e t h a n e . T h e f o r n I a t i o n o f n i t r o t y r o s i n e r e s i d u e , a n d c h a n g e s in t h e A T P a s e a c t i v i t y w i t h i n c r e a s i n g a m o u n t s o f t e t r a n i t r o m e t h a n e u n d e r t h e t w o d i f f e r e n t c o n d i t i o n s . A t p H 8, 15 ° for 2o m i n ( ~ ) a n d a t p H 7, 4" f o r / 15 h (@). -. . . . . . . , r e l a t i v e A T P a s e a c t i v i t y , ; . . . . . , n u m b e r o f n i t r o t y r o s i n e s f o r m e d per m o l e of heavv meromyosin; , n u m b e r o f free S H g r o u p s p e r l o 5 g h e a v y n i e r o m y 0 s i n .
experiment, the cystamine-treated heavy meromyosin in 0. 5 M KC1 and 5o mM borate buffer (pH 7.o), with increasing amounts of tetranitromethane, was allowed to stand in an ice-bath overnight. The dependence of nitrotyrosine residue formation on amount of tetranitromethane is shown in Fig. 5. With the same amount of tetranitromethane more nitrotyrosine residues were formed at pH 8.o and I5 ° for 2o rain than at pH 7.o and 4 ° for I5 h. The difference spectrum of nitrated minus control heavy meromyosin in o. 5 M KC1 and 5 o m M Tris buffer (pH 8.o) was virtually identical with that of 3-nitrotyrosine minus tyrosine which was reported by RIORDAN et al. 17, and provides evidence for the existence of nitrotyrosine residues in the modified heavy meromyosin. About 85% of the decrease in ATPase activity observed on nitration of heavy meromyosin with a 75-fold molar excess of tetranitromethane at pH 7.o and TABLE AN
IV
OPTICAL
ROTATORY
THE DIFFERENT
DISPERSION
PARAMETER
,-b0,
Specific ATPasc tively.
Conditions
tyrosine
. . . . . . . . .
residues per mole of
pH
Temp.
Time
-- b o
8 8 8 7 7 7
15' 15' t5 ' 4:' 4' 4':
20 20 20 15 15 15
(degrees)
heavy meromyosin
2.1 7.i None 2.2 4.3
PREPARATIONS
NITRATED
tINDER
a c t i v i t i e s o f t h e c o n t r o l s t r e a t e d a t p H 8.o a n d 7.o w e r e o . 6 I a n d o.5o, r e s p e c -
Nitro-
None
OF THE
CONDITIONS
t~iochim. Biophys..4 cta, 229-
rain rain rain tl h h
( 1 9 7 I) 3 i ° - 3 2 1
312 312 312 302 302 296
MODIFICATION OF TYROSINE RESIDUES
319
4 ° correlated with the nitration of 6.5 tyrosine residues. At p H 8.0 and IO °, o n the other hand, only 60% of the decrease in activity observed on nitration with a 9-fold molar excess of tetranitromethane correlated with the nitration of 7.1 tyrosine residues. In each instance, on nitration of up to 2 tyrosine residues, the activity fell to about 60% of the control. The content of free SH groups in each preparation recovered to the same level as that of the control, but was lower than that of the original heavy meromyosin. The content of free SH groups in the original heavy meromyosin was 8-8.5 moles per lOs g of heavy meromyosin. Diazotization of two tyrosine residues with D H T caused complete inactivation of ATPase accompanied by a decrease in the --b o value. In marked contrast, the decrease in ATPase activity induced by nitration in both experiments was not accompanied by detectable changes in the --b o value (Table IV). On prolonged treatment of the cystamine-treated heavy meromyosin in the reaction mixture, the --b o value of all preparations decreased very slightly, from 312 to 302, but this does not seem to be significant. DISCUSSION As reported b y SOKOLOVSKY AND VALLEE 14, diazo-derivatives of amino acid residues are destroyed under standard conditions for protein hydrolysis. Thus, the number of diazotized residues in the protein can be determined b y measuring their loss on acid hydrolysis as compared with the native protein. From the results of amino acid analysis of the diazotized enzymes with a relatively small molecular weight29, ~°, it is known that cysteine, tyrosine, lysine and histidine residues couple with DHT. When protein is allowed to react with a large excess of DHT, the methionine content is also decreased. As shown in Fig. 4, Table I and Table I I I , it is clear that two tyrosine residues and about 40 % of the total amino groups, corresponding to 115 lysine residues, coupled with DHT, and heavy meromyosin was completely inactivated. In addition, the formation of less than one residue of bisazohistidine per mole heavy meromyosin was observed. SuzuKI et al. 31 have indicated, in their study of insulin modified by DHT, that monoazohistidine residues also formed before the formation of bisazohistidine residues. It is possible to determine spectroscopically the amount of monoazohistidine residue, which has a spectrum with a m a x i m u m at 359-360 m# and a shoulder with a midpoint at 375 m/~ at p H 8.8 (ref. 14). The product of the reaction with only a large excess of D H T showed a spectrum with a m a x i m u m at 320 m# in this range. No stoichiometric relation was observed between the changes in ATPase activity of the diazotized heavy meromyosin and in its absorbance in the range from 300 to 400 m#. Thus, the complete loss of activity which occurred concomitantly with the formation of two monoazotyrosine residues should not be due to the formation of monoazohistidine residue, if any. B~.R2{NY AND B2~R~NY8~ modified the lysine residues of myosin using Ncarboxy-L-cysteine anhydride and found that ATPase activity was lost. KUBO et al. ~ found that the binding of two moles of trinitrobenzene sulfonate led to a loss of the ATPase activity of myosin. FABIAN AND MOHLRAD33 modified the lysine residues of myosin with trinitrobenzene sulfonate in the presence and absence of ATP, and found Biochim. Biophys. Acta, 229 (1971) 31o-321
];2o
K. UCHIDA, T. HIRATSUKA
a decrease in the K+-activated and the Ca2+-activated ATPase activities. These results suggest that the lysine residues are somehow involved in the functioning of myosin ATPase. When heavy meromyosin was modified with DHT, about 115 lysine residues coupled with D H T and heavy meromyosin was completely inactivated concomitantly with a change in the secondary structure of protein. Nitration of 7 tyrosine residues in heavy meromyosin resulted in a change in activity but n~,t in conformation detectable by optical rotatory dispersion. It appears that the lvsine residues in heavy meromyosin are important for activity. However, the possibility that the lysine residues in heavy meromvosin are the binding site for the substrate seems unlikely. In the present experiments, it is impossible to refer to a role ot7 methionine residues because of the lack of its estimation. STRACHER9 found, in a study' of the photooxidation of myosin, that tile methionine residues were oxidized at a rate too slow to account for the loss in enzymic activity. The important criteria of active site labeling are stoichiometric inactivation and specific protection against inactivation a4. Although treatnlent of heavy meromyosin with D H T caused complete inactivation upon the formation of two moles of monoazotyrosine residue per mole of protein, diazotization in the presence of the substrate did not result in a significantly smaller degree of inactivation than occurred in the absence of substrate. Nitration of tyrosine residues with tetranitromethane under milder conditions resulted in the loss of 85°4, of the activity, and 6 tyrosine residues were nitrated. 2 tyrosine residues per mole of protein were nitrated with tetranitromethane, though about 4 o % of the activity remained. There was no stoichiometric loss of activity for every mole of label covalently attached per mole of protein. It is evident from these results that the tyrosine residue m a y not be involved in the binding of the substrate. From the above discussion, it is concluded that the complete inactivation of heavy meromyosin ATPase by reaction with D H T is due to modification of both tyrosine and lysine residues in heavv meromyosin accompanied by a change in the conformation of the protein molecule. ACKNOWLEI)GMENT
The authors wish to thank Dr. K. Yagi for his discussion and support. This work was supported by a grant from the Muscular Dystrophy Association of America. I~EFERENCES I J. J. BLUM, Arch. Biochem. Biophys., 97 (1962) 309. 2 T. SEKINE AND V~r. V~. KIELLEY, Biochim. Biophys. Acta, 81 (1964) 336. 3 W. YAMASHITA, Y. SOMA, S. KOBAYASHI, W. SEKINE, K. TITANI AND K. NARITA, ,]. Biochem. Tokyo, 55 (1964) 576. 4 M. F. MORALES AND ]c~. HOTTA, J. Biol. Chem., 235 (196o) 1979. 5 A. STRACHER, in W. A. PAUL, E. E. DANIEL, C. M. KAY AND G. MONKTON, Muscle, P e r g a m o n , Oxford, 1965, p. 85. 6 S. KUBO, S. TOKURA AND Y. TONOMURA, J. Biol. Chem., 235 (r96o) 28.35. 7 S. I{UBO, H. TOKUYAMA AND Y. TONOMURA, Biochim. Biophys. Acla, i o o (1965) 4598 K. I-[OTTA, J. Biochem. Tokyo, 5 ° (1961) 218. 9 A. STRACHER, .]. Biol. Chem., 240 (1965) 958 PC. lO F. MORITA ANt) K. YAGI, Biochem. Biophys. Res. Commun., 22 (1966) 297.
Biochim. Biophys. Acta, 229 (1971) 31o-321
MODIFICATION OF TYROSINE RESIDUES
321
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Biochim. Biophys. Acta, 229 (1971) 31o-321