Characterization of the fluorescein isothiocyanate-reactive site of gizzard myosin ATPase

Biochimica etBiophysicaActa 912 (1987) 230-238 Elsevier

230

BBA32808

Characterization of the fluorescein isothiocyanate-reactive site of gizzard myosin ATPase Sudhir Srivastava, Gail Sasser, Darrell L. Peterson and Steven P. Driska Department of Physiology and Biophysics, Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA (U.S.A.) (Received 12 August 1986) (Revised manuscript received 5 January 1987)

Key words: Myosin gizzard; Chemical modification; ATP-binding site; ATPase, actin-activated; (Turkey)

We describe the reaction of fluorescein 5'-isothiocyanate with gizzard myosin, and investigate the effect of this fluorescent modification on ATPase activities. Changes in the ATPase activities upon modification occur rapidly, paralleling the reaction of 'fast reacting lysine residues' during the fast phase of the reaction. The loss in the ATPase activity is linearly correlated with the extent of modification. About 90% of the ATPase activity is lost with the incorporation of 2.6 mol of reagent per mol of myosin. The fluorescent label is mainly incorporated into the heavy chain of the myosin molecule. Using limited tryptic digestion of labeled Sl, we have shown that the fluorescent dye remains in the 18 kDa fragment. The amino acid composition and the partial sequence of the peptide from the N-terminal end is presented. The results presented here suggest the participation of the 18 kDa peptide in the nucleotide binding domain of gizzard myosin.

Myosin is a major contractile protein in muscle. In smooth muscle, it is composed of one pair of heavy chains (molecular weight 200 kDa) and two pair of light chains with molecular weights of 20 kDa and 17 kDa. The 90 kDa segment of the 200 kDa subunit is folded in a globular shape and the rest of the two chains are joined in a coiled-coil a-helix. Like skeletal myosin, the globular portion of the myosin molecule, subfragment-1 (S1), contains the binding site for actin and catalytic site

Abbreviations: $1, subfragment-1; Bz2ATP, 3'-O-(4-benzoyl)benzoic adenosine triphosphate; FITC, fluorescein 5'-isothiocyanate; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography. Correspondence: S. Srivastava, Department of Physiology and Biophysics, P.O. Box 551, MCV Station, Medical College of Virginia, Richmond, VA 23298, U.S.A.

for ATP (for reviews see Refs. 1 and 2). The regulation of smooth muscle contraction via phosphorylation and dephosphorylation of the 20 kDa light chains is well documented [3-5]. However, unlike skeletal myosin, knowledge of the various functional domains of smooth muscle myosin is lacking. In the skeletal system, it has now been shown that the various tryptic fragments of S1 are associated with a particular function; the 20 kDa peptide contains the SH1 and SH2 thiols and the binding site for actin [6-8], whereas the 25 kDa and the 50 kDa regions contribute to the binding site for ATP [9,10]. Although, smooth muscle S1 can also be cleaved by limited proteolysis with trypsin into three major fragments, 29 kDa, 25 kDa and 50 kDa, there is no concrete evidence for the presence of such defined functions for the various tryptic fragments of smooth muscle myosin. The studies on the structure-function relation-

0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

231 ship of smooth muscle myosin have evolved in three directions; chemical modification, proteolytic digestion, and affinity labeling of the myosin head. The approach involving chemical modification of essential residues has previously been used to characterize the active site of gizzard myosin [11-15]. We first reported [11] that smooth muscle myosin contained one rapidly reactive lysyl group whose modification with 2,4,6-trinitrobenzene sulfonate significantly affected the various ATPase activities. Subsequently, we found that trinitophenylated myosin did not require phosphorylation of the 20 kDa light chain for the activation of its Mg2÷-ATPase activity by actin [12]. It was also found that trinitrophenylation shifted the 6S-10S equilibrium towards the 6S species of myosin. Similar effects of thiol modification on the actin-activated Mg2÷-ATPase and on conformation of gizzard myosin have now been reported [13-15]. The amino acid residues at the active site can be determined by labeling with ATP analogs. Recently, an approach using a nitrophenyl azide photoaffinity label and 3'-O-(4-benzoyl)benzoic adenosine triphosphate (Bz2ATP) has been used to characterize the active site of skeletal [9,10] and smooth muscle myosin [16]. In search of an alternate and efficient affinity label for myosin, we have examined the reaction of fluorescein 5'-isothiocyanate (FITC) with gizzard myosin. Our investigation was prompted by recent reports on the successful labeling of many adenine nucleotide binding proteins with FITC [17,18]. In the present report we have examined the reaction of FITC with gizzard myosin in order to gain an insight into the active site. Materials and Methods

Protein purifications. Myosin was prepared from turkey gizzard by the method of Persechini and Hartshorne [19]. Subfragrnent-1 was prepared by the digestion of myosin with papain (Sigma) [20]. S1 was further purified by gel filtration on Sephacryl-300 column (2.5 × 50 cm), pre-equilibrated with buffer containing 0.3 M NaC1, 0.2 mM dithiothreitol, 0.2 mM EDTA and 40 mM Tris-HC1 (pH 7.5). The protein was eluted with the same buffer. Rabbit skeletal F-actin was pre-

pared from acetone-dried powder according to the procedure of Spudich and Watt [21]. Myosin light chain kinase from turkey gizzard and calmodulin from bovine brain were prepared as described in Refs. 22 and 23, respectively. Phosphorylation and A TPase assays. Phosphate (~2Pi) incorporation into myosin was assayed as described in Ref. 24. ['y-32p]ATP was obtained from New England Nuclear. Myosin was phosphorylated by incubation for an indicated time at 25 o C with 10/~g/ml calmodulin, 10/~g/ml kinase, 1 mM [7-32p]ATP, 30 mM Tris-HCl (pH 7.5), and 0.1 mM CaC12. Other conditions are given in the figure legends. ATPase activities in the millimolar range of ATP were determined at 25 °C as described by Ferenczi et al. [25] by using [7-32p]ATP (New England Nuclear) under the following conditions: 4 mM MgC12, 25 mM Tris-HC1 (pH 7.5), 1 mM ATP; 0.6 M KC1, 2 mM EDTA, 25 mM Tris-HCl (pH 7.5), 1 mM ATP. The ATPase reaction mixtures were stopped by the addition of an aliquot (0.5 ml) of the assay mixture to a stoppered plastic funnel (Isolab Quik-Sep column, code QSP) containing I ml of 2% (w/w) charcoal, and 0.5 ml of 1 M perchloric acid and 0.35 M NaH2PO 4. The samples were mixed, by a vortex mixer, kept on ice (to reduce acid-catalyzed hydrolysis of ATP), and filtered by applying slight positive air pressure. The aliquots were subjected to Cerenkov counting. A correction was made for contaminant 32pi in [7-32p]ATP by determining the counts in an aliquot at zero time or in the absence of myosin. Other conditions are given in the figure legends. Sedimentation velocity. Sedimentation studies were carried out in a Beckman model E analytical ultracentrifuge fitted with Rayleigh optics with a helium-neon laser as light source. Rayleigh interferograms were taken on Kodak Technical Pan film TP-135, developed in Dektol. Myosin (2 mg/ml) in various solvent conditions at 25 °C was centrifuged at 40000 rpm in 12-ram light path cells using quartz plain and 1 positive wedge windows. Blank runs were performed at the same speed as the protein samples. During the rotor acceleration period, diffraction patterns were monitored to detect any rapidly sedimenting aggregates. The sedimenting species described in the text were those visible at the final rotor speed.

232

Chemical modification. The reaction of myosin and S1 with FITC were carried out for indicated times at 35 ° C in a solution containing 0.5 M KC1, 40 mM Tris-HC1 (pH 8.0), 0.2 mM EDTA and 2-4 mM MgC12. The reaction was initiated by addition of a 10-fold molar excess of FITC over the myosin concentration. The free FITC was removed by gel filtration on Sephadex G-50 (1 x 10 cm) prequilibrated with 0.5 M KCI and 0.04 M Tris-HC1 (pH 7.5) and 0.2 mM dithiothreitol; longer columns (2 x 50 cm) were used for the preparation of modified S1 for digestion studies. Trypsin digestion and peptide isolation. Modified S1 (2 mg/ml) in 0.1 M KHCO a was digested with trypsin at a protease to protein ratio of 1:100 (w/w) for 20-30 min at 25°C. The digest was then treated with buffer containing 1% SDS, 1% mercaptoethanol, 0.3 M NaCI and 20 mM TrisHCI (pH 7.5) and heated for 5 rain at 100 ° C. The denatured digest was loaded on a Sephacryl S-200 column (1.5 × 200 cm) and eluted with the above buffer. The protein and FITC contents of various fractions were monitored at 280 nm and 492 nm, respectively. The fractions containing the FITClabeled peptide were pooled and used for further purification. Further purification was carried out using one of two procedures: (1) the pooled fractions were concentrated using an Amicon ultrafiltration cell (filter YM 10, molecular weight cutoff 10000), and subjected to SDS-polyacrylamide slab gel electrophoresis. Upon completion of electrophoresis the slab gel was viewed under ultraviolet light. The band containing FITC exhibited fluorescence and was cut out. The peptide was eluted by soaking gel pieces in 0.3 M NaC1 overnight. (2) The pooled fractions were lyophilized and dissolved in 0.1% trifluoroacetic acid and 0.1% morpholine. The sample was applied to Vydac C-18 column (22 mm i.d. × 25 cm, pore size 330 ~,) and chromatographed on a LKB high-performance liquid chromatography system. The solvent gradient was formed by mixing buffer A containing 0.1% trifluoroacetic acid, and 0.1% morpholine, and buffer B containing 0.1% trifluoroacetic acid in 95% isopropanol and 0.1% morpholine at a flow rate of 0.5 ml/rnin. There was one major peak eluted around 33-35% buffer B. The purified

peptide showed one band on SDS-polyacrylamide gel with molecular weight of approx. 17.8 kDa. We routinely used electrophoretic procedures for the peptide preparation. SDS from the purified peptide was removed by precipitating the peptide with a 20 vol. of solvent containing acetone, triethylamine, acetic acid and water at a ratio of 85 : 5 : 5, v/v. Amino acid analysis. The peptide was carboxymethylated [26] and digested extensively in 6 M HCI at 110°C for 24 h using a procedure [27], which employs post-column derivatization of hydrolysate with o-phthalaldehyde for detection [28]. Amino acid analysis was performed on a Durrum MBF D-500 analyzer. The purified peptide was partially sequenced on a gas phase sequenator (Applied Biosystems Protein Sequencer 470A). Other procedures. SDS-polyacrylamide gel electrophoresis was carried out on 7.5-20% polyacrylamide gradient gels using the discontinuous buffer system of Laemmli [29]. Isoelectric focusing was performed according to the procedure described by Driska et al. [30]. Ampholytes (pH 3-10) were obtained from Pharmacia. The molecular weights of the fragments were determined using the following proteins and molecular weight values: phosphorylase b (94 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and lactalbumin (14.4 kDa). Protein concentration was estimated by using Folin [31] and Biuret reagents [32]. FITC was quantitated by dissolving the labeled protein in 2% SDS and 0.1 M NaOH and scanning from 600 nm to 350 nm. The concentration of FITC in standards run in parallel was calculated using molar absorption coefficient 80000 M - l . c m -1 at 492 nm. Bz2ATP was synthesized from [a-32p]ATP according to the procedure of Williams and Colman [33]. This analog was incorporated into myosin as follows. Myosin in 0.5 M KC1, 0.2 mM EDTA, 0.2 mM dithiothreitol, 4 mM MgC12 and 50 mM Tris-HC1 (pH 7.5) at 0 - 4 ° C was exposed to long-wavelength ultraviolet light from a UVSL-58 Minerallight for a specified time. The amount of incorporated label was determined according to the procedure described earlier [16].

233

Results

The reaction of gizzard myosin with FITC proceeded in two phases: an initial fast phase followed by a slow phase. The lysine residues being modified during the fast phase of reaction will be referred to as reactive lysine residues. Samples were withdrawn during this reaction and their EDTA-stimulated (K+)-ATPase activities were measured. The ATPase activity significantly decreased during the reaction, reaching a plateau 30 min after reaction (Fig. 1). Under these conditions, the reaction follows pseudo-first order kinetics, i.e. a semilogarithmic plot of relative myosin ATPase activity (percent active sites remaining) versus time of incubation was linear (see inset of Fig. 1). Analog incorporation into myosin was determined by monitoring the inactivation of the EDTA-stimulated (K+)-ATPase activity at different incubation times. Aliquots from the modification reaction mixture, taken at a given time, were purified from reagents by gel filtration on Sephadex G-50, and the eluted protein was used for ATPase assay and for the estimation of bound FITC using the procedures described in Materials and Methods. The binding of FITC to myosin as a

c 60

.

E x.

~ 2o ~

750 E

1

O-

20 ~ 0 500

zso

20

40 Time (rnin)

E c

~' g

T

60

Fig. 1. Time-course of FITC incorporation (O) and EDTAstimulated (K ÷ )-ATPase activity (A) of gizzard myosin. The reaction mixture contained the following: 50 mM Tris-HC1 (pH 8.0), 0.2 mM EDTA, 4 mM MgC12, 3 mg/ml myosin and a 20-fold molar excess of FITC over myosin head. The reaction mixture for the ATPase assay contained 30 mM Tris-HC1 (pH 7.5), 2 mM EDTA, 0.6 M KC1, 2 mM ATP and 0.25 mg/ml myosin. Inset is the semilogarithmic plot of percent active sites vs. time. ATPase activity of unmodified myosin was considered representing 100 percent intact active sites.

100

- 50

0

1'

2

3

MOl F I T C / m o l myosin

Fig. 2. Relationship between the extent of rapidly reacting lysine residues modification and inhibition of EDTA-stimulated (K ÷ )-ATPase activity.

function of its residual ATPase activity is shown in Fig. 2. It is clear from these data that upon modification of about 2.6 rapidly reacting lysine residues per myosin or 1.3 per myosin head, more than 90% of the active sites were eliminated. The Mg2+-ATPase activities of gizzard myosin both in the presence and absence of actin were significantly decreased. This is in contrast to earlier studies on trinitrophenylation of skeletal and smooth myosin myosin, where Mg2+-ATPase activity was increased and actin-activated (Mg2+)ATPase remained unchanged upon trinitrophenylation [34,35]. It is likely that FITC reacts with different sites on the myosin molecule, but it is clear that FITC reacts rapidly with the active site. This was further substantiated by the findings from the effect of presence of ATP (2-5 mM) during modification reaction (Table I). Inclusion of ATP during the modification reaction decreased the incorporation of FITC into myosin and prevented the loss of the EDTA-stimulated (K+)ATPase activity. The Mg2+-ATPase and actinactivated Mg2+-ATPase activities of myosin also remained unaffected (data not shown). Photoincorporation of Bz2ATP, a photoaffinity analog of ATP, into modified myosin was significantly reduced by 80% (Fig. 3). This analog preferentially reacts with the nucleotide binding site of gizzard myosin [16]. The effect of FITC modification on the actinactivated (MgZ÷)-ATPase activity appears to be different from that of the trinitrophenylation reac-

234 TABLE I

TABLE II

EFFECT OF ATP ON THE FITC INCORPORATION INTO MYOSIN

EFFECTS OF KCI CONCENTRATION IS ON THE SEDIMENTATION RATE OF MODIFIED AND UNMODIFIED MYOSIN

Myosin was reacted for 20 rain in the presence of 20-fold molar excess of FITC and various concentrations of ATP at 35°C. The EDTA (K +)-ATPase activity of myosin was assayed as described in the text. Conditions Unmodified myosin Myosin + no ATP Myosin + 2 mM ATP Myosin+ 5 mM ATP

FITC (tool/ /mol myosin)

ATPase activity a

0.0 1.83 0.34 0.24

0.58 0.06 0.52 0.54

KCL concn. (M) 0.15 0.20 0.35

a The activity is expressed as ~mol Pi/min per mg myosin.

tion. We have s h o w n previously that trinitrophenylation of gizzard myosin removed the requirement for phosphorylation of myosin in the activation of Mg2+-ATPase activity by actin, and that trinitrophenylation favored the 6S conformation of myosin [12]. However, F I T C modification, unlike the trinitrophenylation reaction, did not abolish the requirement for phosphorylation of the 20 k D a light chains in the activation of the Mg2+-ATPase activity b y actin. Smooth muscle myosin can exist either as a folded (10S) or as an extended (6S) molecule [36-38]. Ikebe et al. [38] suggested that that some c o m p o n e n t of this con-

c

E

Q_ N N

15 T i m e (rain)

The solvent conditions were: 30 mM Tris-HC1 (pH 7.5), 1 mM ATP, 4 mM MgCI2, and 0.2 mM EDTA at 25 C. For details, see text.

3O

Fig. 3. BzzATP incorporation into unmodified (I) and modified (1.6 tool FITC/mol myosin) (11) myosin. Myosin (2-3 mg/ml) in 0.5 M KC1, 0.2 mM EDTA, 0.2 mM dithiothreitol, 2.0 mM MgC12, and 40 mM Tris-HCl (pH 7.5) at 0-4°C in the presence of a 10-fold molar excess of the analog was exposed to long-wavelength ultraviolet light for various times.

Sedimentation rate a Unmodified myosin

FITC-myosin

9.8 10.2 5.8

10.2 10.1 6.2

a Sedimentation rates in Svedberg units (S). formation transition is a determinant of enzymatic activity. It was therefore of interest to investigate the effect of F I T C modification on myosin conformation. As shown in Table II, we could not detect any effect of modification on the sedimentation rates of the various myosin species under the various solvent conditions. However, the possibility of some subtle changes in the conformation of myosin not detectable by analytical centrifugation m e t h o d cannot be ruled out. The phosphorylation kinetics of modified myosin remained unaffected after the modification reaction. This indicates that the phosphorylation sites on the myosin molecule were not affected by the modification. In order to localize the FITC-reactive site on the myosin molecule, modified myosin was treated with 1% SDS, 1% mercaptoethanol and 0.5 M NaC1 and subjected to H P L C gel filtration on a T S K G3000 SW column (7.5 × 300 ram). The label was mainly f o u n d in the peak corresponding to the heavy chain of the myosin molecule (see inset of Fig. 4). As reported by us earlier [16], limited tryptic digestion of gizzard S1 also yields three m a j o r fragments with molecular weights of approximately 50 kDa, 29 k D a and 25 kDa. A 5-min tryptic digest of FITC-labeled S1 is shown in Fig. 5. Fluorescence was detected mainly in the 50 k D a fragment and in the undigested 70 k D a heavy chain of S1. We have also found that at 20-rain the 50 k D a peptide was cleaved to smaller fragments and 18 k D a was the major peptide whose appearance coincided with the disappearance of the 50 kDa. Other fragments with small molecular

235 1.0

008

0 005 <

E c 0 0 4 ,,¢ <

8

16

~.4 Time (min]

32

40

Fig. 4. High-performance liquid chromatography elution profile of SDS-treated FITC-modified myosin (1.6 mol FITC/mol myosin) on a gel filtration column (TSK G3000 SW, 7.5 × 300 mm). Protein (solid line) and FITC (e) contents were monitored at 280 nm and 492 nm, respectively. Inset is the SDS-gel electrophoretogram showing a fluorescent band of the myosin heavy chain recovered in the first peak.

weights were not detectable on our gel system (unpublished data). The further characterization of the reactive site was carried out with labeled S1 (1.2 mol F I T C / m o l S1) digested with trypsin for 20 rain. The 20-min tryptic digest was applied to a Sephacryl-200 column. The fractions containing F I T C were pooled and subjected to further purification on a C-18 column as described in Materials and Methods. A b o u t 80% of F I T C was recovered in the 17.8 k D a peptide (we will call this the 18 k D a peptide hereafter). The remaining label was found in fragments smaller than the 18 k D a peptide overlapping with the digested light chains. We attribute this to the 'slow reacting lysine residues' contributing towards the non-specific incorporation of F I T C . A SDS-gel of the 18 k D a peptide is shown in Fig. 6. The percentage of F I T C in the 18 k D a peptide when multiplied by the total F I T C incorporation into S1 yielded a stoichiometry of about 1 tool 18 k D a peptide per mol S1. This peptide appears to be basic as judged

--70

-

-

50

--2'9

j

- - 18 kDa

--25 B

Fig. 5. SDS-polyacrylamide gel electrophoretograms of a 5-min tryptic digest of FITC-labeled SI: (A) gel stained with Coomassie brilliant blue, and (B) the gel illuminated with ultraviolet light. The top most band is the undigested heavy chain (°70kDa) of $1. The numbers shown beside each protein band are the relative molecular weights (× 10-3).

A

B

Fig. 6. SDS-polyacrylamide gel electrophoretograms of the purified 18 kDa peptide: (A) gel stained with Coomassie brilliant blue, and (B) the gel illuminated with ultraviolet light (366 nm). The amounts of protein loaded on the gel (A) and (B) were 20 and 60/~g, respectively.

236 T A B L E Ili A M I N O A C I D C O M P O S I T I O N OF T H E 18 kDa PEPTIDE The data shown are averaged values from determinations on a 24 h acid hydrolysis of three different samples, n.e., not estimated. Amino acid

R e s i d u e s / m o l peptide

Asp Thr Ser Glu Gly Ala Val Met lie Leu Tyr Phe His Lys Arg Pro Cys ~

24.2 7.4 4.9 26.5 14.5 10.4 7.1 5.7 7.4 12.8 3.2 12.3 3.0 8.6 6.8 n.e. 0.8

a Carboxymethylated cysteine.

its mobility on isoelectrofocusing gel (pH range 3-10). The pI value was found to be 8.5. The amino acid composition of this peptide is shown in table III. The N-terminal analysis of the peptide shown below reveals alanine to be the N-terminal residue. A-T-G-N-V-K-A-P-K-D-Q-S-V...

It has been shown that the major tryptic fragments of gizzard myosin, namely 29 kDa, 25 kDa and 50 kDa, are analogous to 25 the kDa, 20 kDa and 50 kDa fragments of its skeletal counterpart [39]. The 23 kDa peptide of skeletal myosin (a proteolytic fragment of the 25 kDa fragment) has been sequenced by Tong and Elzinga [40]. It contain two reactive lysine residues (Lys-83), one in each head, and two types of methylated lysine residues, believed to be involved in binding with the adenine moiety of ATP through the formation of a hydrophobic pocket. We, therefore, compared our partial sequence with the 23 kDa fragment of rabbit skeletal myosin heavy chain. A search of the Microgenic data base (Beckman Instruments) did not reveal any similar sequence in the 203-

amino acid long 23 kDa peptide; match per length was less than 4% and a similar searches failed to reveal any similarity between this peptide and the 20 kDa and 17 kDa light chains. The sequence for the 50 kDa and the 25 kDa peptide of gizzard myosin are not yet known, and therefore no comparison could be made. Discussion

Chemical modification of a protein by a reagent is one of the most direct methods for determining which type of aminoacid residue is responsible for a function of the protein, if it is established that the aminoacid residue in the protein is specifically and stoichiometrically attacked by the reagent under mild conditions. Several such studies have been carried out in the past to gain information on the chemical nature of the active site of skeletal muscle myosin [41] (for a review, see Ref. 42). With the exception of some recent works by us [11,12] and by others [13-15] very little information is available on the chemical structure of the active site of smooth muscle myosin ATPase. Pursuing our long range interest on the active site of smooth muscle myosin, we have employed a fluorescent label, FITC, to characterize the nucleotide binding domain of gizzard myosin. FITC, a specific reagent for lysyl groups, has recently been used to study the nucleotide binding site of the (Na + + K +)-ATPase from lamb and rat kidney [17] and the sarcoplasmic reticulum CaZ+-ATPase from canine cardiac muscle [18]. In the present paper, we report the incorporation of FITC into gizzard myosin and its effects on the various ATPase activities of gizzard myosin. It is clear from the present report that modification of rapidly racting lysyl groups with FITC is both specific and stoichiometric (Figs. 1 and 2). Modification of rapidly reacting lysine residues leads to the loss of various ATPase activities. Hence rapidly reacting lysine residues appear to be at or around the active sites. In previous reports, it was reported that modification of thiol groups [13] and lysine residues [12] of gizzard myosin produced an enhancement of the actin-activated ATPase activity very similar to that produced by phosphorylation of the 20 kDa light chain by light chain kinase. Subsequently, it

237 was found that both of these modifications stabilize the myosin filaments and favor the 6S conformation of myosin [12,15]. In the present studies, Mg 2+-ATPase activities of gizzard myosin both in the presence and absence of actin were decreased significantly by FITC treatment (data not shown) Also, we did not find any effect of FITC modification on the transition of modified myosin from the 10S to the 6S states that occurs on raising the ionic strength (Table I). However, it does not rule out the possibility of subtle conformational changes in the myosin molecule, which may only be detectable with other more sensitive technique. A key point for this experiment is that the modified myosin still retains its characteristic features, i.e. modified myosin like unmodified myosin can undergo the transition between the 6S and 10S states under various solvent conditions. Also, the phosphorylation sites on the light chains are unaffected by this modification. This was confirmed by the phosphorylation kinetics of modified myosin (data not shown). This is in agreement with the earlier reports by us [12] and by Bailin [43] in which modification of lysine [12] and cysteine [43] residues did not affect the extent of phosphorylation of myosin in the presence of myosin light chain kinase and calmodulin. The results presented here raise an important question: why is the effect of FITC modification on the myosin ATPase activity different from that of the TNBS reaction? Although, the present work suggests that FITC and TNBS react with two different sites on the myosin molecule, further work is needed on the complete sequence of sites modified by these two reagents, and this is left to future research. The next objective was to identify an approximate location of the FITC-reactive site on the myosin molecule. The chromatographic elution profile of denatured FITC-labeled myosin clearly shows that the label was mainly incorporated into the heavy chain of the molecule (Fig. 2L). Limited tryptic digestion of modified S1 further reveals that the label was localized in the 18 kDa peptide of the myosin heavy chain (Fig. 6). Limited proteolysis of skeletal S1 produces three fragments of 20, 25 and 50 kDa and they are believed to be involved in the following functions: 25 kDa and 50 kDa contribute to the ATP-binding site [9,10];

and 20 kDa contains two essential thiol groups, SH1 and SH2, and participates in actin binding [6-8]. It has now been shown that these fragments constitute domains of S1 heavy chain [44] Gizzard myosin also yields three fragments of 29 kDa, 25 kDa and 50 kDa which are considered to be analogous to 25 kDa, 20 kDa and 50 kDa tryptic fragments of skeletal S1 [39]. We have recently shown that the 50 kDa peptide may be involved in the ATP binding [16]. However, the roles of other peptides are not clear and the question whether or not they represent domains of smooth muscle S1 remains. The 18 kDa peptide appears to be a proteolytic fragment of the 50 kDa peptide. Our conclusion was based on the following findings: (1) Fractions from the Sephacryl S-200 chromatography containing the 29 kDa and the 25 kDa fragments did not absorb at 492 nm, a wavelength at which FITC absorbs. (2) FITC was mainly incorporated into the 50 kDa peptide as shown by the fluorescence of the 50 kDa peptide in a 5-min tryptic digest of FITC-labeled S1 (Fig. 5). The digestion of S1 with trypsin (20 rain or more) led to a further breakdown of the 50 kDa peptide into an 18 kDa peptide and various smaller fragments (unpublished data). (3) Incorporation of Bz2ATP into FITC modified myosin was significantly reduced (Fig. 3), and we have shown earlier [16] that Bz2ATP was preferentially incorporated into the 50 kDa peptide. This also suggests that FITC was incorporated into the 50 kDa peptide before its cleavage into the 18 kDa peptide. (4) The possibility that the 18 kDa peptide we studied was a proteolytic fragment of the 20 kDa light chain was ruled out because the published sequence of the 20 kDa light chain [44] did not contain a sequence identical to 13 residues at the N-terminal of the 18 kDa peptide we studied. In comparison, the ATP-binding of the skeletal myosin ATPase has been localized in the 25 kDa peptide of the S1 heavy chain [9]. Subsequent studies by Mahmood and Yount [10], using Bz2ATP as a photo-affinity label, have shown that the 50 kDa peptide may also contribute to the ATP binding site. Evidence is increasingly in favor of the active site existing at the interface of two or more fragments. This is clear from the recent work of Okamoto et al. [46] in which they have found that the 17 kDa light chain was preferentially

238

labeled with an ATP analog. Whether such an organization of the active site is true for the smooth muscle myosin ATPase awaits detailed investigations on the various functional domains of smooth muscle myosin. In summary, this report suggests that FITC reacts specifically with the ATPase active site and that the 18 kDa peptide, a proteolytic fragment of the 50 kDa peptide, may be at or around the nucleotide-binding domain of gizzard myosin.

Acknowledgements We express our thanks to Dr. Norman F. Briggs for the use of HPLC facilities in his laboratory, to Dr. Alfred Richards of the Department of Medicinal Chemistry for this assistance in analytical centrifugation studies, and to Dr. Michael B. Cable for helpful discussion during the course of this study. This work was supported by grants from the Virginia Heart Association (S.S.) and from National Institute of Health, H L 24881 (S.P.D.).

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