ARCHIVES
OF BDJCHEMISTBY
Defining
AND
BIOPHYSICS
175, 279-263 (1976)
the “Fast-Reacting” Thiols of Myosin with 1,5 IAEDANS R. TAKASHI,2
Cardiovascular
J. DUKE,
Research Institute,
by Reaction
K. UE, AND M. F. MORALES3
University of California,
San Francisco, California
94143
Received December 17, 1975 The specificity of the fluorescent reagent N-iodoacetyl-N-(5-sulfo-l-naphthyllethylem&amine (1,5 IAEDANS) for a specific thiol group of myosin has been characterized by a comparison with iodoacetamide (IAA) and by observing maximal enhancement of the Ca*+-ATPase activity and inhibition of the K+-EDTA-ATPase activity of myosin. The stoichiometry of the lSH11,5 IAEDANS bound to myosin indicates the presence of two fast-reacting thiols which correspond to the “SH1” groups responsible for the catalytic properties of myosin. Moreover, it has heen unequivocally demonstrated by gel electrophoresis that the fast-reacting thiol is located on the myosin, heavy chain. A single radioactivity-labeled thiol peptide obtained from tryptic digests of myosin labeled with 13H11,5 IAEDANS or &loll-Wlacetamide indicates strongly that the identical thiol was labeled by both reagents.
The Hudson-Weber fluorescent label, 1,5 IAEDANS4 (l), is an important probe of contractile systems (2, 3). Like other analogs of IAA, and like IAA itself, this label maximally activates the Ca2+-ATBase and totally inhibits the K+-EDTAATPase of myosin on reaching a binding ratio of 2 mol of label per mole of myosin (i.e., 1 mol label/m01 S-l moiety). In SDSgel electrophoresis its fluorescence moves 1 This work was supported by Public Health Service Grant (l-Wl-HL16669) and National Science Foundation Grant (GB-24992). 2 Career Investigator Fellow of the American Heart Association. 3 Career Investigator of the American Heart Association. 4 Abbreviations used: lSHll,5 IAEDANS, N-iodo PHlacetyl-N-(5-sulfo-1-naphthyl) ethylenediamine; WXAA, iodoll-Wlacetamide; S-l, subfragment of myosin resulting from papain hydrolysis and containing “heavy chains”; SDS, sodium dodecyl sulfate, SH,, fast reacting thiol in myosin whose ligation radically affects ATPase; NEM, N-ethyl-maleimide; TPCK, cl-tosyl-amide-2-phenylethylchloromethyl ketone (chymotrypsin inhibitor); TES, Ntris(hydroxymethy1) methyld-aminoethane sulfonic acid; BME, beta mercapto ethanol; TCA, trichloroacetic acid; PPO, 2,5 diphenyloxaxole.
exclusively with the mysoin heavy chain or with the S-l heavy chain fragment. Because of its enzymatic effects we have assumed that this label binds to the fasb reacting thiols (“SH1’s”) of myosin; however, since specificity and stoichiometry are so important in fluorescence applications, the ligation of 1,5 IAEDANS is characterized more fully here. The ligation of IAA is likewise characterized, not only for comparative purposes but because there persists dispute over the behavior of IAA, hence over the definition of the SH,‘s. Sekine et al. (4, 5) correctly (as we shall see) inferred the enzymatic consequences of reacting SH1 and provided its first localisation on a peptide map. However, the distribution of radioactivity among several labeled peptides makes firm conclusions regarding stoichiometry from this source difficult. More recently, Trotta et al. (61, using IAA, and Seidel (7) using IAA-spin label, reported two equivalent SH,‘s per mole of myosin ligated by these substances, and Seidel showed that this stoichiometry is attained with maximal enzymatic effects; however, neither study provided peptide map localization. Most re-
279 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
280
TAKASHI
cently Ohe et al. (81, using IAA, separated peptides by ion exchange and gel filtration chromatography but found only 1 mol of bound IAA./mol of native myosin at maximal enzymatic effect, thus questioning the view that the two S-l moieties of myosin are symmetrical. Cur present results setr tle uncertainties about IAA, and show that the SH,‘s of myosin are even better defined with 1,5 IAEDANS. MATERIALS
AND METHODS
Myosin was conventionally prepared from rabbit back muscle (9). [“C]IAA (49 mCi/mmol) was from Amersham-Searle while lsHJ1,5 IAEDANS (16.6 mCi/mmol) was synthesized according to the procedure of Huang et al. (10). Trypsin, TPCK, and IAA were from Sigma, reagents for electrophoresis were from Bio-Rad and ultrapure urea was from Schwarz-Mann. The X-ray film was R. P. Royal X-Omat from Kodak. Protein concentration was rountinely obtained from absorption measurementsassumingE&, = 5.70 for the myosin in 0.6 M KCl. LubeZing of myosin. Myosin (20 mglml) was incubated with 40-fold molar excess of [3H]1,5 IAEDANS or [YJIAA (moles of ligand per mole of S-l moiety) in 0.6 M KCl, 50 mM TES (pH 7.0) at 0°C. Weighed samples were taken as a function of time between 120s and 4 h. The incubation was stopped by a 50fold dilution with 0.6 M KC1 containing loo-fold molar excess BME (moles BME per mol ligand). The ATPase activity (11) and molar binding ratio of ligand to myosin were measured as will be described. Stoichiometry of labeling. The ratios of mole of ligand bound per mole of myosin were measured by two methods, one using Millipore filters (12) and the other gel electrophoresis (13). In the former the weighed samples of labeled protein were precipitated by TCA, filtered, the excess ligand removed by washing, and the protein subsequently solubulized in a scintillator (85% Econofluor (New England Nuclear) 15% BBS-3 (Beckman)) and counted (Beckman LS-150). In the latter method the sample was subjected to gel electrophoresis; the gel was subsequently sliced uniformly and counted. Tryptic digestion of labeled myosin. At a time when the molar ratio of ligand to myosin S-l moiety was 1 to l(20 min for [3H]1,5 IAEDANS and 120 min for [14C]IAA) the labeling was stopped by a 50-fold dilution with 0.03 M KC1 containing a lOO-fold molar excess of BME over ligand. The precipitated myosin was freed from excess ligand and suspended in water and the pH adjusted to 8.5 with KOH. TPCK-trypsin in 1 mM HCl (5 mg/ml) was added in the proportion of 1 mg trypsin per 50 mg myosin and the digestion was allowed to continue for 24 h with trypsin being
ET AL.. added at each 6-h interval. The pH was maintained constant by a Radiometer ‘Ml’1 titrator. The digest was then lyophilized. Sequential labeling. Myosin, incubated with lsH]1,5 IAEDANS and freed from excess ligand as described above was dissolved in 0.6 M KCl, 50 mre TES (pH 7.0) at a concentration of 20 mg/ml. It was then relabeled with [14C]IAA under conditions identical with its previous labeling. Following precipitation and washing, the doubly-labeled myosin was digested as described above. The reverse sequence of labeling was also performed, i.e., myosin labeled with [“CJIAA was subsequently labeled with [3H]1,5 IAEDANS. Peptide mapping. A sample (3 mg) of the lyophilized tryptic digest in water was applied to Whatman 3 MM paper and subjected to descending chromatography in n-butanollacetic acid/water (4/l/5, v/v/v) and subsequently to electrophoresis in formate-acetate buffer (pH 1.9) at 2000 V and 20°C for 50 min. For autoradiography of the carbon-14-labeled peptide the dried paper was placed in contact with the X-ray film at 20°C for 6 days. For fluorography of the tritium-labeled peptide the dried paper was saturated with a 7% (w/v) solution of PPO (2,5 diphenyloxazole) in ether and placed in contact with the X-ray film at dry ice temperature for 25 days (14). With the aid of these films the radioactive spots on the original chromotograms were cut out, eluted with 5% acetic acid, and counted. RESULTS
Effect of labeling on myosin ATPase.
When myosin was labeled with 13H11,5 IAEDANS as shown in Fig. 1, the Ca2+ATPase increased 7-fold in 20 min and remained at a maximum level while the K+EDTA-ATPase in the same interval decreased to less than 10% of its original value. In contrast, the P4CIIAA label reacted much slower, producing similar results in 120 min. Binding ratio of label to myosin. Figure 2 shows that the kinetics of binding of these ligands are very similar to their effect on the ATPase of the myosin. Each myosin S-l moiety maximally binds ca. 1.0 mol of [3H]1,5 IAEDANS in 20 min while the [14C] IAA reacts to the same degree in 120 min. It is clear that the former ligand has the higher specificity for the SH, thiol of the myosin S-l moiety. Location of label on myosin subunits.
Figure 3 shows a comparison of two electrophoretograms of myosin maximally labeled with either [3H11,5 IAEDANS or
“FAST-REACTING”
FIG. 1. Effect of labeling on the ATPase of myosin. Myosin labeled with [3H]1,5 IAEDANS: CaZ+-ATPase (0) and K+-EDTA-ATPase (01, or with [WIIAA: CaZ+-ATPase (A) and K+-EDTAATPase (A). ATPase measured at 25’C in 0.6 M KCl, 50 mu Tris (pH 8.0), 1 mre ATP, and either 10 mM CaCl, or 5 rnr+r EDTA. Myosin concentration was 0.06 mg/ml.
THIOLS
OF MYOSIN
281
marked effect on the ATPase of this enzyme. Location of the labeled peptide in myosin. In order to confirm whether the cysteine residues labeled with [3H11,5 IAEDANS are identical with those labeled with [14C]IAA a study was made of the peptide maps of myosin labeled with each of these ligands. To identity the peptide containing the thiol with [3H11,5 IAEDANS peptide maps of three different myosin preparations were made and both the autoradiographs and fluorographs were examined. Both methods revealed that only one thiol peptide had been labeled with the 13Hj1,5 IAEDANS (Fig. 4A). Subsequent counting of the chromatogram verified this result as 94% of the radioactivity was found in this thiol peptide and 6% at the origin. The peptide map containing the thiol peptide labeled with [14ClIAA is shown in Fig. 4B. From the fact that 90% of the radioactivity was found in the one strongly radioactive spot and only 10% in the other three weak spots we conclude that the
Fro. 2. Kinetics of labeling of myosin. Myosin labeled with [SH]1,5 IAEDANS (0) or with [W]IAA (A). Ratios were determined by counting precipitated protein on Millipore filters.
PCIW, i.e., the ATPase has been maximally altered by binding 1 mol of ligand per mole of S-l moiety. It is clear that the labeled fraction was the heavy chain of the molecule, identified by a comparison with identical gels stained with Coomassie blue. The amount of ligand elsewhere in the gel is negligible. From the amount of ligand indicated by the peak the molar ratio of bound ligand can be calculated. This provides unambiguous evidence of both the location and the number of thiols in myosin whose labeling has such a
FIG. 3. Distribution of label on myosin subunits. Myosin labeled with rHl1,5 L4EDANS for 20 mm (0) or with [‘C]IAA for 120 min (A). Labeling was stopped by lo-fold dilution with 8 M urea, 1% BME, 0.385 M glycine, 50 mM Tris (pH 8.6). A 25 pg sample of labeled myosin was placed on 7.5% acrylamide gel and electrophoresed in 0.1% SDS, 50 mre Trisglycine (pH 8.6). The molar ratios of ligand to myosin heavy chain were calculated with an accuracy of 5% by dividing the total moles of ligand (determined from the disintegrations per minute of the gel slices of the heavy chain and the specific activity of the ligandl by the total moles of myosin applied to the gel, assuming a molecular weight of 2.2 x l(r for the myosin heavy chain (16).
232
TAKASHI
ET AL. DISCUSSION
Q Electrophorssir
--i,
e
FIG. 4. Autoradiograms of labeled myosin. Twodimensional tryptic peptide maps of myosin labeled with only ISH31,5 IAEDANS (A), only [‘4ClIAA (B), first [3H]1,5 IAEDANS then t”ClIAA (C), and tirst [“C]IAA then ISH]1,5 IAEDANS (D). Spots with strong radioactivity are shaded. Spot 1 corresponds to the tritium label while spot 2 corresponds to the carbon-14 label. An autoradiogram of a mixture of A and B showed that the spot En A is not identical with any in B.
thiol peptide in the strongly radioactive spot contains the SH, thiol. It is clear that the mobility of the peptide containing the labeled thiol is different in Figs. 4A and 4B. We attributed this difference to a charge difference in the two ligands under the conditions used for the peptide mapping. To test this interpretation, sequential labeling experiments were performed. Labeling first with 13H31,5 IAEDANS and then with [14ClIAA (Fig. 4C), 94% of the radioactivity was detected in the location corresponding to the tritmm-labeled peptide (3.73 nmol) while only 5% was found in the carbon-1Clabeled peptide (0.234 nmol). When the sequential labeling procedure was reversed (Fig. 4D), 95% of the radioactivity was detected in the spot corresponding to the carbon-lllabeled peptide (3.09 nmol) while only 5% was found in the tritium-labeled peptide (0.176 nmol). These results confirm the fact that the two ligands react specifically with the same thiol peptide in the S-l moiety of myosin. This can be identified as the peptide containing the fast reacting thiol or SH, of myosin.
The peptide mapping data of Sekine et al. (4) employing NEM incubation at room temperature are quite similar to our results (Fig. 4B) using [14ClIAA incubation for 2 h at 0°C. The high specificity of labeling when one uses 1,5 IAEDANS for 20 min at 0°C (Fig. 4A) substantially strengthens the conclusion that the labeling of only one kind of peptide maximally activates the Ca2+-ATBase and inhibits the K+-EDTA-ATBase of myosin (Fig. 1). Furthermore, one may conclude that the reaction of Vys” in Ile-Cys-Arg (15) is causally related to enzyme modification and that this Cys can be identified as the ‘SH1.” In addition, several other matters have been settled. It has been shown that 1,5 IAEDANS labels the same SH1, in fact labels it more effectively than IAA. The gel electrophoretogram proves unambiguously that SH, is on the myosin heavy chain (earlier this had been inferred by Trotta et al. (6) on the basis of dissociation of chains by alkali followed by gel filtration). Finally, when maximal enzymatic modification has just been achieved, 2 mol of label have just reacted with one species of peptide, indicating that myosin yields two such peptides per mole from each of its two heavy chains. This result is in agreement with the spin-label titrations of Seide1 (7) and in disagreement with the more recent work of Ohe et al. (8). ACKNOWLEDGMENTS We are indebted to Drs. David Chung and David E. Garfin for their helpful advice on high-voltage electrophoresis and paper chromatography and are grateful to Dr. Howard M. Goodman for the use of his electrophoresis apparatus and to Dr. Richard P. Haugland for kindly making the 13H11,5 IAEDANS available. REFERENCES 1. HUDBON, E. N., AND WEBER, G. (1973)Biochem istry 12, 4154-4161. 2. MENDELBON,
R., MORALISS,
M. F., AND BOTTS,
12, 2251-2255. 3. NIHEI, T., MENDEISON, R. A., AND Bms, (1974) Biophys. J. 14, 236242.
J.
(1973) Biochemistry
4. SEKINE,
T., BAXNETT,
L. M.,
AND KIELLEY,
W. (1962) J. Biol. Chem. 237, 2769-2772.
J.
W.
“FAST-REACTING” T., AND KIELLEY, W. W. (1964)&o&m. Biophys. Actu. 81, 336345.
5. SEKINE, 6. TIUXTA,
(1968)
P. P., DREIZEN, PFOC.
Nat.
Acd
7. SEIDEL,
J.
(1970) 8. OHE, M.,
Biochemistry
C., SEON,
CHOPEK, B. K.,
P., AND STR~IJER, A. Sci. USA 61,659-666. M., AND GERGELY, J.
9,3265-3272. TXTANI,
K.,
AND TON@
MURA, Y. (1970) J. Biochem. 67, 513-522. 9. TONOMURA, T., A~PEL, P., AND MORALES, M. F. (1966) Biochemistry 5, 515-521. 10. HUANG, K., FAIRCUXJGH, R. H., AND CANTOR, C. R. (1975) J. Mol. Biol. 97, 443-470.
THIOLS
283
OF MYOSIN
11. MORALES, M. F., AND H~TFA, K. (1960) J. Biol. Chem. 235, 1979-1986. 12. SCHA~~NER, W., AND WEIBSMANN, C. (1973) Anal. Biochem. 56, 502-514. 13. PATE&SON, B., AND STROHMAN, R. C. (1970) Bb chemistry 9,4094-4105. 14. RANDERATH, K. (1970) Anal. B&hem. 34, 188205. 15. KIMURA, M., AND KIELLEY, W. W. (1966) Biothem. 2. 345, 188-200. 16. GERSHMAN, P. (1969)
L. C., STRACHER,
A., AND DRJEIZEN,
J. Biol. Chem. 244, 2726-2736.
OF BDJCHEMISTBY
Defining
AND
BIOPHYSICS
175, 279-263 (1976)
the “Fast-Reacting” Thiols of Myosin with 1,5 IAEDANS R. TAKASHI,2
Cardiovascular
J. DUKE,
Research Institute,
by Reaction
K. UE, AND M. F. MORALES3
University of California,
San Francisco, California
94143
Received December 17, 1975 The specificity of the fluorescent reagent N-iodoacetyl-N-(5-sulfo-l-naphthyllethylem&amine (1,5 IAEDANS) for a specific thiol group of myosin has been characterized by a comparison with iodoacetamide (IAA) and by observing maximal enhancement of the Ca*+-ATPase activity and inhibition of the K+-EDTA-ATPase activity of myosin. The stoichiometry of the lSH11,5 IAEDANS bound to myosin indicates the presence of two fast-reacting thiols which correspond to the “SH1” groups responsible for the catalytic properties of myosin. Moreover, it has heen unequivocally demonstrated by gel electrophoresis that the fast-reacting thiol is located on the myosin, heavy chain. A single radioactivity-labeled thiol peptide obtained from tryptic digests of myosin labeled with 13H11,5 IAEDANS or &loll-Wlacetamide indicates strongly that the identical thiol was labeled by both reagents.
The Hudson-Weber fluorescent label, 1,5 IAEDANS4 (l), is an important probe of contractile systems (2, 3). Like other analogs of IAA, and like IAA itself, this label maximally activates the Ca2+-ATBase and totally inhibits the K+-EDTAATPase of myosin on reaching a binding ratio of 2 mol of label per mole of myosin (i.e., 1 mol label/m01 S-l moiety). In SDSgel electrophoresis its fluorescence moves 1 This work was supported by Public Health Service Grant (l-Wl-HL16669) and National Science Foundation Grant (GB-24992). 2 Career Investigator Fellow of the American Heart Association. 3 Career Investigator of the American Heart Association. 4 Abbreviations used: lSHll,5 IAEDANS, N-iodo PHlacetyl-N-(5-sulfo-1-naphthyl) ethylenediamine; WXAA, iodoll-Wlacetamide; S-l, subfragment of myosin resulting from papain hydrolysis and containing “heavy chains”; SDS, sodium dodecyl sulfate, SH,, fast reacting thiol in myosin whose ligation radically affects ATPase; NEM, N-ethyl-maleimide; TPCK, cl-tosyl-amide-2-phenylethylchloromethyl ketone (chymotrypsin inhibitor); TES, Ntris(hydroxymethy1) methyld-aminoethane sulfonic acid; BME, beta mercapto ethanol; TCA, trichloroacetic acid; PPO, 2,5 diphenyloxaxole.
exclusively with the mysoin heavy chain or with the S-l heavy chain fragment. Because of its enzymatic effects we have assumed that this label binds to the fasb reacting thiols (“SH1’s”) of myosin; however, since specificity and stoichiometry are so important in fluorescence applications, the ligation of 1,5 IAEDANS is characterized more fully here. The ligation of IAA is likewise characterized, not only for comparative purposes but because there persists dispute over the behavior of IAA, hence over the definition of the SH,‘s. Sekine et al. (4, 5) correctly (as we shall see) inferred the enzymatic consequences of reacting SH1 and provided its first localisation on a peptide map. However, the distribution of radioactivity among several labeled peptides makes firm conclusions regarding stoichiometry from this source difficult. More recently, Trotta et al. (61, using IAA, and Seidel (7) using IAA-spin label, reported two equivalent SH,‘s per mole of myosin ligated by these substances, and Seidel showed that this stoichiometry is attained with maximal enzymatic effects; however, neither study provided peptide map localization. Most re-
279 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
280
TAKASHI
cently Ohe et al. (81, using IAA, separated peptides by ion exchange and gel filtration chromatography but found only 1 mol of bound IAA./mol of native myosin at maximal enzymatic effect, thus questioning the view that the two S-l moieties of myosin are symmetrical. Cur present results setr tle uncertainties about IAA, and show that the SH,‘s of myosin are even better defined with 1,5 IAEDANS. MATERIALS
AND METHODS
Myosin was conventionally prepared from rabbit back muscle (9). [“C]IAA (49 mCi/mmol) was from Amersham-Searle while lsHJ1,5 IAEDANS (16.6 mCi/mmol) was synthesized according to the procedure of Huang et al. (10). Trypsin, TPCK, and IAA were from Sigma, reagents for electrophoresis were from Bio-Rad and ultrapure urea was from Schwarz-Mann. The X-ray film was R. P. Royal X-Omat from Kodak. Protein concentration was rountinely obtained from absorption measurementsassumingE&, = 5.70 for the myosin in 0.6 M KCl. LubeZing of myosin. Myosin (20 mglml) was incubated with 40-fold molar excess of [3H]1,5 IAEDANS or [YJIAA (moles of ligand per mole of S-l moiety) in 0.6 M KCl, 50 mM TES (pH 7.0) at 0°C. Weighed samples were taken as a function of time between 120s and 4 h. The incubation was stopped by a 50fold dilution with 0.6 M KC1 containing loo-fold molar excess BME (moles BME per mol ligand). The ATPase activity (11) and molar binding ratio of ligand to myosin were measured as will be described. Stoichiometry of labeling. The ratios of mole of ligand bound per mole of myosin were measured by two methods, one using Millipore filters (12) and the other gel electrophoresis (13). In the former the weighed samples of labeled protein were precipitated by TCA, filtered, the excess ligand removed by washing, and the protein subsequently solubulized in a scintillator (85% Econofluor (New England Nuclear) 15% BBS-3 (Beckman)) and counted (Beckman LS-150). In the latter method the sample was subjected to gel electrophoresis; the gel was subsequently sliced uniformly and counted. Tryptic digestion of labeled myosin. At a time when the molar ratio of ligand to myosin S-l moiety was 1 to l(20 min for [3H]1,5 IAEDANS and 120 min for [14C]IAA) the labeling was stopped by a 50-fold dilution with 0.03 M KC1 containing a lOO-fold molar excess of BME over ligand. The precipitated myosin was freed from excess ligand and suspended in water and the pH adjusted to 8.5 with KOH. TPCK-trypsin in 1 mM HCl (5 mg/ml) was added in the proportion of 1 mg trypsin per 50 mg myosin and the digestion was allowed to continue for 24 h with trypsin being
ET AL.. added at each 6-h interval. The pH was maintained constant by a Radiometer ‘Ml’1 titrator. The digest was then lyophilized. Sequential labeling. Myosin, incubated with lsH]1,5 IAEDANS and freed from excess ligand as described above was dissolved in 0.6 M KCl, 50 mre TES (pH 7.0) at a concentration of 20 mg/ml. It was then relabeled with [14C]IAA under conditions identical with its previous labeling. Following precipitation and washing, the doubly-labeled myosin was digested as described above. The reverse sequence of labeling was also performed, i.e., myosin labeled with [“CJIAA was subsequently labeled with [3H]1,5 IAEDANS. Peptide mapping. A sample (3 mg) of the lyophilized tryptic digest in water was applied to Whatman 3 MM paper and subjected to descending chromatography in n-butanollacetic acid/water (4/l/5, v/v/v) and subsequently to electrophoresis in formate-acetate buffer (pH 1.9) at 2000 V and 20°C for 50 min. For autoradiography of the carbon-14-labeled peptide the dried paper was placed in contact with the X-ray film at 20°C for 6 days. For fluorography of the tritium-labeled peptide the dried paper was saturated with a 7% (w/v) solution of PPO (2,5 diphenyloxazole) in ether and placed in contact with the X-ray film at dry ice temperature for 25 days (14). With the aid of these films the radioactive spots on the original chromotograms were cut out, eluted with 5% acetic acid, and counted. RESULTS
Effect of labeling on myosin ATPase.
When myosin was labeled with 13H11,5 IAEDANS as shown in Fig. 1, the Ca2+ATPase increased 7-fold in 20 min and remained at a maximum level while the K+EDTA-ATPase in the same interval decreased to less than 10% of its original value. In contrast, the P4CIIAA label reacted much slower, producing similar results in 120 min. Binding ratio of label to myosin. Figure 2 shows that the kinetics of binding of these ligands are very similar to their effect on the ATPase of the myosin. Each myosin S-l moiety maximally binds ca. 1.0 mol of [3H]1,5 IAEDANS in 20 min while the [14C] IAA reacts to the same degree in 120 min. It is clear that the former ligand has the higher specificity for the SH, thiol of the myosin S-l moiety. Location of label on myosin subunits.
Figure 3 shows a comparison of two electrophoretograms of myosin maximally labeled with either [3H11,5 IAEDANS or
“FAST-REACTING”
FIG. 1. Effect of labeling on the ATPase of myosin. Myosin labeled with [3H]1,5 IAEDANS: CaZ+-ATPase (0) and K+-EDTA-ATPase (01, or with [WIIAA: CaZ+-ATPase (A) and K+-EDTAATPase (A). ATPase measured at 25’C in 0.6 M KCl, 50 mu Tris (pH 8.0), 1 mre ATP, and either 10 mM CaCl, or 5 rnr+r EDTA. Myosin concentration was 0.06 mg/ml.
THIOLS
OF MYOSIN
281
marked effect on the ATPase of this enzyme. Location of the labeled peptide in myosin. In order to confirm whether the cysteine residues labeled with [3H11,5 IAEDANS are identical with those labeled with [14C]IAA a study was made of the peptide maps of myosin labeled with each of these ligands. To identity the peptide containing the thiol with [3H11,5 IAEDANS peptide maps of three different myosin preparations were made and both the autoradiographs and fluorographs were examined. Both methods revealed that only one thiol peptide had been labeled with the 13Hj1,5 IAEDANS (Fig. 4A). Subsequent counting of the chromatogram verified this result as 94% of the radioactivity was found in this thiol peptide and 6% at the origin. The peptide map containing the thiol peptide labeled with [14ClIAA is shown in Fig. 4B. From the fact that 90% of the radioactivity was found in the one strongly radioactive spot and only 10% in the other three weak spots we conclude that the
Fro. 2. Kinetics of labeling of myosin. Myosin labeled with [SH]1,5 IAEDANS (0) or with [W]IAA (A). Ratios were determined by counting precipitated protein on Millipore filters.
PCIW, i.e., the ATPase has been maximally altered by binding 1 mol of ligand per mole of S-l moiety. It is clear that the labeled fraction was the heavy chain of the molecule, identified by a comparison with identical gels stained with Coomassie blue. The amount of ligand elsewhere in the gel is negligible. From the amount of ligand indicated by the peak the molar ratio of bound ligand can be calculated. This provides unambiguous evidence of both the location and the number of thiols in myosin whose labeling has such a
FIG. 3. Distribution of label on myosin subunits. Myosin labeled with rHl1,5 L4EDANS for 20 mm (0) or with [‘C]IAA for 120 min (A). Labeling was stopped by lo-fold dilution with 8 M urea, 1% BME, 0.385 M glycine, 50 mM Tris (pH 8.6). A 25 pg sample of labeled myosin was placed on 7.5% acrylamide gel and electrophoresed in 0.1% SDS, 50 mre Trisglycine (pH 8.6). The molar ratios of ligand to myosin heavy chain were calculated with an accuracy of 5% by dividing the total moles of ligand (determined from the disintegrations per minute of the gel slices of the heavy chain and the specific activity of the ligandl by the total moles of myosin applied to the gel, assuming a molecular weight of 2.2 x l(r for the myosin heavy chain (16).
232
TAKASHI
ET AL. DISCUSSION
Q Electrophorssir
--i,
e
FIG. 4. Autoradiograms of labeled myosin. Twodimensional tryptic peptide maps of myosin labeled with only ISH31,5 IAEDANS (A), only [‘4ClIAA (B), first [3H]1,5 IAEDANS then t”ClIAA (C), and tirst [“C]IAA then ISH]1,5 IAEDANS (D). Spots with strong radioactivity are shaded. Spot 1 corresponds to the tritium label while spot 2 corresponds to the carbon-14 label. An autoradiogram of a mixture of A and B showed that the spot En A is not identical with any in B.
thiol peptide in the strongly radioactive spot contains the SH, thiol. It is clear that the mobility of the peptide containing the labeled thiol is different in Figs. 4A and 4B. We attributed this difference to a charge difference in the two ligands under the conditions used for the peptide mapping. To test this interpretation, sequential labeling experiments were performed. Labeling first with 13H31,5 IAEDANS and then with [14ClIAA (Fig. 4C), 94% of the radioactivity was detected in the location corresponding to the tritmm-labeled peptide (3.73 nmol) while only 5% was found in the carbon-1Clabeled peptide (0.234 nmol). When the sequential labeling procedure was reversed (Fig. 4D), 95% of the radioactivity was detected in the spot corresponding to the carbon-lllabeled peptide (3.09 nmol) while only 5% was found in the tritium-labeled peptide (0.176 nmol). These results confirm the fact that the two ligands react specifically with the same thiol peptide in the S-l moiety of myosin. This can be identified as the peptide containing the fast reacting thiol or SH, of myosin.
The peptide mapping data of Sekine et al. (4) employing NEM incubation at room temperature are quite similar to our results (Fig. 4B) using [14ClIAA incubation for 2 h at 0°C. The high specificity of labeling when one uses 1,5 IAEDANS for 20 min at 0°C (Fig. 4A) substantially strengthens the conclusion that the labeling of only one kind of peptide maximally activates the Ca2+-ATBase and inhibits the K+-EDTA-ATBase of myosin (Fig. 1). Furthermore, one may conclude that the reaction of Vys” in Ile-Cys-Arg (15) is causally related to enzyme modification and that this Cys can be identified as the ‘SH1.” In addition, several other matters have been settled. It has been shown that 1,5 IAEDANS labels the same SH1, in fact labels it more effectively than IAA. The gel electrophoretogram proves unambiguously that SH, is on the myosin heavy chain (earlier this had been inferred by Trotta et al. (6) on the basis of dissociation of chains by alkali followed by gel filtration). Finally, when maximal enzymatic modification has just been achieved, 2 mol of label have just reacted with one species of peptide, indicating that myosin yields two such peptides per mole from each of its two heavy chains. This result is in agreement with the spin-label titrations of Seide1 (7) and in disagreement with the more recent work of Ohe et al. (8). ACKNOWLEDGMENTS We are indebted to Drs. David Chung and David E. Garfin for their helpful advice on high-voltage electrophoresis and paper chromatography and are grateful to Dr. Howard M. Goodman for the use of his electrophoresis apparatus and to Dr. Richard P. Haugland for kindly making the 13H11,5 IAEDANS available. REFERENCES 1. HUDBON, E. N., AND WEBER, G. (1973)Biochem istry 12, 4154-4161. 2. MENDELBON,
R., MORALISS,
M. F., AND BOTTS,
12, 2251-2255. 3. NIHEI, T., MENDEISON, R. A., AND Bms, (1974) Biophys. J. 14, 236242.
J.
(1973) Biochemistry
4. SEKINE,
T., BAXNETT,
L. M.,
AND KIELLEY,
W. (1962) J. Biol. Chem. 237, 2769-2772.
J.
W.
“FAST-REACTING” T., AND KIELLEY, W. W. (1964)&o&m. Biophys. Actu. 81, 336345.
5. SEKINE, 6. TIUXTA,
(1968)
P. P., DREIZEN, PFOC.
Nat.
Acd
7. SEIDEL,
J.
(1970) 8. OHE, M.,
Biochemistry
C., SEON,
CHOPEK, B. K.,
P., AND STR~IJER, A. Sci. USA 61,659-666. M., AND GERGELY, J.
9,3265-3272. TXTANI,
K.,
AND TON@
MURA, Y. (1970) J. Biochem. 67, 513-522. 9. TONOMURA, T., A~PEL, P., AND MORALES, M. F. (1966) Biochemistry 5, 515-521. 10. HUANG, K., FAIRCUXJGH, R. H., AND CANTOR, C. R. (1975) J. Mol. Biol. 97, 443-470.
THIOLS
283
OF MYOSIN
11. MORALES, M. F., AND H~TFA, K. (1960) J. Biol. Chem. 235, 1979-1986. 12. SCHA~~NER, W., AND WEIBSMANN, C. (1973) Anal. Biochem. 56, 502-514. 13. PATE&SON, B., AND STROHMAN, R. C. (1970) Bb chemistry 9,4094-4105. 14. RANDERATH, K. (1970) Anal. B&hem. 34, 188205. 15. KIMURA, M., AND KIELLEY, W. W. (1966) Biothem. 2. 345, 188-200. 16. GERSHMAN, P. (1969)
L. C., STRACHER,
A., AND DRJEIZEN,
J. Biol. Chem. 244, 2726-2736.
