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• W2066706927 abstract "The interaction between the two heads of myosin II during motion and force production is poorly understood. To examine this issue, we developed an expression and purification strategy to isolate homogeneous populations of heterodimeric smooth muscle heavy meromyosins containing heads with different properties. As an extreme example, we characterized a heterodimer containing one native head and one head locked in a “weak binding” state by a point mutation in switch 2 (E470A). The in vitro actin filament motility of this heterodimer was the same as the homodimeric control with two cycling heads, suggesting that only one head of a pair actively interacts with actin to generate maximal velocity. A second naturally occurring heterodimer contained two cycling heads with 2-fold different activity, due to the presence or absence of a 7-amino acid insert near the active site. Enzymatically this (+/–) insert heterodimer was indistinguishable from a (50:50) mixture of the two homodimers, but its motility averaged 17% less than that of the mixture. These data suggest that one head of a heterodimer can disproportionately affect the mechanics of double-headed myosin, a finding relevant to our understanding of heterozygous mutant myosins found in disease states like familial hypertrophic cardiomyopathy. The interaction between the two heads of myosin II during motion and force production is poorly understood. To examine this issue, we developed an expression and purification strategy to isolate homogeneous populations of heterodimeric smooth muscle heavy meromyosins containing heads with different properties. As an extreme example, we characterized a heterodimer containing one native head and one head locked in a “weak binding” state by a point mutation in switch 2 (E470A). The in vitro actin filament motility of this heterodimer was the same as the homodimeric control with two cycling heads, suggesting that only one head of a pair actively interacts with actin to generate maximal velocity. A second naturally occurring heterodimer contained two cycling heads with 2-fold different activity, due to the presence or absence of a 7-amino acid insert near the active site. Enzymatically this (+/–) insert heterodimer was indistinguishable from a (50:50) mixture of the two homodimers, but its motility averaged 17% less than that of the mixture. These data suggest that one head of a heterodimer can disproportionately affect the mechanics of double-headed myosin, a finding relevant to our understanding of heterozygous mutant myosins found in disease states like familial hypertrophic cardiomyopathy. There has been speculation for many years about whether myosin derives any advantage from having two cross-bridge heads. The double-headed structure is clearly not necessary for actin binding or enzymatic activity, because it has long been known that proteolytically derived, single-headed subfragment 1 (S1) 1The abbreviations used are: S1, subfragment 1; HMM, heavy meromyosin; FHC, familial hypertrophic cardiomyopathy; MOPS, 3-[N-morpholino]propanesulfonic acid; DTT, dithiothreitol; BSA, bovine serum albumin, FLAG, epitope tag with amino acid sequence DYKDDDDK; His, epitope tag with amino acid sequence HHHHHH; +insert, smooth muscle myosin heavy chain containing the 7-amino acid insert in loop 1; –insert, smooth muscle myosin heavy chain lacking the 7-amino acid insert in loop 1; WT HMM, wild type homodimeric heavy meromyosin; E470A HMM, E470A homodimeric heavy meromyosin; +insert/–insert HMM, heterodimeric heavy meromyosin with one head containing the 7-amino acid insert and the other head lacking the insert; E470A/WT HMM, heterodimeric heavy meromyosin with one wild type head and one carrying the point mutation E470A; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate.1The abbreviations used are: S1, subfragment 1; HMM, heavy meromyosin; FHC, familial hypertrophic cardiomyopathy; MOPS, 3-[N-morpholino]propanesulfonic acid; DTT, dithiothreitol; BSA, bovine serum albumin, FLAG, epitope tag with amino acid sequence DYKDDDDK; His, epitope tag with amino acid sequence HHHHHH; +insert, smooth muscle myosin heavy chain containing the 7-amino acid insert in loop 1; –insert, smooth muscle myosin heavy chain lacking the 7-amino acid insert in loop 1; WT HMM, wild type homodimeric heavy meromyosin; E470A HMM, E470A homodimeric heavy meromyosin; +insert/–insert HMM, heterodimeric heavy meromyosin with one head containing the 7-amino acid insert and the other head lacking the insert; E470A/WT HMM, heterodimeric heavy meromyosin with one wild type head and one carrying the point mutation E470A; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate. retains both of these abilities (1Margossian S. Lowey S. J. Mol. Biol. 1973; 74: 313-330Crossref PubMed Scopus (137) Google Scholar). Moreover, a single-headed molecule can move actin filaments in the in vitro motility assay (2Toyoshima Y.Y. Kron S.J. McNally E.M. Niebling K.R. Toyoshima C. Spudich J.A. Nature. 1987; 328: 536-539Crossref PubMed Scopus (397) Google Scholar, 3Waller G.S. Ouyang G. Swafford J. Vibert P. Lowey S. J. Biol. Chem. 1995; 270: 15348-15352Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). However, the velocities sustained by one-headed fragments in this assay are generally less than that of double-headed species from the same myosin, and recent measurements in the laser trap have indicated that the unitary step size of a single-headed myosin is only half as great as that of a double-headed molecule (4Tyska M.J. Dupuis D.E. Guilford W.H. Patlak J.B. Waller G.S. Trybus K.M. Warshaw D.M. Lowey S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4402-4407Crossref PubMed Scopus (151) Google Scholar). These observations indicate not only that two heads are more effective than one in moving actin but also that they may work together in some sort of coordinated fashion. One way to gain insight into the nature of potential head-head interactions is to study myosins containing two heads that differ functionally. If each head works independently, then the activity of such a heterodimer should be halfway between that of the faster homodimer and the slower homodimer. On the other hand, if there is an interaction between the heads, the properties of the heterodimer might be closer to that of one of the homodimeric species. To study heavy meromyosin (HMM) molecules with different heads, we developed an expression strategy that involves differential labeling of the constituent heavy chains with FLAG and His tags, co-infection in the Sf9 cell system, and sequential affinity chromatography columns to isolate homogeneous preparations. We assessed two types of smooth muscle HMM heterodimer using enzymatic and mechanical assays. The first was composed of two naturally occurring smooth muscle heavy chain isoforms that differ in vitro by a factor of two in their actin-activated ATPase activity and actin filament motility (5Kelley C.A. Takahashi M. Yu J.H. Adelstein R.S. J. Biol. Chem. 1993; 268: 12848-12854Abstract Full Text PDF PubMed Google Scholar, 6Rovner A.S. Freyzon Y. Trybus K.M. J. Mus. Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (113) Google Scholar). This difference is due to the presence or absence of a 7-amino acid insert in the surface loop near the nucleotide-binding pocket (7White S. Martin A.F. Periasamy M. Am. J. Physiol. 1993; 264: C1252-C1258Crossref PubMed Google Scholar, 8Babij P. Nucleic Acids Res. 1993; 21: 1467-1471Crossref PubMed Scopus (70) Google Scholar), which has been called loop 1 (9Spudich J.A. Nature. 1994; 372: 515-518Crossref PubMed Scopus (421) Google Scholar). Such heterodimers almost certainly exist in nature, because the mRNAs for the +insert and –insert heavy chains are co-expressed in single smooth muscle cells (10Eddinger T.J. Meer D.P. Am. J. Physiol. 2001; 280: C309-C316Crossref PubMed Google Scholar). The second heterodimer contained one wild type (WT) heavy chain and a second head with a mutation in switch 2 (E470A) that prevents the formation of a salt bridge between this residue and R247 in switch 1 of the active site. This mutation slows the ATPase activity of smooth myosin by two orders of magnitude, essentially “locking” it in a weak binding configuration (11Onishi H. Morales M.F. Kojima S. Katoh K. Fujiwara K. Biochemistry. 1997; 36: 3767-3772Crossref PubMed Scopus (27) Google Scholar, 12Onishi H. Kojima S. Katoh K. Fujiwara K. Martinez H.M. Morales M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6653-6658Crossref PubMed Scopus (45) Google Scholar). Thus, the heterodimeric E470A/WT molecule has a much greater disparity in function between the two heads. Enzymatic assays indicated that the two heads of both heterodimers function independently. However, the in vitro motility assay showed that the heterodimeric (+insert/–insert) HMM moved actin filaments 17% more slowly than a 50:50 mixture of the corresponding homodimeric HMMs. In contrast, the heterodimeric E470A/WT HMM showed motility that was not significantly different from WT HMM. These results are consistent with the notion that myosin employs only one head at a time to perform work on the actin filament and, furthermore, suggest that one head of a heterodimer can exert a disproportionate effect on mechanical properties in molecules where both heads are actively cycling. DNA Engineering and Production of Recombinant Baculoviruses— The baculoviral transfer vector pAcSG2 (BD Pharmingen) was modified for the expression of HMM heavy chains with two different epitope tags. For FLAG-tagged species, both N- and C-terminal variants were created. For C-terminal tagging, a BamHI restriction site was added 3′ to an NcoI site in the polylinker, followed by in-frame sequence coding for FLAG (DYKDDDK) and a stop codon. For an N-terminal tag, nucleotide sequence encoding an initiating ATG and the FLAG tag was ligated in a position 5′ to the NcoI site. To produce species fused to an N-terminal hexa-histidine (His) tag, an initiating ATG codon followed by six histidine codons and an NcoI site were added 5′ to a BglII site in the pAcSG2 polylinker. cDNAs encoding the HMM portion of the chicken gizzard myosin heavy chain clone (13Yanagisawa M. Hamada Y. Katsuragawa Y. Imamura M. Mikawa T. Masaki T. J. Mol. Biol. 1987; 198: 143-157Crossref PubMed Scopus (161) Google Scholar) were ligated into these three vectors to express four different HMM heavy chains. First, a C-terminal FLAG tag was attached to a clone encoding the first 1175 amino acids of the +insert heavy chain, whereas a mutant 1168-residue HMM from which the loop 1 insert had been removed (–insert) (6Rovner A.S. Freyzon Y. Trybus K.M. J. Mus. Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (113) Google Scholar) was ligated into the N-terminally His-tagged vector, using the NcoI and BglII sites. Glutamic acid 470 of the 1175-residue cDNA was converted to alanine by site-directed mutagenesis, and this was then cloned into the vector that adds the C-terminal FLAG tag. Finally a wild-type (WT) +insert N-terminally His-tagged construct was also synthesized. Each construct in pAcSG2 was transfected and amplified in Sf9 cells by established methods (14O'Reilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman and Co., New York, New York1992: 124-149Google Scholar). To produce protein, myosin heavy chain viruses were co-infected with a recombinant virus encoding both the smooth muscle myosin essential and regulatory light chains (15Rovner A.S. Freyzon Y. Trybus K.M. J. Biol. Chem. 1995; 270: 30260-30263Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In the case of heterodimers, an N-terminally His-tagged heavy chain virus was co-infected with the appropriate C-terminally FLAG-tagged heavy chain, along with the dual light-chain virus. Preliminary trials were conducted to optimize relative viral ratios so that the expression levels of the FLAG- and His-tagged heavy chains were nearly equal. Purification of HMM Proteins—After 3 days of infection, Sf9 cells were harvested and lysed, and proteins were fractionated by two successive ammonium sulfate precipitations, 0–40% and 40–70%. For the homodimers of C-terminally FLAG-tagged +insert and E470A HMM, as well as for the two types of heterodimer, the 40–70% pellet was dialyzed overnight versus a buffer containing 90 mm NaCl, 10 mm imidazole-HCl (pH 7.4 at 4 °C), 1 mm EGTA, 1 mm NaN3, 1 mm DTT, and 1 μg/ml leupeptin. MgATP was added to a final concentration of 4 mm, the material was clarified by centrifugation, and the supernatant was applied to an anti-FLAG affinity column (M2 antibody, Sigma-Aldrich Chemical). After washing, HMM was eluted using a large molar excess of FLAG peptide (0.1 mg/ml), and peak fractions were pooled. This was the end point for the preparation of FLAG-labeled homodimers. In the case of the two heterodimer preparations, NaCl was added to the FLAG eluate to give a final concentration of 300 mm, and the pH was increased to 8.0 by adding 35 mm MOPS, pH 8.55. This material was then applied to a nickel-charged poly-histidine binding column (Probond, Invitrogen). Nonspecifically bound proteins were washed away using 300 mm NaCl, 10 mm imidazole, 10 mm MOPS (pH 8.0 at 4 °C); then heterodimeric HMM was eluted using 300 mm NaCl, 300 mm imidazole, 10 mm MOPS (pH 8.0 at 4 °C). Peak fractions were pooled and dialyzed overnight versus a buffer containing 50 mm NaCl, 20 mm MOPS (pH 7.4 at 4 °C), and 0.2 mm DTT. The protein was then precipitated by dialysis versus saturated ammonium sulfate, collected by centrifugation, re-suspended, and finally dialyzed into 50 mm NaCl, 10 mm HEPES (pH 7.0 at 4 °C), 5 mm MgCl2, 1 mm EGTA, 1 mm NaN3, 1 mm DTT. The homodimeric N-terminally His-tagged –insert HMM was isolated as follows. Following ammonium sulfate fractionation as above, equimolar F-actin was added to the pelleted material, which was then dialyzed overnight at 4 °C versus a phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.4), containing 7% sucrose (w/v), 1 mm DTT, and 1 μg/ml leupeptin. The actin-bound material was sedimented, then washed with 300 mm NaCl, 10 mm MOPS (pH 8.0 at 4 °C). The HMM was then eluted into the same buffer containing 1 mm MgATP, and loaded onto a Probond column. The –insert HMM was then washed and eluted from this column as described above for the heterodimer. After purification, aliquots of the various HMM preparations were phosphorylated by the addition of the following reagents (concentrations in parentheses): CaCl2 (0.75 mm); calmodulin (7.5 μg/ml); MgATP (1.5 mm); and myosin light chain kinase (3–6 μg/ml). Except for assessments of ADP release rates, all assays were conducted exclusively on phosphorylated proteins. Probes Used in Western Blots—Two 3–8% acrylamide gradient, Tris acetate-buffered NuPAGE gels (Invitrogen) were loaded and run with identical samples from different steps of the heterodimer preparation then transferred to nitrocellulose. One filter was reacted with the anti-FLAG monoclonal antibody diluted to 0.025 μg/ml. The filter was then reacted with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Bio-Rad) diluted 1:3000. To probe for the proteins labeled with the His tag, the duplicate filter was incubated with a horseradish peroxidase-conjugated His-chelating group at a dilution of 1:500 (India His-Probe, Pierce Chemical). Both filters were labeled using diaminobenzamidine in the presence of hydrogen peroxide. Steady-state ATPase Assays—Actin-activated ATPase activity was measured at 37 °C as described previously (16Joel P.B. Trybus K.M. Sweeney H.L. J. Biol. Chem. 2001; 276: 2998-3003Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). NH4+ -activated ATPase was determined at 37 °C, using a buffer containing 400 mm NH4Cl, 2 mm EDTA, 25 mm Tris base (pH 8.0 at 37 °C), 200 mm sucrose, 1 mm DTT, and 1 mg/ml BSA (15Rovner A.S. Freyzon Y. Trybus K.M. J. Biol. Chem. 1995; 270: 30260-30263Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Motility and Average Force Measurements—For all motility experiments, 1.2–3.5 μm HMM was mixed with a 2.5-fold molar excess of F-actin in the presence of 1 mm MgATP, and centrifuged for 25 min at 350,000 × g to remove ATP-insensitive cross-bridge heads. The protein concentration of the supernatant was determined by Bradford (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211946) Google Scholar), and dilutions were made to the appropriate concentrations for either measurement of unloaded maximum velocity, or average force (see below). Polyacrylamide SDS gels run on these supernatants confirmed the accuracy of the concentrations and indicated that the amount of contaminating actin was less than 5%. HMMs were anchored to the coverslip using antibody S2.2 (18Trybus K.M. Henry L. J. Cell Biol. 1989; 109: 2879-2886Crossref PubMed Scopus (34) Google Scholar), and actin filament movement was measured as described previously (16Joel P.B. Trybus K.M. Sweeney H.L. J. Biol. Chem. 2001; 276: 2998-3003Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 19Warshaw D.M. Desrosiers J.M. Work S.S. Trybus K.M. J. Cell Biol. 1990; 111: 453-463Crossref PubMed Scopus (208) Google Scholar). The basic buffer typically used for actin treatment and washing in the flow cell (see below) contained 60 mm KCl, 25 mm imidazole-HCl (pH 7.4 at 30 °C), 4 mm MgCl2, 1 mm EGTA, and 10 mm DTT (buffer A). The final assay buffer additionally contained 0.7% methylcellulose, an oxygen scavenger mixture (containing 0.1 mg/ml glucose oxidase, 0.018 mg/ml catalase, and 2.3 mg/ml glucose), and 1 mm MgATP. In some experiments, the concentration of KCl in the buffer was changed to 25 or 90 mm. Measurements of average force were made on the motility surface using α-actinin as a load, which impeded the movement of actin (20Bing W. Knott A. Marston S.B. Biochem. J. 2000; 350: 693-699Crossref PubMed Scopus (59) Google Scholar). α-Actinin (Sigma) was dialyzed into buffer A with 1 rather than 10 mm DTT and clarified for 25 min at 350,000 × g. Its concentration was determined using BSA as a standard (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211946) Google Scholar). The following components were added sequentially to the flow cell: 1) 25 μg/ml S2.2 antibody for 1 min, wash with buffer A; 2) α-actinin for 1 min, wash with buffer A, including 0.5 mg/ml BSA; 3) HMM for 1 min, wash with buffer A; and finally 4) actin for 2× 30 s. Finally, buffer A containing methylcellulose, oxygen scavengers, and ATP was added, and the filaments were observed under fluorescent illumination. The concentration of α-actinin which just stopped filament movement on the motility surface was determined for different HMM concentrations. The “stopping concentration” of α-actinin was defined by the long, straight, “taut” appearance of the filaments, as well as their failure to move. The precision of these measurements was defined as one-half the concentration difference between the stopping point as defined above and the closest lesser α-actinin concentration at which long, taut filaments were not observed. ADP Release from Acto-HMM—The rate of ADP release was determined by mixing acto-HMM·(100 μm ADP) with 2 mm MgATP, and measuring acto-HMM dissociation either by the decrease in light scattering or the increase in pyrene actin fluorescence. Actin was labeled with pyrene-iodoacetamide as described previously (16Joel P.B. Trybus K.M. Sweeney H.L. J. Biol. Chem. 2001; 276: 2998-3003Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Experiments were done in 10 mm HEPES (pH 7.0), 0.1 m NaCl, 5 mm MgCl2, 1 mm EGTA, 1 mm NaN3, 1 mm DTT at 20 °C using a Kin-Tek stopped-flow spectrophotometer and a 100-watt mercury lamp. For 90° light scattering, the exciting beam was passed through a 294-nm interference filter, and the emission was detected with a 294-nm interference filter. Pyrene actin was excited using a 360-nm interference filter, and emission was detected with a 400-nm cutoff filter. Transients are the average of at least three independent mixings. The signal averaging and fitting was done using Kin-Tek software. Isolation of Homogeneous Heterodimer Preparations—By placing unique epitope tags on the two species of heavy chain included in a given heterodimer, we were able to isolate homogeneous preparations of HMM with two different heads. We prepared and characterized two heterodimers. One was composed of two naturally occurring smooth muscle heavy chain isoforms that differ in their cycling rates by a factor of two due to the presence or absence of a seven amino acid insert in loop 1. The second heterodimer contained the WT inserted heavy chain along with a non-cycling head created by a point mutation in switch 2 (E470A/WT HMM) (11Onishi H. Morales M.F. Kojima S. Katoh K. Fujiwara K. Biochemistry. 1997; 36: 3767-3772Crossref PubMed Scopus (27) Google Scholar, 12Onishi H. Kojima S. Katoh K. Fujiwara K. Martinez H.M. Morales M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6653-6658Crossref PubMed Scopus (45) Google Scholar). The flowchart in Fig. 1 illustrates the stages of this purification for the (+insert/–insert) heterodimer, along with Western blots depicting the amounts of FLAG- and His-reactive material in each step. The sensitivities of our anti-FLAG and anti-His probes were adjusted to give approximately the same amount of staining when reacted with equal amounts of the respective control proteins (Fig. 1B, lane S). The protein that was loaded onto the FLAG column (Fig. 1B, lane 1) contained nearly equal amounts of FLAG-labeled (+insert) heavy chain and His-labeled (–insert) heavy chain. As expected, the flowthrough from the anti-FLAG column contained predominantly His-tagged molecules (Fig. 1B, lane 2). The small amount of FLAG immunoreactivity in this fraction indicated that the column was loaded with more FLAG-labeled material than it had the capacity to bind. Protein eluted from this column contained both FLAG-labeled homodimers of the +insert heavy chain and heterodimers (Fig. 1A, step 3 and Fig. 1B, lane 3). After loading this mixture onto the poly-histidine binding column, a small amount of the His-reactive material was seen in the flowthrough due to supersaturation of the column, along with a large excess of FLAG-tagged protein (Fig. 1B, lane 4). After washing with loading buffer, a more stringent wash with buffer containing 10 mm imidazole-HCl was employed to release any nonspecifically bound FLAG-FLAG homodimer (data not shown). Elution with 300 mm imidazole buffer liberated the final, homogeneous preparation of heterodimeric HMM (Fig. 1B, lane 5), which had equal amounts of FLAG- and His-reactive material in it. Western blots conducted on preparations of the heterodimeric E470A/WT HMM gave the same results (21Kad N.M. Rovner A.S. Fagnant P.M. Warshaw D.M. Trybus K.M. Biophys. J. 2003; 84 (abstr.): 328aGoogle Scholar). NH4+ -ATPase activity was used to quantitatively assess the homogeneity of the heterodimeric E470A/WT HMM preparation, because one of the two heads is virtually inactive (Table I). Homodimeric WT HMM had NH4+ -ATPase activity of 24.2 ± 2.8 s–1, whereas the activity of the homodimeric E470A was barely measurable, even though 50–100 times as much of this protein was used in the assay as the other two species (12Onishi H. Kojima S. Katoh K. Fujiwara K. Martinez H.M. Morales M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6653-6658Crossref PubMed Scopus (45) Google Scholar). The activity of heterodimeric E470A/WT HMM was half that for the homodimeric WT HMM, supporting our contention that this preparation contains exclusively heterodimeric molecules.Table INH4+ -ATPase activity of HMMs with WT and E470A headsHMMRates-1E470A HMM0.01E470A/WT HMM12.0 ± 0.8WT HMM24.2 ± 2.8 Open table in a new tab Actin-activated ATPase of the Heterodimer Is Comparable to a 50:50 Mix of Homodimers—The actin-activated ATPase activity of the heterodimeric (+insert/–insert) HMM was compared with the corresponding homodimers, as well as an equal mix of these two isoforms (Fig. 2). Measurements were made at an actin concentration of 40 μm, which is greater than the K m for each of these species (6Rovner A.S. Freyzon Y. Trybus K.M. J. Mus. Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (113) Google Scholar). The ATPase activity of the homodimeric (–insert) HMM was approximately half that of homodimeric (+insert) HMM, as we have shown previously (6Rovner A.S. Freyzon Y. Trybus K.M. J. Mus. Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (113) Google Scholar). The rate of the heterodimer was the same as an equal mixture of the two homodimers, at a value intermediate between the rates shown by the homodimers. Taken together with the NH4+ ATPase results of Table I, these data indicate that each of the heads in our heterodimers have steady-state ATPase activities that are unaffected by the other head, both in the presence or absence of actin. Rate of ADP Release from Acto-HMM by Two Techniques— The release of ADP from the nucleotide-binding pocket is thought to be the step in the cross-bridge cycle that limits the velocity of actin movement by a given type of myosin (22Siemankowski R.F. Wiseman M.O. White H.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 658-662Crossref PubMed Scopus (317) Google Scholar). Pyrene-labeled actin was used to measure the rate of ADP release from acto-HMM. When pyrene actin-HMM·ADP is mixed with excess ATP, pyrene fluorescence increases at the rate at which ADP leaves the active site and is replaced with ATP (23Criddle A.H. Geeves M.A. Jeffries T. Biochem. J. 1985; 232: 343-349Crossref PubMed Scopus (178) Google Scholar). The time course of this fluorescence increase was best fit by a single exponential that was ∼4-fold faster for the +insert HMM than the –insert HMM (Table II). Phosphorylation had little effect on this rate. When measured over a range of temperatures, the Q10 value for this kinetic step was ∼3. Although our ADP release measurements showed a 4-fold difference between the +insert and –insert species, our current and previous (6Rovner A.S. Freyzon Y. Trybus K.M. J. Mus. Res. Cell Motil. 1997; 18: 103-110Crossref PubMed Scopus (113) Google Scholar) comparisons of velocities by the motility assay indicate a 2-fold difference between these isoforms. These results suggest that other steps in the cross-bridge cycle in addition to ADP release may limit the velocity of actin filament movement.Table IIADP release rates of homodimeric (+insert) and (-insert) HMMs compared to heterodimeric (+insert/-insert) HMM obtained by pyrene fluorescenceProteinPhos stateFitRateχ2SlowFasts-1(-/-) HomodimerDePS9.5 ± 0.50.09(+/+) HomodimerDePS52.3 ± 0.30.02PhosS46.7 ± 0.20.0550:50 Homodimeric mixtureDePD10.5 ± 0.0638.3 ± 0.30.15S16.8 ± 0.201.00(+/-) HeterodimerDePD9.4 ± 0.1637.8 ± 0.800.12S16.3 ± 0.201.10PhosD7.6 ± 0.1633.6 ± 0.670.05S15.2 ± 0.160.52 Open table in a new tab The difference in ADP release rates of the homodimers allowed us to probe the properties of heterodimeric (+insert/–insert) HMM (Table II). As expected, the fluorescence time course of the 50:50 mixture of dephosphorylated homodimers was better fit by an equation with two exponential terms than one, yielding rate constants of about 10 and 40 s–1, similar to the values determined from the homodimers (Table II). The heterodimer, independent of its phosphorylation state, also yielded two rates that were ∼4-fold different and nearly equal in amplitude, suggesting independent head action. ADP release rates were also measured by monitoring the decrease in light scattering of acto-HMM·ADP caused by rapid mixing with excess ATP (Table III and Fig. 3; see “Experimental Procedures”). This technique gave essentially the same results as had been obtained by pyrene fluorescence quenching for the homodimeric HMMs (Fig. 3, A and B), as well as the mixtures of homodimers (Fig. 3C and Table III). However, in contrast to the pyrene data, the light scattering signals for six different preparations of the heterodimeric HMM were better fit by a single exponential with a rate of 14–15 s–1, closer to the value obtained for the homodimeric (–insert) HMM (Fig. 3D and Table III). This observation is the first demonstration that the light scattering signal measures the movement of the entire double-headed HMM molecule away from the mass of the actin filament, whereas pyrene fluorescence gives an independent assessment of the rate of ADP release from each head. Because the slower head will dictate the rate at which the entire HMM can move away from actin, a rate dominated by the slower head is expected and was observed.Table IIIADP release rates of homodimeric (+insert) and (-insert) HMMs compared to heterodimeric (+insert/-insert) HMM obtained by light scatteringProteinPhos stateFitRateχ2SlowFasts-1(-/-) HomodimerDePS8.6 ± 0.020.043PhosS13.6 ± 0.300.018(+/+) HomodimerDePS37.8 ± 0.20.096PhosS43.8 ± 0.30.02150:50 Homodimeric mixtureDePD10.2 ± 0.0433.2 ± 0.200.026S15.9 ± 0.120.240PhosD13.4 ± 0.0943.0 ± 0.400.038S21.2 ± 0.200.160(+/-) HeterodimerDePS14.2 ± 0.070.054PhosS15.4 ± 0.100.084 Open table in a new tab Motility of the Heterodimers—Both the E470A/WT HMM and the +insert/–insert HMM heterodimers were assayed for their ability to move actin in the in vitro motility assay. Homodimeric E470A HMM does not support motility (Fig. 4A). Surprisingly, heterodimeric E470A/WT HMM showed an average velocity (cross-hatched bar, 1.50 ± 0.19 μm/s) in 60 mm KCl buffer that was nearly 95% as great as that of WT HMM (black bar, 1.58 ± 0.20 μm/s). These valu" @default.
• W2066706927 created "2016-06-24" @default.
• W2066706927 creator A5035579449 @default.
• W2066706927 creator A5058503945 @default.
• W2066706927 creator A5086418694 @default.
• W2066706927 date "2003-07-01" @default.
• W2066706927 modified "2023-10-18" @default.
• W2066706927 title "The Two Heads of Smooth Muscle Myosin Are Enzymatically Independent but Mechanically Interactive" @default.
• W2066706927 cites W1515141336 @default.
• W2066706927 cites W1557543954 @default.
• W2066706927 cites W1569283491 @default.
• W2066706927 cites W1569313009 @default.
• W2066706927 cites W1581643045 @default.
• W2066706927 cites W16087226 @default.
• W2066706927 cites W1899282607 @default.
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