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J Biol Chem, Vol. 274, Issue 31, 21575-21580, July 30, 1999

Transient Kinetic Analysis of the 130-kDa Myosin I (MYR-1 Gene Product) from Rat Liver
A MYOSIN I DESIGNED FOR MAINTENANCE OF TENSION?^*

Lynne M. Coluccio and Michael A. Geeves^§

From the Boston Biomedical Research Institute, Boston, Massachusetts 02114 and the  Max-Planck-Institut für Molekulare Physiologie, D-44026 Dortmund, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 130-kDa myosin I (MI¹³⁰), product of the myr-1 gene, is one member of the mammalian class I myosins, a group of small, calmodulin-binding mechanochemical molecules of the myosin superfamily that translocate actin filaments. Roles for MI¹³⁰ are unknown. Our hypothesis is that, as with all myosins, MI¹³⁰ is designed for a particular function and hence possesses specific biochemical attributes. To test this hypothesis we have characterized the enzymatic properties of MI¹³⁰ using steady-state and stopped-flow kinetic analyses. Our results indicate that: (i) the Mg²⁺-ATPase activity is activated in proportion to actin concentration in the absence of Ca²⁺; (ii) the ATP-induced dissociation of actin-MI¹³⁰ is much slower for MI¹³⁰ than has been observed for other myosins (Ca²⁺, second order rate constant of ATP binding, 1.7 × 10⁴ M¹ s¹; maximal rate constant, 32 s¹); (iii) ADP binds to actin-MI¹³⁰ with an affinity of ~10 µM and competes with ATP-induced dissociation of actin-MI¹³⁰; the rate constant of ADP release from actin-MI¹³⁰ is 2 s¹; (iv) the rates of the ATP-induced dissociation of actin-MI and ADP release are 2-3 times greater in the presence of CaCl₂, indicating a sensitivity of motor activity to Ca²⁺; and (v) the affinity of MI¹³⁰ for actin (15 nM) is typical of that observed for other myosins. Together, these results indicate that although MI¹³⁰ shares some characteristics with other myosins, it is well adapted for maintenance of cortical tension.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The myosin superfamily has no fewer than 15 members that share sequence homology in the amino-terminal, so-called "motor," domain, which contains the ATP- and actin-binding sites (1-3). Although qualitatively myosins appear to share the same basic mechanism of coupling ATP hydrolysis to the movement of actin filaments, studies indicate that the ways in which they interact with actin and nucleotide are quantitatively different (4-6). These differences allow for myosins to function in a variety of situations including muscle contraction, vesicle transport, cytokinesis, and mediation of cell surface changes that accompany cell locomotion.

The class I myosins are small, single-headed, nonfilamentous ATPases (see Ref. 7). In lower eukaryotes, class I myosins are involved in cell migration, vesicle transport, and polarity determination, which are processes required for normal cell growth and development (8-12). Biochemical and molecular biological studies show that a myosin I family exists in higher cells (see Ref. 7); one member of this family is the 130-kDa myosin I from rat liver (13), referred to here simply as MI¹³⁰.¹ Partial peptide sequence analysis (14) indicates that the 130-kDa polypeptide is a product of the myr-1 gene cloned from rat brain (15); MM1 is its orthologue from mouse (16). Purified MI¹³⁰ has six associated calmodulins (14) as predicted by the DNA sequence (15). MI¹³⁰ binds F-actin in an ATP-regulated manner (13) and translocates actin filaments in vitro (17). Inhibition of motility observed at high [Ca²⁺] is overcome upon the addition of exogenous calmodulin (17).

Roles for MI¹³⁰ are unknown. In NRK cells, MYR-1 localizes to the plasma membrane and to cell structures involved in movement, such as lamellipodia and membrane ruffles (18). Analyses of subcellular fractions of rat liver suggest that the majority of membrane-associated MI¹³⁰ is found with the plasma membrane and microsomal fractions, but it appears that most of the MI¹³⁰ is associated with the cytoskeleton.²

Divergence in sequence of the carboxyl termini among the mammalian class I myosins might indicate that they are designed to bind different loads and perhaps to perform different functions. Similarly, although homology in the motor domain among the 130-kDa polypeptide and two other mammalian isoforms, namely MM1 (20) or the 110-kDa myosin I (13) and MYR-4 (21) or the 105-kDa myosin I (14), is 70%, the amino acid differences in the motor domain might reflect distinct properties and hence disparate cellular roles for these myosins. As a result, detailed mechanistic analyses may assist in defining the cellular functions of these isoforms.

This report on MI¹³⁰ is the first detailed kinetic analysis of a mammalian myosin I and represents the first step in a long-range plan designed ultimately to compare and contrast the kinetic properties of mammalian class I myosins. The study is made possible by adapting established transient kinetic methods to use the small quantities of myosin I that are available from rat liver. Our analysis shows that MI¹³⁰ interacts with nucleotide and actin in much the same way as other myosins that have been studied. However, it is much slower than any other myosin II, the two Acanthamoeba class I myosins, and avian brush border myosin I (BBMI). Our results suggest that in addition to being very slow kinetically, this myosin I may be designed for efficient tension maintenance.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Proteins and Reagents-- MI¹³⁰ was isolated from rat liver by gel filtration, anion, and cation exchange chromatography as described previously (13). Fractions containing purified MI¹³⁰ were pooled and, in some cases, concentrated in a Centricon 10 microconcentrator (Millipore Corp., Bedford, MA). After protein determination by colorimetry (Bio-Rad protein assay) using serum albumin as a standard, sucrose was added to 3 mg/ml, and the samples were frozen in liquid N₂ prior to storage at 80 °C. Rabbit skeletal myosin subfragment 1 (S1) was prepared by chymotryptic digestion as described by Weeds and Taylor (22). Actin was prepared according to Spudich and Watt (23) and, in some cases, labeled with pyrene at Cys-374 according to Criddle et al. (24). The 2'(3')-O-(N-methylanthraniloyl) derivatives of ADP (mant-ADP) and ATP (mant-ATP) were prepared according to Hiratsuka (25).

Enzyme Kinetics-- All kinetic experiments were performed at 19 °C in 20 mM MOPS, 100 mM KCl, 5 mM MgCl₂, 1 mM EGTA, and 1 mM dithiothreitol at pH 7.0 with a Hi-Tech Scientific SF-61 single mixing stopped-flow system using a 100-watt Xe/Hg lamp and a monochromator for wavelength selection. Pyrene and mant-ADP fluorescences were excited at 365 nm, and emission was detected after passing through a Schott (Mainz, Germany) KV 389 nm cut-off. The stated concentrations of reactants are those present after mixing in the stopped-flow observation cell. Stopped-flow data were fitted to exponentials by a nonlinear least-squares curve fit using software provided by Hi-Tech Scientific.

ATP Hydrolysis-- The release of P_i was measured for 2-3 µg of myosin I using a colorimetric assay described by Pollard (26). The Ca²⁺-ATPase activity of myosin I was measured in 10 mM Tris, pH 7.0, 1 mM dithiothreitol, 5 mM CaCl₂, and KCl concentrations ranging from 30 to 500 mM. The K⁺-EDTA-ATPase activity was measured in 10 mM Tris, pH 7.0, 1 mM dithiothreitol, 2 mM EDTA, and KCl ranging from 30 to 500 mM. The actin-activated Mg²⁺-ATPase activity was determined in 10 mM Tris, pH 7.0, 30 mM KCl, 1 mM dithiothreitol, 1 mM MgCl₂, either 1 mM EGTA or 1 mM EGTA and 1.1 mM CaCl₂, and F-actin from 0-30 µM. All reactions were done at 37 °C. Standard curves were generated with known amounts of phosphate. Controls included samples containing no myosin I. For actin-activated ATPase measurements, the reported values were corrected for activity because of the presence of actin. Data are expressed as s¹ assuming a molecular weight of 232,000 for the myosin I heavy chain and its six associated calmodulins.

Data Interpretation-- As shown in Scheme 1, we interpret the kinetics of myosin I (M) interacting with nucleotide (T, ATP; D, ADP) in terms of the model described by Bagshaw et al. (27), where k_+i and k_i are the forward and reverse rate constants, respectively, and K_i (k_+i /k_i ) represents the equilibrium constant of the ith reaction. Normal characters are used to indicate reactions involving MI¹³⁰, and bold characters refer to reactions involving actin-MI¹³⁰. ATP binds rapidly to myosin in a two-step reaction before ATP is reversibly hydrolyzed on the protein. This results in a conformational change that limits phosphate release and the faster ADP release. The ATP-induced dissociation of actin-MI¹³⁰ and the inhibition of the reaction by ADP have been interpreted in terms of the models developed by Millar and Geeves (28) and Siemankowski and White (29). As shown in Scheme 1B, ATP binds rapidly and reversibly to actin-MI¹³⁰ and is followed by a rate-limiting isomerization (k₊₂) of the complex, which leads to rapid dissociation of actin. ADP competes with ATP for the nucleotide binding site. The dissociation constants of actin for MI¹³⁰, actin for MI¹³⁰.ADP, and ADP for actin-MI¹³⁰ are K_A, K_DA, and K_AD, respectively.

View larger version (12K): [in this window] [in a new window]   Scheme 1.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ATPase Measurements-- The Mg²⁺-ATPase activity of MI¹³⁰ was very low and difficult to quantify with precision, but estimates suggest a k_cat of < 0.01 s¹ in both the presence and absence of Ca²⁺. The Mg²⁺-ATPase activity was significantly activated by actin only in the absence of Ca²⁺. In 30 mM KCl the ATPase rate was approximately linearly related to the actin concentration over the range of 0-30 µM with a steady-state ATPase rate of 0.247 ± 0.003 s¹ at 30 µM actin.

The ATPase activity was also determined in K⁺-EDTA and Ca²⁺, two nonphysiological conditions used historically to characterize conventional myosin II. At 30 mM KCl the k_cat in EDTA of MI¹³⁰ was 1.43 ± 0.021 s¹ and was inhibited at higher KCl concentrations. At 500 mM the activity was decreased to 25%. In contrast, the Ca²⁺-ATPase activity was 0.4 ± 0.02 s¹ and almost independent of KCl concentration up to 500 mM.

Transient Kinetics-- The ATP-induced dissociation of MI¹³⁰-actin complexes can be conveniently followed by monitoring the fluorescence of a pyrene label covalently attached to Cys-373 of actin. Fig. 1A shows the resulting change in the fluorescence signal upon mixing 200 µM ATP with 25 nM MI¹³⁰ and 25 nM phalloidin-stabilized pyrene-actin. At this concentration of ATP the change in the fluorescence can be described by a single exponential with k_obs = 1.6 s¹ and an amplitude of +4%. At concentrations above 500 µM, the reaction is biphasic with both phases having similar amplitudes (Fig. 1B, 1 mM ATP). The low concentrations of MI¹³⁰ used allowed the reaction to be followed over a wide range of ATP concentrations from 25 µM to 10 mM using only a few micrograms of protein. The dependence of the k_obs for both phases of the reaction on ATP concentration is shown in Fig. 1C in both the presence and absence of calcium. The fast phase of both data sets shows a hyperbolic dependence on [ATP] with a maximal observed rate (k₊₂) of 74 and 32 s¹ and 3.2 and 1.9 mM ATP required for half maximal saturation (1/K₁) with and without Ca²⁺, respectively. The values of 1/K₁ are of the order measured for all other myosins studied, but k₊₂ is much slower than any other myosin characterized so far (Table I). The slow phase saturates at a k_obs of 6 and 2 s¹ with and without Ca²⁺, respectively. The k_obs corresponds to the rate constant of ADP dissociation from actin-MI¹³⁰ in each case (see below) and led us to believe that the protein was purified with ADP bound in the nucleotide pocket. The amplitude of the slow phase varied among preparations and was normally in the range of 10-60% of the total amplitude. Extensive treatment with apyrase only partially reduced the amplitude of the slow phase, raising the possibility that the slow phase may have two components (see "Discussion").

View larger version (17K): [in this window] [in a new window]   Fig. 1.   ATP-induced dissociation of pyrene-labeled actin-MI130. A and B, 25 nM MI130 was incubated with 25 nM phalloidin-stabilized pyrene-labeled actin, and pyrene fluorescence was measured immediately following the addition of ATP. A, dissociation of actin-MI130 by 0.2 mM ATP resulted in an increase in fluorescence, and the best fit to a single exponential is superimposed with kobs = 3.1 s1 and an amplitude of 4%. B, increasing the ATP to 1.0 mM gave a biphasic reaction with kobs = 1.8 and 12.9 s1 with amplitudes of 1.7 and 2.8%, respectively. C, the dependence of kobs on [ATP] and Ca2+. The measurements were repeated in either 1 mM EGTA (Ca2+, circles) or 0.1 mM free Ca2+ (+Ca2+, squares) and kobs plotted as a function of [ATP]. The best fits to kobs = K1k+2[ATP]/(1 + K1[ATP]) for the fast phase (closed symbols) are superimposed. The data were well described by hyperbolas with maximal observed rates (k+2) of 74 and 32 s1 and 3.2 and 1.9 mM ATP required for half-maximal saturation (1/K1) with and without Ca2+, respectively. The values for kobs for the slow phase (open symbols) are not fitted.

The influence of ADP on the ATP-induced dissociation of actin-MI¹³⁰ is shown in Fig. 2. Addition of 2.5 mM ATP to apyrase-treated 25 nM pyrene-labeled actin-MI¹³⁰ results in a rapid change in fluorescence. The data for this preparation of protein fit a single exponential with k_obs = 22.5 s¹. If, in place of apyrase, 25 µM ADP was added, the k_obs was reduced to 5.8 s¹. Increasing either ADP or ATP concentration did not change the k_obs (data not shown), suggesting that it is the ADP release rate (k_AD) that is being monitored. At lower ADP concentrations, a biphasic reaction was observed with k_obs of 22 and 6 s¹. Preliminary analysis of the amplitude dependence on ADP concentration suggests an affinity of <10 µM. Establishing the conditions under which the ADP concentration was less than 10 µM was difficult because of the presence of an unknown level of ADP contamination. Repeating the experiment in the absence of Ca²⁺ gave a slower rate of ADP release of 2 s¹ with a similar affinity of <10 µM. This affinity is much tighter than observed for skeletal muscle myosin II and amoeboid myosin Ia (29, 30) but similar to that observed for smooth muscle myosin II (6) and BBMI (Table I). The observation that the total amplitude of the dissociation reaction was almost independent of ADP suggests that the affinity of actin for MI¹³⁰.ADP is much less than 1 µM.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Influence of ADP on the ATP-induced dissociation of actin-MI130. Addition of 2.5 mM ATP to 25 nM pyrene-labeled actin-MI130 in the absence of ADP (i.e. apyrase-treated) resulted in a rapid change in fluorescence. The data fit a single exponential with a kobs = 22.5 s1. Addition of 25 µM ADP reduced the kobs to 5.8 s1.

The amplitude of the ATP-induced dissociation reaction can be used to estimate the affinity of pyrene-actin for a myosin as shown by Kurzawa and Geeves (31). However, such a titration experiment requires a considerable amount of MI¹³⁰, which is limiting because it is present in cells in only small amounts. We therefore used a competition experiment with the well characterized rabbit skeletal muscle myosin S1 to provide an estimate of the affinity of MI¹³⁰ for actin. The addition of 50 µM ATP to 25 nM pyrene-actin and 50 nM MI¹³⁰ in the stopped-flow spectrofluorometer led to a dissociation reaction with a k_obs of 0.74 s¹ (Fig. 3). The addition of 50 µM ATP to 25 nM pyrene-actin and 50 nM S1 gave a k_obs of 80 s¹. Both reactions had a similar amplitude of 3.8%, suggesting a comparable affinity if both myosins quench the pyrene fluorescence to a similar extent. When 25 nM actin was mixed with 50 nM MI¹³⁰ and 200 nM S1 before mixing with ATP, a biphasic reaction was observed with 68% of the reaction occurring at 80 s¹ (the k_obs for A·S1 dissociation) and 32% at 0.79 s¹ (the k_obs for A·MI¹³⁰ dissociation). The amplitudes of the dissociation reaction were dependent upon the S1 concentration as shown in Fig. 3B. It is important to note that in Fig. 3B it is the concentration of the proteins in the syringe that is plotted, because the system is at equilibrium before the ATP is added. This usage is the opposite of the normal convention used for all of the other figures. When the fast and slow reactions have the same amplitude (i.e. the two myosins bind the same amount of actin in the mixture), then K_d^S1/K_d^MI130 = [S1]/[MI¹³⁰] = ([S1]₀  0.5[A]₀)/([MI¹³⁰]₀  0.5[A]₀). This equation was valid at 100 nM S1 in the absence of ADP and at approximately 300 nM S1 in the presence of 30 µM ADP. Under the experimental conditions, the affinity of S1 for actin is 30 nM in the absence of ADP (31) and 600 nM in the presence of ADP, leading to an affinity of pyrene-actin for MI¹³⁰ of 13 nM (ADP) and 60-110 nM (+ADP). The presence of Ca²⁺ had little influence on the measured affinities (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   ATP-induced dissociation of actin-MI130 and actin-S1. A, the addition of 50 µM ATP to 25 nM pyrene-actin and 50 nM MI130 resulted in a dissociation reaction with kobs of 0.74 s1. The addition of 50 µM ATP to 25 nM pyrene-actin and 50 nM S1 gave a kobs of 80 s1. Both reactions had a similar amplitude of 3.8%. When 25 nM pyrene-actin was premixed with 50 nM MI130 and 200 nM S1, a biphasic reaction was observed with 68% of the reaction occurring at 80 s1 and 32% at 0.79 s1. B, dependence of the fast and slow amplitudes of the dissociation reaction on [S1] in the syringe (see "Results") and ADP. The fast and slow reactions have the same amplitudes at 100 nM S1 in the absence of ADP and at approximately 300 nM S1 in the presence of 30 µM ADP, leading to an affinity of pyrene-actin for MI130 of 13 nM (ADP; KA) and 60-110 nM (+ADP; KDA) (see "Results").

Mixing 2.5 µM mant-ATP with 250 nM MI¹³⁰ led to a 3.3% increase in fluorescence when the reaction was monitored by exciting the intrinsic protein fluorescence at 295 nm and measuring the energy transfer to the mant group (Fig. 4A). The observed reaction occurred at 0.28 s¹, but it could only be followed over the concentration range of 1-2.5 µM mant-ATP. No reaction could be seen by monitoring the mant fluorescence directly. We could detect no reaction when monitoring mant-ADP binding over a similar concentration range. Addition of 5 µM ATP to 100 nM MI¹³⁰ resulted in a 1.2% increase in intrinsic protein fluorescence at 0.56 s¹ (Fig. 4B). The reaction could be observed over the ATP concentration range of 1-15 µM (Fig. 4B, inset), and the data were consistent with a second order rate constant, K₁k₊₂, of 0.1 × 10⁶ M¹ s¹. Thus, both ATP and mant-ATP bind with a similar rate constant and five times faster than the apparent rate of binding to actin-MI¹³⁰. No calcium dependence of the reaction was detected (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Rate of mant-ATP binding to MI130. A, mixing 2.5 µM mant-ATP with 250 nM MI130 led to a 3.3% increase in fluorescence when the reaction was monitored by exciting the intrinsic protein fluorescence at 295 nm and monitoring the energy transfer to the mant group. The observed reaction occurred at 0.28 s1, but it could only be followed over the concentration range of 1-2.5 µM. No reaction could be seen by monitoring the mant fluorescence directly. B, rate of ATP binding to MI130. The addition of 5 µM ATP to 100 nM MI130 resulted in a 1.2% increase in intrinsic protein fluorescence with kobs = 0.56 s1. Inset, the plot of kobs versus [ATP] with a best-fit straight line superimposed. The intercept is undefined because of the errors on the data, but the slope defines the second order rate constant, K1k+2, of 0.1 × 106 M1 s1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The ATPase activity and transient kinetics data presented here show that MI¹³⁰ interacts with both ATP and actin in a manner similar to that of other well characterized myosins, with the exception that many of the events are much slower. Although restricted in the quantity of available purified myosin I, the current studies were possible because of (i) the high sensitivity of pyrene-labeled actin to interactions between actin and myosin I complexes and (ii) the use of phalloidin to stabilize F-actin, thereby allowing many transient measurements using only microgram quantities of protein (31).

A major observation resulting from these studies is that the ATP-induced dissociation of actin-MI¹³⁰ (K₁k₊₂ = 0.023 × 10⁶ M¹ s¹) is slower than has been determined for other myosins. As compared with BBMI, the closest known relative to MI¹³⁰ (15), the rate of ATP-induced dissociation of the actin-MI¹³⁰ complex is 10% in the absence of Ca²⁺ (32). This is largely attributable to a smaller value of k₊₂, as the 1/K₁ is comparable with that observed for BBMI. Across the range of myosins listed in Table I, k₊₂ for BBMI is similar to amoeboid myosin Ia (33) and Dictyostelium myosin II (5) and much slower than the muscle myosins (30). Thus, MI¹³⁰ is by far the slowest myosin so far characterized. Similarly, the rate of ATP binding to MI¹³⁰ in the absence of actin is 10% that of BBMI, which in this regard more closely resembles all of the other myosins listed in Table I. In contrast, both the dissociation rate constant for ADP from A·M·D (k_AD) and the affinity of ADP for A·M (K_AD) are comparable for MI¹³⁰ and BBMI, suggesting that these differences are an intrinsic property of the protein and not caused by the method of preparation.

What is responsible for the slow phase observed in the ATP dissociation of actin-MI¹³⁰ is unknown. Although the rate is similar to that of ADP dissociation, it could not be eliminated with extensive treatment with apyrase. Indeed, actin-MI¹³⁰ treated with 40 µM ADP to eliminate the fast phase, then treated with apyrase, resulted in restoration of the original fast phase, showing that the ADP had been removed; however, the slow phase remained. This slow component may represent a contaminating myosin in the preparation, damaged MI¹³⁰, MI¹³⁰ without a full complement of calmodulin, or an alternately spliced version of MI¹³⁰. The examination of the kinetic properties of MI¹³⁰ expressed in baculovirus will soon allow us to rule out some of these possibilities.

The affinity of MI¹³⁰ for actin is similar to that observed for all myosins examined so far. Interestingly, the affinity of MI¹³⁰ for actin is reduced 5-7-fold in the presence of ADP. This behavior is similar to that displayed by smooth muscle myosin S1 (6) and differs markedly from skeletal muscle (30) and cardiac muscle myosins (29). Thus, coupling between the ADP and actin binding sites resembles that found with smooth muscle myosin II and Dictyostelium cytoplasmic myosin II (<10-fold (5)) and differs from that of skeletal muscle and cardiac muscle myosin II (>20-fold). It has been proposed that this low level of coupling between actin and ADP binding is a property of myosins designed for more efficient bearing of tension rather than fast contraction (6), and so it may not be surprising that members of the myosin I family should have more in common with smooth muscle and cytoplasmic myosin IIs than with fast skeletal muscle myosin II (see Fig. 5 and below).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Proposed free energy diagram of attached MI130 cross-bridges. The diagram shows the parabolic free energy diagrams for elastically distorted myosin cross-bridges based on those of Hill (19) and Cremo and Geeves (6). The following biochemically defined pathway is illustrated: A-M·D·Pi (the pre-force attached or A-state); loss of phosphate to form A·M·D (more strongly attached, rotated, or rigor-like, R-state); and loss of ADP to form A·M (also an R-state). The position of the minimum G value for the preforce generating, attached or A-state (A-M·D·Pi) is arbitrarily set to zero, and the minimum for the postforce R-state (A·M·D) is displaced by +5 nm, the cross-bridge throw. This produces a positive strain on the cross-bridge. Loss of ADP from A·M·D to A·M results in a higher minimum G value (i.e. work is done to displace ADP at cellular ADP concentrations of 10-20 µM) and displaced to the right by 3 nm in the case of smooth muscle myosin II or 5 nm for MI130. Note that the displacement of ADP from A·M·D is energetically prohibited unless its cross-bridge loses its strain. Assuming a similar elasticity, this strain inhibition is more pronounced for MI130 than for smooth muscle myosin II because of the larger displacement but similar change in the minimum G.

The ATPase results described here are consistent with those from an earlier look at the ATPase activity of MI¹³⁰ (13). Also, although performed under slightly different buffer conditions, the rate of ATP hydrolysis of MI¹³⁰ resembles qualitatively and quantitatively that previously observed for BBMI. Conzelman and Mooseker (34) reported rates of 0.644 s¹ in K⁺-EDTA (575 mM KCl), 1.07 s¹ in Ca²⁺, and 0.1 s¹ in Mg²⁺, with a 2.5-3.5-fold activation in Mg²⁺ in the presence of actin (Ca²⁺- and Mg²⁺-ATPases in 75 mM KCl), similar to those reported by Krizek et al. (35) (1.3 s¹ in K⁺-EDTA, 0.96 s¹ in Ca²⁺, and 0.04 s¹ in Mg²⁺ and a 2-fold activation of the Mg²⁺-ATPase in the presence of actin; all in 50 mM KCl). On the other hand, ATP hydrolysis by the amoeboid myosin I family is significantly faster than MI¹³⁰ and more closely resembles that of skeletal muscle myosin II (33). This resemblance is not surprising because there is significant diversion in sequence between the mammalian and amoeboid class I myosins. One particularly noteworthy point is that the presence of Ca²⁺ eliminates the actin-activated Mg²⁺-ATPase activity of MI¹³⁰. The motor activities of BBMI and MI¹³⁰ have been shown to be inhibited by increasing Ca²⁺, which causes dissociation of calmodulin (17, 36), although there is evidence that it is the binding of Ca²⁺ to calmodulin that inhibits motility of a related mammalian myosin I, MM1 (37).

In contrast to the inhibitory effect on both the actin activation of the steady state Mg²⁺-ATPase activity and motility, the transient kinetic parameters measured show no major sensitivity to Ca²⁺ (2-3-fold maximum) and, in fact, both K₁k₊₂ and k_AD are slightly accelerated by Ca²⁺. MI¹³⁰ appears similar to smooth muscle myosin II (Ref. 38; regulated by phosphophorylation) or the scallop muscle myosins (Ref. 39; regulated by Ca²⁺ binding to the light chains) in that the rate of P_i release from M·D·P_i or A·M·D·P_i is primarily affected, although effects on ADP release are also reported. Other events such as ATP binding, actin binding, or ATP hydrolysis on the protein are relatively little affected.

Another feature that MI¹³⁰ shares with smooth muscle myosin II and BBMI is that the molecules produce movement in two steps, as revealed by an optical tweezers transducer (40, 41). The second step is significantly greater for MI¹³⁰ and BBMI (5.5 nm) than for smooth muscle myosin II (2-3 nm). It has been proposed that the first step in the power stroke is associated with P_i release, whereas the second step represents ADP release. As expected, the time between the mechanical steps (300 ms, MI¹³⁰; 120 ms, BBMI) is in good agreement with 1/k_AD (500 ms MI¹³⁰, 125 ms, BBMI) given the differences in experimental conditions. The ADP release step corresponds to the ADP-dependent structural changes observed in three-dimensional reconstructions of smooth muscle actomyosin II (42) and actin-BBMI (43, 44) generated by cryoelectron microscopy and helical image analysis and by spectroscopy for smooth muscle myosin S1 (45, 46). This argument predicts that a similar or greater structural change should be observed for MI¹³⁰; studies to address this issue are in progress.

Cremo and Geeves (6) proposed that the high affinity of ADP for A·M, the structural change accompanying ADP release, and the weak coupling between actin and ADP affinities for smooth muscle myosin S1 could be indicative of a strain-sensing ADP release mechanism rather than the source of an additional power stroke in vivo (Fig. 5). These features of smooth muscle myosin II are common to MI¹³⁰. Because the free energy of ADP release from actin-MI¹³⁰ is small (K_AD < 10 µM) and may even be positive at cellular ADP concentrations, a large input of energy would be required to remove ADP from a myosin head bearing positive strain if the cross-bridge displacement is to be large (Fig. 5).

The amount of energy required to dissociate ADP can be estimated assuming the elastic energy of the MI¹³⁰ cross-bridges is similar to that of skeletal muscle myosin II (0.5 milli-Newton m¹). The energy (E) in the elastic element is related to the stiffness (k') and the imposed stretch (x) by E = k'x²/2. If the cross-bridge is already stretched by 5 nm (the size of the cross-bridge throw corresponding to the crossover of the A-M·D·P_i and A·M·D parabolas), then an extra 3- or 5-nm stretch is required to release ADP for the smooth and MI¹³⁰ cross-bridges, respectively. The ratio of the extra spring energy, k'(x²)/2, to the thermal energy, RT, then defines the extent of the strain-induced change in K_AD. K'_AD = K_AD exp(k'(x²)/2RT), where K_AD is the equilibrium constant in the absence of strain, R is Boltzman's constant, and T is absolute temperature (47). This equation predicts that ADP would bind approximately 10-fold more tightly for an isometric smooth muscle cross-bridge bearing maximum tension and 100-fold more tightly for MI¹³⁰. If this change in equilibrium constant can be assigned to a strain-dependent inhibition of the rate of ADP release (the simplest assumption), then k_AD is slowed from 20 to 2 s¹ for an isometric smooth muscle cross-bridge and from 2 to 0.02 s¹ for MI¹³⁰, giving an average lifetime for the isometric cross-bridge of 0.5 and 50 s, respectively. If such a mechanism is contributing to the slow turnover of the myosin, then a Ca²⁺-induced change in conformation of calmodulin, leading to a reduction in the stiffness of the calmodulin binding domain or lever arm, could provide a simple way to reduce the strain in the cross-bridge without sliding. This strain reduction would lead to faster release of ADP and cross-bridge detachment by ATP.

The large displacement of the head and the tight affinity of ADP for actin-MI¹³⁰ may indicate that MI¹³⁰ is designed primarily to bear a load rather than to shorten. ADP would effectively remain trapped on MI¹³⁰ cross-bridges bearing a significant load. This role would be also consistent with the slow kinetics of MI¹³⁰. The hypothesis outlined here and in Fig. 5, namely that the slow release of ADP from myosin is a strain-sensing mechanism, is entirely consistent with the data and represents a testable hypothesis. The prediction is that the time between the two steps of the mechanical event observed with the laser trap for the interaction of MI¹³⁰ with actin (40) will increase with the stiffness of the trap.

Previous studies show that MI¹³⁰ is present at the plasma membrane and in association with cell protrusions such as lamellipodia and cell ruffles (15). In rat liver, 30% of the MI¹³⁰ is membrane-bound with 70% presumably in association with the cytoskeleton.² Additionally, in vitro studies have shown that MI¹³⁰ cross-links actin filaments (13). Given the kinetic and structural properties of this isoform, it is plausible to believe that MI¹³⁰ is involved in the maintenance of tension within the cytoplasm. Other evidence for a similar role of a myosin I comes from studies in Dictyostelium in which over-expression of MyoB results in a decrease in the fluidity of pseudopodia (12).

	ACKNOWLEDGEMENTS

The hospitality extended to L. M. C. by Max-Planck-Institut-Dortmund is greatly appreciated. We are grateful to Dr. Justin Molloy, University of York, for helpful discussion.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM56130 and a grant from the March of Dimes (to L. M. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Current address and to whom correspondence should be addressed: Dept. of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ UK. Tel: 44-1227-827597; Fax: 44-1227-763912; E-mail: m.a.geeves@ukc.ac.uk.

² M. F. Balish, E. F. Moeller III & L. M. Coluccio, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: MI¹³⁰, 130-kDa myosin I; BBMI, brush border myosin I; S1, subfragment 1; mant, 2'(3')-O-(N-methylanthraniloyl); MOPS, 4-morpholinepropanesulfonic acid; A, actin; M, myosin; D, ADP.

	REFERENCES

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