The Rate of Release of ATP from Its Complex with Myosin

Transcription

1 Eur. J. Biochem. 92, (1978) The Rate of Release of ATP from Its Complex with Myosin Jeffrey W. CARDON and Paul D. BOYER Molecular Biology Institute and Department of Chemistry, University of California, Los Angeles (Received June 26, 1978) An approach previously published from this laboratory for measurement of the rate of dissociation of ATP from its complex with myosin has been carefully evaluated. The procedure has been found valid, and the off constant (21 "C, I =.21 M, ph 7.) is 1 x s-'. Other data for the rate of ATP binding give a Kd for myosin ATP of 6 x lo-" M. Reasons for the apparent discrepancy between this value and that reported by others have been examined. When various factors are appropriately taken into account, this discrepancy is eliminated. The mechanism of the Mg2 -I -activated myosin ATPase has been studied in some detail. At present a seven-step scheme [Eqn (l)] has become generally accepted [1]: M + ATP A M.ATP A M*. ATP + M"". ADP.Pi HOH A M*.ADP.P~ 4 M*.ADP R M. ADP 2+ M PI + ADP (11 where the asterisks denote intermediate states which, among other things, show increased myosin fluorescence. The free energy change associated with the hydrolysis of ATP on the enzyme (step 3) is only about kj/mol, compared with kj/mol in solution at ph 7 [2]. Steps 4-7 have been found to have either positive or small negative free energy changes [3,4], suggesting that ATP binds with a large negative free energy change. This was independently demonstrated by two experimental approaches first reported in These approaches depended on the labelling of bound and medium ATP by medium 32Pl, as can readily be accounted for by the scheme of Eqn (1). The interpretations given previously and herein are not, however, dependent on a particular hydrolysis scheme. Mannherz et al. [4] and Goody et al. [5] measured the extent of M". ATP formation as a function of P, concentration. This, in combination with the known value of the equilibrium constant for ATP hydrolysis and the previously measured ADP dissociation constant [3] allowed calculation of a value for the ATP binding constant, equivalent to KlK2 for Eqn (1). Wolcott and Boyer [6,7] directly estimated the first-order rate of release of ATP from M*. ATP by the rate of release of [32P]ATP from M*.ATP, labelled by exposing myosin or myosin subfragment 1 (myosin S1) to ADP, ATP and "Pi. Upon mixing with 32Pi a relatively rapid formation of a small amount of ["PI- ATP was noted followed by a slower release of ["PI- ATP to the medium. Combined with the fluorimetrically determined association rate of ATP and myosin, K1k2 [8], this gave a calculated value for the ATP binding constant. Both laboratories agree that the ATP dissociation constant is small, i.e. that ATP binds with a large negative change in free energy. However, there has been a troublesome disagreement as to the actual value of this binding constant, the initial value of Mannherz et al. for myosin S1 (Kd z M) [4] being about 1-fold smaller than that calculated by Wolcott and Boyer [6]. Also, Wolcott and Boyer reported a near linear dependency of M". ATP formation from 32Pi on the concentration of 32Pi [7], while Mannherz et al. noted a hyperbolic relationship [4]. Part of this discrepancy was eliminated in the later contribution of Goody et al. [5] by using a smaller and more appropriate value for the equilibrium constant of ATP hydrolysis and by a different analysis of the data that did not require a hyperbolic dependence of M*.ATP formation on Pi concentration. Their later results confirmed the apparent linear relation between M*. ATP formation and Pi concentration and gave an estimated Kd of 3 x lopi2 M. This still left a factor of about 3 between the two values. The purpose of the present research was to explore the probable sources of the difference between the two measurements of the ATP binding constant. The approach of Wolcott and Boyer has been examined in more detail. The value they reported and the validity of their interpretation has been confirmed. Also, we have confirmed the data reported by Mann-

2 444 ATP Release from Myosin Iierz et al., eliminating experimental discrepancy as a source of error. However, more adequate consideration of the effects of differences in experimental conditions on the equilibrium constant for ATP hydrolysis and on the apparent Kd for ADP brings the results from tlie two approaches into reasonable agreement. MATERIALS AND METHODS Myosin subfragment 1 was prepared by the method of Cooke 191 with digestion of myofibrils either by chymotrypsiii or by papain, or by the method of Weeds and Taylor [lo]. In some experiments, myosin S1 was separated into A1 and A2 fractions [lo]. Similar experimental results were obtained with all preparations. Actin was prepared from an aceione powder of a muscle extract [ll]. Myosin and acetone powder were kindly provided by Dr J. Sleep of these laboratories. [32P]ATP was recovered from the reaction mixture after quenching in 1 M perchloric acid containing 1 mm EDTA by adsorption onto charcoal and separation from other nucleotides on BioRad AGI X4, essentially as described elsewhere [12]. Reactions were started by the addition of 32P; to a reaction mixture containing enzyme and ATP (or ADP) that had been incubated for about 5 min. Ionic strength was controlled by the addition of KCl. The measurement of appearance of 32Pi into ATP was conducted in the presence of a regenerating system (5 units of pyruvate kinase/ml and an appropriate amount of phosphoenolpyruvate) to maintain a constant ATP concentration. Under these conditions, hydrolysis of ATP is balanced by its reformation, and after the initial more rapid formation of [32P]ATP, the measurement is essentially one of 32Pi $ ATP exchange. In those experiments in which ATP was separated from myosin S1 without quenching, the first step was application of the reaction mixture to a column of DEAE-cellulose (Whatman DE-23). The column was washed with low-ionic-strength buffer to remove myosin S1 and ATP was eluted in 4 ml of.4 M KCI, 5 mm imidazole ph 7.. The samples were then treated with charcoal and worked up in the usual manner. The course of [32P]ATP accumulation can be described by Eqn (2), where x = amount of [32P]ATP, B = amount of [32P]ATP bound to myosin S1, 1' = steady-state rate of ATP hydrolysis, k = rate constant for the dissociation of ATP from myosin S1 and c = amount of AT!? in the medium: dxjdt = kb - v (X- B)/c. The first term represents the release of myosin-s1- bound [32P]ATP to the medium. The loss of medium [32P]ATP through hydrolysis is described by the second term, since (x - B)/c is the fraction of the ATP pool that is labelled and thus the fraction of the steadystate rate which is due to the hydrolysis of labelled ATP. The solution to the equation is:.x= B+ ['y 1 (1- e-l''r), Values of k were obtained by a least-squares fit of the data to Eqn (2). At early times Lqn (2) reduces to: s = B + kbt (3) where 'early times' (vf < c) means when only a small fraction of the ATP pool has been hydrolyzed by the myosin S1 and regenerated. Obviously, tlie larger the concentration of ATP, the larger will be this linear region. This linear approximation was used in the earlier studies of Wolcott and Boyer [5]. RESULTS The Rate of ATP Relmsefi-om M*. ATP The release of ATP from its complex with myosin as measured by the procedure of Wolcott and Boyer [7] is subject to experimental error because only a small fraction of the myosin has ATP bound and the radioactivity released as ATP is small in comparison to the total 32P present. Consequently results are subject to troublesome scatter. Experimental error might thus be one possible explanation for differences in evaluation of the equilibrium for ATP binding using this approach or that of Mannlierz et al. [4]. To check this possibility, experiments of the type of Wolcott and Boyer have been repeated a number of times with different myosin S1 preparations. Results of some of these measurements are presented later in this paper as part of experiments to check effects of other parameters. Data from these and other trials that appeared reasonably free from experimental difficulty give for five different experimental series a value of (4 i 1) x s-' for the apparent rate constant of Sl-M*. ATP dissociation at 21 "C, 5 inm KCI, 1 mm Mg2', ph 6.5 and total I=.18 M. This is in reasonably good agreement with the value of 2.5 x s-' reported by Wolcott and Boyer under similar conditions. We thus confirm the value they obtained for measurement of the rate of [32P]ATP formation from 32Pi after the initial 'burst' of T2P]ATP formation. We also confirm the previous value obtained for the rate of dissociation of ATP from myosin of 1.3 x s-l. Whether this difference between myosin and myosin S1 represents an effect of the additional polypeptide chain or other factors is uncertain. Other recent work in this laboratory has shown the presence of a contaminating ATPase in myosin preparations

3 I J. W. Cardon and P. D. Boyer 445 that is absent from myosin S1 preparation [13] and this contaminant could conceivably contribute to Pie ATP exchange. Demonstration that [ 32P]ATP Is Released to the Medium The interpretation of Wolcott and Boyer was based on the likelihood that [32P]ATP appearing after the initial more rapid 'burst' in ATP labeling resulted from release of [32P]ATP to the medium and its mixing with unlabeled ATP already present. However, because the total molarity of myosin S1 considerably exceeded that of the [32P]ATP formed, the possibility existed that the slower increase in [32P]ATP formation reflected an unexpected increase in the amount of bound ATP. Assessment of this possibility was approached in two ways. First, the effect of concentration of unlabeled medium ATP on the accumulation of [3ZP]ATP was measured. The accumulation represents a balance between the [32P]ATP formed from Pi and released to the medium and the hydrolysis of part of the total medium ATP. As is readily evident, and as noted by examination of Eqn (2), increase in medium ATP concentration should increase the accumulation of 32Plabelled ATP, as discussed in Methods. At high ATP concentration, the accumulation of [32P]ATP should follow Eqn (3) for a longer time, whereas at low ATP concentration, accumulation should reach a plateau sooner, as described by Eqn (2). This prediction is verified by the data of Fig. 1, where in the presence of.5 mm added medium ATP, the accumulation of [32P]ATP soon levels off. In contrast, in the presence of 8 mm added ATP, accumulation of [32P]ATP continues for the duration of the experiment. A second, more direct approach involved separation of the myosin S1 together with any bound components on a suitable column. Because the release of ATP is relatively slow, this can readily be accomplished. Results are shown in Table 1. Early in the reaction sequence most of the [32P]ATP is enzyme bound with relatively little [32P]ATP retained on the column. With increased incubation time, within experimental error, the increase in total [32P]ATP was accounted for by the increase in medium ["'PIATP that bound to the column. The characteristics of the [32P]ATP formation made it likely that the initial ["PIATP formed was enzyme-bound. This was assumed in earlier work [5,6] and the above results justify this assumption. The Effect of Actin on the Rate of ATP Release Another possibility that needed consideration was that a small amount of actin contamination caused a greater rate of ATP release from the myosin-atp complex. The magnitude of any effect of actin was 25 I, I I I 2 a ii CI N 5 1 I I I I Tme (min) Fig. 1. The dependence of P,+ATP exchange on (ATP]. Reaction mixtures (1 ml) at 21 C contained 2.3 mg myosin S1, 5mM KHzP4, 5mM KCI, 1mM MgCIZ, 1mM phosphoenolpyruvate, 5 units pyruvate kinase and ATP as indicated. The reaction medium was brought to ph 6.5 by the addition of Tris prior to the addition of myosin S1. Reactions were quenched at the times indicated with an equal volume of 1 M perchloric acid containing 1 mm EDTA Table 1. Measurenient of free and myosin-bound [32P]ATP afier incubation with 32Pi A I-ml incubation mixture at 21 "C, brought to ph 6.5 with Tris, contained 5 mm KH2P4, 5 mm KCI, 5 mm neutralized ATP, 1 mm neutralized phosphoenolpyruvate, 1 nim MgC12, 5 units of pyruvate kinase and 4.8 mg myosin S1. A trace of 32Pi (approx..1 mci) was added, then samples quenched at times indicated by addition of an equal volume of 1 M perchloric acid to give a measure of total [3ZP]ATP formed or by application to a column to give a measure of free ATP (see Materials and Methods). Values given are for duplicate or triplicate determinations Incubation time 32P[ATP] total free mln pmol ~ , 96 67, 64, , 112 3, 29, 29 tested by adding increasing amounts of actin to the myosin S1 before measuring 32P i incorporation into ATP. Results are shown in Fig.2 and 3. As evident from the data of Fig. 2, in order to account for a tenfold increase in the ATP off rate, an actin contamination of.7 mg/mg myosin S1 would be needed. Presence of much less actin would be anticipated from the method of preparation. Also, if contamination by actin were responsible for an increased ATP release rate, the apparent rate constant for ATP release should increase with increasing concentration of myosin S1 because the effective molarity of actin would be increased. However, in a set of Pi+ATP exchange experiments at ph 7. with S1 concentrations of 8 pm and 2 pm, the calculated rate constant was the same (1 x lop4 s-i). These results show that the rate of release of ATP from myosin S1 as observed

4 446 ATP Release from Myosin I I I I 25 1 I I I -a O k 15 a k- a r loo N I Time (min) Fig. 2. The stimulation of Pi+ATP exchange by actin. Conditions were the same as in Fig. I, except 2.5 mm ATP, 4 mm phosphoenolpyruvate and I mg myosin S1. () No actin; ().35 mg actin/ml; ().7 mg actin/ml I I, L I I I I I Time (min) Fig. 4. Thedependence?/Pi+ ATPexchange on [M$"]. Conditions were the same as in Fig. 1 except 1.S mm ATP, 6 mm phosphoenolpyruvate, 3 mg myosin S1 and MgC12 as indicated were used. () 4 mm MgCL: () 25 mm MgCl2 8 /O I L a 41 / / / I I Time (min) 25 3 Fig. 3. The sfimulution of P,S ATP excharge by artin. Conditions were the same as in Fig. 2, except 2 mg myosin S1:ml. () No actin; ().9 mg actin/ml; ().18 mg actin/ml by Wolcott and Boyer and in this paper is not in error because of actin contamination. The EfJect of Mg2-I on the Rate of ATP Release A possible objection to the results of Wolcott and Boyer IS that the observed rate of ATP release could result from participation of cations other than Mg2' in the catalysis. For example, Ca2' accelerates ATP hydrolysis by myosin and could also accelerate ATP release. To assess this, measurement was made of the effects of a 1-fold increase in Mg" concentration. Because presence of free + Mg2 strongly inhibits activatiop of myosin ATPase by other metals, increase in the + MgZ concentration should inhibit any catalysis due to other metals. Results, given in Fig. 4, show that instead of inhibition a small increase occurs in the extent of [32P]ATP accumulation. This may reflect in part inhibition of ATP hydrolysis activity by 25 mm Mg2+. We conclude that the observed rate of ATP release results from an Mg-activated catalysis. The Equilibrium Dependency of M ' ATP Formation on [PJ This dependency served as the basis for the cdculation by Mannherz et al. of the equilibrium con- 'i-f,,,, 1 OO [pi] (mw Fig. 5. The dependence of myosin-hound [y-3zp]atp synthe.sis on /Pi] at constunl [ADP]. Reaction mixtures of 1-ml volume at 21 "C contained 1.7 mg myosin S1, 2 mm imidazole ph 7., 1 mm MgC12, 1 mm ADP and KH2P4 (adjusted to ph 7. with Tris) as shown; ionic strength was adjusted to.2 M with KCI. All points are duplicates; the range falls within the symbols stant for ATP binding by myosin. As a further check on the possible reasons for discrepancy between this approach and that of Wolcott and Boyer, we repeated such measurements under conditions used for our P, $ATP exchange measurements. Results of measurements with an intact myosin S1 preparation and one separated into the A1 and A2 fractions [lo] are given in Fig. 5 and 6. These results were treated by the approach of Mannherz et al., that is the slope of the double-reciprocal plot of 1/[M*.ATPI vs l/[p,] should be KhydKdlKADPEtr where Khyd is the equilibrium Constant for ATP hydrolysis, & is the ATP dissociation constant, K A D is ~ the dissociation constant for ADP, and E, is the total molar concentration of catalytic sites. With use of the value of 8 x lo4 M for Khyd [2] and the value of KADp of 1 pm as used by Goody et al. [5], the value of Kd from the experiment shown in Fig. 5 is 1.O x lo-" M. Both A1 and A2 fractions of myosin S1 show the same dependency of M 'ATP formation on [Pi], giving a value for Kd of.5 x lo-' M (Fig. 6).

5 J. W. Cardon and P. D. Boyer , 1 IPiI (mm) Fig. 6. The dependence oj' myosin-bound [y3'p]atp synthesis on [el ar constant [ADP]. Conditions were the same as for Fig.5 except that subfragment A1 () or A2 () of myosin S1 was used. All points are duplicates; range is shown or falls within the symbols ll[adp] (pm-l) Fig. 7. The apparent Kd./or ADP. Double-reciprocal plot showing the extent of [y3'p]atp synthesis as a function of [ADP]. Reaction volumes of 2 ml at 21 "C contained.4 mg myosin S1, 5 mm KHzP4, 5 mm KCI, 1 mm MgCl2 and ADP. Tris was added to bring the ph to 6.5. Reactions were quenched after 5 min with 1 ml of 1 M HC14. All points are replicates with the ranges shown I The Apparent Binding Constant Of'ADP to Myosin. Pi Another possible reason for the apparent discrepancy between results of Goody et al. and of Wolcott and Boyer might be a different value of the binding constant of ADP under our conditions. Pi is known to have an antagonistic effect on ADP binding [14], and thus we measured the Kd for ADP at different Pi concentrations, maintaining the ionic strength by the addition of KC1. For these experiments, the amount of the 'burst' in ATP formation, as initially detected by Wolcott and Boyer, was estimated at different ADP concentrations. Because of the high affinity of ADP for myosin or myosin S1, the myosin S1 concentration needed to be considerably lower than for experiments with excess ADP when 32Pi concentration effects were measured. A much smaller fraction of total 32Pi added was bound and the experiments were somewhat limited by the amount of radioactivity required (about.5 mci for each experimental point) and the lower accuracy of final measurements. The results of a typical experiment are shown in Fig. 7; these results indicate a & for ADP at ph 6.5 of 17 pm. Results of several other experiments at ph 7 are summarized in Table 2. They suffice to show a definitive increase in the apparent Kd for ADP as Pi is increased. The affinity of Pi for myosin has been estimated by its competition with the rate of ATP binding to give an apparent Kd of 1.5 mm [14]. In contrast, from the increase in medium PieHOH exchange with increase in Pi concentration and the effect of Pi on M.ATP formation (Wolcott and Boyer [7], Goody et al. [5] and this paper), the effective Kd for Pi is clearly greater than 5-1 mm. This value reflects the binding of Pi in the presence of ADP; bound ADP is required for the exchange of Pi oxygens [15] and for formation of M. ATP. These results indicate that Table 2. Estimation of'the apparent Kd,fiw myo.sin. ADP Incubation mixtures of 2 ml at 21 "C contained.4 mg myosin S1, 1 mm MgC12, 2-5 pm ADP and KHzP4 as indicated. KCI was added to adjust the total ionic strength to.2 M. Tria was added to adjust the ph to 7. except in.1 mm KI12P4, where 1 mm imidazole, brought to ph 7. with HCI, was used as buffer. The apparent K d was estimated by the dependence of myosin-bound [32P]ATP formation on ADP concentration. A trace of 32Pi was added and the reaction quenched after 5 min by the addition of 1 ml 1 M perchloric acid containing 1 mm EDTA [Pi1 mm Apparent & PM presence of ADP on the myosin increases the dissociation of Pi over 1-fold. Because of equilibrium relationships the dissociation of ADP would thus also be expected to be increased over 1-fold by the presence of bound Pi. However, the data of Table 2 indicate only about a 5-fold increase. Other factors may limit increase in the apparent Kd for ADP as Pi concentration approaches 5 mm, and thus mask the effect of increases in bound Pi. At this stage, it appears clear that all factors involved in the binding measurement are not satisfactorily accounted for. DISCUSSION The results presented in this paper confirm the value of the rate of release of ATP from its complex with myosin and myosin S1 as measured by the approach of Wolcott and Boyer [6,7]. Examination of various factors that may have resulted in misinterpretation of the earlier experiments give confidence that their approach leads to a valid measurement. If,

6 445 J. W. Cardon and P. D. Boyer: ATP Release from Myosin as seems even more likely because of the straightforward methodology involved, the value of Klkz of close to 1 x lo6 M-ls-' holds for the experimental conditions used [S], then the dissociation constant of ATP from M*.ATP is about lo-'' M. Although this value is still considerably larger than the most recent value of 3 x M reported by Goody et al. [S], the discrepancy is lessened by consideration that their experiments were done at a different ionic strength and ph. As pointed out by Goody et al., data of the type reported by Wolcott and Boyer [7] and given in this paper can be used for calculation of the dissociation constant for M*. ATP by their approach. When we use their calculation procedure for our data, obviously eliminating any differences in ionic strength or ph, we obtain a value for the dissociation constant of 1 x lo-" M. This value makes use of the same Kd for ADP as used by Goody et al. [S], namely one based on measurement of fluorescence change in the absence of Pi [3]. This is an appropriate value for their experimental conditions. Our measurements indicate that the Kd for ADP under our conditions is somewhat larger. Using our value of 4 pm in.1 mm Pi (which should be a good estimate of the value in the absence of Pi if Kpi = 1.5 mm [14]), the dissociation constant of ATP from myosin calculated by the method of Goody et al. would be 4 x lo-" M. Some additional correlations of our results with other data for the myosin-atp system are of interest. The results of Fig.2 may be used to estimate an apparent second-order rate constant for the association of actin with M*. ATP of about 2.1' M-' s-'. Sleep and Hutton [16] obtained a value of 2.7~ lo4 M-' s-' for this constant at ph 7, 4 mm KC1. White and Taylor [17] showed that the association rate of actin with M**. ADP. Pi decreases by a factor of about 13 in going from 4 mm KC1 to 1 mm KCI. Assuming that actin binding to M*. ATP is equivalently affected by ionic strength, the apparent second-order rate constant at 1 mm KC1 would be about 2 x lo3 M-' s-l. Our experiments were conducted at ph 6.5 and.2 M ionic strength, and the ph decrease and ionic strength change might further lower the rate constant. There is thus reasonable agreement between the two approaches to the rate of actin combination with M". ATP. Also, from the results of Fig.5, we can estimate directly the value of the product K3K4K5, assuming that M*.ADP % E,. This assumption seems to be fairly accurate since the plot of [32P]ATP versus Pi concentration is linear. Under this assumption the slope of this plot would be E,IK3&Ks. This gives a value for this product of 1 M. The results of Table 2 give us a means of estimating the value of K6K7 as 4 pm. Strictly speaking, KADP should be equal to K6K7/(l + K6); but if, as seems reasonable, K6 is small then KADP z K6K7. This is at least a lower limit. These two values, combined with our measurement of k-2 s-' at ph 7.) and the previously measured value of K1k2 allow 11s to compare our results directly with the work of Rosing and Slater [2] on the equilibrium constant of ATP hydrolysis. At ph 7, 25 "C, 1 mm MgC12 and high ionic strength, their value for this constant is 7-8 x 14 M. Our calculated value is 6 x lo4 M which, considering the system, is fairly good agreement. The correct value of the binding constant for ATP is also interesting in relation to the actomyosin scheme in that it must be consistent with constants measured for the binding of actin to myosin S1 and the binding of ATP to actomyosin S1. The thermodynamics of this scheme have been discussed in detail elsewhere [16]. This work was supported in part by U.S. National Science Foundation grant PCM J.W.C. is a U.S. National Science Foundation Predoctoral Fellow. REFERENCES 1. Trentham, D. R. (1977) Biochem. Soc. Trans. 5, Rosing, J. & Slater, E. C. (1972) Eiochim. Biophys. Acta, 267, Bagshaw, C. R., Eccleston, J. F., Eckstein, F., Goody, R. S., Gutfreund, H. & Trentham, D. R. (1974) Biochem. J. 141, Mannherz, H. G., Schenk, H. & Goody, R. S. (1974) Eur. J. Biochem. 48, Goody, R. S., Hofmann, W. & Mannherz, H. G. (1977) Eur. J. Biochem. 78, Wolcott, R. G. & Boyer, P. D. (1974) Biorhem. Biuphys. Res. iommun. 57, Wolcott, R. G. & Boyer, P. D. (1975) J. Supramol. Struct. 3, Johnson, K. A. & Taylor, E. W. (1978) Biochemistry, 17, in the press. 9. Cooke, R. (1972) Biochem. Biophys. Res. iommun. 49, Weeds, A. G. & Taylor, R. S. (1975) Nature (Lnd.j 257, Gergely, J. (1964) Biochem. Muscle Contraction, Proc. Symp. p Smith, D. J., Stokes, B.. & Boyer, P. D. (1976) J. Biol. ihem. 251, Sleep, J. A,, Hackney, D. D. & Boyer, P. D. (1978) J. Biol. ihem. in the press. 14. Bagshaw, C. R. & Trentham, D. R. (1974) Biochem. J. 141, Swanson, J. R. & Yount, R. G. (1966) Biochem. Z. 345, Sleep, J. A. & Hutton, R. L. (1978) Biochemistry, 17, in the press. 17. White, H; D. & Taylor, E. W. (1976) Biochemistry, 15, J. W. Cardon and P. D. Boyer, Molecular Biology Institute, University of California, 45 Hilgard Avenue, Los Angeles, California, U.S.A. 924