Assembly and kinetic properties of myosin light chain isozymes from fast skeletal muscle

J. Mol. Biol. (1983) 170, 403-422

Assembly and Kinetic Properties of Myosin Light Chain Isozymes from Fast Skeletal Muscle STYLIANI C. PASTRA-LANDIS~, TED HUIATT~ AND SUSAN LOWEY

Rosenstiel Basic Medical Sciences Research Center Brandeis University, Waltham, Mass. 02254, U.S.A. (Received 31 December 1982, and in revised form 7 July 1983) Myosin' from chicken pectoralis muscle consists of isozymes that differ in their alkali light chains. I t is possible to isolate alkali 1 (A1) and alkali 2 (A2) homodimers of native myosin by immunoadsorption methods, and to compare their steady-state kinetics as well as their assembly into synthetic filaments under a variety of ionic conditions. Bipolar filaments of the isozymes formed at low salt concentrations had a narrow length distribution and did not differ from controls made from unfractionated myosin. Chicken myosin also assembles into highly homogeneous minifilaments similar to those formed by rabbit myosin in a citrate/Tris buffer. Analytical ultracentrifugation and electron microscopy showed that Al-homodimer, A2-homodimer and unfractionated myosin assembled into 0.3 pm short, bipolar minifilaments, which were indistinguishable from one another in size and shape. The steady-state myosin ATPase activity of the two homodimeric isozymes was identical in K+(EDTA) and Ca 2+ assay media. The actomyosin Mg2+ ATPase measured at 25 and 55 mM-KC1 (pH 8.0) showed only minor differences in both Vm~xand K~pp. Actomyosin activity was also determined for the more homogeneous minifilament preparations of the isozymes and these, as well, produced essentially indistinguishable kinetic parameters. Thus we find no evidence to support the hypothesis that a particular alkali light chain of myosin can affect either the structure of the filaments or the steady-state rate of ATP hydrolysis.

1. I n t r o d u c t i o n Myosin from v e r t e b r a t e fast skeletal muscle consists of two 200,000 M r h e a v y chains, which form both the structural coiled-coil a-helical rod and the two globular heads t h a t bind actin and hydrolyze ATP. In addition, each myosin head contains two pairs of light chains in chemically distinct classes (Weeds & Lowey, 1971; Lowey & Risby, 1971). Light chains of M r 19,000 (LC2f) can be r e m o v e d selectively upon reaction with NbS2§ (from which the earlier n a m e D T N B - l i g h t t Present address: Chemistry Department, Wheaton College, Norton, Mass. 02766, U.S.A. Present address: Muscle Biology Group, Iowa State University, Ames, Iowa 5001 l, U.S.A. § Abbreviations used: NbS2, 5,5'-dithiobis (2-nitrobenzoic acid); A1, alkali-I light chain or i C l f ; A2, alkali-2 light chain or LC3f; DTNB light chain or LC2f (removable by treatment with NbS2); S-1 (A1) or S-1 (A2), subfragment-I containing alkali I or alkali 2; HMM, heavy meromyosin; LMM, light meromyosin; EGTA, ethyleneglycol-bis-fl-aminoethyl ether)-N,N'-tetraacetic acid. 403 0022-2836/83/300403-20 $03.00]0 © 1983 Academic Press Inc. (London) Ltd.

404

S.C. PASTRA-LANDIS, T. HUIATT AND S. LOWEY

chains was derived) and their removal does not significantly influence the actinbinding or enzymatic functions of myosin in vitro (Gazith et al., 1970; Weeds & Lowey, 1971; Margossian et al., 1975; Holt & Lowey, 1975). The other class of light chains in fast skeletal myosin have molecular weights of 21,000 (A1 or LClf) and 17,000 (A2 or LC3f), and are commonly known as the alkali light chains, because they were originally dissociated from the parent molecule at alkaline pH (Kominz et al., 1959). These are chemically homologous but phenotypically distinct, and in the rabbit muscle they have an identical amino acid sequence over 141 C-terminal residues; they differ only at the N terminus, where A1 has a unique 41-residue additional peptide, referred to as the difference peptide Al, which is absent from A2 (Frank & Weeds, 1974). The eight residues (42 to 49) between A1 and the extensive common sequence include five amino acid replacements between Al and A2, and constitute the 42 difference peptide. Extensive homology exists between light chains of the same chemical class from different species, especially when they are derived from the same muscle type. For instance, comparison of rabbit and chicken A2 reveals only a 16% conservative substitution rate, 24 changes in 149 amino acid residues, distributed uniformly throughout the primary structures (Maita et al., 1981). The N-terminal peptide of chicken A1 had only four substitutions in 38 residues when compared to the rabbit; specifically, the proline-rich nature of the A1 peptide is maintained in both chicken and rabbit (Maita et al., 1980). Alkali light chains can occur in non-integral ratios that differ from species to species, or among adult muscles of the same species; these ratios appear to change systematically during development, suggesting the existence of isozyme populations (Lowey & Risby, 1971; Sarkar, 1972; Hoh, 1979; Benfield et al., 1983). It has been shown that myosin exists as the A1- and A2-homodimers and the A1,A2heterodimer (Hoh, 1978; Lowey et al., 1979). Myosin A1 or A2 light chains have been located within single isolated fast fibers, within single myofibrils and even within individual native filaments (Weeds et al., 1975; Pette & Schnez, 1977; Gauthier & Lowey, 1979; Silberstein & Lowey, 1981). Even before the characterization and localization of these light chain isozymes was firmly established, their possible involvement in regulation of the hydrolytic activity of myosin, and in the process of myosin self-assembly, was hypothesized (Weeds, 1969; Stracher, 1969; Dreizen & Gershman, 1970). Initial fractionation of subfragment-1, the globular head portion of the molecule, into two populations, each containing either an Al or an A2 light chain, was achieved by conventional ion-exchange chromatography (Yagi & Otani, 1974). The separated S-1 isozymes exhibited identical K+(EDTA), Ca 2+ and Mg 2+ ATPase activities, but the steady-state kinetics in the presence of actin and at low ionic strength (6 m~-KC1) revealed that. S-I (A2) had a Vmax value double that of S-l (A1) and Kapp for actin was also considerably higher (Weeds & Taylor, 1975). These observations on subfragment-1 were subsequently extended to include HMM isozymes fractionated by use of an ADP affinity column (Wagner, 1977). Thus it was established that the kinetic differences were not artefacts due to the use of a single-headed proteolytic subfragment of myosin, but applied equally to two-headed species. Experiments with hybrid subfragment-I molecules

ASSEMBLY AND KINETICS OF MYOSIN ISOZYMES

405

formed by alkali light chain exchange in 4.7 M-NH4CI confirmed that the observed differences in activity were due to the particular light chain present (Wagner & Weeds, 1977). However, the kinetic differences ascribed to distinct light chains at low salt concentrations were no longer observed when the ionic strength was increased to 25 mM-KC1 and up to physiological salt conditions (Wagner et al., 1979). The availability of purified antibodies specific for the difference peptides in the A1 and A2 light chains has made possible the fractionation of myosin into individual homodimers of high purity, with little cross contamination by the heterologous light chain (Holt & Lowey, 1977; Silberstein & Lowey, 1981). Unlike proteolytic subfragments, these isolated native molecules retain all the essential properties of myosin, so that one can search for differences between the isozymes both in enzymatic activities and in the structural interactions of myosin; namely, the process of filament assembly. Here we report experiments that explore the functional purpose of the "essential" alkali light chains of myosin. Possible differences in the natural heavy chain associated with a particular light chain were investigated by twodimensional electrophoretic peptide mapping of limited chymotryptic digests. Filament formation by the myosin homodimers was studied under a variety of solvent conditions, including the "minifilament" buffer system of Reisler et al. (1980). Within the limitations of these techniques, we find that the filaments formed from isozymes are indistinguishable from each other, and that the type of alkali light chain present in myosin does not affect its steady-state kinetic properties at moderate ionic strengths. Preliminary accounts of this work have been presented at the 24th and 25th Annual Meetings of the Biophysical Society and the 1981 Woods Hole Symposium of the Society of General Physiologists (Pastra-Landis & Huiatt, 1980; PastraLandis et al., 1981,1982).

2. Materials and M e t h o d s

(a) Preparation of proteins Muscle proteins were prepared from adult White Rock chicken pectoralis muscle and all preparative procedures executed at 4°C. Myosin was prepared as described by Holtzer & Lowey (1959) and stored prior to subsequent chromatographic treatment in 50% (v/v) glycerol, 0.5 M-KCI, 25 raM-potassium phosphate (pH 7.2) at -20°C. Ion-exchange chromatography was used as a final purification step before all experiments. Two chromatographic procedures were used: a DEAE-Sephadex A-50 column equilibrated in 0-15mM-potassium phosphate (pH7-5), l0 mM-EDTA (Offer et al., 1973) or a DEAE-cellulose DE-52 column equilibrated in 20 mM-sodium pyrophosphate (pH 7.5) (Lowey & Risby, 1971). In each case, the protein was eluted with a linear gradient from zero to 0"5 M-KCIin the corresponding buffer. Myosin from fractions in the first 2/3 of the eluted peak was free of actin; these fractions were pooled and the myosin was precipitated after overnight dialysis against 5 mM-potassium phosphate (pH 6"5), 40 mM-KCI and resuspended in high salt buffer at a concentration of l0 mg/ml. HMM was prepared by digesting a myosin solution (10mg/ml) in 10raM-sodium

40(i

S.C. PASTRA-LANDIS, T. HUIATT AND S. LOWEY

phosphate (pH 7"0), 0"6 m-NaCl for 10min at room temperature with a-chymotrypsin included at 0-10 mg/ml {Weeds & Taylor, 1975; Holt & Lowey, 1977). The reaction was terminated by addition of phenyl methane sulfonyl fluoride to 0"5 mm. After dialysis against 5 raM-sodium phosphate (pH 6.5), 40 mM-NaCl, the soluble HMM was separated from the precipitated undigested myosin and LMM. Further purification was accomplished b y fractionating between 42~o and 58% saturated ammonium sulfate. The protein was resuspended in 10 ram-potassium phosphate (pH 7.0), 0"l m-KCI, 0.2 mm-EGTA. Actin was prepared according to Spudich & Watt (1971). Just before the final polymerization step, a quick-freeze procedure was used and the G-actin at 10 mg/ml was stored as frozen pellets. Sucrose, included as a cryoprotectant, was dissolved in the protein solution to 20 mg/mt, and the solution was added dropwise into liquid nitrogen at a rate no faster than 20 drops/min. The frozen pellets were transferred to precooled small plastic vials, sealed and stored at -80°C. Before use, the pellets were thawed, dialyzed against 2 mM-Tris, 0.2 mm-CaC12, 0.5 mM-dithiothreitol, 0.2 mM-ATP (pH 8.2), and then polymerized with 2 mm-MgCl2, 10 mM+KC1 at room temperature for l h+ After centrifugation at 160,000g for 2.5 h, the F-actin pellet was resuspended in appropriate buffer for kinetic assays. F-actin prepared by this procedure gave comparable results to freshly prepared F-actin, Protein concentrations were determined using the following extinction coefficients: myosin E ~ = 5.0; heavy meromyosin E ~ = 6-0; and actin E ~ = 6-2 (Margossian & Lowey, 1982). Absorbance was routinely corrected for light-scattering. (b) Isozymefractionation by immunoadsorption chromatography Antibodies were purified and coupled to Sepharose 4B as described by Silberstein & Lowey (1981). Column-purified chicken pectoralis myosin was fractionated on immunoadsorbents composed of anti-A1 (5 mg antibody/5 ml packed gel) or anti+A2 antibodies {6 mg antibody/4 ml packed gel). Typically, 3 mg of myosin were fractionated by 5 mg of coupled antibody. Myosin was applied at a concentration of ~5 mg/ml in 0.5 i-NaCl, 0.015 re-sodium phosphate (pH 7.0) at 4°C to a 0-7 cm x 5 cm column equilibrated in the same buffer. Following protein application, the column was clamped for 15 min to allow for complete binding of antibody, and the protein eluted at a flow-rate of 40 ml/h, monitored using an Altex model 152 dual wave-length ultraviolet light detector. After the unretained fraction was eluted (Vo) and the baseline stabilized, the bound myosin was released (Ve) by buffer containing 4 M-guanidine hydrochloride (Holt & Lowey, 1977). The column was quickly re-equilibrated using column buffer with 0.02°/o (w/v) NaN 3 to retard bacterial growth, and stored at 4°C. The binding capacity of used anti-A columns decreased slowly, presumably because of repeated exposure to 4 M-guanidine hydrochloride. Unretained fractions (Vo) eluted at a concentration of 0-2 to 0-4 mg/ml were used for all kinetic and assembly studies. Retained fractions eluted with guanidine hydrochloride were freed of denaturant by dialysis against l0 raM-sodium phosphate (pH 7.0) before analysis by l or 2-dimensional gel electrophoresis. (c) Gel electrophoresis One-dimensional gel electrophoresis was performed according to Laemmli (1970), in 1.2 mm-thick polyacrylamide slab gels. Routinely, a 12-5~o separating gel was used in conjunction with a 5% stacking gel for optimum separation of the myosin light chains. (d) Formation of myosin filaments Synthetic myosin filaments were formed by a modification of the rapid dilution procedure of Wachsberger & Pepe {1980). Myosin solutions, 0"12 to 0"25 mg/ml, in 0-5 m-KCl buffered with either l0 mi+imidazole (pH 7-0) or l0 mm-potassium phosphate

ASSEMBLY AND K I N E T I C S OF MYOSIN ISOZYMES

407

(pH 7-0), were diluted with the appropriate buffer to a KCI concentration of 0.3 M over a period of 1 rain, left on ice for 30 min, and then diluted to 0.1 M-KCI over 5 min. In some experiments, MgCl 2 was added to the buffers to give a concentration of 5 mM. All experimental steps were done a t 4°C using pre-cooled solutions. Negatively stained samples of the filaments were prepared for electron microscopy immediately after the final dilution.

(e) Preparation of myosin minifilaments Myosin minifilaments were formed by the procedure of Reisler et al. (1980). Myosin solutions in 0-6 M-KCI, 10 mM-potassium phosphate (pH 7-0) were clarified by centrifugation at 100,000 g for 2 h to remove any large aggregates. The supernatant was dialyzed against 200 vol. 5 raM-sodium pyrophosphate (pH 8-0) for 8 h, with one buffer change. The dialyzed sample was centrifuged at 57,000 g for 15 min, and the supernatant dialyzed against 10 raM-citric acid, 35 mM-Tris (pH 8"0) for 10 to 12 h with one additional buffer change. The resulting minifilament preparation was clarified by centrifugation a t 9000g for 10 min. When minifilaments were prepared from the isolated isozymes the myosin solution was clarified before it was applied to the immunoadsorbent column, and the unretained void volume column fractions containing the purified homodimers were pooled and immediately dialyzed against 5 raM-sodium pyrophosphate (pH 8"0). As a control, a separate sample of unfractionated myosin was diluted to approximately the same protein concentration as the isozyme samples and dialyzed in parallel against the same solutions. The protein concentration in minifilament solutions was determined either by the Biorad Protein Assay (Biorad Laboratories, Richmond, California) using chicken pectoralis myosin as a standard, or spectrophotometrically after minifilaments were depolymerized by addition of 3 M-KCI to a final concentration of 0"6 M. (f) Analytical ultracentrifugation Sedimentation velocity experiments on myosin minifilaments were done with a Beckman model E analytical ultracentrifuge equipped with a photoelectric scanner. The 2-place AnD rotor and schlieren optics were used for samples with protein concentrations greater than 1.5 mg/ml. For more dilute samples, the 4-place A n F rotor was used in combination with the photoelectric scanner, operated at wavelengths of 280 or 236 nm. Use of the A n F rotor allowed minifilament samples formed from control myosin and the 2 homodimers to be examined in a single experiment. All runs were done at 20°C at a rotor speed of 20,000 revs/min. Observed sedimentation coefficients were corrected to s2o.w values using d a t a on the density and viscosity of the solvent provided by Dr E. Reisler. (g) Electron microscopy Synthetic myosin filaments were negatively stained by placing a drop of the filament suspension (20 to 50/~g/ml) on a glow-discharged, Formvar/carbon-coated grid. The grids were left undisturbed for 60 s, rinsed with 5 drops of distilled water, and negatively stained with 2°/o (w/v) aqueous uranyl acetate. Washing of the grids with water before staining prevented the formation of salt deposits on the grid, and resulted in more even staining of the filaments over large areas of the grids. To ensure t h a t this washing procedure did not alter the f l a m e n t structure, samples of the filaments were also directly stained without washing. Alternatively, they were fixed for 60 s by placing on the grid a drop of 2"5~o (v/v) glutaraldehyde, in the same buffer as the myosin filaments. The fixation was followed by washing and staining. Using either of these procedures, we saw no difference in the length or appearance of the filaments when compared to the samples treated with an initial water wash. Samples of myosin minifilaments were fixed in solution with glutaraldehyde by dilution of 0-1 ml of minifilament preparation, a t a concentration of 0.6 mg/ml, with an equal

408

S . C . P A S T R A - L A N D I S , T. H U I A T T AND S. LOWEY

volume of ice-cold 5% (v/v) glutaraldehyde, l0 raM-citric acid, 35 mM-Tris (pH 8-0). Samples were fixed in ice for 5 min, diluted to a concentration of 20 to 30pg/ml and negatively stained as described for myosin filament preparations. Glutaraldehyde solutions were prepared immediately before use in order to prevent inactivation by the Tris buffer. Rotary shadowing of minifilaments with platinum was done by the method of Tyler & Branton (1980). Minifilaments were fixed with glutaraldehyde as described for negatively stained specimens, diluted to a concentration of 20 $g/ml with 70O/o glycerol, l0 raM-citric acid, 35 mM-Tris (pH 8-0), and sprayed onto a freshly cleaved mica surface. The mica was placed in an Edwards Vacuum evaporator and the chamber evacuated to 10 -5 torr (1.33 x l0 -6 kPa). The samples were rotary shadowed with platinum at an angle of 6 ° and finally coated with a layer of carbon. Replicas were floated onto a water surface and collected on 400-mesh Cu grids, Grids were examined in a Phitips EM301 electron microscope operated at an accelerating voltage of 80 kV. Filament lengths were measured with a ruler on prints at a magnification of about 30,000 x for filaments, or 87,000 x for minifilaments. Diameters of minifilaments were measured on images of electron microscope negatives, enlarged approximately 25 x . The final magnification in each case was determined by measurement of the 39.5 nm spacing of tropomyosin paracrystals (Caspar el al., 1969) on negatives taken at the same time as the particular filament or minifilament pictures and enlarged by the same procedure. (h) A T P a s e activity ATPase activities were measured with a p H - s t a t at pH 8"0 using l0 mM-NaOH to titrate the proton released upon ATP hydrolysis. Conditions for the K + - A T P a s e assays were 0.66M-KCI, 1 mM-EDTA, 2.5mM-ATP; and for the Ca2+-dependent ATPase measurements, 0-25 M-KC1, 2-5 mM-CaCI 2, 2 mM-Tris, 2.0 mM-ATP. Enzyme, in 0-5 M-KCI, 50 raM-potassium phosphate (pH 7.5) was added (50 to 100 pg), and the reaction started by the addition of ATP. All measurements were made at 25°C in a volume of 2 ml. F o r assays in the presence of actin, the medium contained varying concentrations of KCI, 2.5 mM-ATP, 3-75 mM-MgCl 2 and actin concentrations in the range of 5 to 50 pM. Stock myosin and actin solutions were mixed under high salt conditions to avoid the formation of viscous actomyosin, and water added to the final 2 ml volume immediately before initiating the reaction by the addition of ATP. As far as possible, the assay circumstances, sequence of addition of stock solutions and timing were reproduced carefully from one assay to the next. Minifilaments were assayed by measuring the amount of phosphate released by the method of Fiske & Subbarow (1925). For K+(EDTA) or Ca2+-dependent activity, minifilament stock solution was added to the high salt medium and therefore dissociated myosin monomers were assayed. The actin-activated activities were measured in l0 mM-KCI, l0 mM-citric acid, 35 mM-Tris (pH 8-0), a medium in which the minifilament structure remains assembled. Measurements were made at 25°C and a volume of 2 ml. The reaction was terminated at 5 to l0 rain by the addition of 10°/o (w/v) cold trichloroacetic acid. The precipitated protein was pelleted by centrifugation at 2000g for l0 min and phosphate measurements were made on the supernatant.

3. Results Column-purified myosin was fractionated into the Al- and A2-homodimers u s i n g i m m u n o a f f i n i t y c h r o m a t o g r a p h y w i t h a n t i b o d y t o t h e difference p e p t i d e s , A1 a n d A2, a s t h e i m m o b i l i z e d l i g a n d ( S i l b e r t s e i n & L o w e y , 1981). T h e u n r e t a i n e d p o r t i o n o f m y o s i n , for e x a m p l e , A l - h o m o d i m e r s f r o m t h e a n t i - A 2 c o l u m n , e x h i b i t e d full e n z y m a t i c a c t i v i t y , a n d w a s u s e d for t h e a s s e m b l y s t u d i e s . T h e

ASSEMBLY AND KINETICS

OF M Y O S I N I S O Z Y M E S

409

material bound to the column was eluted with 4 M-guanidine hydrochloride. Because these eluted fractions were denatured, they were used only for peptide mapping of the heavy chains. The guanidine was quickly washed off the columns, which retain their capacity to fractionate myosin over multiple (about 20) uses. Since a portion of the heterodimer appears in the initial unretained fractions, the remainder from the 30°/o found in native mixtures binds to the antibody columns and appears in the eluted fractions (Lowey et al., 1979). As a result, both the native state homodimer preparations (Fig. l) and the denatured fractions are 80 to 85°/o pure, a result comparable to that achieved in the artificial homodimer experiments (Pope et al., 1981). (a) Myosin filaments A major advantage of the isolated natural homodimers is that they carry the native heavy chain. Therefore, possible differences in the assembly of the two isozymes could be examined by forming synthetic myosin filaments from control myosin and the A1- or A2-isozymes. Filaments were formed by a two-step rapid dilution method (Wachsberger & Pepe, 1980) because filaments formed by this procedure exhibited a relatively narrow length distribution. Assembly studies were done with myosin solutions buffered at pH 7-0 with either l0 mM-potassium

Unfroctionoted

AI

A2

myosin

myosin

myosin

Alkali I

DTNBJ Alkali 2

Fro. 1. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of unfractionated chicken pectoralis myosin and the Al and A2 homodimers isolated by immunoadsorption chromatography. The fractions shown were unretained by the affinity columns (Vo): 12.5% polyacrylamide gels were run according to the method of Laemmli (1970). Only a small amount (<20%) of the heterologous light chain is found in the purified isozymes.

410

S. c. PASTRA-LANDIS, T. HUIATT AND S. LOWEY

phosphate or l 0 mM-imidazole, with or without added 5 mM-MgC12. The filaments formed in the presence of Mg2+, in phosphate or imidazole buffer, were all similar in appearance. Examples of filaments formed using 10 mM-imidazole (pH 7-0), 5 mM-MgCl2 are shown in Figure 2. The filaments were tapered at both ends and were clearly bipolar. Although a well-defined central bare zone was not discernible in a majority of the filaments, a reversal of the polarity of the myosin heads, which project from the filament shaft, can be seen near the center of several of the filaments in each micrograph. Filaments formed from all the myosin preparations were identical in appearance. Filaments formed in phosphate buffer without Mg2+ were significantly shorter (mean length 0.74 to 0.77 #m) than those formed in imidazole (l.00 to 1-05#m), and often appeared branched near the ends. The disorganizing effects of phosphate on assembly were reversed by inclusion of Mg2*. Comparison of the lengths of filaments found for the three myosin preparations under the various conditions used (Fig. 3) demonstrated no differences between the isozymes and control myosin. In addition, similar results were obtained with mixtures of the two isolated isozymes. ATPase activities were measured for the isolated native myosin isozymes eluted in the unretained fraction of the immunoaffinity columns. The K+(EDTA) and Ca2+-dependent ATPases were identical and equal to those of unfractionated myosin, confirming the expectation that the fractionated samples retained the full enzymatic activity of the starting material. In the K + (EDTA) assay, the turnover number for Al-myosin was 10.3s -1 per head, and for A2-myosin 10.2s -I per head in one preparation; unfractionated enzyme assayed in parallel had an activity of 9-8 s -1 per head. The range of values obtained for representative different preparations is given in Table 1. Actin-activated ATPase measurements were made at a variety of ionic strength conditions: 25 mM, 55 mM and 100 mM-KC1, and actin concentrations in the range 5 to 50 pM. Kinetic constants derived from the actin-activated data were obtained from regression line analysis of Lineweaver-Burk plots, such as those shown in Figure 4. The values listed in Table 2 demonstrate, once again, that no striking kinetic differences between A1 and A2-myosin can be observed in steady-state measurements, even for the actin-activated Mg2+ATP hydrolysis. Small differences in the calculated kinetic parameters either among the isozymes and the unfractionated material, or among different preparations are observed regularly and are most likely due to the difficulty in obtaining reliable actomyosin activity measurements under conditions where the state of aggregation of the myosin is unknown and certainly heterogeneous: The actin-activated assays cannot be done readily at a significantly lower ionic strength, or at higher actin concentrations, because the actomyosin solutions become exceedingly viscous and adequate stirring for a sensitive electrode response in the pH-stat becomes very difficult. (b) Myosin minifilaments Minifilaments, described by Reisler et al. (1980) for rabbit myosin, exist in a highly homogeneous state of assembly, and therefore this system can provide for more reliable kinetic analyses. With rabbit myosin, minifitaments had been

•. ~-,~ ".

'

. i~:~:' ~.

, .?~:,. -.

.

~ ~ : : ~ : ~ Fro. 2. E l e c t r o n

,~.. ~:,\~.: :?

mierographs

,~'..,:~-,

:~!i ~

~:!.!!:Jill

~:~:

of negatively stained synthetic

myosin filaments

formed from

(a)

unfraetionated myosin and the isolated (b) AI and (c) A2 homodimers. The scale bar represents 1,0/~m x 2l ,000.

S. C. PASTRA-LANDIS. T. HUIATT AND S. LOWEY

412

hi myosin

Unfroctionoted

60 40

f.x= 0.77

A2 myosin } .; = 0.74

x= 0.76

I1, ,A_

I0 mM-KPI

20 0

60

: 0"97

= 0"95

•~ = I'01

I

40

L

20

E o •-:

I

0

~6

I0 mM-KP i + 5 mM-MgCI 2

.~ 60 ~; = 1"05

iT= 1"00

} = I'05

40

I0 mM -imidozole

20

60

20 0

0"5

I'0

x=l'12

•~ = 1"06

-; : 1-13

I

J

1"5 2-0

2,,

0.5

1.0

I-5 2"0

I0 mM-imidozole + 5 mM-MgCI 2

0-5

I'0 I-5 2'0

Length (/~m)

Fin. 3. H i s t o g r a m s showing the lengths of s y n t h e t i c filaments formed from unfractionated myosin and the A1 a n d A2 homodimers. F i l a m e n t s were formed in a variety of buffers as s h o w n on t h e right. For the purpose of comparison, t h e ordinates were normalized to 200 filaments per histogram. Between 200 a n d 400 filaments were actually measured in each case.

formed from a 5 mg/ml solution of monomeric myosin in p y r o p h o s p h a t e buffer, after extensive dialysis against 10 mM-citric acid, 35 mM-Tris (pH 8.0). Our results demonstrate t h a t chicken pectoralis myosin also assembled into minifilaments under these conditions. Figure 5(a) shows the schlieren pattern obtained in the analytical ultracentrifuge at two different protein concentrations; the minifilaments sedimented as a hypersharp b o u n d a r y with little evidence of myosin monomers and gave an intrinsic sedimentation coefficient s~0,, of 30.4 S (Fig. 5(b)), similar to the value of 32.3 S obtained by Reisler et al. (1980) for

413

ASSEMBLY AND K I N E T I C S OF MYOSIN ISOZYMES TABLE 1

Myosin A TPase activities Experiment

Myosin

K +(EDTA)

Ca2+

l

Unfractionated AI A2 Unfractionated AI A2 Unfr'actionated A1 A2

9.8 + 0.8 10.3-+ l-0 10.2_+0.3 10.8_+ 1.7 9.8___1-5 10.3_+ 1-4 10.0-+ 1-5 9.8_+ 1.3 10-6-+0-8

1.1 +_()-1 1-5_+0-2 1,3_+0-2 1.0_+0-1 0.9_+0-1 0.9+_0-1 I, 1-4-0-2 1.5±0-3 1-1 -+0-1

2 3

The activity is expressed in mol Pi per mol protein active sites s- 1 r a b b i t myosin. A s s e m b l y into minifilaments also t o o k place f r o m u n f r a c t i o n a t e d material and the isolated i s o z y m e s at v e r y dilute protein c o n c e n t r a t i o n s of 0.1 to 0-2 mg/ml, such as those o b t a i n e d from t h e i m m u n o f r a c t i o n a t i o n (Fig. 5(c)). I n this case, the sequence of buffer changes, which precedes a s s e m b l y into minifilaments, t o o k place over the 24 hours i m m e d i a t e l y following m y o s i n f r a c t i o n a t i o n , in o r d e r t o a v o i d loss o f a c t i v i t y in t h e dilute solutions. Nevertheless, citrate is a d e f o r m i n g buffer (Shaltiel et al., 1966) a n d we f o u n d t h a t K + ( E D T A ) A T P a s e m e a s u r e m e n t s in high salt (0-66 M-KCI) before a n d after the minifilament a s s e m b l y precedures s h o w e d at least a 30O/o r e d u c t i o n in a c t i v i t y ; this was o b s e r v e d b o t h for the p r e p a r a t i o n s s t a r t i n g a t 0-2 m g / m l a n d those at 5 mg/ml. E l e c t r o n m i c r o g r a p h s o f a t y p i c a l p r e p a r a t i o n o f chicken m y o s i n minifilaments are shown in Figure 6 for b o t h (a) n e g a t i v e l y stained a n d (b) r o t a r y s h a d o w e d I

0.6 ~

~

.......

.

" o,,°

J

>, 0-4 0.2

0

I

I

0.04

0.08 (o)

0't2 0 l/[ac'~in! (/ZM-I )

I

I

0-08

0-04

__

)'12

lb)

Flo. 4. Lineweaver-Burk plot of actin-activated ATPase data for myosin isozymes at 2 salt concentrations: (a) 25 mM-KCt, (b) 55 mM-KCI. Rates are expressed as tool phosphate/mol myosin s- 1 The enzyme concentration was 75 to 90 nM. Unfractionated myosin (O), Al-myosin (0) and A2 myosin (~). Kinetic parameters were obtained from regression line analysis.

414

S. C. PASTRA-LANDIS, T. HUIATT AND S. LOWEY TABLE 2

Kinetic constants of myosin isozymes Myosin Unfractiona~d (3 experiments) Al-isozyme (3 experiments) A2-isozyme (3 experiments) Unfractionated A 1-isozyme A2-isozyme Unfraetionated Al -isozyme A2-isozyme

KCI concn ( m r s )

Aetin-aetivated ATPase

Vm,~ (s- ')

K.pp (~M)

25

3.7-4.9

7-6-9-8

25

3-5-4.9

7.2-8.2

25 55 55 55 100 100 100

2.6-3.9 2.8 2-5 t .8 0-90 1.7 1.4

4.7-8-1 31 19 19 21 24 25

The activity is expressed in mol Pi per mol protein active sites s- l

specimens. The short bipolar filaments exhibit a clearly defined central bare zone, and projections of individual myosin molecules. F o r the negatively stained samples, filament lengths averaged 3 1 6 + - 3 3 n m ( m e a n + s t a n d a r d deviation, n = 182). The d i a m e t e r of the central bare zone was 7.4+_0.7 nm (n = 176). A high background level of dissociated myosin was seen in unfixed samples, suggesting t h a t the minifilaments were disassembled either due to the effects of the stain or due to the low protein concentrations used. I n the r o t a r y shadowed samples, filament lengths averaged 382 +_32 nm (n = 7I) and diameters were 15-5-t- 2.0 nm (n = 85). These m e a s u r e m e n t s were not corrected for metal deposition. A t t e m p t s to obtain shadowed p r e p a r a t i o n s of unfixed minifilaments were unsuccessful; only myosin molecules and a few small aggregates were seen on grids from such preparations, once again indicating disassembly at low protein concentrations. Most i m p o r t a n t is the observation that, as with the synthetic filaments, the isozymes do not differ in their assembly behavior. Minifilaments remained stable at concentrations of 0.1 to 0-2 mg/ml even over a two-week period; no a p p a r e n t dissociation could be detected by their sedimentation behavior after storage. H o m o g e n e o u s minifilament p r e p a r a t i o n s were used to extend our actinactivated A T P a s e studies; indeed, a m a r k e d i m p r o v e m e n t in the quality of d a t a is observed (Fig. 7). Assay d a t a on the a c t i v i t y of the minifilaments in l0 m i - K C l and l0 mM-citric acid, 35 mM-Tris (I ~ 90) show noticeably less scatter in double reciprocal plots when c o m p a r e d to d a t a obtained with filamentous myosin a t c o m p a r a b l e ionic strength in 90 mM-KC1. D a t a representative of the isozyme minifilament p r e p a r a t i o n s are shown in Figure 8 and the calculated p a r a m e t e r s are given in Table 3. Again we found no differences due to distinct alkali light chains. In fact, the kinetic p a r a m e t e r s of homogeneous minifilaments resembled those of the more heterogeneous myosin filaments formed a t low ionic strength,

ASSEMBLY

AND KINETICS

OF MYOSIN ISOZYMES

(o)

415

(b)

30 [I

*

,

I 0

2-0

o~. ZO iO

3.0

Concentration (mg/m[)

(c)

0"4.~ AI myosin

: i ' i i

0.4~" Utnfracti°noted myosin' !.... ili-i

J :

:

+

oJ

0"4+r o-2

A2 myosin

ii

llI

,

i

i

!

i

ii! "

tl

:

i

Fro. 5. Velocity sedimentation of chicken myosin minifilaments. All experiments were done at 20°C at a rotor speed of 30,000 revs/min. (a) Schlieren patterns obtained with minifitaments formed from myosin at an initial concmttration of about 5 mg/ml before dialysis against the series of buffers used to prepare minifilaments. The final protein concentration and corrected sedimentation coefficients were: upper pattern, 2.70 mg/ml, s20.w 16.6 S; lower pattern, 2.02 mg/ml, s2o., 19.1 S. Sedimentation is from left to right. The first frame was taken 24 min and the second frame at 136 min after reaching 30,000 revs/min. (b) Extrapolation of the sedimentation coefficient to zero concentration. Closed symbols represent points obtained from minifilament preparations formed from myosin at an initial concentration around 5mg/ml. Open symbols represent the data obtained from the isozyme experiment shown in (c), plotted on the same graph for comparison. Unfractionated myosin ((:)), Al-myosin (i--I), and A2-myosin (/x). Points at concentrations above 1-5 mg/ml were obtained using schlieren optics; other points were obtained with the photoelectric scanner. (c) Photoelectric scanner traces of sedimentation velocity patterns of minifitaments formed from a typical isozyme experiment, using myosin or purified homodimers at an initial concentration of 0-12 to 0.25 mg/ml. Absorbance was measured at a wavelength of 236 nm. The direction of sedimentation was from left to right. Experimental parameters were: Al-myosin 0.]2 mg/ml, scan initiated 36 min after reaching speed, s2o, w 27-6S; A2-myosin 0.]8mg/ml, scan initiated 4 0 m i n after reaching speed, sz0,, 27-0S; unfractionated myosin 0.22 mg/ml, scan initiated after 44 min, s2o., 25-9 S. 14

!ii

(a)~

"

" : " :~

;p" • ' . ~ i

'

~

i>

Flo. 6. Electron micrographs of minifilaments formed from unfraetionated chicken pectoralis myosin at an initial concentration of 5 mg/ml. Minifilaments were fixed with glutaraldehyde as described in Materials and Methods before negative staining or rotary shadowing. (a) Minifilaments negatively stained with uranyl acetate. The scale bar represents 0.3/~m x 62,000. (b) Minifilaments after rotary shadowing with platinum. An individual myosin molecule head can be seen extending from the shaft of the minifilament on the left. The scale bar represents 0-3/~m × 75,000. E

2.0

I-5

-~ 1.0 >

0-5

0

/

o

I

0,C)5

0'~10

°

0 0.05 I/[ocfinl (/~M-~)

0.10

0.15

FIG. 7. Lineweaver-Burk plot of the actin-activated ATPase activities of myosin and myosin minifilaments assayed at comparable ionic strengths. Myosin filaments (O) were assayed at a myosin concentration of 92 nM in an assay medium containing 85 mM-KCI, 2.5 mM-ATP, 3.75 m~1-MgCl2. Minifilaments formed from unfractionated myosin (O) were assayed in the Tris-citrate buffer containing l0 mM-KCI, 2-5 mM-ATP and 3.75 mM-MgCl2.

ASSEMBLY AND KINETICS

2'0

i

'

417

OF MYOSIN ISOZYMES I

i

if"

~5

~'~

I'0

0-5

I

0

0"04

I .......

0"08

0"12 0 I/[actin] (p.M-I)

....

I

1

0-04

0"08

0"12

Flo. 8. Lineweaver-Burk plot of actin-activated ATPase data from minifilaments formed from purified myosin isozymes. Unfraetionated myosin (O), Al-myosin (R), and A2 myosin (A). Two separate experiments are shown to indicate the quality and reproducibility of the method.

suggesting that in both cases the steric hindrance caused by assembly had approximately the same influence on kinetic behavior. Heavy meromyosin processed in parallel through the Tris-citrate buffer system shows somewhat higher kinetic parameters, Vmax= 5 s- 1 per head and Kapp = 60 mM, as expected for soluble myosin subfragments. 4. Discussion

The availability of native chicken myosin isozymes, isolated by immunoaffinity chromatography, has allowed a new examination of the structural and TABLE 3

A ctomyosin A TPase activities with min~filaments

Myosin Unfractionated A 1-isozyme A2 -isozyme Unfractionated A 1-isozy me A2-isozyme Unfractionated Unfractionated Unfractionated

Stock myosin concentration (mg/ml)

Vm,x (s- 1)

K~pp (gM)

0.22 0.15 0-17 0-22 0-12 0-18 0.17 2.7 4.5

2-7 2.7 2-3 1"8 2-5 1-6 3-0 3.1 2.4

44 51 37 29 33 32 32 45 28

418

S.C. PASTRA-LANDIS. T. HUIATT AND S. LOWEY

biochemical functions of the two distinct alkali light chains in fast vertebrate skeletal muscle. Our experiments show that the alkali light chains, A1 and A2, do not directly regulate the ATPase activity or modulate filament formation and architecture. Filament assembly studies can only be done using these native homodimers because the heavy chains of the artificial molecules (Pope et al., 1981) are identical by the nature of their preparation. Although the precise length of synthetic filaments (see also Whalen et al., 1981; Pinset-H~rstrSm & Truffy, 1979) depended on the solvent conditions of the polymerization process, under the same set of conditions, no differences were seen between filaments formed from unfractionated myosin or from the isolated homodimers. In addition, "minifilaments", closely resembling those obtained from rabbit myosin, are indistinguishable in appearance whether made from isolated myosin homodimers or from the control mixed population. Even more significant is the fact that the homodimers sediment as a hypersharp boundary at identical rates, indicating that the two isozymes must have the same length distribution. In agreement with the results for subfragment-1 and HMM (Weeds & Taylor, 1975; Wagner, 1977), the native myosin isozymes have identical K+(EDTA) and Ca 2+ ATPase activities. The actin-activated ATPase activity, determined using either heterogeneous myosin filaments or the more homogeneous myosin minifilament system, also shows only small differences, generally not significant above the expected level of experimental error. At 55 mM-KCI, or in the minifilament buffer system (I ~ 90), the kinetic parameters were very similar. At lower ionic strength (25 mM-KCI), only small differences were found from one experiment to the next, and never were these differences in either Vmax or Kapp larger than twofold. A limitation imposed on this investigation stems from the use of immunoadsorbents to fractionate myosin. With yields of 1 mg protein at 0.1 to 0.3mg/ml, only a small number of assays can be performed with each preparation, so that the opportunity to collect many points for more reliable double reciprocal plots does not exist. The high viscosity of actomyosin solutions also precludes the collection of data at the high actin concentrations necessary to make the extrapolation to Vmax more certain. Furthermore, it is difficult to perform meaningful actomyosin assays at the very low ionic strength of 6mM-KCI, where kinetic differences between the soluble S-1 isozymes were originally observed (Weeds & Taylor, 1975). The importance of small differences in Vmx and K~pp for the intact native myosin isoforms is difficult to evaluate, since these differences probably lie within the limits of experimental error. Nevertheless, it should be noted that the Vm~x of Al-myosin was usually somewhat higher than that of A2-myosin (see Tables 2 and 3; and Silberstein & Lowey, 1981), whereas differences in the opposite direction are found for subfragment-I isozymes. The possibility exists that these small differences arise from the differential stability of the isozymes and are not related to their intrinsic kinetic properties (Mrakovcic-Zenic et al., 1981). The enzymatic behavior of chicken myosin minifilaments is similar to that of myosin filaments, and unlike the soluble proteolytic subfragment HMM. This result is in contrast to the reports by Reisler on the rabbit protein, in which soluble HMM and homogeneous minifilament preparations were found to exhibit

ASSEMBLY AND KINETICS OF MYOSIN ISOZYMES

419

identical catalytic parameters (Reisler, 1980). It is not clear whether the discrepancy is a question of animal species, or some particular experimental difference in the assays. A further consideration should be added to a discussion of minifilaments, because citrate buffer systems (Shaltiel et al., 1966) are known to be deforming agents, For example, in the case of rabbit muscle phosphorylase b, imidazolium citrate at high concentrations is effective in deforming the protein to allow the release of pyridoxal 5'-phosphate, although no irreversible denaturation occurs, since the apoenzyme can be fully reactivated. An analogous deformation of myosin by the minifilament buffer system, while not interfering with assembly behavior, may well explain differences in kinetic parameters obtained by different laboratories with different species, since small conformational changes at the active site would affect kinetic results. Although the concentration of citrate used is quite low, myosin is unusually susceptible to buffer conditions. We have indeed observed such sensitivity exhibited by the F-actin used for ATPase assays. If F-actin is stored in the minifilament buffer system for longer than 24 hours before use, it appears to be unstable and appreciably higher Kapp values are obtained from double reciprocal plots. For this reason, F-actin was stored as a pellet until resuspended in Tris-citrate buffer on the day of use. The myosin heavy chains associated with a distinct alkali light chain in isolated myosin homodimers did not show a parallel heterogeneity when analyzed by twodimensional gel electrophoresis of partial chymotryptic digests (data not shown). This result is consistent with earlier findings on myosin heavy chains analyzed by one-dimensional electrophoresis (d'Albis etal., 1979). The two-dimensional method has proven to be highly sensitive in comparative studies of myosin heavy chain isozymes from fast and slow muscles or from developing fibers in embryonic, neonatal and adult muscles (Whalen et al., 1979; Benfield et al., 1983). Heavy chain heterogeneity for fast muscle myosin had initially been shown in N-terminal peptides (Starr & Offer, 1973) and by isoelectric focusing on polyacrylamide gels (John, 1980). Partial amino acid analysis of the N-terminal peptides from fractionated subfragment-1 isozymes (Pope et al., 1977) demonstrated no selectivity in the interactions between the heavy chains and the light chains. Our results are consistent with those found for subfragment-1, in that they also provide no evidence for specific association of a particular heavy chain isozyme with a particular alkali light chain. The results of our kinetic and assembly experiments show no measurable functional differences between myosin light chain isozymes; light chains do not appear to regulate the ATPase activity and do not serve to control filament formation and architecture. A variety of results in agreement with this conclusion have accumulated from a number of laboratories. Artificial myosin homodimers enriched in either Al or A2 light chains, by exchange in high salt conditions, exhibit nearly identical rates of actin-activated ATP hydrolysis (Pope et al., 1981). Hybrid myosins, prepared by using subunits from slow ox cardiac myosin and fast rabbit skeletal myosin, gave an actin-activated ATPase activity that was determined primarily by the heavy chain present (Wagner, 1981). This result strongly supports the idea that light chains are not involved in modulating

420

S.C. PASTRA-LANDIS, T. HUIATT AND S. LOWEY

activity, especially when one considers that the cardiac LC1 is quite different from the fast muscle LC1, being only 30% homologous (Malta et al., 1980), although the substitutions are conservative and the total chemical composition of the light chains is quite similar. Furthermore, the alkali light chains of myosin subfragment-1 at elevated temperature, 37°C, and in l0 mM-Mg2+ ATP, seem to exchange freely, so that at physiological conditions of ionic strength and temperature the interaction between subunits of the soluble subfragment-1 is very labile (Burke & Sivaramakrishnan, 1981). However, the significance of this result needs to be tested both in native myosin molecules and the intact filament lattice. In another study, subfragment-1 heavy chains were stripped of "essential" light chains by use of immunoaffinity chromatography, following high salt dissociating conditions (Wagner & Giniger, 1981). The denuded subfragment-1 heavy chains hydrolyze ATP and bind F-actin reversibly; however, full activity of the native enzyme is not retained and, at the very least, alkali light chains are essential for the stability of subfragment-1. Our failure to discover any functional differences between myosin molecules containing closely related but distinct light chains should not be taken to mean that the light chains have no function in muscle. Instead, these experiments serve to remind us of the limited scope of our assays and methods for analyzing myosin function. It is only within the last year that we have come to realize that under certain conditions myosin can assume a stable folded conformation, instead of the extended shape we have always associated with the myosin structure (Trybus et al., 1982; Onishi & Wakabayashi, 1982; Suzuki et al., 1982). Phosphorylation of the regulatory (or LC2) light chain in this new form enhances the assembly of smooth and non-muscle myosin into filaments (Suzuki et al., 1978; Scholey et al., 1980). Even in skeletal myosin, where so far no convincing role for the LC2f light chain has been found, Cooke etal. (1982) have shown recently that phosphorylation of the light chain in a well-ordered filamentous array leads to a twofold change in the ATPase activity; in contrast, the activity of the isolated actomyosin system is unchanged by phosphorylation (Morgan et al., 1976). These are only two examples of new insights into the role of the light chains. Although in both cases the regulatory LC2 class of light chain is involved, there are already indications in the molluscan system that the essential and regulatory light chains are in close proximity to each other and may be involved in cooperative head-to-head interactions (Hardwicke et al., 1982; Walliman et al., 1982). It is conceivable that in the near future a functional role will be found for the A1 and A2 light chains, be it in filament assembly during development or in the replacement of filaments during regeneration, or yet in the maintenance of the filament lattice in a mature muscle. We thank P. A. Benfield for the 2-dimensional electrophoretic analyses of chymotryptic digests, and D. D. LeBlanc for the antibody columns used in the myosin fractionation. E. Reisler kindly provided the densities and viscosities of buffers for the minifilament sedimentation experiments. This work was supported by grants from the National Institutes of Health (5 ROl AM!7350), the National Science Foundation (PCM 782 2710), and the Muscular Dystrophy Association (to S.L.). T.W.H. was the recipient of a National Research Service award (5 F32 AM06176-03) from the National Institutes of Health.

ASSEMBLY AND KINETICS OF MYOSIN ISOZYMES

421

REFERENCES Benfield, P. A., Lowey, S., LeBlanc, D. D. & Waller, G. (1983). J. Muscle Res. Cell Motil. 4, in the press. Burke, M. & Sivaramakrishnan, M. (1981). Biochemistry, 20, 5908-5913. Caspar, D. L. D., Cohen, C. & Longley, W. (1969). J. Mol. Biol. 41, 87-I07. Cooke, R., Franks, K. & Stull, J. T. (1982). F E B S Letters, 144, 33-37. d'Albis, A., Pantaloni, C. & Bechet, J.-J. (1979). F E B S Letters, 106, 81-84. Dreizen, P., & Gershman, L. C. (1970). Biochemistry, 9, 1688-1693. Fiske, C. H. & Subbarow, Y. (1925). J. Biol. Chem. 66, 375-400. Frank, G. & Weeds, A. G. (1974). Eur. J. Biochem. 44, 317-334. Gauthier, G. F. & Lowey, S. (1979). J. Cell Biol. 81, 10-25. Gazith, J., Himmellfarb, S. & Harrington, W. F. (1970). J. Biol. Chem. 245, 15-22. Hardwicke, P. M. D., Walliman, T. & Szent-GySrgyi, A. G. (1982). J. Mol. Biol. 156, 141-152. Hoh, J. F. Y. (1978). F E B S Letters, 90, 297-300. Hob, J. F. Y. (1979). F E B S Letters, 98, 267-270. Holt, J. C. & Lowey, S. (1975). Biochemistry, 14, 4609-4620. Holt, J. C. & Lowey, S. (1977). Biochemistry, 16, 4398-4402. Holtzer, A. & Lowey, S. (1959). J. Amer. Chem. Soc. 81, 1370-1377. John, H. W. (1980). Biochem. Biophys. Res. Commun. 92, 1223-1230. Kominz. D. R., Carroll, W. R., Smith, E. N. & Mitchell, E. 1%. (1959). Arch. Biochem. Biophys. 79, 19 l - 199. Laemmli, U. K. (1970). Nature (London), 227, 680-685. Lowey, S. & 1%isby, D. (1971). Nature (London), 234, 81-85. Lowey, S., Benfield, P. A., Silberstein, L. & Lang, L. M. (1979). Nature (London), 282, 522-524. Malta, T., Umegane, T., Kato, Y. & Matsuda, G. (1980). Eur. J. Biochem. 107, 565-575. Malta, T., Umegane, T. & Matsuda, G. (1981). Eur. J. Biochem. 114, 45-49. Margossian. S. S. & Lowey, S. (1982). Methods Enzymol. 85, 55-71. Margossian, S. S., Lowey, S. & Barshop, B. (1975). Nature (London), 258, 163-164. Morgan, M., Perry, S. V. & Ottaway, J. (1976). Biochem. J. 157, 687-697. Mrakovcic-Zenic, A., Oriol-Audit, C. & 1%eisler, E. (1981). Eur. J. Biochem. 115,565-570. Offer, G., Moos, C. & Starr, R. (1973). J. Mol. Biol. 74, 653-676. Onishi, H. & Wakabayashi, T. (1982). J. Biochem. (Tokyo), 92, 871-879. Pastra-Landis, S. C. & Huiatt, T. W. (1980). Fed. Proc. Fed. Amer. Soc. Exp. Biol. 39, 2169. Pastra-Landis, S. C.~ Huiatt, T. W. & Lowey, S. (t981). Biophys. J. 33, 242a. Pastra-Landis, S. C., Huiatt, T. W. & Lowey, S. (1982). In Basic Biology of Muscles: A Comparative Approach (Twarog, B. M., Levine, R. T. & Dewey, M. M., eds), Society of General Physiologists Series, vol. 37, pp. 1-10, 1%owen Press, New York. Pette, D. & Schnez, U. (1977). F E B S Letters, 83, 128-130. Pinset-H~rstrSm, I. & Truffy, J. (1979). J. Mol. Biol. 134, 173-188. Pope, B. J., Wagner, P. D. & Weeds, A. G. (1977). J. Mol. Biol. 109, 470-473. Pope, B., Wagner, P. D. & Weeds, A. G. (1981). Eur. J. Biochem. 117, 201-206. Reisler, E. (1980). J. Biol. Chem. 255, 9541-9544. Reisler, E., Smith, C. A. & Seegan, G. (1980). J. Mol. Biol. 143, 129-145. Sarkar, S. (1972). Cold Spring Harbor Symp. Quant. Biol. 37, 14-17, Scholey, J. M., Taylor, K. A. & Kendrick-Jones, J. (1980). Nature (London), 287, 233-235. Shaltiel, S., Hedrick, J. L. & Fischer, E. H. (1966). Biochemistry, 5, 2108-2116. Silberstein, L. & Lowey, S. (1981). J. Mol. Biol. 148, 153-189. Spudich, J. A. & Watt, S. (1971). J. Biol. Chem. 246, 4866-4871. Starr, R. & Offer, G. (1973). J. Mol. Biol. 81, 17-31. Stracher, A. (1969). Biochem. Biophys. Res. Commun. 35, 519-525. Suzuki, H., Onishi, H., Takahashi, K. & Watanabe, S. (1978). J. Biochem. (Tokyo), 84, 1529-1542.

422 S.C. PASTRA-LANDIS, T. HUIATT AND S. LOWEY Suzuki, H., Kamata, T., Onishi, H. & Watanabe, S. (1982). J. Biochem. (Tokyo), 91, 1699-1705. Trybus, K. M., Huiatt, T. W. & Lowey, S. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 6151-6155. Tyler, J. M. & Branton, D. (1980). J. Ultrastruct. Res. 71, 95-102. Wachsberger, P. P~. & Pepe, F. A. (1980). J. Cell Biol. 85, 33-41. Wagner, P. D. (1977). F E B S Letters, 81, 81-85. Wagner, P. (1981). J. Biol. Chem. 256, 2493-2498. Wagner, P. D. & Giniger, E. (1981). Nature (London), 292, 560-562. Wagner, P. D. & Weeds, A. G. (1977). J. Mol. Biol. 109, 455-473. Wagner, P. D., Slater, C. S., Pope, B. & Weeds, A. G. (1979). Eur. J. Biochem. 99, 385-394. Walliman, T., Harwicke, P. M. D. & Szent-GySrgyi, A. G. (1982). J. Mol. Biol. 156, 153-173. Weeds, A. G. (1969). Nature (London), 223, 1362-1364. Weeds, A. G. & Lowey, S. (1971). J. Mol. Biol. 61,701-725. Weeds, A. G. & Taylor, R. S. (1975). Nature (London), 257, 54-56. Weeds, A. G., Hall, P~. & Spurway, N. C. S. (1975). F E B S Letters, 49, 320-324. Whalen, R. G., Schwartz, K., Bouveret, P., Sell, S. M. & Gros, F. (1979). Proc. Nat. Acad. Sci., U.S.A. 76, 5197-5201. Whalen, R. G., Sell, S. M., Bulter-Browne, G. S., Schwartz, K., Bouveret, P. & PinsetH~rstrSm, I. (1981). Nature (London), 292, 805-809. Yagi, K. & Otani, F. (1974). J. Biochem. 76, 365-373. Edited by H. E. Huxley