J. Mob. Biol. (1977) 116, 883-890
Crystal Forms of a,-Tropomyosin a,-Tropomyosin crystals can be distinguished from those obtained from unfractionated tropomyosin by phase contrast microscopy and by t,heir electron microscopic appearance after thin sectioning and negative staining. The overlapping of two conventional kite-shaped tropomyosin nets gives projections that are similar to those obtained from the new a,-tropomyosin crystals. Both symmetrical and unsymmetrical overlap patterns were observed. These crystals ma) prove to be useful for X-ray diffraction studies because of their presumably higher protein densities.
purpose of this paper is to describe and interpret the structures of two unique crystalline forms of rabbit muscle a,-tropomyosin. Tropomyosin and its complex with subunit T of troponin have the remarkable property of being able to crystallize in a variety of forms. Most of these are known only by t,heir electron microscopic appearance in the dried and negatively stained state, since X-ray diffraction analysis is made difficult by their abnormally high water content (N95oj,). In many cases a 40 nm repeat distance, or linear measure, is immediately apparent on the micrographs (Fujime-Higashi & Ooi, 1969 ; Caspar et al., 1969; Greaser et al., 1972 ; Yamaguchi et al., 1974). Since this is also approximately the length of the rod-like molecules of tropomyosin, one may infer that upon drying, the molecules in t,he crystals become oriented nearly parallel to the supporting grid. This interpretation has been confirmed in the one case for which X-ray diffraction dat’a are available, that of the crystal form which shows a kite-shaped net in two dimensions (Cohen et al., 1971). Tropomyosin from skeletal muscle is heterogeneous, giving rise to two major bands when examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Weber & &born, 1969; Greaser & G-ergely, 1971; Spudich & Watt, 1971). Cummins & Perry (1973) have separated two fractions of tropomyosin on CM-cellulose in 8 Murea : the one which migrated most rapidly on gels containing sodium dodecyl sulfate was designated as GCand the slower one as /I. Hydroxylapatite chromatography also separat’es tropomyosin into two fractions, with one fraction containing only c( subunits (as defined by Cummins & Perry) but with the other containing an equal mixture of ccand /I subunits (Eisenberg & Kielley, 1974; Yamaguchi et aZ., 1974). These fractions were formerly referred to as single band and double band tropomyosin, respectively (Yamaguchi et al., 1974). Thus a,-tropomyosin is equivalent t,o single band tropomyosin and up-tropomyosin corresponds to tropomyosin which gives a double band with equal amounts of each type of subunit. The two crystal forms to be considered here are both only obtained from purified a,-tropomyosin. The preparative methods and other aspects of the experimental procedures have all been given in detail in previous papers (Greaser et al.? 1972 ; Yamaguchi et al., 1974). Since the new crystal forms of a,-tropomyosin have only been observed after negativc staining and thcp could therefore have arisen by an altered mode of collapse on The
883
884
M. L.
(a)
GREASER
ET
AL.
(b)
FIG. 1. Crystals of unfractionated and a,-tropomyosin. Unfractionated and hydroxylapatitepurified a,-tropomyosin were dialyzed against several changes of a solution containing 0.2 BIKCl, 0.01 M-sodium acetate (pH 5.6) at 4°C. Crystals were allowed to grow for 10 to 14 days, and observed by phase contrast microscopy. (a) Unfractionated tropomyosin, magnification 200 x ; (b) cc,-tropomyosin, magnification 200 x .
the grids, it is necessary to establish that they are in fact crystalline in nature. Figure 1 contains phase contrast micrographs of crystals of unfractionated tropomyosin and purified cc,-tropomyosin. The unfractionated tropomyosin crystals had regular, straight edges and were typically rhombic or coffin shaped. This crystal shaFe has been described by many other groups before (Bailey, 1948 ; Tsao et al., 1955 ; FujimeHigashi & Ooi, 1969; Caspar et al., 1969; Ooi & Fujime-Higashi, 1971. The a,-tropomyosin crystals, however, were usually either irregular hexagons or had poorly defined shape (Fig. l(b)). The structure of these crystals in the third dimension was difficult, to discern, perhaps due to their being thicker or having a greater density. Figure 2 shows the appearance of tropomyosin crystals after thin sectioning. Crystals of unfractionated tropomyosin gave either a series of parallel lines with a periodicity of approximately 20 nm or displayed the kite-shaped nets which are seen after negative staining (Fig. 2(a)). This pattern is similar to that observed previously (Fujime-Higashi & Ooi, 1969). Most of the a,-tropomyosin crystals gave different patterns, with either pairs of parallel lines or closely woven networks (Fig. 2(b) and (c)). The average spacing between the lines was about 10 nm, half of that found in the conventional crystals. The 10 nm spacing was never seen in the unfractionated tropomyosin crystals, despite the large number of random orientations observed. The unique patterns from the a,-tropomyosin preparations in thin sections indicate that they are true three-dimensional crystals and that the new patterns observed et al., 1974) are not the result of a different pattern by negative staining (Yamaguchi of crystal collapse. Figure 3 is a micrograph of a negatively stained a,-tropomyosin crystal in the familiar kite-shaped net pattern. Less than 10% of the crystals appeared in this form. No significant differences could be detected between this pattern and that given by the extensively studied unfractionated tropomyosin preparations. The sum of one long and one short arm of a kite is approximately 40 nm.
(b
FIG. 2. Fixed and embedded preparations Crystals were grown as described in the 0.25 ~-sucrose, 60 mM-potassium cacodylate end then post-fixed in 2% osmium tetroxide uranyl acetate for 1 h, dehydrated in ethanol slxtions (approx. 50 to 60 nm) were stained rnagnificat-ion 300,000 x ; (b) az-tropomyosin, Inagnification 300,000 X .
of unfractionated and cr,-tropomyosin crystals. legend to Fig. 1 and fixed in 3% glutaraldehyde, (pH 7.4) for 3 h at 0°C. The samples were rinsed for 2 h. After rinsing t,he blocks were soaked in 1 o. and propylene oxide, and embedded in Epon. Thin with lead citrate. (a) Gnfractionated tropomyosin, magnification 300,000 x ; (c) cc,-tropomyositr,
886
M. L.
GREASER
ET AL.
FIQ. 3. Kite-shaped net from an az-tropomyosin crystal. An cc,-tropomyosin preparation was dialyzed against several changes of 0.2 M-KCl, 0.01 Msodium acetate (pH 6.6) at 4°C for 2 days. A drop of the crystal suspension was then placed on a carbon-coated grid and negatively stained with 1% uranyl acetate. Less than 10% of the crystals observed gave this conventional kite-shaped pattern; magnification 600,000 x .
Figure 4 shows the appearance of a second crystal form of qtropomyosin, which is the most abundant form obtained with conventional crystallization methods (Greaser et al., 1972). It has proven possible to analyze this structure in terms of two identical, overlapping kite-shaped nets having the same characteristics as the net of Figure 3. This is illustrated in the schematic overlay drawing on Figure 4. Translation of one of the nets, by an amount which bears no simple relationship to the unit cell dimensions, results in its superposition on the other net. Bright spots on the micrograph correspond either to vertices of the kites or to crossing points. In all cases, the long arm of one net crosses a short arm of the other net. Two crossing points and two vertices give the pattern of parallelogram-shaped spots. As can best be seen in the overlay drawing, the direction of the longer dimension of these parallelograms alternates in successive locations. This crystal type will be referred to as an unsymmetrical double kite net.
LETTERS
TO
THE
Sri7
EDITOR
The second crystal form of interest was obtained originally from mixtures of x,tropomyosin plus TN-Tt (Fig. 5). This pattern has also recently been observed among crystals of highly purified qtropomyosin. Once again, a pair of overlapping kitcshaped nets simulates the observed pattern, as shown in the overlay on Figure 5. Jn this case, the translation of one net by exactly half the width of a kite brings it into superposition with the other net. Whereas in Figure 4 long arms of the kites always intersected short arms, in Figure 5 long arms cross long arms, and short arms cross short arms. Bright spots correspond to kite vertices or crossing points. The curious rows of bars or bridges (indicated by arrows in Fig. 5) would be formed by the crossings of the long arms; these must also be the points at which the TN-T is bound, since TN-T, just as whole troponin, binds to the centers of the long arms in the simple kite-shaped nets (Cohen et al., 1971; Yamaguchi et al., 1974). Addition of
FIG. H~droxylspatit,e-purified Magnification 600,000 t Abbreviation
used:
4. The
unsymmetrical a,-tropomyosin
double kite was treated
x TN-T,
troponin
subunit
T.
net of cc,-tropomyosin. as described in the
legend
to
Fig.
:I.
888
M. L.
GREASER
ET
AL.
FIG. 6. The symmetrical double kite net of a,-tropomyosin. This pattern has been observed among crystals of purified a,-tropomyosin alone aa well as in mixtures of al-tropomyoain plus TN-T. The crystal shown was prepared from a mixture of al-tropomyosin plus TN-T that had been dialyzed against 0.1 M-sodium acetate, 0.06 M-(NH&SO~ (pH 643) for 3 days. The arrows indicate the bar-like region where TN-T binds; magnification 600,000 x .
TN-T to u,-tropomyosin crystal suspensions in which some of the crystals had a pattern like that in Figure 5 resulted in a thickening of the bar regions of these crystals and therefore supported the interpretation suggested above. These crystals will be referred to as symmetrical double kite nets. The shape of the kite-shaped structures varies considerably both between the different kinds of crystals and between different crystals of the same type (Table 1). The new a,-tropomyosin crystals usually gave more acute net angles than the single kite type. In each case the sum of the lengths of one long and one short arm was approximately 40 nm. Thus the ratio of the long diagonal of a kite to that of its short diagonal increases with a decrease in net angle (Table 1). The models presented suggested a new interpretation of the location of troponin and TN-T in the a,-tropomyosin crystals compared to that given previously (Yamaguchi et al., 1974). In both of the new a,-tropomyosin crystal types (Figs 4 and 5) the
LETTERS
TO
THE
TABLE
EDITOR
sx9
1
Dimensions of a,-tropomyosin
crystals
Mean net angle? (“)
Range (‘)
(‘/Jx
76 68 73
73-77 59-77 6:3-78
I.26 1.42 l.y‘J ,I
Type A. Kite B. Unsymmetrical double kite C. Symmetrical double kite
t The means and ranges were obtained from 6 plates of each type. $ Ratio of t,he length of the long diagonal to the short diagonal of a kite ((‘ohen et rtl., 1972).
vertices are more prominent than the cross-over points. Thus the dominant bright lines of points that appear when troponin is bound to the unsymmetrical double kite net correspond to vertices and the less prominent points (arising from the intersection of a short and a long arm) must be the region of troponin binding. Similarly, the pairs of bright lines which appear on the symmetrical double kite crystals also arise from the double kite vertices. In summary, crystals of the a,-tropomyosin and unfractionated tropomyosin can be distinguished from each other using phase contrast and electron microscopy. Models of the a,-tropomyosin crystals constructed from two overlapping kite-shaped nets are consistent with the crystal appearance in thin sections and after negative staining. The apparent doubling of the protein density in the cc,-tropomyosin crystals compared to crystals of unfractionated tropomyosin suggests that they may be especially well suited for X-ray diffraction analysis. This work was supported by the College of Agricultural and Life Sciences and by thrl Research Committee of the University of Wisconsin, Madison; by the Campbell Inst)itute for Food Research ; and by grants from the National Institutes of Healt,h. This is Muscle Biology Laborat,ory manuscript no. 85. University of Wisconsin Muscle Biology Laboratory 1805 Linden Drive Madison, Wise. 53706, U.S.A. Kooeived
2 June
1975, and in revised
M. L. GREASER M. YAMAGUCHlt ti. VAKDERKOOI:
form
4 April
1977
REFERENCES Bailey, K. (1948). Biochem. J. 43, 271-279. Caspar. D. I,. D., Cohen, C. & Longley, W. (1969). J. Mol. Biol. 41, 87-107. Cohen, C., Caspar, D. L. D., Parry, D. A. D. & Lucas, R. M. (1971). Cold Spring Symp. Quant. Biol. 36, 205-216. Cummins, P. & Perry, S. V. (1973). Biochem. J. 133, 765-777. Eiseuhorg, E. & Kielley, W. W. (1974). J. Biol. Chem. 249, 4742-4748. Fujime-Higashi, S. & Ooi, T. (1969). J. Microsc. (Paris), 8, 535-548. t Present address: Iowa State University, $ Prevent address: Chemistry Department, Z1.S.A.
Ames, Iowa 60010, U.S.A. Northern Illinois Universit,y,
De Kalb,
Harbor
111. 60115,
890
M. L.
GREASER
ET
AL.
Greaser, M. L. & Gergely, J. (1971). J. Biol. Chem. 246, 422tF-4233. Greaser, M. L., Yamaguchi, M., Brekke, C., Potter, J. & Gergely, J. (1972). Cold Spring Harbor Xymp. Quant. Biol. 37, 235-244. Ooi, T. & Fujime-Higashi, S. (1971). Advan. Biophys. 2, 113-153. Spudich, J. A. & Watt, S. (1971). J. Biol. Chem. 246, 4866-4871. Tsao, T. C., Tan, P. H. & Peng, C. M. (1955).&i. Sin. 5, 91-111. Weber, K. & Osborn, M. (1969). J. Biol. Chem. 244, 4406-4412. Yamaguchi, M., Greaser, M. L. & Caasens, R. G. (1974). J. Ultra&u&. Res. 48, 33-58.
Crystal Forms of a,-Tropomyosin a,-Tropomyosin crystals can be distinguished from those obtained from unfractionated tropomyosin by phase contrast microscopy and by t,heir electron microscopic appearance after thin sectioning and negative staining. The overlapping of two conventional kite-shaped tropomyosin nets gives projections that are similar to those obtained from the new a,-tropomyosin crystals. Both symmetrical and unsymmetrical overlap patterns were observed. These crystals ma) prove to be useful for X-ray diffraction studies because of their presumably higher protein densities.
purpose of this paper is to describe and interpret the structures of two unique crystalline forms of rabbit muscle a,-tropomyosin. Tropomyosin and its complex with subunit T of troponin have the remarkable property of being able to crystallize in a variety of forms. Most of these are known only by t,heir electron microscopic appearance in the dried and negatively stained state, since X-ray diffraction analysis is made difficult by their abnormally high water content (N95oj,). In many cases a 40 nm repeat distance, or linear measure, is immediately apparent on the micrographs (Fujime-Higashi & Ooi, 1969 ; Caspar et al., 1969; Greaser et al., 1972 ; Yamaguchi et al., 1974). Since this is also approximately the length of the rod-like molecules of tropomyosin, one may infer that upon drying, the molecules in t,he crystals become oriented nearly parallel to the supporting grid. This interpretation has been confirmed in the one case for which X-ray diffraction dat’a are available, that of the crystal form which shows a kite-shaped net in two dimensions (Cohen et al., 1971). Tropomyosin from skeletal muscle is heterogeneous, giving rise to two major bands when examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Weber & &born, 1969; Greaser & G-ergely, 1971; Spudich & Watt, 1971). Cummins & Perry (1973) have separated two fractions of tropomyosin on CM-cellulose in 8 Murea : the one which migrated most rapidly on gels containing sodium dodecyl sulfate was designated as GCand the slower one as /I. Hydroxylapatite chromatography also separat’es tropomyosin into two fractions, with one fraction containing only c( subunits (as defined by Cummins & Perry) but with the other containing an equal mixture of ccand /I subunits (Eisenberg & Kielley, 1974; Yamaguchi et aZ., 1974). These fractions were formerly referred to as single band and double band tropomyosin, respectively (Yamaguchi et al., 1974). Thus a,-tropomyosin is equivalent t,o single band tropomyosin and up-tropomyosin corresponds to tropomyosin which gives a double band with equal amounts of each type of subunit. The two crystal forms to be considered here are both only obtained from purified a,-tropomyosin. The preparative methods and other aspects of the experimental procedures have all been given in detail in previous papers (Greaser et al.? 1972 ; Yamaguchi et al., 1974). Since the new crystal forms of a,-tropomyosin have only been observed after negativc staining and thcp could therefore have arisen by an altered mode of collapse on The
883
884
M. L.
(a)
GREASER
ET
AL.
(b)
FIG. 1. Crystals of unfractionated and a,-tropomyosin. Unfractionated and hydroxylapatitepurified a,-tropomyosin were dialyzed against several changes of a solution containing 0.2 BIKCl, 0.01 M-sodium acetate (pH 5.6) at 4°C. Crystals were allowed to grow for 10 to 14 days, and observed by phase contrast microscopy. (a) Unfractionated tropomyosin, magnification 200 x ; (b) cc,-tropomyosin, magnification 200 x .
the grids, it is necessary to establish that they are in fact crystalline in nature. Figure 1 contains phase contrast micrographs of crystals of unfractionated tropomyosin and purified cc,-tropomyosin. The unfractionated tropomyosin crystals had regular, straight edges and were typically rhombic or coffin shaped. This crystal shaFe has been described by many other groups before (Bailey, 1948 ; Tsao et al., 1955 ; FujimeHigashi & Ooi, 1969; Caspar et al., 1969; Ooi & Fujime-Higashi, 1971. The a,-tropomyosin crystals, however, were usually either irregular hexagons or had poorly defined shape (Fig. l(b)). The structure of these crystals in the third dimension was difficult, to discern, perhaps due to their being thicker or having a greater density. Figure 2 shows the appearance of tropomyosin crystals after thin sectioning. Crystals of unfractionated tropomyosin gave either a series of parallel lines with a periodicity of approximately 20 nm or displayed the kite-shaped nets which are seen after negative staining (Fig. 2(a)). This pattern is similar to that observed previously (Fujime-Higashi & Ooi, 1969). Most of the a,-tropomyosin crystals gave different patterns, with either pairs of parallel lines or closely woven networks (Fig. 2(b) and (c)). The average spacing between the lines was about 10 nm, half of that found in the conventional crystals. The 10 nm spacing was never seen in the unfractionated tropomyosin crystals, despite the large number of random orientations observed. The unique patterns from the a,-tropomyosin preparations in thin sections indicate that they are true three-dimensional crystals and that the new patterns observed et al., 1974) are not the result of a different pattern by negative staining (Yamaguchi of crystal collapse. Figure 3 is a micrograph of a negatively stained a,-tropomyosin crystal in the familiar kite-shaped net pattern. Less than 10% of the crystals appeared in this form. No significant differences could be detected between this pattern and that given by the extensively studied unfractionated tropomyosin preparations. The sum of one long and one short arm of a kite is approximately 40 nm.
(b
FIG. 2. Fixed and embedded preparations Crystals were grown as described in the 0.25 ~-sucrose, 60 mM-potassium cacodylate end then post-fixed in 2% osmium tetroxide uranyl acetate for 1 h, dehydrated in ethanol slxtions (approx. 50 to 60 nm) were stained rnagnificat-ion 300,000 x ; (b) az-tropomyosin, Inagnification 300,000 X .
of unfractionated and cr,-tropomyosin crystals. legend to Fig. 1 and fixed in 3% glutaraldehyde, (pH 7.4) for 3 h at 0°C. The samples were rinsed for 2 h. After rinsing t,he blocks were soaked in 1 o. and propylene oxide, and embedded in Epon. Thin with lead citrate. (a) Gnfractionated tropomyosin, magnification 300,000 x ; (c) cc,-tropomyositr,
886
M. L.
GREASER
ET AL.
FIQ. 3. Kite-shaped net from an az-tropomyosin crystal. An cc,-tropomyosin preparation was dialyzed against several changes of 0.2 M-KCl, 0.01 Msodium acetate (pH 6.6) at 4°C for 2 days. A drop of the crystal suspension was then placed on a carbon-coated grid and negatively stained with 1% uranyl acetate. Less than 10% of the crystals observed gave this conventional kite-shaped pattern; magnification 600,000 x .
Figure 4 shows the appearance of a second crystal form of qtropomyosin, which is the most abundant form obtained with conventional crystallization methods (Greaser et al., 1972). It has proven possible to analyze this structure in terms of two identical, overlapping kite-shaped nets having the same characteristics as the net of Figure 3. This is illustrated in the schematic overlay drawing on Figure 4. Translation of one of the nets, by an amount which bears no simple relationship to the unit cell dimensions, results in its superposition on the other net. Bright spots on the micrograph correspond either to vertices of the kites or to crossing points. In all cases, the long arm of one net crosses a short arm of the other net. Two crossing points and two vertices give the pattern of parallelogram-shaped spots. As can best be seen in the overlay drawing, the direction of the longer dimension of these parallelograms alternates in successive locations. This crystal type will be referred to as an unsymmetrical double kite net.
LETTERS
TO
THE
Sri7
EDITOR
The second crystal form of interest was obtained originally from mixtures of x,tropomyosin plus TN-Tt (Fig. 5). This pattern has also recently been observed among crystals of highly purified qtropomyosin. Once again, a pair of overlapping kitcshaped nets simulates the observed pattern, as shown in the overlay on Figure 5. Jn this case, the translation of one net by exactly half the width of a kite brings it into superposition with the other net. Whereas in Figure 4 long arms of the kites always intersected short arms, in Figure 5 long arms cross long arms, and short arms cross short arms. Bright spots correspond to kite vertices or crossing points. The curious rows of bars or bridges (indicated by arrows in Fig. 5) would be formed by the crossings of the long arms; these must also be the points at which the TN-T is bound, since TN-T, just as whole troponin, binds to the centers of the long arms in the simple kite-shaped nets (Cohen et al., 1971; Yamaguchi et al., 1974). Addition of
FIG. H~droxylspatit,e-purified Magnification 600,000 t Abbreviation
used:
4. The
unsymmetrical a,-tropomyosin
double kite was treated
x TN-T,
troponin
subunit
T.
net of cc,-tropomyosin. as described in the
legend
to
Fig.
:I.
888
M. L.
GREASER
ET
AL.
FIG. 6. The symmetrical double kite net of a,-tropomyosin. This pattern has been observed among crystals of purified a,-tropomyosin alone aa well as in mixtures of al-tropomyoain plus TN-T. The crystal shown was prepared from a mixture of al-tropomyosin plus TN-T that had been dialyzed against 0.1 M-sodium acetate, 0.06 M-(NH&SO~ (pH 643) for 3 days. The arrows indicate the bar-like region where TN-T binds; magnification 600,000 x .
TN-T to u,-tropomyosin crystal suspensions in which some of the crystals had a pattern like that in Figure 5 resulted in a thickening of the bar regions of these crystals and therefore supported the interpretation suggested above. These crystals will be referred to as symmetrical double kite nets. The shape of the kite-shaped structures varies considerably both between the different kinds of crystals and between different crystals of the same type (Table 1). The new a,-tropomyosin crystals usually gave more acute net angles than the single kite type. In each case the sum of the lengths of one long and one short arm was approximately 40 nm. Thus the ratio of the long diagonal of a kite to that of its short diagonal increases with a decrease in net angle (Table 1). The models presented suggested a new interpretation of the location of troponin and TN-T in the a,-tropomyosin crystals compared to that given previously (Yamaguchi et al., 1974). In both of the new a,-tropomyosin crystal types (Figs 4 and 5) the
LETTERS
TO
THE
TABLE
EDITOR
sx9
1
Dimensions of a,-tropomyosin
crystals
Mean net angle? (“)
Range (‘)
(‘/Jx
76 68 73
73-77 59-77 6:3-78
I.26 1.42 l.y‘J ,I
Type A. Kite B. Unsymmetrical double kite C. Symmetrical double kite
t The means and ranges were obtained from 6 plates of each type. $ Ratio of t,he length of the long diagonal to the short diagonal of a kite ((‘ohen et rtl., 1972).
vertices are more prominent than the cross-over points. Thus the dominant bright lines of points that appear when troponin is bound to the unsymmetrical double kite net correspond to vertices and the less prominent points (arising from the intersection of a short and a long arm) must be the region of troponin binding. Similarly, the pairs of bright lines which appear on the symmetrical double kite crystals also arise from the double kite vertices. In summary, crystals of the a,-tropomyosin and unfractionated tropomyosin can be distinguished from each other using phase contrast and electron microscopy. Models of the a,-tropomyosin crystals constructed from two overlapping kite-shaped nets are consistent with the crystal appearance in thin sections and after negative staining. The apparent doubling of the protein density in the cc,-tropomyosin crystals compared to crystals of unfractionated tropomyosin suggests that they may be especially well suited for X-ray diffraction analysis. This work was supported by the College of Agricultural and Life Sciences and by thrl Research Committee of the University of Wisconsin, Madison; by the Campbell Inst)itute for Food Research ; and by grants from the National Institutes of Healt,h. This is Muscle Biology Laborat,ory manuscript no. 85. University of Wisconsin Muscle Biology Laboratory 1805 Linden Drive Madison, Wise. 53706, U.S.A. Kooeived
2 June
1975, and in revised
M. L. GREASER M. YAMAGUCHlt ti. VAKDERKOOI:
form
4 April
1977
REFERENCES Bailey, K. (1948). Biochem. J. 43, 271-279. Caspar. D. I,. D., Cohen, C. & Longley, W. (1969). J. Mol. Biol. 41, 87-107. Cohen, C., Caspar, D. L. D., Parry, D. A. D. & Lucas, R. M. (1971). Cold Spring Symp. Quant. Biol. 36, 205-216. Cummins, P. & Perry, S. V. (1973). Biochem. J. 133, 765-777. Eiseuhorg, E. & Kielley, W. W. (1974). J. Biol. Chem. 249, 4742-4748. Fujime-Higashi, S. & Ooi, T. (1969). J. Microsc. (Paris), 8, 535-548. t Present address: Iowa State University, $ Prevent address: Chemistry Department, Z1.S.A.
Ames, Iowa 60010, U.S.A. Northern Illinois Universit,y,
De Kalb,
Harbor
111. 60115,
890
M. L.
GREASER
ET
AL.
Greaser, M. L. & Gergely, J. (1971). J. Biol. Chem. 246, 422tF-4233. Greaser, M. L., Yamaguchi, M., Brekke, C., Potter, J. & Gergely, J. (1972). Cold Spring Harbor Xymp. Quant. Biol. 37, 235-244. Ooi, T. & Fujime-Higashi, S. (1971). Advan. Biophys. 2, 113-153. Spudich, J. A. & Watt, S. (1971). J. Biol. Chem. 246, 4866-4871. Tsao, T. C., Tan, P. H. & Peng, C. M. (1955).&i. Sin. 5, 91-111. Weber, K. & Osborn, M. (1969). J. Biol. Chem. 244, 4406-4412. Yamaguchi, M., Greaser, M. L. & Caasens, R. G. (1974). J. Ultra&u&. Res. 48, 33-58.
