Two homogeneous myosins in body-wall muscle of Caenorhabditis elegans

Cell, Vol. 10. 721-728,

April

1977, Copyright

0 1977 by MIT

Two Homogeneous Myosins Caenorhabditis elegans Frederick Henry F. Department Stanford Stanford,

H. Schachat, Harriet E. Harris Epstein of Pharmacology University School of Medicine California 94305

and

Summary Myosin purified from the body-wall muscle-defective mutant E675 of the nematode, Caenorhabditis elegans, has heavy chain polypeptides which can be distinguished on the basis of molecular weight. On SDS-polyacrylamide gels, bands are found at 210,000 and 203,000 daltons. This is in contrast to myosin from the wild-type, N2, which has a single heavy chain band at 210,000 daltons. Both heavy chains of E675 are found in body-wall muscle (Epstein, Waterston and Brenner, 1974). When native myosin from E675 is fractionated on hydroxyapatite, it is separated into myosin containing predominantly one or the other molecular weight heavy chain and myosin containing a mixture of the heavy chains. Comparison of the CNBr fragments of myosin that contains predominantly 210,000 dalton heavy chains with those of myosin that contains predominantly 203,000 dalton heavy chains reveals multiple differences. These differences are not explained by the difference in molecular weight of the heavy chains, but may be explained if each type of heavy chain is the product of a different structural gene. Furthermore, because there are fractions which exhibit >60% 210,000 or >60% 203,000 dalton heavy chain, there is myosin which is homogeneous for each of the heavy chains. Although N2 myosin has only a single molecular weight heavy chain, it too is fractionated by hydroxyapatite. By comparing the CNBr fragments of different myosin fractions, we show that N2, like E675, has two kinds of heavy chains. E190, a body-wall muscle-defective mutant in the same complementation group as E675, is lacking the myosin heavy chain affected by the e675 mutation. This property has allowed us to determine by co-purification of labeled El90 myosin in the presence of excess, unlabeled E675 myosin that most, if not all, of the myosin that contains two different molecular weight heavy chains is due to the formation of complexes between homogeneous myosins and not to a heterogeneous myosin. Introduction Starr and Offer (1973) reported that heavy chains of rabbit fast skeletal muscle myosin had two different

in Body-Wall

Muscle of

amino-terminal amino acid sequences. Although alternative hypotheses were considered, they proposed as the most probable explanation that there are at least two structural genes for rabbit fast skeletal muscle myosin heavy chains. Epstein et al. (1974) in their studies on myosin from the body-wall muscle-defective mutant E675 of the nematode, Caenorhabditis elegans, also found evidence for myosin heavy chain multiplicity in a single muscle type. SDS-polyacrylamide gel electrophoresis of actomyosin extracts from E675 revealed two different myosin heavy chains which were assigned masses of 210,000 and 203,000 daltons. In contrast, actomyosin extracts from the wild-type, N2, showed only a single 210,000 dalton heavy chain. Both molecular weight heavy chains of E675 were located in body-wall muscle. As the most straightforward explanation of these observations, they proposed that the heavy chains of bodywall muscle myosin in wild-type C. elegans were specified by two nonallellic structural genes, and that the mutation in E675 resulted in the conversion of one of the 210,000 dalton gene products to 203,000 daltons. In this paper, we confirm this proposal of Epstein et al. (1974). Further, we show that each of the different body-wall muscle myosin heavy chains associates with a like heavy chain in the native myosin molecule, which is dimeric with respect to the heavy chains. Chromatography of purified E675 myosin on hydroxyapatite resolves myosins homogeneous with respect to either the 210,000 or the 203,000 dalton heavy chains. Comparison of the CNBr peptides of these heavy chains suggests that they are the products of different structural genes. Using the CNBr fragments to detect the different heavy chains, we show that both are present in the wild-type and that its myosin fractionates similarly on hydroxyapatite. Analysis of another mutant, E190, in the same gene as E675 shows that it lacks the homogeneous myosin affected by the e675 mutation. This property allowed us to determine, by co-purification of El90 and E675 myosins, whether fractions from the E675 hydroxyapatite chromatography containing myosin with both heavy chains were due to a heterogeneous myosin species (one which has one of each type of heavy chain). The results indicate that most, if not all, of this mixed material is due to complexes between the two homogeneous myosin species. Results The E675 Myosin Heavy Chains Co-purify Figure 1 D shows the two predominant heavy chains of purified E675 myosin which can be distinguished by SDS-polyacrylamide gel electrophoresis. There

Cdl 722

is also a minor band at 206,000 daltons which Epstein et al. (1974) found in both E675 and N2, and which is located in pharyngeal muscle. Because of its molecular weight, the 206,000 dalton species was putatively identified as a myosin heavy chain. Figure l A-l D show the heavy chain composition of E675 myosin at various stages of its purification. The ratio of 203,000 to 210,000 dalton bands as determined by densitometry of 35S-labeled polypeptides remains constant, within experimental error, during the purification, varying from 1.91 in the homogenate to 2.12 in the Sepharose-purified myosin with a mean and standard deviation of 1.96 t 0.05. The 206,000 dalton band varies between 7% and 10% of the total of the three bands. The ratio of the two predominant heavy chain bands varies from preparation to preparation, but the 203,000 dalton heavy chains is always the major species. The 203,000 to 210,000 dalton ratio was 1.77 ? 0.17 in five homogenates analyzed by densitometry of Coomassie brilliant blue stain or of 35Sautoradiographs. Separation of Myosins Homogeneous for the 203,000 and 210,000 Dalton Heavy Chains When native E675 myosin is chromatographed on hydroxyapatite, species homogeneous for the 203,000 and 210,000 dalton heavy chains are resolved. Figure 2A shows the chromatogram when 600 pg of Sepharose-purified E675 myosin are eluted with a linear phosphate gradient from 0.005 M to 0.5 M with constant 0.6 M KCI, 0.001 M dithiothreitol, 0.0005 M phenylmethylsulfonylfluoride and 0.1% Triton X-100 at a constant pH of 6.5. The profile is complex with three apparent regions: a leading shoulder, fractions 70-89 (region I); a central peak, fractions 92-96 (region II); and a trailing shoulder, fractions 98-110 (region Ill). Since the different molecular weight heavy chains are clearly resolved by SDS-polyacrylamide gel electrophoresis, we are able to quantitate the separation of the two chains by densitometry. Inspection of Figure 3, an autoradiograph of the SDS-polyacrylamide gels on the ?S-myosin from the hydroxyapatite fractions, reveals that region I contains myosin which has predominantly 210,000 dalton heavy chain and region III myosin containing predominantly 203,000 dalton heavy chain. Fractions 75-80 contain myosin that has >80% 210,000 dalton heavy chains, and fractions 100-I 10 contain myosin with >80% 203,000 dalton heavy chains. This demonstrates that native E675 myosin, before electrophoresis under denaturing conditions, contains molecules that are homogeneous for either the 210,000 or 203,000 dalton heavy chains. Figure 26 shows the fractionation of the 210,000 and 203,000 dalton chains separately. The peak fractions are 87 and 103, respectively. Assuming that

A

B

C

D

210 kd

FRONT

Figure

1. Co-purification

of E675 Myosin

Heavy

Chains

Autoradiograph of samples from the purification of Yi-E675 myosin electrophoresed on 4.5% polyacrylamide-SDS gels to show the 210,000 and 203,000 dalton heavy chain polypeptides. (A) the homogenate; (8) the actomyosin extract; (C) the 100,000 supernatant 4; (D) purified myosin. Purification fractions refer to Harris and Epstein (1977).

the separation of chains reflects the separation of three myosin populations-a 210,000 dalton homogeneous, a 203,000 dalton homogeneous and a mixed myosin-we estimate that 24% of the material is homogeneous for the 210,000 dalton heavy chain, 52% is homogeneous for the 203,000 dalton heavy chain and 15% can be represented as myosin containing the two heavy chains. The remaining 8% is 206,000 dalton material and has a peak fraction 74. Its co-purification with myosin and co-migration with myosin on hydroxyapatite lend further support for its identification as a pharyngeal myosin heavy chain. To confirm the identification of two homogeneous myosins, fractions from regions I and III were

Two

Nematode

Body-Wall

Myosins

723

I

14-

I

I

1

1

1

"d'

’

""1

A

12-

8 6 4

T6 -

I

2 0 1011

I ’ -

B

1 ’ ?

’

1 ’ 1

‘D

8642-

ing buffer for the chromatography. Upon rechromatography, this myosin elutes with a maximum at fraction 87 (Figure 2C). When myosin from region III (fractions 101-108) composed of 67% 203,000 dalton heavy chain is similarly treated, it rechromatographs with a maximum at fraction 103 (Figure 2D). These maxima correspond to the maxima of 210,000 and 203,000 dalton myosin heavy chains from the densitometric analysis of the original chromatography (Figure 2B). Thus the homogeneous myosins of E675 rechromatograph true. When myosin containing both molecular weight heavy chains from region II (fractions 92-97) was rechromatographed, it was clear that the 203,000 dalton homogeneous myosin was a major component. The second maximum of the rechromatography at fractions 98-103 corresponds to the elution position of the 203,000 dalton homogeneous myosin. Densitometric analysis of SDS-polyacrylamide gels of the rechromatographed fraction is consistent with this interpretation. However, the absence of 210,000 dalton homogeneous myosin in the rechromatography suggests that the heavy chains do not exchange between myosin molecules during hydroxyapatite chromatography.

ol 14 70

90 FB A:,b?d

10 -

7 ,o x

B 8

8642-

Figure

2. Hydroxyapatite

Chromatography

FRACTION NO. of E675 Myosin

Chromatograms of hydroxyapatite chromatography of %-E675 myosin and rechromatography of fractions of the initial chromatography. Hydroxyapatite columns were run at 2-3 ml/hr. and 1 ml samples were collected. (A) chromatography of E675 myosin: 50 AI samples from each fraction counted as in Experimental Procedures. (6) densitometric analysis of the chromatography based on gels of each of the fractions (Figure 3). (O-O) represents the 210,000 dalton heavy chain; (O-O) the 203,000 dalton heavy chain. Ratios of the chains were determined by densitometry. and the fraction of counts due to each was plotted. (C). (D) and (E) are the rechromatography of fractions 73-81. 92-97 and 101-106, respectively. 100 ~1 samples from each fraction were counted as in Experimental Procedures.

rechromatographed. Myosin from region I (fractions 73-81) composed of 84% 210,000 dalton myosin heavy chain was dialyzed against the start-

Both Homogeneous Myosins Contain 18,000 and 16,000 Dalton Light Chains Harris and Epstein (1977) showed that two light chains of 18,000 and 16,000 daltons are observed in SDS-polyacrylamide gel electrophoresis of N2 myosin. Both these light chains are present in E675 myosin. Figure 4 shows the light chains of Sepharose-purified myosin and those of myosin from the fractions of regions I and Ill which were pooled for rechromatography. The material in region I contains some diffuse material of mass <16,000 daltons. Harris and Epstein (1977) observed this in their characterization of the light chains and attributed it to degradation. Besides retaining its light chains, E675 myosin after hydroxyapatite chromatography is able to form filaments, to bind to the surface of paramyosin paracrystals and to form short bipolar assemblies (data not presented). Cross-linking experiments show that the myosin heavy chains are associated as dimers. These observations indicate that the myosin is structurally intact after hydroxyapatite chromatography. The Homogeneous Myosins Different CNBr Peptides We compared peptides produced by CNBr cleavage of each of the homogeneous myosins of E675. Figure 5 shows a comparison of the peptides on the SDS-urea gel system of Epstein and Wolff (1976). By molecular weight, there are at least six clear differences labeled A-F, most probably reflecting

Cell 724

Figure

3. Separation

of Homogeneous

Autoradioaraph of 4.5% polyacrylamide-SDS 210,000 and iOS,OOO daltbn heady chains.

Myosins

from

E675

gel of the E675 myosin -

differences in the position of methionine. We estimate the masses of these peptides as 47,000, 31,000, 3700, 2300 and 2000 daltons, respectively, as determined by their F&and the apparent molecular weight against Fir relationship determined by Epstein and Wolff (1976). Fragments A, D, E and F are characteristic of the 203,000 dalton homogeneous myosin; fragments B and C are characteristic of the 210,000 dalton homogeneous myosin. Since this separation of CNBr fragments is based principally on size, it should be noted that any band may consist of multiple peptides. For convenience, we refer to any such band as a peptide fragment. This pattern of differences may be explained if the 203,000 and 210,000 dalton heavy chain polypeptides are the products of different structural genes. If the 203,000 dalton heavy chain is either an amino-terminal or a carboxyl-terminal fragment of the 210,000 dalton heavy chain of E675, it would show at most several missing fragments and a single shorter fragment. That is clearly not the case, since the 203,000 dalton heavy chain CNBr fragments contain the largest peptide and a greater number of characteristic fragments. The Wild-Type Also Contain Two Homogeneous Myosins Figure 6 shows the preparative fractionation of myosin from the wild-type, N2, on hydroxyapatite. The elution profile is complex and asymmetric, indicative of multiplicity of myosin. In a double-label experiment using %-E675 myosin and 3H-N2 myosin, we found that the breadths of the fractionations are similar. Although SDS-polyacrylamide gels show no evidence of multiplicity of myosin heavy chains, the CNBr fragments characteristic of the different heavy chains of E675 myosin are present in N2. We have used those characteristic CNBr fragments to analyze the subunit composition of N2 myosin. As in E675, the myosin of N2 contains two homogeneous species with respect to the two different heavy chains. Myosin from the trailing edge (fractions 81-86) yields CNBr fragment A, characteristic of the 203,000 dalton heavy chain of E675, whereas

from

fractions

of the hydroxyapatite

chromatography

showing

the

myosin from the leading edge (fractions 59-63) yields CNBr fragment B, characteristic of the 210,000 dalton heavy chain of E675. Figure 5 is representative of those differences. In these onedimensional gels, we have been unable to detect differences between respective hydroxyapatite fractions of E675 and N2. From these results, it is evident that myosin from both N2 and E675 contains two different heavy chains that are most probably the products of different structural genes. El90 Is Lacking One Homogeneous Myosin The hydroxyapatite elution profile of myosin from the uric-54 mutant El90 is strikingly different from that of either E675 or N2 myosins. Figure 7 shows that El90 myosin elutes over a smaller number of fractions than either E675 or N2 myosin. It appears to be completely contained within region I, and its profile is remarkably similar to the rechromatography profile of the samples from region I of E675 (Figure 2D). Since regions II and III contain material from the gene which specifies the 203,000 dalton myosin heavy chain of E675, this observation implies that this chain is either absent at our level of detection or has an altered elution profile. Analysis of Myosin Containing Both Heavy Chains There are two possible explanations for the composition of region II of the E675 myosin hydroxyapatite chromatogram which contains both the 210,000 and 203,000 dalton heavy chains. It may either be heterogeneous myosin, a myosin with one 203,000 and one 210,000 dalton heavy chain, or an association between myosins homogeneous for the 203,000 and 210,000 dalton heavy chains. We consider the existence of a different predominant heavy chain containing 210,000 dalton myosin species an improbable explanation, because such a myosin would have to yield the same pattern of CNBr fragments as the 210,000 dalton species of region I and yet be undetectable in E190. We have attempted to distinguish between these explanations with the following experiment. Since El90 contains only the 210,000 dalton homogene-

Two

Nematode

Body-Wall

Myosins

725

Figure

4. Light

Chains

of Homogeneous

Myosins

from

E675

Autoradiograph of 15% polyacrylamide-SDS gel to show the 16,000 and 16,000 dalton light chains of E675 mysoin samples as fractionated on hydroxyapatite. (A) purified myosin; (8) myosin from region I of hydroxyapatite chromatography (fractions 73-61); (C) myosin from region Ill of the hydroxyapatite chromatography (fractions 101-106).

ous myosin, ?S-El90 myosin is used as a marker for that myosin in the presence of excess unlabeled E675 myosin. 0.5 g of labeled El90 nematodes were added to 8 g of unlabeled E675 nematodes, and myosin was purified from the mixture. Figure 7 shows the results of the chromatography of this mixture of myosins on hydroxyapatite. Unlike El90 myosin purified alone, El90 myosin purified from the mixture of nematodes does not elute solely in region I. Some 26% elutes as a population in regions II and III. The El90 profile now appears similar to that of the 210,000 dalton heavy chain of E675 myosin, as determined by densitometry (Figure 28). In contrast, El90 myosin purified alone chromatographs like the rechromatography of the region I material from E675. The fraction of the El90 myosin eluting as a population in regions II and III is comparable to the 21% of E675 210,000 dalton heavy chain eluting in those regions. This implies that most, if not all, of the myosin eluting with both heavy chains is due to association of the two homogeneous myosins, not to a heterogeneous myosin.

Figure 5. Comparison Myosins

of

CNBr

Peptides

from

Homogeneous

Urea-SDS-polyacrylamide gel of CNBr peptides of homogeneous myosins showing the characteristic fragments A, B. C, D. E and F. (A) myosin from region I of the hydroxyapatite chromatogram; (B) myosin from region Ill of the hydroxyapatite chromatogram.

Under conditions similar to ours, Reisler et al. (1973) have shown that a significant fraction of rabbit skeletal muscle myosin molecules associate as dimers. Although it can be argued that the myosin heavy chains might exchange and that their distribution in our isolated myosin is different from the in vivo distribution, we believe that this is an improbable explanation. The structure of the myosin rod, with its heavy chains in a coiled-coil helix, is very stable. It is improbable that they dissociate over the 150 nm length of the coiled-coil interaction in these experiments, a necessary condition for exchange. Tonomura, Sekiya and lmamura (1962) show that under the ionic strength conditions of our proce-

Cdl 726

FRACTION I 62

01

I 06

I 70

I 74

I 78

I 82

I 66

I 90

1

FRACTION Figure

6. Fractionation

Chromatogram

of N2 Myosin

of hydroxyapatite

Figure

7. Hydroxyapatite

Chromatograms sence (O-O)

Chromatography

of El90

Myosin

of %-El90 myosin on hydroxyapatite in the aband presence (O-O) of cold E675 myosin.

on Hydroxyapatite

fractionation

of ?3-N2

dures, the physical properties associated coiled-coil interaction are not affected.

myosin.

with the

Discussion Our ability to resolve two myosins from E675 homqgeneous with respect to the different molecular weight heavy chains by hydroxyapatite chromatography has enabled us to study myosin multiplicity in C. elegans. We show that these two homogeneous myosins of E675 account for at least 85% of the total myosin and, on the basis of our studies with E190, perhaps all the myosin. Comparison of the CNBr peptides from the homogeneous myosins reveals multiple differences in their primary structure. These differences cannot be accounted for by the difference in molecular weight between the two types of chains. They support the proposal by Epstein et al. (1974) that these heavy chains are the products of different structural genes. We have shown that peptides characteristic of these two heavy chains are present in the wild-type nematode, N2. The 203,000 dalton heavy chain is due to the effect of the e675 mutation, and we call that myosin heavy chain the uric-54-affected heavy chain. As further evidence that the 203,000 dalton myosin heavy chain in E675 is an uric-54-affected gene product, our analysis of the hydroxyapatite profile of purified myosin from another mutant in the same gene as E675, E190, shows that it is lacking. Consistent with this observation, H. F. Epstein and J. A. Wolff (personal communication) show that the El90 myosin is lacking the CNBr peptides characteristic of the uric-54-affected myosin heavy chain. These studies show that two uric-54

mutations have different effects on only one of the myosin heavy chains. This supports the suggestion of Epstein et al. (1974) that uric-54 is the locus of a myosin heavy chain structural gene. The simplest model consistent with our results is that the heavy chains of body-wall muscle myosin are specified by two structural genes, and that the products of those genes each associate with a like gene product in the formation of homogeneous myosins. In E675 or N2, we are not able to exclude the possibility that heterogeneous myosin molecules (a myosin with each of its heavy chains specified by a different gene) exist, but comparison of the 21% of E675 210,000 dalton heavy chain in the central region with the 26% of El90 myosin in that region in the presence of E675 myosin shows that such a form cannot be more than a small percentage of the total myosin. Epstein et al. (1974) located 210,000 and 203,000 dalton heavy chains in the body-wall muscle of the nematode since they both were found in dissected body walls. Clearly, the 203,000 dalton heavy chain of E675 is in all body-wall muscle, since the mutation which results in its appearance produces the disruption of the myofilament lattice in each of those cells. 210,000 dalton heavy chains are found in both pharyngeal and body-wall muscle, and may be the same in both tissues, as proposed by Epstein et al. (1974). However, there is clearly a 210,000 dalton component in body-wall muscle cells, since E190, which we show is lacking the myosin affected in E675, has residual thick filaments in its disrupted body-wall myofilament lattice. For these reasons, we postulate that both homogeneous myosins in E675 are present in all the body-wall muscle lattices, although there may be other, minor, 210,000 dalton myosin heavy chain polypeptides.

Two 727

Nematode

Body-Wall

Myosins

Clear evidence for multiplicity of myosin heavy chains in a single tissue has been presented by Starr and Offer (1973). These studies show that there were two different amino-terminal amino acid sequences from rabbit fast skeletal muscle myosin. They found approximately a 2:l ratio of the different amino-terminal sequences, similar to the ratio of heavy chains in E675. The similarity of the ratios of two myosins in phylogenetically and functionally diverse muscles from rabbits and nematodes suggests a relationship to some fundamental property of thick filaments. One interesting possibility is that the two myosins participate in a vernier mechanism for in vivo length determinaion of thick filaments as proposed by Huxley (1963) and Huxley and Brown (1967). In this model, the different myosins form distinct strands of slightly different axial periodicities within the same filament, and when the periodicities coincide, growth stops. Accordingly, the observed ratios of the two myosins are consistent with the three-stranded and six-stranded models for thick filaments of Squires (1973) that are applicable to rabbit skeletal and nematode body-wall muscle structures, respectively. We are attempting to test such hypotheses by using antibody specific to one of the homogeneous myosins in contrast to mixed antibody in order to determine the distributions of the two myosins with the myofilament lattice and individual thick filaments. The expression of at least two structural genes for myosin heavy chains and resultant formation of two homogeneous myosins with a definite stoichiometry in body-wall muscle cells raises important questions concerning the regulatory mechanisms responsible for these phenomena. Are these relations maintained during development? Do specific genetic alterations of muscle structure affect this regulation? To investigate such controls, we are currently studying the synthesis and degradation of the two myosins at different stages of the nematode life cycle and in mutants producing alterations at different levels of myofibrillar organization. Experimental

Procedures

Nematode Strains N2 and uric-54 mutants E675 and El90 of C. elegans by Brenner (1974) and Epstein et al. (1974).

are described

Animal Growth and Yf Labeling Gram quantities of animals were grown in liquid culture by the procedures of Harris and Epstein (1977). For “S labeling, E. coli NA22 was grown on the sulphaterestricted media of Bretscher and Smith (1972) supplemented with carrier-free 3sS-sulphate (New England Nuclear) to a specific activity of 1.25 Ci/mmol. E675 worms were grown for 4-5 days on NP agar [l mM MgCI,. 2.5% Difco Bacto Agar, 5 *g/ml cholesterol, 25 mM potassium phosphate, 20 mM NaCl (pH S)] 9 cm petri plates with lawns of these labeled bacteria. These radiolabeled worms were harvested by washing them off the plate in 25-50 ml of M9 salts (5.6 g/l Na2HPOI, 3 g/l NaH,POI,

0.5 g/l NaCI, 1 .O g/l cylinder at 4°C for vested worms were dithiothreitol (DTT). -20°C.

NH&I) and allowed to settle in a graduated 1 hr. This settling was repeated twice. Harstored in 50% glycerol 50% M9 buffer, 1 mM 1 mM phenylmethylsulfonylflouride (PMSF) at

Purllkatlon of Myostn Myosin from both radiolabeled and unlabeled preparations purified by the procedure of Harris and Epstein (1977).

was

Hydroxyapatlte Chromatography of Myosin Purified myosin was chromatographed on a 1 cm x 15 cm column of hydroxyapatite prepared by the method of Bernardi (1971), equilibrated with 0.6 M KCI, 0.005 M potassium phosphate, 1 mM DTT, 0.5 mM PMSF. 0.1% Triton X-100 (pH 6.5) (Sigma Chemical). Myosin was applied in the Sepharose column buffer and washed with l-2 vol of 0.6 M KCI. 5 mM potassium phosphate, 1 mM DTT, 0.5 mM PMSF, 0.1% Triton X-100 (pH 6.5), and the column was developed with a 200 ml linear gradient arising from equal volumes of 0.6 M KCI, 0.001 M DTT, 0.0005 M PMSF, 0.1% Triton X100, 0.005 M potassium phosphate (pH 6.5) and 0.6 M KCI, 0.001 M DTT. 0.0005 M PMSF, 0.1% Triton X-100, 0.5 M potassium phosphate (pH 6.5). Generation and Analysis of CNEr Fragments Appropriate fractions from hydroxyapatite chromatography were pooled, and Triton X-100 was removed by the procedure of Halloway (1973). Samples were then dialyzed exhaustively against 1% acetic acid. After lyophilization, 50-150 wg samples were resuspended in 150 ~1 of 70%fonic acid, and 25 ~1 of 0.5 g/ml CNBr in 70% formic acid were added. Cleavage was allowed to proceed for 46 hr at 1°C in the dark. The reaction was stopped by the addition of 10 vol of distilled water, and the protein fragments were recovered by lyophilization. Samples, approximatey 50 pg, were electrophoresed on the urea-SDS-polyacrylamide gel system of Epstein and Wolff (1976). and stained and destained according to the procedures of Epstein et al. )1974). Gel Electroporesis Hydroxyapatite samples were analyzed in 4.5%. 10% or 15% acrylamide-SDS gels by the procedures of Epstein et al. (1974). Autoradiography and Densltometry Gels were autoradiographed by the methods of Epstein et al. (1974) using DuPont Cromex-4 (Pickering Medical Supplies). Densitometry was performed at 600 nm on an RFT scanning densitometer (Transidyne General, Ann Arbor, Michigan). Peak areas were quantitated by weight. Protein Determinations Protein concentrations were determined by a fluorescence assay using fluorescamine. 50-100 /LI of sample containing 10-100 ng of protein were added to 2.0 ml of 0.75 M K,HPO,. While vortexing, 0.5 ml of 0.25 mg/ml of fluorescamine (Roche Diagnostics) in acetone was added. The fluorescence was determined on a Perkin-Elmer 512 Fluorescence Spectrophotometer (excitation wave length at 390 nm and emission wave length at 490 nm) and compared to standard bovine serum albumin solutions. Countlng of Radioactive Samples Aqueous samples were applied to GF/C glass fiber filters (Whatmann) and dried under a heat lamp. They were counted in plastic scintillation vials after the addition of 6 ml of scintillation fluid (42 ml liquifluor per liter toluene. New England Nuclear) to counting accuracies of 1% for the peak fractions. Acknowledgments We are indebted to David Sedgewick Triton X-100 in our chromatography

for his suggestion and to Janice Wolff

to use for her

Cell 728

work on initial aspects of this study. The work was performed during the tenure of a Muscular Dystrophy Association Fellowship to F.H. S., a McCormick Fellowship through Stanford University School of Medicine to H.E.H. and a Mellon Foundation Fellowship to H.F.E. The research was supported by grants from the National Institute of Aging, from the American Heart Association through the Santa Clara County chapter and from the Muscular Dystrophy Associations of America, and by an institutional grant from the American Cancer Society. Received

November

16, 1976; revised

December

23. 1976

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A. E. (1972).

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95-139.

77, 71-94.

M. S. and Smith,

Epstein, H. F., Waterston, Biol. 90, 291-300. Halloway,

in Enzymology27,

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