ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 185, No. 2, January 30, pp. 488-495, 1978

Some Physical

Properties

JIN-JYI Department

CHANG”

of Chemistry, Received

of Paramyosin

Washington July

ALFRED

AND

Uniuersity,

21, 1977; revised

from Earthworms’ HOLTZER

St. Louis,

October

Missouri

63130

10, 1977

Paramyosin was prepared from earthworms (Lumbricus terrestris) by two different methods that have been used in the past. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows that the older method yields slightly degraded material (mostly p- and y-paramyosin) while the newer method yields essentially intact, i.e., a-, paramyosin. Physical studies, particularly circular dichroism, light scattering, and sedimentation velocity show that the native molecule is a double (Yhelical coiled coil of molecular weight 200,000, length 1200 A, and diameter 20 A. These properties are the same as reported previously for molluscan paramyosin. Also like clam paramyosin, the worm protein molecule loses its helix content and dissociates into its two constituent polypeptide chains upon exposure to sufficient concentration of Gdn.HCl. Furthermore, the same partially denatured states can be reached from either native or completely denatured proteins, indicating that they are all equilibrium states. However, the Gdn HCl-induced denaturation profile for the worm paramyosin is quite different from the clam. The helix content of worm paramyosin diminishes monophasically with increasing concentration of Gdn HCl, showing that the molecule does not possess a region of special stability such as its clam analog boasts. This conclusion is supported by experiments on papain digestion of worm paramyosin, wherein no resistent core is seen.

The protein paramyosin is of great interest in biology. Found in many invertebrate muscles (l-6), it is thought to be essential for their specialized ability to maintain tension for long periods with little expenditure of energy. Paramyosin molecules form the core of the thick filaments (which are longer and thicker in these muscles) with myosin molecules forming the surface (7, 8). The exact function of the paramyosin is not clear, and elucidation of the question is as important from the point of view of biological evolution as from that of muscle physiology and biochemistry. Physical studies of paramyosin have hitherto been mostly confined to paramyosin from molluscan (usually the clam Mercenaria mercenaria) adductor and show that, overall, the molecule is rod-

like, -1200 A long, -20 A wide, and has a molecular weight of -2 x lo5 (9-11). Furthermore, each molecule consists of two completely a-helical chains, supercoiled (9-13). The two chains separate in denaturing media (10, 11, 13) and re-join upon restoration of benign conditions (11, 13). Detailed studies of denaturation of paramyosin from M. mercenaria show that, whether induced by Gdn. HC13 (11, 14, 15) or thermally (15, 16), the process is biphasic, about 30% of the molecule being particularly stable. This stable core of clam paramyosin possessesextra resistance to proteolytic digestion W&18), and, after digesting away the rest of the molecule, the core can be isolated and studied separately (15, 16), and was found to come from the N-terminus (15).

1 This investigation was supported by Research Grant GM-20064 from the Division of General Medical Sciences, U.S. Public Health Service. * Present address: Department of Microbiology, Vanderbilt University, Nashville, Tennessee 3’7232.

3 Abbreviations used: Gdn HCI, guanidine hydrochloride; SDS, sodium dodecyl sulfate; CD, circular dichroism; PMSF, phenyl methyl sulfonyl fluoride: DTT, dithiothreitol; Mops, morphalinopropane sulfonic acid huffer. 488

0003-9861/78/1852-0488$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

PHYSICAL

PROPERTIES

OF

Although the body wall muscle of annelids (usually Lumbricus terrestris) has received considerably less attention, it seems clear that the basic thick and thin filament structure is present (19, 201, that some thick filaments have the large diameters appropriate to tonic muscle (211, that tonic contractions are possible (191, and that the muscle contains paramyosin (4, 22, 23). Almost no physical chemical experiments have been done, however, on the earthworm paramyosin, although SDS-gel electrophoresis indicates a polypeptide chain molecular weight of -lo5 (22, 23) and an interpretation of the structure of certain compound segments seen in the electron microscope suggests 1235 A for the molecular length (23). To provide a better picture of the annelid paramyosin molecule, we have prepared paramyosin from Lumbricus terrestris by both the older (4) and a more recent (24) method; characterized it by several means, including SDS-gel electrophoresis, light scattering, CD, and sedimentation rate; and determined its Gdn. HCl denaturation profile and its susceptibility to proteolytic digestion. MATERIALS

AND

METHODS*

Reagents Gdn.HCl, ultrafine grade, and SDS were from Schwarz/Mann. (NH&SO.,, enzyme grade, from Mann Research Laboratories, was dissolved in boiling water, filtered (coarse frit), and crystallized at 5°C. To each volume was added 0.01 vol of concentrated NH,OH and 0.01 vol of 0.01 M EDTA; the resulting solution was used as “saturated ammonium sulfate.” The protease inhibitor PMSF was from Sigma Chemical Co. Paramyosin

Preparations

Clam paramyosin. This protein was prepared essentially as described by Johnson et al. (24). Details have been given elsewhere (25). Earthworm paramyosin. This protein was prepared by two different methods. In the first method, essentially that of Kominz et al. (41, a dried ethanolether powder is made, from which paramyosin is extracted and purified by successive isoelectric and (NH&SO, precipitations. In the second method, essentially that of Johnson et al. (241, the muscle is

3 For a fuller

account,

see Ref.

(26)

EARTHWORM

PARAMYOSIN

489

washed, the protein extracted, and the paramyosin precipitated by addition of ethanol, redissolved, and reprecipitated at low ionic strength. Details are given elsewhere (26). In all procedures, live earthworms (Lumbricus terrestris, trade name “Canadian night crawlers”) were obtained from a bait shop. Each worm was slit longitudinally from end to end and all the internal organs and dirt were cleaned out. The body walls were then extracted. The final purified protein solution was dialyzed vs (KCl),.,(K[PO,I),~.,,,(7.4).” Preparations by the first method had an absorbance ratio A2BJA28,, 10.60. When SDS-gel electrophoresis showed a small amount of low molecular weight (-40,000) material, an additional (NH&SO, precipitation eliminated the contaminant (26). Two preparations were made [designated, respectively, PM(W1) and PM(WP)] in this way. In order to assess the effect of partial enzymatic degradation [as described for the clam paramyosin (27, 2811 in the worm case, a third preparation [PM(W311 was made in the same manner except that protease inhibitors, 3 x 10m4 M PMSF and 0.01 M EDTA, were present in all solutions (27). The second method, that of Johnson, et al. (24), was employed with slight modification (25, 271, the major one being that all solutions contained protease inhibitors as above (271. Two such preparations were made lPM(W5) and PM(W6); PM(W4) was a preparation irrelevant for the present paper]. The first of these showed a satisfactory A260/AZBU; the second showed APGO/APHO = 0.78, which dropped to a satisfactory 0.53 after one (NH&SO, precipitation [from (KCl), ,(K[PO& (7.4), 19% saturation]. Protein concentrations in KC1 solutions were first determined by the micro-Kjeldahl method without distillation (26, 29) using 18.4% as the nitrogen content (4). On this basis, the extinction coefficient of worm paramyosin in (KCl),,(K[PO,I),,,,and (DTT) ,,,U,,0s(7.4) at 277 nm is 0.302 ml/mg-cm this was used to assess concentrations. The corresponding value for clam paramyosin is 0.324 ml/ mg-cm (11). For solutions containing large amounts of nonprotein nitrogen (from Gdn . HCl), an exact dilution method was used. The protein was dissolved in and dialyzed vs (KCI),,.,(K[PO,I), ,,(DTT),,.,,,,,,,(7.4), and the concentration determined by absorbance. A known volume (usually 2 ml) was then dialyzed vs

5 KIPO,l is used to signify a mixture of potassium phosphates. The frequent necessity of referring to complex aqueous solvent media makes shorthand notation desirable. We designate such solvent by writing the chemical formula (or name) of each component (omitting water) with its molarity as subscript, followed by a parenthetical specification of pH.

490

CHANG

500 ml of the desired Gdn. alysis, the protein solution transferred to a volumetric with dialysate washes, and the mark. This method was tions. The error is less then Digestion

of Proteins

with

AND

HCl

solution. After diwas quantitatively flask (usually 5 ml) dialysate was added to tested on dummy solu1%.

Papain

Essentially, the method of Cowgill (17) was employed for both clam and worm paramyosins. Papain was purchased from Sigma Chemical Co. Paramyosin solutions (5-6 mg/ml) were prepared in Gdn. HCl) ,.,, (EDTA) ,,.,y,,,, (Mops),, ,,,(L-cysteine),,.,,,(DTT),,,,,(7.4). A volume of papain suspension containing a weight of enzyme equal to 2% of that of paramyosin was added at room temperature. Aliquots (2 ml each) were removed from the reacting solution at 10, 30, 50, 75, and 90 min and the reaction in each was stopped by pipetting into 0.084 ml of 1 M HCl. The solutions were then first dialyzed vs 250 ml of 0.02 M HCl to remove small fragments and then prepared for SDS-gel electrophoresis. Controls were done in exactly the same way but without papain. Physical

Measurements

in (KCl),.,(K[PO~I),,,,,,(DTT),,.,,,,,j(7.4) was taken as of 100% helix

(and

called

[0&,),

and

that in (Gdn~HC~),,,(KCl),,.,(K[PO,l),, ,(DTT),.,,5(7.4) as 0% helix, The fraction

i.e., helix

previously (9, 11). Light scattering cells with no back reflection were used with 436nm light over the angular range 30” to 135”. Dust-free water for washing cells and pipets was obtained as before (31). Solutions were cleaned by ultracentrifugation and the solvent by filtration through an ultratine frit. Data were treated as described earlier (9). The refractive index increment of worm paramyosin in (KCl),,,(KIPO,l),,, (DTT),,,,,,,(7.4) at room temperature was measured with a Brice-Phoenix differential refractometer at a wavelength of 436 nm. The instrument was calibrated with standard KC1 solutions (32~. Some runs were also made at pH 2, specifically in (KCl),,,,,,(HCl),, ,,, (DTT),, ,,,,,(2.0), but the refractive increment was not determined in that medium. Two separate determinations (at protein concentrations of 5.21 and 5.99 mg/ml, respectively) gave an average value for the refractive increment of 0.208 ml/g and differed by only 1%. This value is substantially different from that reported earlier (0.179 ml/g) for clam paramyosin (9). We therefore repeated the measurement on clam paramyosin, obtaining 0.176 ml/g, which indicates that the difference is real. SDS-Polyacrylamide

CD. Protein solution concentrations were in the range 2 to 3 mg/ml and excess ellipticity over dialysate was measured in the O.l-mm cell from -250 to 190 nm in a Jasco J-20 spectropolarimeter. The mean residue molecular ellipticity [#I, was calculated from 101 = 11.58/cl, wherein H is the ellipticity in degrees, c is the protein concentration in grams per cubic centimeter, and I is the pathlength in centimeters; 11.5 is the average mass in grams of 1 dmol of residues for both clam and worm paramyosin (4). Helix content was calculated from the trough at 222 nm rather than 208 nm because the former is less affected by the solvent absorption at high concentrations of Gdn.HCl. The value of [0J at 222 nm characteristic

HOLTZER

random coil was obtained

(and called as: fh =

[&,J,).

{[&,,] -

L%221e~/{[H2221h - M,,,l,}. Ultracentrifugation. A Beckman Model E ultracentrifuge was used. The experiments were done at 52,000 rpm and 14°C with Schlieren optics. The sedimentation coefficients were corrected to water at 20°C as usual. We used, for the apparent partial specific volume of worm paramyosin, 0.730 ml/g, i.e., the value for clam paramyosin (30). The intrinsic sedimentation coefficient was obtained by the usual reciprocal plot. Light scattering. The instrumentation and techniques are essentially the same as those employed

Gel Electrophoresis’

The gels used were 5.6%, prepared according to Fairbanks et al. (33), and were polymerized to have a length of 10 cm in tubes with 5 mm i.d. The electrophoresis cell was from Bio-Rad (Richmond, Calif.). Runs were at room temperature at 8 mA/gel in some experiments and 3 to 4 mA in others. Bromophenol blue was the tracking dye and Coomassie blue the stain. The proteins used for calibration were: clam paramyosin (P-chain, 100,000); phosphorylase a (92,500); bovine serum albumin (68,000); pyruvate kinase (57,000); fumarase (48,500); enolase (42,000); and rabbit tropomyosin (a-chain, 32,800; P-chain, 35,000). Paramyosin and tropomyosin were prepared in this laboratory; others were purchased from Sigma Chemical Co. The calibration plot of log M vs mobility is linear (26). For worm paramyosin, the gels were scanned with a Gilford spectrophotometer 240 (Gilford Instrument, Oberlin, Ohio) at 540 nm with a lo-cm cuvette. The amount of protein in each band, assumed to be proportional to the amount of dye bound, was determined using a Gaussian lineshape. RESULTS

SDS-Polyucryhnide

Gel Electrophoresis

Sample results are shown in Fig. 1, in which PM(W3) is compared with the p (M, = 100,000) and y (M, = 94,000) chains

PHYSICAL

PROPERTIES

OF EARTHWORM

PARAMYOSIN

491

tially undegraded (i.e., a-1 worm paramyosin of molecular weight 100,000. There is a very small band (~4%) at higher molecular weight (120,000) which may be myosin tails. Light Scattering In all runs, the protein concentration was sufficiently low (co.4 mg/ml) that second, and higher, virial coefficient terms are negligible. Consequently, no extrapolation to zero concentration is necessary, and a plot of KcIRO vs sin2 (e/2) provides all the information available from the data bc (34). Such plots for worm paramyosin are linear over the entire accessible angular range (26). In this, they resemble closely the results for clam paramyosin (9). Four runs were made using protein prea b pared ci la Kominz et al. [PM(W2) or FIG. 1. SDS-polyacrylamide gel electrophoresis. From left to right: (a) clam paramyosin showing /3- PM(W3)l at pH 7.4. These gave an average chain (lo-” M = 100) and y-chain (10m3 M = 94); value for (M),, the weight average molecworm paramyosin [PM(W2)]; and phosphorylase a ular weight (341, of 180,000, with a total (10m3 M = 92.5). The amount of protein applied is spread of 15,000; and an average value of -3 pg; (b) phosphorylase a; worm paramyosin (S),, the “light scattering average root [PM(WG)I. Amount of protein applied is -5 pg. mean square radius of gyration” (34) of 323 A with a total spread of 18 A. Three similar experiments at pH 2 provide no of clam paramyosin and with phosphorylase a (M = 92,500); and PM(W6) with molecular weight values (because refracphosphorylase a (M, = 92,500). The main tive increment was not measured), but band in PM(W3) is of slightly higher mo- iwe 4% = 293 A with a spread of 11 A. lecular weight than phosphorylase a, and The internal consistency is very good. there are two lower molecular weight On the other hand, protein prepared ci bands present in smaller amounts. The la Johnson et al. [PM(WG)I gave (M), = chain composition of worm paramyosin 200,000 and (S),, = 349 A, at pH 7.4. prepared by this method is: 93.2% M, = Thus, the lesser degradation, in this prep95,000; 4.5% M, = 85,000; 2.3% M, = aration, as deduced from the electrophore80,000. Evidently, degradative processes sis, is also evident from the light scatterduring preparation (27, 28, 22) are even ing. more serious in the worm than the clam. Furthermore, no differences were observed Sedimentation Velocity The plot of l/s,,,, vs concentration for between preparations PM(Wl), PM(W2), and PM(W3), so in the case of worm para- PM(W3) in (KCl),.,(K[PO,l),.,,(DTT),.,,,,(7.4) is shown in Fig. 2. The intercept myosin and the method of Kominz et al., protease inhibitors are ineffective in pre- gives [.s~~,~]= 3.35 S. This value is close to that reported for clam paramyosin (9, venting some degradation. On the other hand, the main band in 11). PM(W6) is of slightly higher molecular weight than phosphorylase a, and since Circular Dichroism no bands of lower molecular weight apCD spectra of worm paramyosin in pear, we find, in agreement with Weisel benign medium [(KCl),.,(K[PO,l),.,,(22, 231, that the method of Johnson et al. and in various denaturing W-M’) o.00,,s(7.4)1 (241, used with inhibitors, produces essen- media are shown in Fig. 3. In the benign --

_L_

492

CHANG

AND

FIG. 2. Concentration dependence of sedimentation coefficient of worm paramyosin lPM(W3)l. Runs were made at 14°C in (KCl),,(K[PO,l),,,(7.4). Intercept gives [.s~,,,~ ] = 3.35 s. (DT’h.,,,

0 F

I

I

200

HOLTZER

worm paramyosin. The differences are within expectations for comparison among different laboratories and among different highly a-helical proteins; clearly, native worm paramyosin is essentially 100% (Yhelix. Figure 3 also shows the expected decline in helix content with increasing concentration of Gdn 3HCl. The Gdn *HCl denaturation profile is shown in Fig. 4 for the worm protein at pH 7.4 and the corresponding curve for clam protein is given for comparison. The difference is striking. Unlike the clam case, the worm paramyosin denaturation is monophasic. Some data for denaturation at pH 2 are also shown on Fig. 4, from which it is apparent that, like the clam case, the worm paramyosin helix is more stable at acidic than at near-neutral pH, although the denaturation is monophasic for worm paramyosin at pH 2 as at pH 7.4. In interpreting denaturing experiments, it is essential to discover whether the

I

225

250

r\(nm)

FIG. 3. CD spectra

of worm paramyosin [PM(W2)1 as function of Gdn . HCl concentration at room temperature. Gdn.HCl molar concentration as marked. Other constituents are WCl),WPO,I),,,,(DTT10,,,,,(7.4), where x varies from 0.6 at low Gdn.HCl to 0.2 at high. Exact composition of each medium is given elsewhere (261. IGdn.HUI

medium, deep troughs at 208 and at 222 nm, characteristic of the a-helix, appear. Furthermore, the absolute magnitude of the mean residue molar ellipticity is consistent

with

100%

a-helix:

this

quantity

has been reported as [&J = -39,000 degcm2/dmol for clam paramyosin (35); we find -42,000 for clam and -46,000 for

FIG. 4. Fraction Circles, 7.4; filled PM(W3) pH 7.4 smoothed Dashed paramyosin

helix vs molarity of Gdn.HCl. PM(W2) at pH 7.4; squares, PM(W3) at pH triangle, PM(W6) at pH 7.4; filled circles, at pH 2. Solid curve is smoothed curve for worm paramyosin data. Dotted curve is curve for pH 2 worm paramyosin data. curve is previous result for pH 7.4 clam (11, 14).

PHYSICAL

PROPERTIES

OF

EARTHWORM

TABLE

493

PARAMYOSIN

I

RENATURATION OF WORM PARAMYOSIN Denatured

Renatured

in”

in”

(Gdn. HClk,, (HCl) ,,.,,, (2.0)

(KC1),,,,Z(HW, ,,,(2.0)

(Gdn HCIL.,, (HCl), ,.,,, (2.0) (an. HClk,,(HClh,, (2.0) (Gdn.HClX, ,,(KC1),,,(7.4) (Gdn HCl),.,,(KCl1,,,, (7.4)

(Gdn HCl),.,, (HCl),, ,!, (2.0) (KC&,,, (HCl),, ,,, (2.0) (KCl),, ,j(7.4) (KCI),,,,, (7.4)

‘I In addition to the constituents contained 60 rnM K[PO,l as well.

listed,

all solutions

contained

Direct, Direct, Anneal, Anneal, Direct,

25°C 25°C 5°C 5°C 5°C

0.5 mM

DTT,

0.80 0.16 0.95 1.16 0.99 and

1.00 0.21 1.00 1.00 1.00

solutions

at pH 7.4

states being observed are equilibrium states. To this end, several renaturation experiments were performed. These are summarized in Table I in which are compared the fraction helix observed on renaturation (fhohs)and that anticipated from denaturation (fh”“‘>. The message is clear. Even if the necessary, somewhat lengthy, dialyses are performed at room temperature the reversibility is 80%; if they are done at 5”C, reversibility is complete. Furthermore, this is true whether or not the annealing step, necessary in renaturing clam paramyosin (111, is introduced. Thus, the states recorded in Fig. 4 are equilibrium states.

Digestion

of Proteins

with Papain

Some results of the digestion experiment are shown in Fig. 5. A pronounced difference between clam and worm paramyosins is clearly manifest. In the clam protein, after 10 min of digestion, about five bands, some quite heavy, are seen in the vicinity of the tracking dye position; after 30 min, except for the virtual disappearance of the slowest band and some shift in the relative amounts of the others, the same pattern persists, as indeed it does even after as long as 90 min (26). In the worm protein, on the other hand, almost no material is seen even in as short a time as 10 min. Clearly, then, the entire worm protein has essentially been reduced to very small fragments, which have been lost on dialysis, in the same time as is required for the most susceptible regions in the clam protein. That is, the worm paramyosin does not possessa papain-resistant region such as clam paramyosin has.

a

b

c

d

e

f

FIG. 5. SDS-polyacrylamide gel electrophoresis of papain digests. (a) clam paramyosin control; (b) clam paramyosin lo-min digest; (c) clam paramyosin 30-min digest; (d) worm paramyosin [PM(WZ)l control; (e) worm paramyosin [PM(WB)l lo-min digest; (f) worm paramyosin [PM(WZ)l 30min digest. In (d) the low molecular weight (10e3 M = 40) contaminant band showing between the main worm paramyosin complex of bands and the tracking dye seems very heavy for two reasons: First, this experiment was done before the need for an extra (NH&SO, precipitation was fully appreciated (see Methods), and second, the initial protein concentration (i.e., of the controls) must be rather high in order to see anything in the digests. In this case, about 20 pg of control protein was applied.

494

CHANG

AND

DISCUSSION

As seen above, the preparative method of Johnston et al. (24) yields essentially undegraded worm paramyosin (i.e., a-paramyosin (27)) and the molecular parameters for this protein are therefore best assessed from our results on PM(W6). The light scattering results on PM(W6) indicate an (it!) of very near 200,000 and an (S),, of 349 x. However, the electrophoresis indicates a small amount (~5%) of a contaminant with a chain molecular weight of 120,000, the value for the bulk of the material being 100,000. We must assess the effect of this contaminant on the average values obtained. Since the molecular weight is about double the chain weight, we first hypothesize that the contaminant shares this twochain structure and therefore has a molecular weight of 240,000. Using the experimental (M), and assuming 5% contaminant, we then calculate 198,000 for the (Yparamyosin component. On this hypothesis, then, the effect of the contaminant is negligible. If, on the other hand, the contaminant remains single in benign media (M, = 120,000), a similar calculation gives 204,000 for worm a-paramyosin; again, the contaminant is negligible. We conclude that the molecular weight of earthworm paramyosin is very close to 200,000 and therefore indistinguishable from clam paramyosin. A similar argument can be made for the experimental (.!& (34), which is 349 A. Assuming the contaminant to be a double a-helical entity with paramyosin’s linear density, we get 344 A for the 5’ of the a-paramyosin. Since any other reasonable assumption about the contaminant leads to an even lower contribution, it cannot be significant. Since 344 A translates to a rod length of 1200 A, in striking agreement, not only with results for the clam, but also with that (1235 A) deduced from a hypothesis concerning the arrangement of worm molecules in certain arrays seen in the electron microscope (231, it is clear that the proposed model of the arrays must be correct. Our light scattering data from the somewhat degraded preparations lPM(W2) and

HOLTZER

PM(W3)l agree extremely well with those above. Using the relative amounts from the gel scans and doubling the chain molecular weight for each component, we calculate an (M), of 188,000, which agrees with the experimental value of 180,000. Furthermore, using the same gel data and the S/M ratio from experiments on undegraded protein (i.e., S/M = 344 Al 198,000), leads to a predicted radius of 328 A, in agreement with the observed value of 323 A. The diameter of the molecular rods can be estimated from our data in two ways (9). Firstly, using the specific volume of the molecule (with fi = 0.730 ml/g (30) and LIM = 1200 A/200,000), gives d = 19.6 A. Second, using the intrinsic sedimentation coefficient, and estimating the axial ratio (to which the calculation is insensitive) at 1200/20 = 60, gives d = 19.9 A. Thus, the two methods are self-consistent, confirming the rod shape, and in agreement with the diameter obtained for clam paramyosin (9) and other double a-helical proteins. The number of polypeptide chains in the molecule, deduced from the ratio of the light scattering and electrophoresis molecular weights, is confirmed by comparison of the molecular length with the total a-helix length. The degree of polymerization is 200,000/115 = 1739, giving a helix length of 1.48 x 1739 = 2573 A or 2573/1200 = 2.14 chains, i.e., two chains. However, a big difference between clam and worm paramyosin is apparent in comparing their stabilities toward denaturation by Gdn * HCl. The monophasic curve (Fig. 4) for the worm paramyosin shows definitively that this protein does not possess a helical region of extra stability. This conclusion is independent of whether worm a-paramyosin is used (Fig. 4) and is confirmed by the demonstrated absence of a papain-resistant core (Fig. 5). Explanation for this difference may be sought in differences in the amino acid composition. However, using the published amino acid analyses (4), we find (26) that whether the helix-forming index (36) or the conformation parameter (37) are used, there is little to choose between worm and clam

PHYSICAL

PROPERTIES

OF

paramyosin, i.e. those statistical correlations of occurrence frequency in globular proteins do not work any better for the comparison of clam and worm paramyosin than they do for the different regions of the clam paramyosin molecule itself (26, 38). This difference in the homogeneity of helix stability within the molecules of clam and worm paramyosin raises interesting questions as to the biological evolution of the paramyosins. The molecular morphology and physiological behavior of earthworm and molluscan tonic muscle are not the same, and questions also therefore arise concerning the possible role of the extra stable region of the clam paramyosin molecule in the biological context. In that connection, it is perhaps relevant that our preliminary studies of lobster indicate that, like the worm, its paramyosin does not possess a region of extra stability. ACKNOWLEDGMENTS The authors thank Dr. David Kominz for an informative exchange of correspondence regarding the precise preparative procedures employed in Ref. (4); Professor Oscar Chilson for very useful discussions and advice on gel electrophoresis; and Professor George Johnson for use of the gel scanning spectrophotometer. REFERENCES 1. HALL, C. E., JAKUS, M., AND SCHMITT, F. (1945) J. App. Phys. 16, 459. 2. BAILEY, K. (1957) Biochim. Biophys. A& 24, 612. 3. IKEMOTO, N., AND KAWAGUTI, S. (1967) Proc. Japan. Acad. 43, 974. 4. KOMINZ, D. R., SAAD, F., AND LAKI, K. (1957) in Conference on the Chemistry of Muscular Contraction, Japan, p. 66, Igaku Shoin Ltd., Tokyo, Japan. 5. WATERSTON, R., EPSTEIN, H., AND BRENNER, S. (1974) J. Mol. Biol. 90, 285. 6. HARRIS, H., AND EPSTEIN, H. (1977) Cell 10, 709. 7. SZENT-GYORGYI, A. G., COHEN, C., AND KENDRICK-JONES, J. (1971) J. Mol. Biol. 56, 239. 8. SQUIRE, J. M. (1973)J. Mol. Biol. 77, 291. 9. LOWEY, S., KUCERA, J., AND HOLTZER, A. (1963) J. Mol. Biol. 7, 234.

EARTHWORM 10. 11.

12. 13. 14.

15. 16. 17. 18. 19. 20. 21.

22. 23. 24. 25. 26. 27. 28.

29.

30. 31. 32. 33. 34. 35. 36. 37. 38.

PARAMYOSIN

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