Subunit structure of glyceraldehyde-3-phosphate dehydrogenase

J. Mol. Biol. (1965) 13, 885-893

Subunit Structure of Glyceraldehyde-3-Phosphate Dehydrogenase WILLIAM

F.

HARRINGTON AND GERTRUDE

M.

KARR

McCollum-Pratt Institute, The Johns Hopkins University Baltimore 18, Maryland, U.S.A. (Received 4 June 1965) The molecular weight of glyceraldehyde-3-phosphate dehydrogenase isolated from both pig and rabbit muscle has been determined from short-column sedimentation equilibrium experiments, giving 145,000 ± 6000 for both species. Velocity sedimentation, viscosity and sedimentation equilibrium studies of the enzyme in aqueous 5 M-guanidine-HCl solutions reveal that in this solvent glyceraldehyde-3-phosphate dehydrogenase is dissociated into monodisperse subunits of molecular weight 36,300 ± 1500.

1. Introduction Harris & Perham (1963; Harris,' 1964) have recently demonstrated that the chemical subunits of glyceraldehyde-3-phosphate dehydrogenase are identical polypeptide chains with unit molecular weight of 35,000 to 36,000. The number of subunits linked together by non-covalent forces to form the quaternary structure of the native enzyme is still uncertain. A molecular weight of about 120,000 has been estimated from sedimentation and diffusion measurements (Taylor, Lowry & Keller, 1956; Elias, Garbe & Lamprecht, 1960) and from approach-to-equilibrium sedimentation experiments (Elias et al., 1960). On the other hand, a higher molecular weight of 137,000 to 140,000 was reported by Dandliker & Fox (1955; Fox & Dandliker, 1956) based on their light-scattering and sedimentation-diffusion studies (see also Elodi, 1958). This latter value is consistent with the proposal (Harris, 1964) that the native GPDHt molecule consists of four identical polypeptide chains. In the studies to be reported below, the molecular weights of GPDH, isolated from pig and from rabbit muscle, have been determined by equilibrium sedimentation measurements of the protein in dilute salt systems. Velocity sedimentation, viscosity and equilibrium sedimentation determinations have also been made on GPDH in 5 M-guanidinium chloride, since this solvent appears to be very effective in dissociating fibrous and globular proteins into their fundamental, covalently-linked subunits (Kielley & Harrington, 1959; Woods, Himmelfarb & Harrington, 1963; Marler, Nelson & Tanford, 1964). Our investigations reveal a molecular weight of 145,000 ± 6000 for both the pig and rabbit enzymes in their "native" state and 36,300 ± 1500 for the subunit polypeptide chain in aqueous 5 M-guanidine-HCI. When this evidence is taken in conjunction with the information presented in the preceding paper (Harris & Perham,

t Abbreviation used: GPDH, glyceraldehyde-3-phosphate dehydrogenase. 885

886

W. F. HARRINGTON AND G. M. KARR

1965) it seems probable that the quaternary structure of GPDH consists of four identical polypeptide chains.

2. Experimental Methods Glyceraldehyde.3.phosphate dehydrogenase was prepared from pig muscle by the method of Elodi & Szorenyi (1956) and recrystallized 3 times from ammonium sulfate containing redistilled ,B-mercaptoethanol (0'005 M) in the presence of 0·001 M-EDTA. The enzyme was prepared from rabbit skeletal muscle by the method of Cori, Slein & Cori (1948). (We are indebted to Dr J. 1. Harris for the pig muscle preparation and to Dr J. H. Parks for the rabbit muscle preparation.) The enzymes were stored as crystalline suspensions in saturated ammonium sulfate (0,001 M-EDTA) at 4°C until used. Solutions used for physical studies were obtained by dialysis (4°C) of a portion of the stock solution against several changes of pH 7·4 buffer (0'15 M·NaCI-0·05 M-NaH 2PO cO'002 M.EDTA, adjusted with NaOH) and the resulting solutions concentrated as required by dialysis against the buffer under reduced pressure. (a) Dissociation in aqueous guanidine-HCl

Glyceraldehyde-3-phosphate dehydrogenase was dissociated into its subunit polypeptide chains in aqueous 5 M-guanidine-HCl. Dialyzed solutions (see above) were treated with a tenfold molar excess ofN-ethyl maleimide (based on one titratable - SH group per 12,000 grams (Koeppe, Boyer & Stulberg, 1956; Benesch, Lardy & Benesch, 1955» to eliminate the possibility of disulphide bridge formation between the unfolded polypeptide chains. The mixture was allowed to stand overnight at 4°C, then was made 5 M with respect to guanidine-HCl and dialyzed against several changes of 5 M-guanidine-HCI for several days at room temperature in a closed container mounted on a magnetic stirrer. (b) Protein concentrations

The concentration of protein in the various solvent systems was estimated from the extinction coefficient at 280 mp.. A value of € = 1000 cm 2g- 1 was assumed for the extinction coefficient in dilute salt systems (Dandliker & Fox, 1955; Velick, Hayes & Harting, 1953). The extinction coefficient of pig GPDH in 5 M-guanidine-HCI was determined (using density data on aqueous guanidine-HCI solutions (Kielley & Harrington, 1960» by adding a weighed amount of the solid salt to an enzyme solution of known concentration. The absorption at 280 mp' was compared immediately after mixing with that of the solvent without enzyme. In 5 M-guanidine-HCI, € = 959 ± 8 cm 2g- 1 at 280 mp.. The extinction coefficient of the rabbit enzyme in 5 M-guanidine-HCI was not determined. (c) Chemicals

All chemicals were reagent grade and the water deionized and then glass distilled. Guanidine-HCI was purified by treating a methanol solution at 40°C with activated charcoal (Norit), followed by recrystallization at - 10°C. The crystals were vacuum dried. (d) Viscosity measurements

Viscosity measurements were made in an Ostwald viscometer with an average shear gradient of 300 sec -1 and an outflow time of 90 sec for 2 ml. of water. Measurements were made at 25°C and the temperature was maintained within ± O·OI°C. Viscosity results are reported as reduced viscosities, 7]sp/c, where 7]sp is the specific viscosity and c is the protein concentration in g/100 ml. (e) Sedimentation studies The Spinco model E ultracentrifuge was used for sedimentation velocity and sedimentation equilibrium experiments. The Rayleigh interference optical system of the machine, aligned by the procedure of Richards & Schachman (1959), was employed for the equilibrium sedimentation studies, and the schlieren optical system for velocity sedimentation. Double-sector cells with a 12-mm optical path were used for equilibrium studies;

SUBUNITS OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

887

these were equipped with sapphire win dows for the high-speed runs. Photographic plates (Spectroscopic II G) were analyzed with the aid of a Nikon model 6 optical microcomparator, The molecular weight of native GP DH was d etermined from two general types of sedimen tation equilibr ium experim en ts with rotor tempe rat ures between 5 and 9°C. Lowsp eed equilibriu m m easurem ents wer e performed with I-mm liquid columns layered ov er F C43 (perfluorotcibut.ylarnine, Minnesota Mining and Manufacturing Co .) and employed rot or velo cities of 5227 or 6991 r ev Jtn ui . Initial concentrations, Co in fringes, of the protein solution were determined separately b y means of a double-sector synt he t ic boundary cell. The time required for equilibrium was est imat ed by calculation (Van Holde & Baldwin, 1958) to be 5 to 6 hr under these conditi ons. but expe rimen ts were routinely performed for 15 hr. In some ex pe r ime nts. fringe patterns were ph otographed after an additional sedimen t a t ion for 10 hr t o ens ure attainment of equilibrium; the fringe pattern was found to be indistinguishable fr om t hat m easured after 15 hr. The apparent weight aver age m olecular weight. M w • of the protein was calculated from the equation: (1)

wh ere l' is the distance from the center of rotation to the center of the liquid column, Co is the initial concentration, dc/dr is the value of the concentration gradient at r, R is the gas constant, T the absolute t emperature. w the angular velocity in radians/sec, p the density of the solution and V the partial specific volume. The concentration gradient at the midpoint was determined by est imat ing the tangent to a c aga int r plot laid out on 25 em x 38 em graph paper. The concen t rat ion at the midpoint was found to be within 1 % of the initial concentration, Co. for the column heights and speeds used in this study (see Yphant.is, 1960). Molecular weights were al so est ima te d using the high-speed equilibrium procedure outlined by Yphantis (1964) . In t hese expe r iment s. 3-mm liquid columns were layered ov er FC43 (0,105 ml. prot ein solut ion and 0·05 ml. FC43) and rotor velocities of 16.200 rev./ min wer e employed for the native enzyme in the dilute salt systems. High-speed equilibrium studies of the enzyme in 5 )l-guanidine-HCI at rotor sp eed s between 39.460 and 44.770 rev.] min were al so carried out with 3 -rnm liquid columns. In these exper im en t s considerable care was taken in loading the cells t o ens ure that the volume of FC43 and also the volume of solvent and solution in ea ch sector of the cell were the same. At, the high concentrations of salt and relatively high rotor velo cities employed. significant differences in salt distribut ion will result from unequal liquid columns. Molecular weights wer e calculated from t he equat ion : 2RT dine M w = (1 _ Vp)w 2 dr 2 •

(2)

Aft er alignment of the photographic plate. the vertical fringe displacement (proportional directly to protein concentration, c.) was measured as a function of distance. r, from the cente r of rotation. Values were taken every 0·1 mm (plate co-ordinate) and were plotted on 40 em x 50 em graph paper as a function of 1'2 to permit a choice of the zero reference level near the meniscus. Only net fringe displacement points greater than 100 p, were used in estimating the molecular weight (Yphantis, 1964). Sedimentation velocity studies on the native enzyme in dilute salt systems were performed at low temperature (4 to 8°C). All sed imenfat ion vel ocity experiments of the protein in aqueous guanidine-HCI solut ions were conduc ted at room temperature with ro tor speeds of 59,7 80 rev.fmin. The obse rve d sedimentati on coefficients were corrected to values corresponding to a solven t with the viscosity and density of water at 20°C (82 0 • w). (f) Densities of solutions A 25-m!. Leach pycn omet er was used for t he determination of de nsity of solvents used in equilibrium and sedimentation velocit y studies. Densities wer e determined in the t empera-

888

W. F. HARRINGTON AND G. M. KARR

ture range of the ultracentrifuge experiments and estimated at the temperature of the run by interpolation. (g) Partial specific volumes

The partial specific volume of GPDH isolated from various sources has been measured by a number of workers. Taylor et al. (1956) have reported values of V = 0·729 ml.jg at 5°C and V = 0·740 ml.jg at 20°C from measurements utilizing density-gradient columns on the enzyme derived from rabbit muscle. Fox & Dandliker (1956) derived a value of 0·725 ml./g (temperature not specified) for the same species from pycnometric studies. A value of V = 0·737 ml./g has been reported by Elodi (1958) for both the rabbit and pig enzyme at 20°C. We assume a value of V = 0·729 ml.jg for both the pig and rabbit enzyme at low temperature in all calculations. Density measurements of solutions of ribonuclease (Kielley & Harrington, 1960), myosin (Woods et al., 1963) and y-globulin (Marler et al., 1964) in guanidine-HCI indicate that the apparent specific volume of these proteins, 4>', in 5 M-guanidine-HCI is about 1 to 2% lower than the measured thermodynamic Vin dilute salt systems. 4>' is the apparent specific volume of the protein in a three-component system as defined by Casassa & Eisenberg (1960,1961). All sedimentation studies in guanidine-HCI reported below have been performed at 25°C, and subsequent calculations utilized a value of 4>' = 0·729 ml.jg for GPDH in 5 M-guanidine-HCI at this temperature.

3. Results and Discussion Velocity sedimentation studies on the native GPDH isolated from both pig and rabbit at concentrations in the range of 1 % protein revealed that the enzyme sedimented as a single symmetrical boundary. However, a trace of more rapidly sedimenting material can be detected in the pig enzyme from a careful examination of the base line of the schlieren patterns. The low-speed equilibrium experiments also demonstrate that the major enzyme component is contaminated with a small amount of high molecular weight component. Although log c against r 2 plots appeared to be sensibly linear over most of the liquid column, upward curvature, indicative of particle

45 (·65

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49 FIG. 1. Molecular weight determination from a sedimentation equilibrium experiment on native pig GPDH at low speed. The left ordinate gives the logarithm of the protein concentration in fringes and the abscissa represents the square of the distance (cm'') from the axis of rotation. The right ordinate gives the protein concentration directly in fringes (40 fringes = 10 mg/ml.). Initial concentration of GPDH was 0'93% in 0·15 M-NaCl, 0·05 M-NaH 2PO., 0·002 M-EDTA (adjusted to pH 7·4). The speed was 5227 rev.jrnin and the time of sedimentation was 15 hr at a temperature of 6°C. Column height, 1 mm.

SUBUNITS OF GL YCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

889

heterogeneity, was always observed near the base of the column. A typical plot is presented in Fig. 1. The apparent weight average molecular weight, M w , ofthe protein at various positions in the cell at equilibrium was calculated from the slopes of log c against r 2 plots according to equation (2) and gave M m = 140,000 and M b = 166,000, evaluated from the limiting slopes at the meniscus and base of the cell, respectively. The molecular weight determined at the midpoint of the liquid column (equation (1)) was generally in good agreement with that estimated from the limiting slope of log c against r2 at the meniscus trace. Thus it would appear that the contaminant is of relatively high molecular weight as compared to the major enzyme component, and consequently most of its mass is distributed near the base of the liquid column. This interpretation is supported by the results of the high-speed equilibrium experiments (16,200 rev.fmin). Plots of log c against r 2 derived from these studies (Fig. 2) were

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FIG. 2. Molecular weight determination from sedimentation equilibrium experiment on native pig GPDH at high speed. The left ordinate gives the logarithm of the fringe displacement above the solvent base line, and the abscissa represents t.he square of the distance (cm 2 ) from the axis of rotation. The right ordinate gives the fringe displacement directly in 1-" Initial concentration of pig GPDH was 0·01 % in 0·15l\I-NaCl, 0·05 M-NaH 2P0 4 , 0·002 M-EDTA. The speed was 16,200 rev.Jmin and the time of sedimentation was 15 hr at a temperature of 5·5°C. Column height, 3 mm.

completely linear over the readable range-i.e. from a fringe displacement of about 100 fL until the fringes could no longer be resolved at the column base. At the high speeds used in these studies, each equilibrium distribution spans approximately a tenfold range in concentration. Evidently at these rotor speeds the contaminating component is effectively sedimented out of solution and makes no detectable contribution to the mass distribution in the liquid column. Molecular weights evaluated from the equilibrium sedimentation results are summarized in Table 1. Within the experimental precision ofthese measurements, the molecular weight of GPDH from pig muscle was found to be independent of protein concentration over a 100-fold range. The weight average molecular weights estimated by the two methods are in good agreement, giving an average value of M w = 146,000 for the principal component from pig and M w = 144,000 for the rabbit enzyme. 59

W. F. HARRINGTON AND G. M. KARR

890

1

TABLE

Molecular weight of glyceraldehyde-3-phospliate dehydrogenase Low-speed equilibrium

1. Pig

High-speed equilibrium

Concn (%)

Mw

Concn (%) (initial)

Mw

0·93 0·40 0·32 0·21

156,000 140,000 150,000 140,000

0·01 0·01 0·01

139,000 144,000 156,000

0·01 0·01 0·01 0·01 0·01 0·01 0·02 0·02

140,000 140,000 141,000 144,000 151,000 151,000 134,000 147,000

2. Rabbit

High-speed equilibrium studies (16,200 rev.jmin) employed 3-mm columns of protein solution. Low-speed experiments (5227 to 6991 rev.jmin) were made with I-rnm columns of solution. Solvent was 0·15 M-NaCI, 0·05 M-NaH2P0 4 , 0·002 M-EDTA. Temperature, 6 to 9°C. V = 0·729 ml.jg.

Molecular weight of the subunit

The addition of guanidine-HCI to a solution of GPDH caused a marked change in the sedimentation velocity pattern. In 5 M-guanidine-HCl the boundary characteristic of the native enzyme is completely transformed into a much slower sedimenting schlieren peak which broadens much more rapidly than that of the native enzyme in dilute salt systems. Plate I demonstrates that, in this solvent system, the pig enzyme treated with N-ethyl maleimide migrates as a single symmetrical boundary with no indication of the presence of more rapidly or more slowly sedimenting material. A plot of 1jS2o,w against concentration (Fig. 3) is quite linear over the concentration range examined and yields by least-square analysis the equation:

1jS2o,w = 0·549 + 0·326 c; sgo,w = 1·82 s where the concentration, c, is given in gjlOO mi.

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6

8

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Fig. 3. The sedimentation against concentration dependence of pig GPDH in 5 M-guanidineHCl. The reciprocal of the sedimentation coefficient corrected to a standard state of water of 20°C is given on the ordinate and the concentration is in gjm!. All runs were made at 59,780 rev.jmin at temperatures between 22 and 25°C.

PLATE I. Sedimentation velocity patterns for pig GPDH (treated with N-ethyl maleimide) in ;) ~l-guanidine-HCl. Top: Wed/!e cell. 0·35% pig GPDH. Bottom: Conventional cell. 0·67% pig GPDH. Schlieren patterns were obtained 7 hr after attaining a speed of 59,780 rev.fmin at an angle of 60° for the schlieren diaphragm.

[facing p. 890

SUBUNITS OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

891

The sedimentation behaviour in guanidine-HCl should be compared to that in dilute salt systems. Fox & Dandliker (1956) have reported the concentration dependence of the sedimentation coefficient of rabbit muscle GPDH in 0·1 ionic strength phosphate buffer (pH 6,55) to be

+

I/S 2 0 ,w= 0·130 O'Oll c; sgo,w = 7·71 s (4) Thus the absolute value of the sedimentation coefficient decreases about fourfold in 5 M-guanidinium chloride, whereas the slope of the I/S against c plot is markedly enhanced consistent with a drastic alteration in the conformation of the macromolecules (see, for example, Schachman, 1959). Since a decrease in sedimentation coefficient 0·6

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can be attributed to a dissociation of the GPDH molecules, to an increase in the frictional coefficient or to various combinations of dissociation and disorganization of individual subunits, further information was sought from viscosity studies. Solutions of the enzyme treated with N-ethyl maleimide in 5 M-guanidinium chloride were dialyzed against the solvent for several days and the final concentration of protein determined from the optical density (280 mu: see Experimental Methods). This solution was diluted (by weight) with dialysate to give a series of concentrations for viscosity measurements. Results are presented in Fig. 4, from which the reduced viscosity was found to vary as a function of concentration according to the equation: TJsp/c

= 0·345 + 0·080 c.

(5)

The intrinsic viscosity, [TJ] = 0·345 dl./g is much greater than that expected for compact globular proteins ([TJ] = 0·04 dl.jg) and, in agreement with thesedimentation results, indicates a drastic unfolding of the polypeptide chains of the native enzyme. Assuming 4>' = 0·729 cc/g, sgo. w = 1·82 s, [7]] = 0·345 dl.jg and fJ = 2·5 X 106 , the value generally accepted for a random chain, the molecular weight estimated from the Scheraga-Mandelkern equation (1953) is 37,500 (corrected for the N-ethyl maleimide content). As Eisenberg (1962) has demonstrated, the molecular weight calculated from the Scheraga-Mandelkern treatment is independent of any preferentially bound solvent if measurements of sedimentation and viscosity parameters are made on dialyzed solutions and the term (1- V'p) is replaced by (1-g,'p). A number of high-speed sedimentation equilibrium runs (39,460 to 44,770 rev.jmin) were also made on GPDH in 5 M-guanidine-HCl. All of the log c against r 2 plots

w.

892

F. HARRINGTON AND G. M. KARR

obtained from analysis of the Rayleigh fringe patterns of these studies were quite linear (see Fig. 5 for a typical graphical illustration of the logarithm of the fringe displacement as a function of the square of the distance from the center of rotation), indicating a relatively high degree of homogeneity with respect to size. 750 2·8

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FIG. 5. Molecular weight determination from sedimentation equilibrium experiment on pig GPDH in 5 M·guanidine-HCl at high speed. Ordinates and abscissa are labeled as in Fig. 2. Initial concentration of pig GPDH, 0·01 % in 5 M-guanidine-HCl. The speed was 42,040 rev./min and the time of sedimentation was 19 hr at a temperature of 25°C. Column height, 3 mm.

Problems inherent in the determination of molecular weights by equilibrium sedimentation of protein in 5 M-guanidinium chloride have been considered in an earlier paper (Wood et al., 1963). The major difficulty in molecular weight determination in three-component systems involves preferential binding of solvent (either water or guanidinium chloride); but as Casassa & Eisenberg (1960,1961) have shown, this problem can be circumvented through dialysis and use of the apparent specific volume parameter, c/>', in place of the thermodynamic apparent specific volume, V. Another difficulty arises from the non-ideality of the protein-guanidinium chloride system. Since very low concentrations of protein were employed in the present studies (0·01 %) errors resulting from non-ideality are probably not serious. Table 2 summarizes the results of the high-speed equilibrium studies on both pig and rabbit GPDH in 5 M-guanidinium chloride. A molecular weight, M w = 36,500, was estimated for the pig enzyme, and M w = 35,000 for the rabbit enzyme in this solvent. The most likely interpretation of these results is that native glyceraldehyde-3phosphate dehydrogenase (Mw 145,000) possesses a tetrameric structure and that it dissociates into subunits (Mw 36,000) in 5 M-guanidine-HCl. The subunit formed in this way undoubtedly corresponds to the structural monomer in the enzyme, which has been shown by Harris & Perham (1965) to be a single protein chain with a molecular weight of 36,000. Evidence for the tetrameric structure of GPDH is also provided by the results of X-ray crystallographic analysis of the crystalline enzyme from lobster muscle (Watson & Banaszak, 1964). Allison & Kaplan (1964) have shown that the

SUBUNITS OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE TABLE

893

2

M olecular weight of glyceraldehyde-3-phosphate dehydrogenase subunit in 5 M-guanidine-HCl

Temperature 25°C. maleimide,

Concn (%)

Mw

Rev.jmin

1. Pig

0·01

2. Rabbit

0·015

36,200 35,900 38,500 38,700 35,300 35,300 39,300 33,000 35,000

42,040 42,040 42,040 42,040 39,460 44,770 42,040 42,040 42,040

V=

0·729 ml./g. Molecular weights were corrected for the bound N-ethyl

sedimentation constants of GPDH's from rabbit, ox, man, chicken, turkey, pheasant, halibut, sturgeon, lobster, Escherichia coli and yeast (average sgo. w value, 7·44 ± 0·15 s) are closely similar. This result suggests that they also have molecular weights similar to those which we have determined for the pig and rabbit muscle enzymes. This study was aided by U.S. Public Health grant no. AM-04349. REFERENCES Allison, W. S. & Kaplan, N. O. (1964). J. si«. Ohern; 239, 2140. Benesch, R. E., Lardy, H. A. & Benesch, J. (1955). J. BioI. Chern: 216, 663. Casassa, E. F. & Eisenberg, H. (1960). J. Phys. Chem. 64, 753. Casassa, E. F. & Eisenberg, H. (1961). J. Phys. Chem. 65, 427. Cori, G. T., Slein, M. W. & Cori, C. F. (1948). J. tu«. Chern, 173, 605. Dandliker, W. B. & Fox, J. B., Jr. (1955). J. BioI. Chem. 214, 275. Eisenberg, H. (1962). J. Chem, Phys. 36, 1837. Elias, H. G., Garbe, A. & Lamprecht, W. (1960). Z. physiol. Chem, 319, 22. Elodi, P. (1958). Acta Physiol. Acad. Sci. Hung. 13, 199. Elodi, P. & Szorenyi, E. (1956). Acta Physiol. Acad. Sci. Hung. 9, 339. Fox, J. B., Jr. & Dandliker, W. B. (1956). J. tu«. Chem. 218, 53. Harris, J. I. (1964). Nature, 203, 30. Harris, J. I. & Perham, R. N. (1963). Biochem. J. 89, 60P. Harris, J. I. & Perham, R. N. (1965). J. Mol. BioI. 13, 876. Kielley, W. W. & Harrington, W. F. (1960). Biochim. biophys. Acta, 41, 401. Koeppe, O. J., Boyer, P. D. & Stulberg, M. P. (1956). J. BioI. Chem. 219, 569. Marler, E., Nelson, C. A. & Tanford, C. (1964). Biochemistry, 3, 279. Richards, E. G. & Schachman, H. K. (1959). J. Phys. Chern; 63, 1578. Schachman, H. K. (1959). Ultracentrifugation in Biochemistry, chap. 4. New York: Academic Press. Scheraga, H. A. & Mandelkern, L. (1953). J. Amer. Ohem, Soc. 75,179. Taylor, J. F., Lowry, C. & Keller, P. J. (1956). Biochim. biophys. Acta, 20, 109. Van Holde, K. E. & Baldwin, R. L. (1958). J. Phys. Ohem, 62, 734. Velick, S. F., Hayes, J. E., Jr. & Harting, J. (1953). J. BioI. Ohem, 203, 527. Watson, H. C. & Banaszak, L. J. (1964). Nature, 204, 918. Woods, E. F., Himmelfarb, S. & Harrington, W. F. (1963). J. st«, Chem. 238,2374. Yphantis, D. A. (1960). Ann. N.Y. Acad. Sci. 88, 586. Yphantis, D. A. (1964). Biochemistry, 3, 297.