The N, O-Diacetylmuramidase of Chalaropsis Species

Lysozyme Chalaropsis is a single polypeptide chain with an apparent weight average molecular weight of 19,100 f 900. The ultraviolet absorption is typical for proteins with an E ::, = 24.8 + 0.4, and its isoelectric point is weakly alkaline at 7.53. The sedimentation constant (si,,,) of 2.27 f 0.14 exhibits no apparent concentration dependence, a phenomenon consistent with a symmetrical sedimenting mass. The secondary structure, as jud.ged by optical rotatory dispersion in the ultraviolet, exhibits significant structural differences as compared to hen egg white lysozyme.

Chalaropsis is a single polypeptide chain with an apparent weight average molecular weight of 19,100 f 900. The ultraviolet absorption is typical for proteins with an E ::, = 24.8 + 0.4, and its isoelectric point is weakly alkaline at 7.53. The sedimentation constant (si,,,) of 2.27 f 0.14 exhibits no apparent concentration dependence, a phenomenon consistent with a symmetrical sedimenting mass.
The secondary structure, as jud.ged by optical rotatory dispersion in the ultraviolet, exhibits significant structural differences as compared to hen egg white lysozyme.
The specificity of a fungal Chalaropsis species, crystalline bacteriolytic enzyme has been established recently as a p-1,4-N-acetylmuramidase (l), a specificity identical with hen egg white lysozyme.
However, the Chalaropsis lysozyme (henceforth designated lysozyme Ch) also exhibits @-1,4-N, O-diacetylmuramidase activity, in contrast to egg white lysozyme. This specificity difference offers a unique opportunity for comparative enzyme structure analysis.
A physical characterization of lysozyme Ch is described in this communication.

EXPERIMENTAL PROCEDURES
Lysozyme Ch-Twice crystallized lysozyme Ch prepared as previously described (I) was used in these studies.
B&&on Coe$%ent-The absorption spectrum of lysozyme Ch was determined in a Cary model 14  (il/1u) for the entire liquid column determined by the relationship: (1) in which R is the gas constant, T the absolute temperature, 7 the partial specific volume, p the buffer density, w the angular velocity, b the base and a the meniscus of the liquid column, and C,, the concentration gradient in fringes between b and a at equilibrium. The Rayleigh interference system was used for all determinations with fringe positions recorded on Spectroscopic II G photographic plates (Kodak) and analyzed with a Nikon model 6 microcomparator at 50x magnification.
A value of 5.62 cm was used as the position of the counterbalance wire from the center of rotation. Extensive dialysis was used to ensure equilibration of solvent and solution.
Runs were made for 20 to 24 hours, equilibrium being assured by a lack of change in fringe concentration gradient on sequential photographs. Z-average molecular weight (M,) for the low speed runs were calculated from the M, (b) and M, (a) obtained from the base and meniscus slopes, respectively, of the In c against x2 plots, by the relationship: where Cb and C, are the fringe concentrations at the base and meniscus.
The type of aggregation phenomena (dimer as opposed to higher order aggregate) observed in the low speed sedimentation experiments was evaluated by the following expression which relates M, to the content of dimer in a monomer-dimer equilibrium : (5) in which Ml in this case was taken as M, (CL) and Cl and CZ are the concentrations of monomer and dimer, respectively. A single high speed experiment was made in guanidine.HCl to establish whether the experimentally determined M, was the monomeric weight.
Guanidine. HCl was recrystallized from methanol after decolorizing with Norite. The enzyme was reduced with dithiothreitol and extensively dialyzed against 5 M guanidine.HCl containing 1 mM dithiothreitol. There is still some degree of uncertainty regarding the partial specific volume of proteins in guanidine.HCl.
Values ranging from no apparent change (4) to a 1 to 2 y0 decrease in the apparent partial specific volume (5) have been reported.
Nevertheless, the results in this case allow a clear assessment of whether or not there is subunit structure.
Solution Densities and Partial SpecQic Volumes-Leach pycnometers with a volume of 50 ml were used at 20" for all density determinations.
The apparent partial specific volume (papi,) was determined in water.
Isoelectric Point-The isoelectric point of lysozyme Ch was determined by the isoelectric focusing technique (6). The temperature was maintained at 20" f 0.5 by a Tamson circulating water bath.
Two separate experiments were performed with a pH gradient of 3 to 10 in one case and 5 to 8 in another, and a protein concentration of 4 mg per run. Measurements of pH were performed with a Radiometer pHM4d meter and a combined electrode calibrated to ho.01 pH unit. Activities were measured by the lysis of cell-free staphylococcal cell wall suspensions (7).
Optical Rotatory Dispersion-Measurements of the optical rotatory dispersion from 350 rnE.1 to 210 rnp for lysozyme Ch were made on a Cary model 60 recording spectropolarimeter.
Experiments were carried out at 27" with a l-cm path length cell in both sodium phosphate (pH 6. 5 Table  I).

Partial Specific Volume
The partial specific volume for lysozyme Ch was determined by density measurements to be 0.726 ml per g. This value was used in all subsequent calculations of molecular weight.

Sedimentation Studies
Sedimentation Velocity-High speed sedimentation velocity runs (Fig. 1) over the concentration range of 2.5 mg per ml to 15 mg per ml showed a symmetrical peak with a value of 2.27 f 0.14 for the s&,~ at infinite dilution (Fig. 2). However, careful examination of the schlieren base line on occasion revealed a trace of more rapidly sedimenting material.
No dependence of sedimentation coefficient on protein concentration was observed, indicative of a symmetrical sedimenting mass.
Sedimentation Equilibrium-Low speed equilibrium sedimentation also revealed evidence of a small amount of high molecular weight material as evidenced by upward curvature of the In c against x2 plot at the base of the liquid column, although a linear relationship exists over most of the liquid column. This phenomenon was observed in both acetate and phosphate buffers. Fig. 3 shows a typical low speed run for lysozyme Ch. ilnalyses at the isoelectric point were impossible because of the low solubility of the enzyme at pH 7.5 in buffered solutions.
Weight average and Z-average molecular weights evaluated from the low speed equilibrium results are summarized in Table I. No apparent concentration dependence was observed.
Taking the meniscus M, average, the M, weight would indicate approximately 10% aggregation if the higher molecular weight species consists entirely of dimers. The lack of a contaminating peak of this magnitude in the sedimentation velocity analyses argues for a small amount of higher order aggregation.
In contrast to the low speed equilibrium runs, the distribution of lysozyme Ch in high speed ultracentrifugation analyses showed a linear In c against x2 relationship over the entire liquid column in both phosphate and acetate buffers (Fig. 4). Values  (Table II) cluster around a JJ, = 20,000 * 700.
A single experiment with lysozyme Ch in 5 M guanidine.HCI with t.he enzyme sulfhydryls in the reduced state revealed a ~~tmw = 20,000, a value indicative of a single polypeptide chain for the native enzyme.
Isoelectric Point-Two determinations of the isoelectric point with the use of the technique of isoelectric focusing yielded values differing by only 0.01 pH unit. Sharp, symmetrical boundaries were obtained with this technique as indicated by Fig. 5. The observed isoelectric point was 7.53 at 20".
Optical Rotatory Dispersion-The optical rotatory dispersion of lysozyme Ch reveals significant differences in secondary structure as compared to egg white lysozyme (Fig.  6). The latter possesses the typical Cotton minima at 233 rnp ([m'] of -3400"), a feature common to the majority of globular proteins. Treatment of the data for egg white lysozyme by the Moffitt equation and a X0 of 212 yielded an excellent linear correlation and a b0 of -145 + 8 in close agreement with the data of Tomimatsu and Gaffield (11 However, these optical rotatory parameters are quite similar to a small group of globular proteins reported by Jirgensons (12).

DISCUSSION
The physical properties of lysozyme Ch examined in this study and contrasted to hen egg white lysozyme are summarized in Table III.
It is evident that the molecular structure of this fungal muramidase shows considerable differences from the well known egg white lysozyme.
The apparent molecular weight of lysozyme Ch is approximately one-third higher than the established value for egg white lysozyme.
The previous demonstration of a high degree of homogeneity of these lysozyme Ch preparations (I) indicates that the high molecular weight material observed in low speed equilibrium experiments (Fig. 3) is most likely the result of a small amount of aggregation.
However, since the value cited is a weight average molecular weight, the chemical weight may be slightly reduced.
The lack of concentration dependence in the sedimentation velocity experiments is indicative of a spherical sedimenting mass. As compared to hen egg white lysozyme there is a large difference in isoelectric point, a property related to the primary structure. Other differences in these two muramidases may also be noted by the apparent alterations in secondary structure as observed with optical rotatory dispersion studies. The observed X, falls within the range commonly associated with denatured proteins (15). In addition, as compared to egg white lysozyme, lysozyme Ch exhibits a shift in its Cotton minima from 233 rnp to 227 rnp and a low or positive 6,,, a feature suggested by Jirgensons to be applicable to the presence of fi structure (12). Although nonprotein moieties may be responsible for the optical rotatory behavior, preliminary chemical studies indicate that no carbohydrate is present.