TNE JOURNAL OF BIOLOGICAL CHE&~ISTRY Vol. 247, No. 23, Issue of December 10, pp. ii50-7758, 1972

Printed in U.S.A.

Physical Properties of Acinetobacter Glutaminase-

Asparaginase with Antitumor Activity*

(Received for publication, June E,l972)

JOHN S. HOLCESBERG,: DAVID C. TELLER, JOSEPH ROBERTS,$ AND WILLIAM C. DOLOWY

From the Departments of Medicine, Pharmacology, Biochemistry and Experimental Animal Medicine, University of V7ashingtou School of Medicine, Seattle, Washington 98195

SUMMARY

Acinetobaclet glutaminase-asparaginase has been shown to consist of 4 subunits (molecular weight 33,000) by sedi- mentation equilibrium in 5.5 M guanidine HCl and electro- phoresis in sodium dodecyl sulfate on polyacrylamide gels after cross-linking the protein with dimethyl suberimidate.

Moving boundary velocity experiments showed that most of the native enzyme sediments as the tetramer (SZO+ = 7.42 f 0.03 S). On the other hand, equivalent boundary calculations always showed a smaller ~20,~. Analytic sedi- mentation equilibrium experiments revealed a tetramer- dimer dissociation with a dimer molecular weight of 69,000 f 3000. The molecular weight on calibrated Sephadex G-200 and Bio-Gel P-200 was 97,000 and 93,000, respectively, which may indicate reversible dissociation.

The hydrolysis of S-diazo-4-oxonorvaline was used to determine the sedimentation coefficient of the active species. Sedimentation of the enzyme in 5-diazo-4-oxonorvaline showed complex patterns with ultraviolet optics due to pro- tein absorbance. A new double sector cell was devised which allows layering of enzyme into both sectors simul- taneously, cancelling the absorbance of the enzyme. The SZ~,~ value for the species which degraded 5-diazo-4-oxonor- valine was 7.6 f 0.2 S. By matching zone sedimentation and active enzyme experiments, enzyme species smaller than tetramer were shown to have 4% or less of the activity of the tetramer.

The isolation, crystallization, and catalytic properties of Acinetobacter glutaminase-asparaginase have recently been re- port,ed (1). Preliminary physical studies of this enzyme revealed a lower molecular weight by gel filtration than by sed- imentation velocity. These observations suggested that the

* This investigation was supported by United States Public Health Service grants CA 11881 and CA 25976 from the National Cancer Institute and GM 46363 and GM 13401 from the National Institute of General Medical Sciences. A preliminary report of some of this work was reported at the Federation of American Societies for Experiment,al Biology, April 1972 (2).

$ Career Development Awardee, United States Public Health Service.

enzyme may dissociate under certain conditions. The physical properties of this enzyme were of particular interest as part. of our evaluation of various characteristics which ma)- alter its effectiveness as a cancer chemotherapeutic agent. Dissociation of the enzyme into active smaller fragments mav increase its effectiveness by increasing the distribution of the enzyme in the animal host. During the course of these investigations, we have devised some new ultracentrifuge techniques which may be applicable to other enzymes.

EXPERIMENTAL PROCEDURE

Jfaterials and !Methods-Crystalline bovine serum albumin was purchased from General Biochemicals. Human y-globulin was Cohn fraction II from Schwarz Bioresearch and Mann Re- search Laboratory. Ovalbumin, trypsin, and lysozyme were from Worthington. Glutamic dehydrogenase was from Sigma.

Ultrapure guanidine HCl was from Heico; 6 M solutions were made in 0.01 M sodium phosphate buffer, and the pH was adjusted from 6 to 7 with NaOH.

L-5-Diaao-4-oxonorvaline was a gift from Dr. R. E. Handschumacher, Yale University School of Medicine. Escherichia coli asparaginase was a gift from Dr. Morris Zim- merman, Merck, Sharp & Dohme, received as a lyophilized preparation, Lot ABI-2. which had 200 i. u. per mg of powder.

FC-43 was obtained from Minnesota Mining & Manufacturing Company. Saturation of reaction mixtures with this oil pro- duced no inhibition of the enzyme activity. This material was not used in the sedimentation equilibrium studies, but was nec- essary for the active enzyme experiments.

Glutaminase-asparaginase was isolated from Acinetobacter glutaminasificans (ATCC 27197), prepared as previously de- scribed (l), and stored as a lyophilized powder at 4”. ,411 en- zyme used had a specific activity greater than 150 iu per mg of protein. Enzyme activity was assayed with glutamine or as- paragine by ammonia formation (1). Activity with 5-diazo-4- oxonorvaline was monitored spectrophotometrically (3).

Sephadex G-200 obtained from Pharmacia was sieved through loo-, 200-, and 270-mesh screens for 3 hours. The 200- to 270- mesh fraction was hydrated and equilibrated with buffer. Each gel was packed by gravity in a 0.9 x 60 cm K6 Pharmacia col- umn. Protein solutions (0.5 ml) were layered on the columns and eluted by downward flow at less than 15-cm hydrostat,ic pressure at 1.6 to 4.1 ml per hour per cm2. The 0.5- to l-ml eluates were monitored for absorbance and enzyme activity.

7750

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from

7751

Blue dextran, bovine serum albumin, human y-globulin, and ovalbumin were used as standards. Buffers used were 0.05 M

Tris-HCl (pH 7.5) or 0.01 to 0.02 M sodium phosphate (pH 7.3) containing 0.15 to 0.2 M NaCI.

Disc gel electrophoresis in sodium dodecyl sulfate was per- formed by layering 20 to 40 pg of denatured protein on 5y0 acryl- amide gel containing 0.1 y0 sodium dodecyl sulfate and phosphate buffer (pH 7.0), and subjecting to electrophoresis in the same buffer (1, 4). Protein was denatured in 1 y0 sodium dodecpl sulfat,e and 1% 2-mercaptoethanol for 2 hours at 37” prior to electrophoresis. Protein was stained by Coomassie brilliant blue R250 (Colab) in trichloroacetic acid.

Dimethyl suberimidate was prepared by the method of Davies and Stark (5) and stored desiccated at 4”. Cross-linking was done by incubating 0.1 mg of enzyme with 0.24 or 0.6 mg of dimethyl suberimidate in 0.2 M triethanolamine buffer (pH 8.3) for 3 hours at room temperature. Aliquots were removed and denatured in sodium dodecyl sulfate and subjected to electro- phoresis in the usual system.

Ultracentrifugation Stud&s-All sedimentation studies were performed with a Beckman-Spine0 model E analytical ultra- centrifuge equipped with schlieren and interference optical sys- tems, as well as an ultraviolet absorption scanning system. The ultraviolet scans could be linked to both an Z, y plotter and a PDP-I2 computer (6).

Sedimentation Equilibrium-High speed sedimentation equi- librium studies were performed by standard methods (7) with six-chambered Yphantis centerpieces and Teflon window liners (8). The ultracentrifuge drive had minimal low and high fre- quency procession. Kodak II-G photographic plates were used to record the interference pictures. White light patterns of the reference holes and monochromatic pictures of the channels were superimposed by a swinging gate mask apparatus (8). Plates were analyzed on a Nikon 6C microcomparator with a fringe mask which allows reading of several fringes simultaneously by reading maximal light transmission on an oscilloscope (9). Base- line data were obtained at the end of a run by vigorous shaking of the cells and rerunning at low speed. Analysis of the data utilized base-line smoothing (8) and high speed equilibrium sedimentation programs on a CDC 6400 computer (7, 10).

Prior to ultracentrifugation, enzyme solutions were either dialyzed or passed through a Sephadex G-200 column. The buffer used was the dialysate or the Sephadex buffer. After dialysis of the enzyme in the 6 M guanidine solution, the density of the dialysate was 1.137 measured with a lo-ml pycnometer. The density of this solution indicated that the guanidine was only 5.5 M. A value of 0.740 ml per g for the partial specific volume of the enzyme was calculated (11) based on the amino acid composition (1) and was used for both sedimentation equi- librium calculations and sedimentation velocity corrections.

Sedimentation Velocity-Viscosity and density of buffers were usually determined from International Critical Tables, 1926- 1933. The relative viscosity at 20” of the solution containing 3% sucrose, 0.02 M sodium phosphate (pH 7.3), and 0.2 M NaCl was determined to be 1.105 with a capillary flow viscometer (12). This is identical with the value calculated from standard tables.

For sedimentation velocity studies, the absorption optics were utilized exclusively. Data collection and analysis were per- formed by computer programs written in machine language for the PDP-12 computer. Three types of sedimentation velocity experiments were performed.

First, moving boundary experiments were conducted and sed- imentation coefficients were computed from the inflection point

of the boundary and from equivalent boundary calculations (6). Disparity between sedimentation coefficients calculated by these two methods can be used to detect heavy or light materials in the preparation. The error bars graphed in the figures for t,his method are computed from uncertainty in the concentration (6).

Second, active enzyme sedimentation velocity experiments were performed to estimate the size of t’he enzymatically active species (13). The results of these experiments were calculated in several ways. The sedimentation coefficient was estimated from time differences eit’her by the maximum ordinate meas- urements, or from equivalent boundary measurements (14). In the equivalent boundary method, sedimentation and diffusion of the substrate was neglected during the 2-min time increment between scans. This was done because control experiments utilizing the sedimentation of n-glyceraldehyde 3-phosphate dehydrogenase yielded low sedimentation coefficients and high diffusion coefficients for this enzyme when a substrate sedimen- tation-diffusion correction was applied. This equivalent bound- ary program also computes the mass-average concentration difference between any two specified limits in the data

where A& is the mass average difference in substrate eoncen- tration between times t and t + At, r is the radial position, and A&’ is the time difference of substrate at that position. The limits of integration, r1 and TZ, can be set anywhere by the op- erator. This latter aspect of the program was used to calculate the activity of material sedimenting behind the major zone of enzyme.

Because of the very slow turnover of 5-diazo-4-oxonorvaline by Acinetobacter glutaminase-asparaginase (l), it was necessary to use quite concentrated solutions for the active enzyme studies (0.5 to 1.0 mg per ml). Due to the superposition of absorption bands of enzyme and substrate an artifact was generated when enzyme was layered on one side of the centerpiece only (Fig. 1). Consequently, a centerpiece with two layering channels was designed which is similar to the Beckman centerpiece (part no. 331359), except scribe line channels were made on the cen- tripetal and centrifugal extrema of the central rib of the center- piece. FC-43 (0.02 to 0.03 ml) was used to provide a false bottom in the cell. Solvent (0.3 to 0.31 ml) containing diazo- oxonorvaline was placed in the solution sector, and the same quantity of solvent without diazo-oxonorvaline was placed in the solvent sector. Because of the thermodynamic requirement for equality of pressures at the centrifugal scribed channel, menisci were matched automatically, cancelling absorbance due to pro- tein (Fig. 2). When such a centerpiece was not used for the active enzyme sedimentation method, spuriously low sedimen- tation coefficients were observed due to the subtraction of en- zyme absorbance (Fig. 1). Experimentally, this decrease was 0.5 S. This problem was simulated by a computer program which presented the effects of sedimentation and diffusion of protein but not the effects of sedimentation and diffusion of sub- strate. The artifact produced a decrease of 0.1 S by this calcu- lation.

The third type of velocity experiment performed was meas- urement of the zone sedimentation in a self-generated increase gradient of density (15). Sedimentation coefficients were cal- culated by the maximum ordinate and equivalent boundary methods. The enzyme concentrations and buffers for these experiments were identical with those used in the active enzyme

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from

7752

FIG. 1. Absorbance patterns in active enzyme experiment. The solid lines in the upper figure trace the absorbance of 5-diazo-4- oxonorvaline at three consecutive time points at 4-min intervals in an experiment with enzyme layered ident,ically into each sector. The center figure shows the approximate protein absorbance in each sector at each of these times. The dotted line in the upper ftgure is a sum of the protein and 5-diazo-4-oxonorvaline absorb- ance at each time point. This is the pattern seen if the enzyme is layered only into the sector containing 5-diazo-4-oxonorvaline. The lower Jigure shows the absorbance difference derived by sub- stracting consecutive scans. The solid lines illustrate the absorb- ance difference of 5-diazo-4-oxonorvaline when the protein absorb- ance does not interfere. The dotted line is absorbance difference of the sum of protein and 5-diazo-4-oxonorvaline shown in the upper figure. This absorbance difference is distorted and has a negative component. DONV, 5-diazo-4.oxonorvaline.

studies. For the equivalent boundary calculations, the moment method of Vinograd and Bruner (16) was used. In order to confirm that the sedimentation coefficients were not seriously in error due to theoretical approximations, a computer program was constructed which computed both the moments

and

‘2 ‘2 <logR> =

s log r C rdr/

s C rdr

‘1 r’t

R is an approximation to the equivalent zone position, whereas the latter equation is exact (17). The difference of d log R/dt from d < log R > / dt was 0.4% for rr = r (meniscus) and rz zz r (C = 0) beyond the boundary. Therefore, the approximate

theory is satisfactory for these experiments. This computer pro- gram also calculates the mass average concentration between two arbitrary points, ~1 and r2 by the integral

Co = 1:’ C rdr/l:’ rdr (2)

where C is the optical density at point r. Both Equations 2

FIG. 2. Ability of the ultracentrifugation cell to cancel protein absorbance. A, 5 ~1 of human r-globulin, 1 mg per ml, was placed in one well and water was placed in the other. Each sector con- tained 0.32 ml of 0.02 M sodium phosphate buffer (pH 7.3) contain- ing 0.2 M NaCI and 3% sucrose. FC-@ (0.03 ml) was layered on the bottom of each cell. Scans were taken at C-min intervals at 280 nm after the rotor reached 60,600 rpm. Part B is identical to A except that the protein was layered into each well. Protein absorbance is completely cancelled. XIR and XOR are inner and outer reference lines, respectively. X, is the meniscus, and FC-4s is the artifact due to this oil.

and 1 were used to compute the specific activity of materials trailing centripetal to the major optical density zone in these experiments.

RESULTS

Moleczdar Weeight of Subunits-The electrophoretic migration of proteins denatured in sodium dodecyl sulfate has been shown to be a linear function of the logarithm of the subunit molecular weight (4). In this system with bovine serum albumin, glu- tamic dehydrogenase, trypsin, E. coli asparaginase, and lysozyme as standards, the glutaminase-asparaginase has a migration identical with E. coli asparaginase which corresponds to a mo- lecular weight of 33,000 f 200 g per mole (N = 6).

Dimethyl suberimidate produces cross-linked proteins by amidination (5). Fig. 3 shows the results of reaction of the enzyme with the reagent, followed by electrophoresis in sodium dodecyl sulfate. The gel on the left is an untreated control. The cross-linked enzyme clearly shows four bands. These bands have appropriate migrations for tetramer, trimer, dimer, and monomer. Identical experiments with E. coli asparaginase also show this pattern of 4 subunits.

Equilibrium sedimentation on the denatured enzyme was performed in 5.5 M guanidine HCl. The enzyme was dialyzed for 3 days at room temperature in 100 volumes of 5.5 M guanidine HCl and 0.01 M sodium phosphate buffer (pH 7.0). The pro- tein at 0.5,0.75, and 1.5 mg per ml was sedimented to equilibrium at 36,000 rpm at 20”. Rayleigh plates at 48 hours were analyzed by plotting- log C as a function of r2 and by computer analysis, assuming V was unchanged in the solvent and equal to 0.74 ml per g. Molecular weight was 32,000 f 2,000 g per mole.

The results of these subunit molecular weight studies indicate that the glutaminase-asparaginase molecule is composed of 4 subunits with a monomer molecular weight of about 33,000.

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from

7753

‘2 ! I I 1 I I I I I I I

u L 4 6 8 IO 12 6’

0 2 4 6 8 IO 12 Concentration (fringe) Concentration (fringe)

FIG. 3. (left). Electrophoretic migration of proteins denatured. in sodium dodecyl sulfate. Gel on the left is an untreated control Gel on the tight clearly shows four bands of the cross-linked en- zyme after reaction with dimethyl suberimidate.

FIG. 4 (right). Molecular weight distributions for Acinetobacter glutaminase-asparaginase. A, weight average molecular weight (MU). B, number average molecular weight (M,,). Data pre- sented by open symbols and solid lines were taken from a photo- graph at 26 hours at 16,000 rpm and 20”. The filled symbols and dashed lines were data from a photograph at 43 hours at the same speed and temperature. The buffer was 0.01 M sodium phosphate, 0.2 M NaCl, 3 mM NaNa (pH 7.0). The numbers on the lines indi-

Gel Filtration-Calibrated Sephadex G-200 columns gave symmetrical single peaks for the native enzyme. Molecular weight was calibrated by plotting the linear relationship be- tween the logarithm of molecular weight and the ratio of elution volume to void volume. Human y-globulin, bovine serum albumin, and ovalbumin were used as standards. The average molecular weight of the enzyme from 13 experiments was 97,000 f 9600 g per mole. These experiments were routinely done in high salt concentration. The elution volume and absorbance profile of the enzyme was not changed by addition of 0.01 to 0.02% NaNa, saturation of the column with bovine serum al- bumin, change in temperature from 5 to 25”, successive rechro- matography of the peak tubes, or dialysis, relyophilizing, and resuspension. The specific activity of the eluate was 80 to 96% of the initial enzyme solution. Gel filtration on a Bio-Gel P-200 column gavean average molecular weight for threedeterminations of 93,000 f 9000 g per mole.

Gel filtration of E. coli asparaginase on Sephadex G-200 gave a

cate the channel pairs of the Yphantis centerpiece; 1 is the centrip- etal channel pair. The lines were calculated from dimer molecu- lar weight of 69,000 g per mole with initiJ concentrations (mg per ml) and Kz values (X 1OP liter/mole) as follows. At 26 hours: A- - -A, channel 1, initial concentration of 0.75, apparent Kt 706; l - - -0, channel 2, initial concentration of 0.50, apparent KP 257; w--m, channel 3, initial concentration of 0.25, apparent Kz 114. At 43 hours: A-A, channel 1, initial concentration of 0.75, apparent KZ 369; O-O, channel 2, initial concentra- tion of 0.50, apparent KZ 178; III----~, channel 3, initial concen- tration of 0.25, apparent Kt 72. KZ is the association constant for dimer-tetramer equilibrium.

molecular weight of 106,000 zt 6000 g per mole (iV = 8). This value is the same as that obtained by other workers (18-20), but is lower than that calculated by sedimentation equilibrium (18, 21, 22).

Sedimentation Equilibrium-Fig. 4 shows a typical experiment in which the molecular weight for each channel is plotted at various fringe displacements. Solid symbols represent the cal- culations after 26 hours at 36,000 rpm at 20”. The numbers correspond to the sectors with initial concentrations of enzyme equal to 0.75, 0.5, and 0.25 mg per ml for numbers 1, 2, and 3, respectively. The figure shows that dissociation occurred in each cell. The molecular weight average distributions (Mn and M,) for each sector are similar but do not superimpose. In addition, the highest molecular weights are observed for the highest initial loading concentrations. This result is opposite that expected for an irreversibly dissociated solute where the lowest initial loading concentration produces the highest mo- lecular weight distributions (23). It is also inconsistent with

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from

complete chemical equilibrium among subunits. With chemical equilibrium the curves of molecular weight averages as a function of concentration should superimpose for all loading concentra- tions (24, 25).

The open symbols of Fig. 4 represent the same run at 43 hours. Clearly, more dissociation has occurred in each cell. Such a pattern is consistent with a slow time-dependent irreversible dissociation of the tetramer to the dimeric form. The irrevers- ible dissociation is sufficiently slow to allow accurate calculations assuming equilibrium at the 26 and 43-hour time points.

A computer program has been developed to analyze the high speed equilibrium data of dissociating protein systems (10). This program calculates the root mean square error of fitting the data to a dimer-tetramer dissociation for various molecular weights of dimer. The data in Fig. 4 were analyzed by this method. Fig. 5 depicts the error plots for each channel for dimer molecular weights from the 26-hour pattern. Various

patterns are seen, but each has a clear minimum error. Four additional high speed sedimentation equilibrium ex-

periments were analyzed by this computer program. In each the enzyme solution was obtained from the peak tubes of Sephadex columns. Initial concentrations were 0.34 to 1.10 mg per ml. Dissociation was observed in each sector. The data were analyzed with the same computer program illustrated in Fig. 5. These calculations gave an average dimer molecular weight of 69,000 f 3,000 g per mole.

Sedimentation Velocity Stud&s-The results of moving bound- ary sedimentation velocity experiments on the enzyme at various concentrations are shown in Fig. 6. The solid circles are results of experiments in 0.2 M NaCl and 0.01 to 0.02 M NaP04 buffer (pH 7.0 to 7.4). The solid line is drawn from a linear least square

0” 60 70 80 90 60 70 80 90

molecular wgtlt Ii 10-31 g/mole

FIG. 5. Search for minimal molecular weight from Acinetobacter glutaminase-asparaginase dat,a (86 hour data, Fig. 4) at increments of 1000 g per mole. This figure illustrates various different shapes, but each has a clear minimum. Dashed lines represent deviations based on all of the data. Solid lines are the deviations of data from concentrations greater than one fringe. The left panels are number average molecular weight deviations and the right panels are weight average molecular weight deviations.

analysis of these data. The si,, zu value is 7.42 f 0.03 S (r\i = 33). The concentration dependence, k, is defined by the equation, s2o.w = so,,,, (l-kc), h w ere C is concentration (26). The value for k is 0.031 f 0.006 liter per g, which is higher than that ob- served for most globular proteins (27), but in the range reported for Erwinia asparaginase (26).

Several experiments were performed in the same buffer plus 3% sucrose. These data points are shown by the open circles

in Fig. 9. These values are in the same range as those without sucrose, demonstrating that sucrose does not significantly alter the hydrodynamic propert’ies of the enzyme.

The sedimentation experiments often showed a complex ab- sorption profile, (Fig. 7). The major absorbance inflection point was used to calculat’e t’he sedimentation coefficients dc- scribed in Fig. 6. The minor absorbance profile suggested a slower sedimenting species. In order to analyze this compo- nent an equivalent boundary program was developed for the PDP-12. This program subtract.s the base-line after all protein has sedimented, measures all of the remaining sedimenting ab- sorbance, and creates an equivalent sharp boundary (29). The migration of this boundary with time was used to calculate the value of Bzo,~, the weight average sedimentation coefhcient. Fig. 8 illustrates the results from t,his type of experiment. The upper figure shows the sedimentation coefficients calculated from the boundary and migration of the major absorbance pattern. Below is the .%o,~ from the same data utilizing the equivalent boundary program. The error bars in this figure are computed from the uncertainty in concentrations. Clearly, the sedimen- tation coefficients are lower when all of the absorbing material is used in the calculations. These &o,W values decrease with de- creasing initial concentration. This pattern is consistent with

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Concentration (mq/ml)

FIG. 6. Sedimentation coefficient as a function of concentration for moving boundary of the enzyme in 0.01 to 0.02 M phosplmte- 0.2 M NaCl, pH 7.3 (0). Same buffer containing 3% sucrose (0).

FIG. 7. Absorbance profile at 280 nm during moving boundary sedimentation of glutaminase-asparaginase. Upper scan was

taken 40 min after rotor reached 60,000 rpm. Lozuer scan taken at 164 min was used as a base-line.

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from

7755

72 T T i 8

7’. P

Y P

0 2 4 6 8 IO 12 14 16 18 Concentraf~on ( mg/ml)

FIG. 8. Sedimentation coefficient as a function of concentration for moving boundary experiments by absorption optics in 0.62 M

phosphate-O.2 M NaCI, pH 7.3, at about 20” and 60,000 rpm. A, ~20.~ calculated from the inflection point of the boundary. B, equivsIent boundary calculations from the same data as in A.

a reversible concentration dependent dissociation of the en- zyme .

Active Enzyme-Experiments and computer programs were next designed to test whether dissociated species of the enzyme pos- sess enzymatic activity. The enzyme degrades 5-diazo-4-oxo- norvaline with the loss of absorbance at 274 nm (1). The spec- trophotometric assay at 25” with this substrate yields Vn,,, of 0.1% relative to glutamine and K, = 8 x 10e5 M for this re- action. All active enzyme ultracentrifuge experiments report- ed here were performed at 280 nm.

In initial experiments, the active enzyme sedimentation studies were performed by the method of Cohen and Hahn (13), but led to spuriously low values of the sedimentation coefficient as discussed under “Experimental Procedure.” On the other hand, with the double layering, meniscus matching centerpiece, meas- urement of the maximum ordinate of the substrate difference peaks in eleven experiments yielded a sedimentation coefficient of 7.6 & 0.2 S for the reactive species (2). The same .sz~,~ was obtained in experiments at 7” and 20 to 23” and in runs in buffer with and without 3% sucrose. Similar active enzyme experi- ments with E. coli asparaginase gave the same sedimentation coefficient.

Because of the dissociation noted with the Acinetobacter en- zyme, it was important to determine whether other forms of the enzyme also possessed activity. The available unequivocal methods for such a determination include (a) measurement of the molecular weight of the active species (141, or (b) meas- urement of the specific activity of the enzyme in various portions of the sedimenting zone.

Programs were written for the PDP-12 computer to compute molecular weights from sedimentat.ion and diffusion coefficients of the active species according to approximations to the theories of Cohen et al. (14). By first using differences of the concen- tration distributions between 2-min intervals, without correcting for sedimentation and diffusion of substrate, this method gave an acceptable molecular weight of 128 & 20 x lo3 g per mole for the active species of n-glyceraldehyde 3-phosphate

TABLE I Comparison of sedimentation coeficients in consecutive active

enzyme and zone sedimentation experiments

RUIl

1506 1507 1508

1509 1510 1511 1512 1513 1514 Averages

-

.-

Peak0

TYPO

~20,W S.?D,W

Active enzyme 7.77 7.57

Zone 6.91 6.25 Active enzyme 7.44 7.70 Zone 6.93 6.41 Active enzyme 7.28 7.53 Moving boundary 7.33 6.93 xk 0.07

Zone 6.85 6.41

Active enzyme 7.49 7.63 Zone 7.06 6.24 1 mg/ml of zone 6.94 f 0.096.33 f 0.10 1 mg/ml of active enzyme 7.49 f 0.207.61 f 0.07 0.4 mg/ml of moving bound- 7.33 6.93 f 0.07

nry

a Peak s20,w values are calculated from the migration of the peak absorbance. The ~20,~ values are calculated from migration of the midpoint of the equivalent boundary.

dehydrogenase of rabbit muscle (Sigma Lot 3IC9590). Nev- ertheless, these calculations failed completely for glutaminase- asparaginase. Since all of the errors in the theoretical approxi- mations tend toward low molecular weights, and errors in the estimate of diffusion coefficient are large, this method was par- ticularly unsatisfactory for the determination of active smaller forms of the enzyme. Consequently, the specific activity of various portions of the sedimentation velocity profiles was meas- ured.

Eight sedimentation velocity experiments were performed, alternating active enzyme and zone sedimentation experiments. The results of this series of experiments are presented in Table I. A moving boundary experiment was also conducted so that re- sults could be compared to other preparations of enzyme re- ported in this paper. In all experiments an attempt was made to accelerate the ultracentrifuge to 60,000 rpm in the same time period. Runs 1513 and 1512 came closest to the following cri- teria: (a) identical acceleration times and (b) absence of anom- alies in the active enzyme sedimentation. These two experi- ments were used to measure the specific activity of material trailing the major zone of optical density in the zone velocity studies.

The movement of the zone in the zone velocity studies is shown in Fig. 9 for run no. 1512 at lo-min intervals beginning at 10 min after the rotor reached 60,000 rpm (the first time used in the moment calculations). Two characteristics are noteworthy. First, there is appreciable absorbance in the supernatant cen- tripetal to the zone, and second, the zone is asymmetric after long periods of time. All zone experiments on this enzyme showed these characteristics. The asymmetry of absorbance in zone sedimentation could arise from large dependence of S upon the concentration (16, 30). With kCo approximately 0.1, slight asymmetry at late times might be noted (30) ; however, in the zone experiments reported here, kCo was only 0.031. Thus the trailing absorbance and asymmetric zones have a different origin. This behavior is consistent with equilibrium dissociation of the enzyme during the experiment. The enzyme dissociates in both the leading and trailing edges of the zone, but the leading edge reassociates when the major concentration band reaches it.

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from

7756

FIG. 9. Zone sedimentation of 1 mg per ml of glutaminase- asparaginase. Scans were taken at 280 nm at 2.min intervals. The figure shows scans at lo-min intervals beginning 10 min after the rotor reached 60,000 rpm.

TABLE II

Activity of small materials relative to major zone

1 20'-26' (20' + 26')/2 23'

‘

20'-26' (22' + 24')/2 23'

3 30'-36' (32' + 34')/2 33'

Calculation

Activity 0.256 25.4 Protein 9.8 40.4 Specific activity 0.0262 0.630

Activity 0.179 24.9 Protein 9.7 44.4 Specific activity 0.0184 0.562

Activity 0.407 22.6 Protein 9.92 30.2 Specific activity 0.041 0.748

Concentration

TOP Zone regiona regid

I

a

Zelative specific .ctivity*

%

4.1

3.3

5.5

= Concentrations are mass average vaIues as defined by Equa- tions 1 and 2 under “Experimental Procedure.” These regions are illustrated in Fig. 10.

6 Relative specific activity is the specific activity in the top re- gion divided by the specific activity in the zone region.

Table II shows that the calculation is accurate to about 1%. Actually, the precision is probably less than this since negative activities are observed in this upper region of the cell when the calculation was made between 38 and 44 min. Nonetheless, the experiment can be used to establish the upper limit of activity of species smaller t,han tetramer (7.6 S).

The trailing edge dissociates, but the concentration is so low that little or no reassociation occurs.

The trailing material should consist of dimer and monomer (if dissociation proceeds that far) while the major density should be an equilibrium mixture of monomer, dimer, tetramer, and larger species (18).

The computer programs allow one to compute the mass average substrate concentration difference (Equation l), between two radial points in an active enzyme experiment and align a zone experiment such that the mass average protein concentration (Equation 2) can be computed between the same two radial points. In this way, it was possible to calculate the specific and relative activities of material trailing behind the zone of runs 1512 and 1513 of Table I. This analysis is presented in Table II.

Any protein which dissociates to smaller species is left behind the zone in the region called “top” in the table. Material in this “top” region of the cell can reassociate to form tetramer. If we assume that only the tetramer is active, then activity in this region may be solely due to a 47, reassociation of the enzyme. On the other hand, this activity may be due to dissociated species. These species then have a combined activity less than or equal to 4 & 1% that of the tetramer.

DISCUSSION

The present studies have shown that the glutaminase-as- paraginase from Acinetobacter consists of 4 subunits each with a molecular weight of approximately 33,000 g permole. This subunit size is very similar to other asparaginases from E. coli, Alcaligenes euthrophes, and Erwinia caratovora (28, 31, 32).

Fig. 10 illustrates how this calculation was made. The top The molecular weight of the tetramer of the Acinetobacter of the figure shows the difference in the 5-diazo-oxonorvaline enzyme can be estimated by doubling the dimer molecular weight. absorbance pattern between 26 and 20 min after the rotor reached This value of 138 f 6 X lo3 g per mole is also quite similar to 60,000 rpm. This difference reflects the enzyme activity in this the other asparaginases.

FIG. 10. Upper jigure shows the difference spectrum of B-diazo- 4-oxonorvaline between 26 and 20 min after the rotor reached 60,000 rpm. The lower figure shows the protein absorbance in a zone sedimentation experiment at these same time points. X, is meniscus position which was matched by the computer for the specific activity calculations (see text). XL,, is an arbitrary posi- tion marking the top of the major activity. The top region ex- tends from X, to Xi,. The zone region extends from Xt, to the lower end of the absorbance.

time interval. The point marked Xtop was chosen as the super- natant region in this figure and the integral of Equation 1 eval- uated between r, = x, and r2 = xtop. The values of A?% are given in Table II for two replicates of this calculation. The 20- and 26-min zone sedimentation experiments were then aligned by matching the meniscus with that in the active en- zyme experiment. The same points of rl and rz relative to the meniscus were selected for the integrals of Equation 2. (Slight mismatching of meniscus positions should contribute only minor errors.) Since the concentration decreases with time in thrse zone sedimentations, the average of the extreme times (Table II, Entry 1) or the midpoint times (Table II, Entry 2) were taken as the protein concentrations for the time interval. Spe- cific activities were expressed as the relative mass averages, A&/??0 where c, is an average concentration between the two times in the table.

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from

On the other hand, the stability of the tetrameric form of our enzyme differs greatly from the other enzymes. Most physical studies of E. coli asparaginase show no dissociation in the pH range between 4.5 and 9.25 (22). At a pH above 11.8, rapid dissociation occurs to monomeric or smaller species (22, 28, 31). Possible dimeric species with ~20,~ of 3.3 and 3.2s have been seen after ultracentrifugation at pH 3.0 and 11.2, and after dialysis of the urea-denatured enzyme (33). The Erwinia asparaginase appears to be even more stable since it dissociates and loses en- zymatic activity only slowly at pH 11.9 (22). In contrast, the Acinetobacter enzyme dissociates into smaller subunits at neutral pH during gel filtration and ultracentrifugation. These ob- servations are clearly dissociation and not aggregation since the weight average sedimentation coefficients calculated in both the zone and moving boundary experiments are always less than the movement of the masimum or of the inflection point. We

believe that the enzyme undergoes both a slow irreversible disso- ciation and a more rapid reversible dissociation.

The irreversible dissociation of the tetramer enzyme is well established by the sedimentation equilibrium results. First, the patterns of molecular weight averages as a function of con- centration at different positions in the cell are not superimposable for different initial concentrations. Second, the molecular weight averages decrease with increasing time as is expected for a slow, irreversible dissociation.

The evidence for a simultaneous reversible dissociation is less strong. The Sephadex G-200 and Bio-Gel P-200 molecular weights are low, but these results might also be due to binding to the column matrix. However, equal binding strengths of the tetramer to two such dissimilar matrices seems improbable. These low molecular weights are clearly not due to irreversible dissociation since the specific activity of the eluate in gel filtration is nearly the same as the initial enzyme.

The zone sedimentation velocity experiments show that 20 to 25% of the optical density trails the major zone (Table II). The lighter material and the asymmetric profile of the zone ap- pears within 1 hour. In contrast, many hours are required to show the irreversible dissociation in the sedimentation equi- librium experiments (Fig. 4). Finally, the appearance of the decreasing weight average sedimentation coefficient, S~O,~, with decreasing initial concentration shown in Fig. 8B is consistent with a reversible dissociation.

AActive enzyme experiments were performed with 5-diazo-4- oxonorvaline because its ultraviolet absorbance could easily be followed during sedimentation. Inhibitor and kinetic studies indicate that 5-diazo-4-oxonorvaline, glutamine, and asparagine probably have the same active site on both the Acinetobacter

glutaminase-asparaginase (1) and E. coli asparaginase (3). Therefore, we presume that these results apply to the major substrates.

The active enzyme experiments indicated that the tetramer of the Acinetobacter enzyme is the only species which appreciably degrades 5-diazo+oxonorvaline. The sedimentation coefficient of the active species is the same for both the Acinetobacter and E.

coli enzymes, and equal to that reported for the tetramer of E.

coli asparaginase (10). Furthermore, in the active enzyme ex- periments, the SZO,~ o f the peak was the same as that of the in- tegrated absorption difference. If slower sedimenting species had activity, the .SZO,~ calculated from the integrated absorption difference should be smaller than that of the peak. Second, direct calculation of the specific activity for 5.diazo-4-oxonorva- line degradation in the region of the cell containing dissociated mwiw WR.S comnared with that of the area containing the major

7757

activity band. These calculations showed that the slower sed- imenting species had only 4yo or less of the activity of the tetramer.

It is not surprising that the molecular weight calculation of the active enzyme failed. These calculations are very sensitive to the tails of the optical density zone (14). Furthermore, the computer programs use several theoretical approximations which tend toward low molecular weights. Finally, although the cell designed to match menisci works well for the relatively insen- sitive calculation of sedimentation coefficients, it has not been tested for the calculation of diffusion coefficients which would be extremely sensitive to minor mismatch of menisci.

Several asparaginases and glutaminases have been described with molecular weights below 90,000 (34-36). The molecular weights of the active forms have not been reported for any of these enzymes. The techniques used in this paper may be quite useful in further evaluating these potential antitumor-agents, since enzymes of smaller size may be distributed outside the vascular space (37, 38).

REFERENCES 1. ROBERTS, J., HOLCENBERG, J. S., AND DOLOWY, W. C. (1972)

J. Biol. Chem. 24’7, 84-90 2. HOLCENBERG, J. S., TELLER, D. C., ROBERTS, J., AND DOLOU-Y,

W. C. (1972) Fed. Proc. 31, 473 3. JACKSON, R. C., AND HANDSCHUMACHER, R. E. (1970) Biochem-

istry 9, 3585-3590 4. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-

4412 5. DAVIES, G. E., AND STARK, G. R. (1970) Proc. Nat. Acad. Sci.

U. S. A. 66, 651 6. TELLER, D. C. (1972) Methods Enzymol., Vol. 27, chap. 14 7. TELLER, D. C., HORBETT, T. A., RICHARDS, E. G., AND SCHACH-

MAN, H. K. (1969) Ann. N. Y. Acad. Sci. 164, 66 8. HORBE:TT, T. A., AND TELLER, D. C. (1972) Anal. Biochem. 46,

86-99 9. TELLER, D. C. (1967) Anal. Biochem. 19, 256-264

10. HOAGLAND, V. D., JR., BND TELLER, D. C. (1969) Biochemistry a, 594

11. MCMECKIN, T. L., GROVES, M. L., AP\‘D HIPP, N. J. (1949) J. Amer. Chem. Sot. ‘71, 3298

12. SCHACHMAN, H. K. (1957) Methods Enzymol. 4, 32 13. COHEN, Il., AND HAHN, L. W. (1965) C. R. Hebd. Seances Acad.

14.

15.

16. 17.

18.

19.

20.

21.

22.

23.

24.

25. 26.

27.

Sci. 260, 2077 COHEN, R., GIRAUD, B., AND MESSIaH, A. (1967) Biopolymer

6, 203-225 VINOGRAD, J., BRUNER, R., KENT, R., AND WEIGLE, J. (1963)

Proc. Nat. Acad. Sci. U. S. A. 49, 902 VINOGRAD, J., AND BRUNER, R. (1966) Biopolymer 4, 131-156 SCHUM.~KER, V. N., AND ROSENBLOOM, J. (1965) Biochemistry

4, 1005-1011 LABOUREUR, P., LANGLOIS, C., LABROUSSE, M., BOUDON, M.,

EMERAUD, J., SAMAIN, J. F., AGERON, M., AND DUMESNIL, Y. (1971) Biochimie 63, 1147

YELLIN, T. O., AND WRISTON, J. C., JR. (1966) Biochemistry 6, 1605-1612

ARENS, A., RAVENBUSCH, E., IRION, E., WAGNER, O., BAUER, K., AND KAUFMANN, W. (1970) Hoppe-Seyler’s 2. Physiol. Chem. 361, 197

STAERIC, J., H.PUPT, H., AND KRANZ, T. (1970) Ezperientia 26, 131

SCKOLTAN, W., AND LIE, SIE YING (1971) Biochim Biophys. Acta 236, 105-126

HARRIS, C. E., KOBES, R. D., TELLER, D. C., AND RUTTER, W. J. (1969) Biochemistry 8, 2442

GUGGENHEIM, E. A. (1957) Thermodynamics, North-Holland Publishing, Amsterdam

ADAMS, E. T., JR. (1964) Proc. Nat. Acad. Sci. U. S. A. 61,509 SCHACHMAN, H. K. (1959) Ultracentrifugation in Biochemistry,

Academic Press, N. Y. CREETH, J. M., AND KNIGHT, C. G. (1965) Biochim. Biophys.

Acta 102, 549-558

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from

7758

28. CAMMACK, K. A., MARLBOROUGH, D. I., AND MILLER, D. C. (1972) Biochem. .J. 126. 361

29. GOLDBERG, R. J. (1953) j. Phys. Chem. 67, 194 30. DISHON, M., WEISS, G. H., AND YPHANTIS, D. A. (1969) Ann.

N. Y. Acad. Sci. 164, 33 31. FRANK, B. H., PEKAR, A. H., VEROS, A. J., AND Ho, P. P. K.

(1970) J. Biot. Chem. 246, 3716-3724 32. ALLISON, J. P., MANDY, W. J., AND KITTO, G. B. (1971) Fed.

Proc. 30, 1297 33. SHIFRIN, S., LUBORSKY, S. W., AND GROCHOWSKI, B. J. (1971)

J. Biol. Chem. 246, 7708-7714

34. BROOME, J. D. (1971) Colloques International Sur L’aspara- ginase, Centre Nat. Rech. Sci. (Paris) 19’7,95

35. RAMADAN, M. E. A., ASMAR, F. E., AND GREENBERG, D. M. (1964) Arch. Biochem. Biophys. 108, 143

36. KIRSHBAUM. J.. WRISTON. J. C.. JR.. AND RATYCH. 0. T. (1969) B&h&. Biophyys: Acta i94, 161-169 ’

37. Ho, D. H. W., CARTER,. C. J. K., THETFORD, B., AND FREI, E. III (1971) J. Clin.. Pharmacol. Z’her. 11.408: (1971) Cancer Chemoiher. Rep. 66, 539

_ *. I

38. MAYERSON, H. S., WOLFRAM, C. G., SHIRLEY, H. H., JR., AND WASSERMAN, K. (1969) Amer. J. Physiol. 198,155

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from

John S. Holcenberg, David C. Teller, Joseph Roberts and William C. DolowyActivity Glutaminase-Asparaginase with AntitumorAcinetobacterPhysical Properties of

1972, 247:7750-7758.J. Biol. Chem. 

  http://www.jbc.org/content/247/23/7750Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/247/23/7750.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from