Purification by affinity chromatography and preliminary characterization of ornithine decarboxylase from simian virus 40-transformed 3T3 mouse fibroblasts

ARCHIVES

OF

BIOCHEMISTRY

Purification Characterization

AND

BIOPHYSICS

184, 408-415 (1977)

by Affinity Chromatography and Preliminary of Ornithine Decarboxylase from Simian Virus 40Transformed 3T3 Mouse Fibroblastsl

ROBERT J. BOUCEK, JR.* Departments

of * Pediatrics

AND

KENNETH

and ? Biochemistry, Vanderbilt Tennessee 37232 Received

June 24, 1977; revised

J. LEMBACH?

University August

School of Medicine,

Nashville,

8, 1977

Ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) has been purified from simian virus 40-transformed 3T3 mouse fibroblasts by a procedure utilizing affinity chromatography as the principal step. Selective elution of the enzyme from a pyridoxamine 5’-phosphate-agarose affinity matrix with the use of pyridoxal Y-phosphate effected a single-step purification of approximately 500-fold, with a significantly higher overall recovery of activity (30 to 45%) than achieved with previous procedures. In the presence of optimal protein concentrations, the enzyme from transformed fibroblasts exhibited a significantly higher specific activity than reported previously for the decarboxylase purified from liver. The apparent affinities of the fibroblast enzyme for substrate and cofactor were similar to those reported for the decarboxylases purified from other tissues. With the use of sodium dodecyl sulfate-gel electrophoresis, the subunit molecular weight of the purified ornithine decarboxylase was demonstrated to be approximately 55,000, while the apparent molecular weight of the active enzyme in vitro as determined by gel filtration was approximately 110,000.

The activity of ornithine decarboxylase (EC 4.1.1.17, L-ornithine carboxy-lyase) , the first enzyme of polyamine biosynthesis in eucaryotic cells, has been shown to increase markedly following growth stimulation (l-5). In addition, a close correlation between the level of this enzyme and growth rate has been demonstrated in a series of Morris rat hepatomas (61, while we and others have previously shown that transformed fibroblasts exhibit significantly higher levels of ornithine decarboxylase than untransformed fibroblasts cultured in vitro under similar conditions (7, 8). Since ornithine decarboxylase regulates the biosynthesis of the polyamines, which appear to be significant factors in nucleic acid metabolism (91, it is of importance to characterize this enzyme and the mechanisms which regulate its activity. ’ This investigation was supported by USPHS Grants CA-12810 from the National Cancer Institute and lF32HL05233 from the National Heart and Lung Institute and by a grant-in-aid from the Tennessee Heart Association to R.J.B. Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

A definitive analysis of the mechanisms controlling the level of ornithine decarboxylase requires methodology to quantitate both the amount of enzyme protein and the activity. The purification of this enzyme from SV40*-transformed mouse fibroblasts was therefore initiated to obtain specific antibody for use in evaluating more thoroughly the serum-induced increase in ornithine decarboxylase seen in these cells (7) and to compare with the enzyme from normal tissues. Although the purification of ornithine decarboxylase from rat liver and prostate has been reported (10-131, previous procedures require several steps and yield low recovery of active enzyme.‘In view of the relatively limited amount of starting material available with in vitro systems, and the reported instability of the enzyme (lo), it was apparent that a new method was essential for purification of ornithine decarboxylase from cultured cells. A unique e Abbreviations used: DEAE, diethylaminoethyl.

SV40,

simian

virus

40;

ISSN 0003-9861

PURIFICATION

OF ORNITHINE

and simplified purification procedure involving affinity chromatography as a key step is described. Purification to apparent homogeneity of ornithine decarboxylase from SV40-transformed 3T3 mouse fibroblasts has been achieved. The purified enzyme from these transformed cells exhibits characteristics similar to those described for the decarboxylase from other sources, with the exception that the specific activity appears to be significantly greater than that reported for the enzyme from normal tissues. The subunit molecular weight of the purified enzyme was found to be approximately 55,000, while the apparent molecular weight of the active enzyme in vitro as determined by gel filtration was 110,000. Stabilization of the purified enzyme was achieved with added protein or polyethylene glycol . METHODS Cell culture and induction of ornithine decarboxylase. An SV40-transformed 3T3 cell line, SVlOl, which was originally obtained from Dr. Howard Green, Department of Biology, Massachusetts Institute of Technology, was used in these studies. Stock cultures were maintained in Dulbecco’s medium supplemented with 5% (v/v) calf serum. The medium contained 50 pgiml of chlortetracycline and was buffered at pH 7.4 with 0.02 M N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid and 0.01 M N-tris(hydroxymethyl)methylglycine. The concentration of NaHCO, was 1.0 g/liter. For preparation of ornithine decarboxylase, cells were grown in disposable plastic roller bottles (490 cm’, Corning) to a density of approximately 3 to 4 x 10”/cmZ. The cells were then provided with medium containing 0.2% (v/v) calf serum and, following a 20- to 24-h interval, cultures were stimulated by a nutritional shift-up to medium containing 5% (v/v) serum (7). The previous methodology was modified to optimize the stimulation of ornithine decarboxylase activity by increasing the pH of the shift-up medium to 8.3 and by the addition of methylisobutylxanthine (0.1 mM). Cells were harvested 4 to 5 h following the serum shift-up, at which time the enzyme activity is maximal. The extracellular medium was decanted, the cell monolayers were washed with phosphate-buffered saline (calciummagnesium free), and the cells were removed from the roller bottles with 1 mM EDTA in buffered saline. After chilling to 4”C, the cells were harvested by centrifugation at 1OOOgfor 5 min and the cell pellets were stored at -70°C. The enzyme activity was stable under these conditions for at least 2 months.

DECARBOXYLASE

409

Assay of ornithine decarboxylase activity. Enzyme activity was assayed at 37°C in the presence of saturating concentrations of cofactor, pyridoxal 5’phosphate, and substrate, L-ornithine, according to previously described methods (71, with the exception that 0.025 M Tris-HCl, pH 7.5, containing 5 mM dithiothreitol and 0.1 mM EDTA was used as the incubation buffer. Protein was quantitated by the method of Lowry et al. (14) using crystalline bovine serum albumin as standard. Protein samples for analysis were first precipitated with 5% trichloroacetic acid at 4°C to eliminate dithiothreitol, which interferes with the assay (10). Similar concentrations of crystalline bovine serum albumin were precipitated in an identical manner to determine the standard curve. Enzyme activity is expressed as nanomoles of CO, liberated per hour per milligram of protein. The formation of putrescine has been shown previously to be equivalent to the evolution of CO, under the conditions of the assay (7). Preparation of the affinity matrix. Pyridoxamine 5’-phosphate was coupled with the activated agarose matrix according to the directions provided by the supplier. One gram of lyophilized Affi-Gel 10 was suspended in 25 ml of 0.10 M sodium phosphate buffer, pH 7.0, containing 200 mg of pyridoxamine C’-phosphate. The coupling reaction was allowed to proceed for approximately 24 h at 4°C with rotational mixing of the suspension. Unreacted groups on the agarose matrix were then coupled with 1 M ethanolamine under the same conditions and the gel was washed extensively on a Buchner funnel with 1 M NaCl in 0.01 M sodium phosphate buffer, pH 7.0. Polyacrylamide gel electrophoresis. Homogeneity of the purified enzyme was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a discontinuous system with 10% resolving gels (pH 8.9) and 3% stacking gels (pH 6.8) (15). The samples and standards were precipitated with cold 5% trichloroacetic acid and centrifuged for 20 min at 4°C and 4000g. The pellets were washed once with chilled acetone to remove residual acid, solubilized in Tris-HCl buffer (pH 6.8) containing sodium dodecyl sulfate (2%, w/v), dithiothreitol (1 mM), and 2-mercaptoethanol (l%, v/v), and heated to 100°C for 5 min. After cooling, the samples were layered directly onto the stacking gel and electrophoresed at 2.5 to 3.0 mA/gel at 20°C for 2-3 h. All gels were fixed with 12.5% (w/v) trichloroacetic acid in 50% (v/v) ethanol for 15 min at 80°C and were stained for protein with 0.02% Coomassie brilliant blue. The gels were destained at 37°C with 7% (v/v) acetic acid in 6% (v/v) methanol and were scanned for absorbency at 550 nm. MATERIALS Dulbecco’s modified Eagle’s medium and calf serum were from Grand Island Biological. Aff-Gel

410

BOUCEK

AND

10, an N-hydroxysuccinimide ester of agarose, was obtained from Bio-Rad, and DEAE-cellulose (DE52) was from Whatman. Crystalline bovine serum albumin, DNase I, pyridoxamine 5’-phosphate, and a-casein were purchased from Sigma; dithiothreitol and pyridoxal5’-phosphate were obtained from Calbiochem. Aldrich Chemical supplied acrylamide, bisacrylamide, tetramethylethylenediamine, and methylisobutylxanthine. L-[ l-‘4C10rnithine (sp act, 59 Ci/mol) was obtained from AmershamSearle. Protein standards for molecular weight calibration were nurchased from Boehringer, and polyethylene glycol was from Serva. RESULTS

Preparation Cellulose

of Cell Extracts Chromatography

and DEAE-

The starting material for a typical preparation of the enzyme was 10 to 15 g wet weight of SVlOl cells (approx 3 x lo9 cells) containing approximately 1 g of total protein. Although the method could readily accommodate larger quantities of starting material, the accumulation of cells was a limiting factor in the present studies. All operations were performed at O4°C unless otherwise noted. SVlOl cultures were stimulated by serum shift-up at 37°C as described under Methods to increase the level of ornithine decarboxylase activity (‘7). The cells were then harvested and resuspended in 0.025 M TrisHCl, 0.1 mM EDTA, 5.0 mM dithiothreitol, pH 7.5 (“starting buffer”), at a cell density of approximately 5 x lo7 cells/ml. Cell extracts were prepared by two cycles of freezing and thawing and were centrifuged at 20,OOOgfor 10 min at 4°C. The resulting supernatant fraction (Fraction I), which contained virtually all of the ornithine decarboxylase activity, was used for DEAE-cellulose chromatography. The recovery of protein in Fraction I was approximately 300-500 mg. Fraction I was diluted to a protein concentration of 3 to 5 mg/ml with starting buffer and was adsorbed onto a DEAEcellulose column (2.5 x 7.0 cm) previously equilibrated with the same buffer. The column was washed with 2 vol of the buffer followed by elution with a linear gradient (500 ml) of from 0 to 0.3 M NaCl in the starting buffer. Elution of ornithine decarboxylase activity occurred at a salt concentration of approximately 0.2 M, as

LEMBACH

previously reported for the enzymes from rat liver and prostate (10-13). The active fractions were pooled and the enzyme was precipitated by the addition of solid ammonium sulfate to 55% saturation. After stirring for 30 min at 4”C, the suspension was centrifuged at 20,OOOgfor 20 min. The protein pellet was resuspended in 25 mM Tris-HCl, pH 7.2, 0.1 mM EDTA, and 5.0 mM dithiothreitol (affinity column loading buffer) and was dialyzed for 18 h against 100 vol of the same solution. The enzyme activity eluted from the DEAE-cellulose column was quantitatively recovered during salt precipitation and dialysis. The purification and recovery of the enzyme to this step were at least comparable to previous reports, as shown in Table I. The DEAE fraction (Fraction II) was stable at -70°C for at least 3 months. Affinity

Chromatography

A column (1.5 x 7.0 cm) of the agarose affinity matrix with coupled pyridoxamine 5’-phosphate was prepared and equilibrated with the affinity column loading buffer containing added 0.1 mM ATP and 1 mM CaCl,. Crystalline ATP was added to Fraction II to give a final concentration of 1.0 mM, and, after 30 min at 4°C this fraction was applied to the affinity column at a relatively slow flow rate (2 to 4 ml/h). Approximately 10 to 20% of the total enzyme activity added was not retained by the column. Readdition of this unadsorbed activity to the same column failed to achieve further adsorption of the activity, despite the fact that additional studies indicated that the column capacity was not being exceeded under these conditions. After application of the sample, the column was washed successively with 12 column volumes of the loading buffer containing 0.1 mM ATP, 1 mM CaCl,, and 15 mM NaCl, followed by 12 column volumes of loading buffer plus 15 mM NaCl. Ornithine decarboxylase was then eluted optimally with a linear gradient of pyridoxal 5’-phosphate (0 to 10 PM) in loading buffer containing 15 mM NaCl. For preparative purposes, the gradient can be replaced by a step change to 10 PM pyridoxal phosphate. The rationale for the addition of Ca’+

PURIFICATION

OF ORNITHINE TABLE

-

411

DECARBOXYLASE I

COMPARISON OF METHODS FOR PURIFICATION OF ORNITHINE DECARBOXYLASE ’ Specific activity” Purification step Friedman et al. (11) (regenerating rat liver)

1. High-speed supernatant (Fraction I) 2. Ammonium sulfate (20 to 50% saturation) 3. DEAE-cellulose (Fraction II) 4. Gel filtration 5. Gel filtration 6. Isoelectric precipitation 7. Sucrose density gradient 8. Affinity chromatography (Fraction III)

Janne and Williams-Ashman (10) (rat prostate)

1.09

2.06

2.99 (65%)

5.70 (91%)

14.22 (30%) 190 (6.7%)

117 (56%) 601 (30%)

On0 et al. (12) (thioacetamidetreated rat liver) 2.16

Present method hiXIU~st~p,16.9

31.2 96.0 (53%) 606 (32%) 2,676 (9.3%) 3,552 (4.7%) 11,604 (1.3%)

210 (69%)

100,000 (45%)

‘I The indicated results are considered to be representative of the purification method. The starting material is indicated in parentheses below the respective authors. b The specific activity is given as nanomoles of CO, per hour per milligram of protein. The values in parentheses are the overall net recoveries.

and ATP to the loading buffer was based on the observation in early purification efforts that actin appeared to be the single major contaminant of ornithine decarboxylase eluted from the affinity column. This identification of actin was based on: (a) the apparent molecular weight by sodium dodecyl sulfate-gel electrophoresis lactin constitutes a majority of proteins in the 40,000-50,000 molecular weight class in this cell line (1611; (b) the ability of the contaminant to complex in solution with DNase I (EC 3.1.4.5) la previously reported property of actin (1711; and (c) its binding and elution from a DNase I-coupled Sepharose column by a previously reported method for purification of actin (18). ATP and Ca’+ were added to the affinity column loading buffer to enhance the polymerization of actin and to compete with any actin binding to the phosphate groups on the affinity column. In this way the initial binding of actin to the column was decreased and its elution during the wash was facilitated. The affinity of ornithine decarboxylase for the pyridoxamine phosphate matrix was judged to be specific by the following criteria: (i) Affi-Gel 10 not coupled with pyridoxamine 5’-phosphate did not selectively bind the enzyme; (ii) the addition of low concentrations (1 FM) of pyridoxal phosphate to Fraction II prevented the

subsequent binding of ornithine decarboxylase to the affinity column; (iii) the adsorbed enzyme could be eluted by pyridoxal phosphate alone (0.25 mM); and (iv) neither phosphate buffer (0.2 M, pH 6.0) nor ATP (0.1 mM) caused elution of the enzyme activity once bound. As shown in Table I, a single-step purification of approximately 500-fold with a recovery of about 50 to 60% was achieved by affinity chromatography. The overall recovery of activity (30-45%) represents a marked improvement as compared with previous procedures. Sodium dodecyl sulfate-gel electrophoresis of the pooled ornithine decarboxylase fractions from the affinity column (Fraction III) is shown in Fig. 1. Only a single major protein band was observed in Fraction III, although the limited amount of protein applied to the gel (10 to 20 pg) would not have allowed the detection of minor contaminants. Electrophoretic analysis of the fractions eluted from the affinity column indicated that the appearance of this protein coincided exactly with the elution of ornithine decarboxylase activity. The specific activity of ornithine decarboxylase in Fraction III varied from 100 to 160 pmol of CO,/h/mg of protein in different preparations. This range in specific activity probably reflects the inherent variation in protein determination at the low protein concentrations

412

BOUCEK

AND

LEMBACH

after approximately 50 min of incubation at 4”C, while at 37°C the activity was reduced by 63% within 5 min (data not shown). As shown in Table II, low concentrations of albumin, as well as of other proteins and polyethylene glycol, were found to stabilize the purified enzyme. The enzyme activity observed following prior incubation at 4°C with albumin or casein was in fact greater than that of the control assayed under identical conditions. Since the addition of albumin (0.01 to 1 mg/ml) to the assay mixture was also found to increase the observed activity of the purified enzyme by three- to fivefold, the activity of the Fraction III enzyme was routinely assayed in the presence of excess albumin (1 mg/ml), which was added during the lo-min preincubation with pyridoxal 5’-phosphate. Properties

of the Purified

Enzyme

To compare the properties of ornithine decarboxylase from transformed fibroblasts with the enzyme from other sources, the kinetic parameters of the Fraction III enzyme were calculated by linear regression analysis of the double-reciprocal plots of activity versus concentrations both in the presence and in the absence of albumin (1 mg/ml) and are given in Table III. It may be noted that the enzymatic reaction FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Fraction III. Fraction III (right), containing approximately 20 pg of protein, was concentrated and electrophoresed in 10% sodium dodecyl sulfate gels at 2.5 mA/gel for 2.5 h at 20°C. The gels were stained with Coomassie blue. The bovine serum albumin and ovalbumin standard gel is also shown for comparison. There was a single major band from Fraction III. The direction of migration was from top to bottom.

measured. The final specific activity, however, is at least eightfold greater than that previously reported for ornithine decarboxylase isolated from thioacetamidetreated rat liver (12). Stability ylase

of Purified

Ornithine

Decarbox-

The activity of the purified Fraction III enzyme was found to decrease by 50%

TABLE

II

STABILITY OF PURIFIED ORNITHINE DECARBOXYLASE AT 4°C” PreincubaPreincubation condition Per;E;pmy of tio;;fn4FcWal 18 h

1 week

None Albumin (0.1 mg/ml) Ovalbumin (0.1 mg/ml) o-Casein (0.1 mg/ml) Polyethylene glycol (O.l%, w/v)

0.4 217 96 204 60

Albumin

113

(0.1 mg/ml)

o Purified enzyme (Fraction III) was stored at 4°C for the specified time intervals with the additions indicated. Aliquots were then assayed in duplicate and compared with the control value obtained by assay of the enzyme prior to storage. All assay mixtures contained 0.1 mg/ml of albumin and duplicate analyses agreed to within 10% or better.

PURIFICATION TABLE KINETIC Kinetic

III

PARAMETERS OF PURIFIED DECARBOXYLASE u parameter

K,,, (ornithine) (pyridoxal K,, phosphate) V max

5’-

OF ORNITHINE

ORNITHINE

-Albumin

+Albumin

0.125 rnM 3.62 /LM

0.066 mM 1.0 PM

252

490

413

DECARBOXYLASE

dithiothreitol, as has been previously shown to occur with ornithine decarboxylase from rat prostate (10). The molecular weight of the active enzyme was evaluated by gel filtration of Fraction II on a Bio-

” The activity of the purified enzyme (Fraction III) was measured in duplicate with various concentrations of substrate and cofactor in the presence and absence of albumin (1 mg/ml). Double-reciprocal plots of l/v vs l/concentration were analyzed by linear regression analysis of the data to obtain the kinetic parameters. The activity is expressed as nanomoles of CO, per hour per milliliter of Fraction III enzyme.

was linear during the assay period (20 min) in the presence or absence of albumin. For the determination of the Km for pyridoxal phosphate, an appropriate correction was made for the concentration of this compound added with the Fraction III enzyme. The K, values with respect to both ornithine and pyridoxal phosphate were not appreciably affected by albumin (Table III). In addition, these kinetic parameters were similar to those reported for the rat liver and prostate enzymes, which were determined under comparable assay conditions (10-12, 20, 21). The activity of the Fraction III enzyme was not affected by the addition of ATP, GTP, cyclic AMP, or cyclic GMP (0.1 nM to 0.1 mM) to the assay mixture or by putrescine at concentrations of from 0.05 to 2 mM (data not shown). The molecular weight of the single protein species observed by sodium dodecyl sulfate-gel electrophoresis of Fraction III was estimated to be 55,000 by comparison (15) of its electrophoretic mobility ratio with those of molecular weight standards simultaneously electrophoresed (Fig. 2A). If dithiothreitol was removed from Fraction III by dialysis, which resulted in almost complete loss of enzymatic activity, subsequent sodium dodecyl sulfate electrophoresis in the absence of reducing agents revealed a major protein band with an estimated molecular weight of 110,000. The fibroblast enzyme thus appears to form an inactive dimer in the absence of

\

,a’

0

I I

FIG. 2. Log of molecular

) v,/x

10

=

weight versus migration during gel electrophoresis (A) and gel filtration (B). The numbered points represent the following molecular weight standards: 1 and 2, dimer and monomer of bovine serum albumin; 3, catalase; 4, y-globulin heavy chain; 5, actin (presumptive); 6, aldolase; 7, deoxyribonuclease I; 8, y-globulin light chain; 9, chymotrypsinogen A; 10, cytochrome c. The starred point represents ornithine decarboxylase. The linear relationship (r = 0.99) between log molecular weight and R, or V,/V, was used to calculate the apparent molecular size of ornithine decarboxylase. A: Simultaneous electrophoresis on sodium dodecyl sulfate-acrylamide gels of Fraction III and standards as described in the text. After staining with Coomassie brilliant blue, the destained gels were scanned at 550 nm with a Gilford linear recording spectrophotometer. The R, is the calculated ratio of the migration distance of the protein band to the tracking dye. B: Gel filtration on Bio-Gel A-0.5m, of Fraction II and of the standards was performed as described in the text. The elution volume (V,) was determined and expressed as a ratio (V,/V,) of the void volume (V,) measured with blue dextran.

414

BOUCEK

AND

Gel A-0.5m column (1.5 x 55 cm) at 4°C in Tris-HCl (25 mM, pH 7.51, EDTA (0.1 mM), dithiothreitol (2.5 mM), NaCl (0.05 MI, and polyethylene glycol (O.l%, w/v) after concentration dialysis overnight of Fraction II against this same buffer. The recovered activity (80%) eluted in a single peak with a molecular weight of approximately 110,000 estimated by comparison with the V,/V, of various standard proteins run on the same column under identical conditions (Fig. 2B). Polyethylene glycol was added to the column buffer to improve the stability of the enzyme during chromatography. DISCUSSION

The unique feature of the method described in this report for the purification of ornithine decarboxylase is the affinity chromatography step, which achieved a significant purification (approx 500-fold) and high recovery of active enzyme (Table I). Although the pyridoxamine phosphateagarose matrix utilized in the purification procedure is not a specific adsorbent for ornithine decarboxylase, the relatively low affinity of this apoenzyme for cofactor (lo), as compared with other pyridoxal phosphate-requiring enzymes, apparently allows selective elution of the decarboxylase from the affinity matrix. The mild requirements for the elution of ornithine decarboxylase (approx 4 pM pyridoxal phosphate, 15 mM NaCl) may be compared, for example, with the conditions necessary to elute tyrosine aminotransferase from a similar affinity matrix. In the latter case a transition to pH 4, 0.5 M NaCl and 10 mM pyridoxal phosphate was necessary for elution of the enzyme (19). Experience with this affinity matrix would suggest that the decarboxylase binds to the cofactor prior to the formation of the Schiff’s base intermediate and in the absence of the substrate. This binding of cofactor prior to substrate has been postulated to be the first step in the conversion of apo- to holoenzyme (23). The pyridoxamine phosphate-agarose matrix, which is readily prepared in a one-step coupling procedure, should be applicable to the isolation of ornithine decarboxylase from

LEMBACH

other sources, since partially purified enzyme of specific activity comparable to that of Fraction II has been achieved from other systems (Table I). A potentially significant observation is the finding that the specific activity of the decarboxylase from SVlOl cells is at least eightfold greater than that previously reported for the enzyme purified from rat liver (12). The variation in specific activities could reflect an intrinsic difference in the ornithine decarboxylase from cultured cells as compared with tissues or between normal and virus-transformed cells. The latter possibility is consistent with the higher activity of enzyme previously found in crude homogenates of transformed versus nontransformed fibroblasts (7, 8) and in various hepatomas (6). Estimations of the molecular weight of the ornithine decarboxylases purified to date have varied from 65,000 (10) to 160,000 (22). With the use of sodium dodecyl sulfate-gel electrophoresis, we have demonstrated that the subunit molecular weight of the purified fibroblast enzyme is approximately 55,000 (Fig. 2A), whereas by gel filtration in the absence of pyridoxal phosphate the active enzyme was found to exhibit an apparent molecular weight of 110,000 (Fig. 2B). Our estimation of the subunit molecular weight of the fibroblast enzyme thus differs from the 90,000 molecular weight species found by sodium dodecyl sulfate-gel electrophoresis of an immune-precipitable protein obtained from a rat liver homogenate with monospecific anti-ODC antibody (24). The molecular weight of the active enzyme estimated by gel filtration (110,000) is similar to the value of 100,000 found for the rat liver enzyme (12), but differs from the molecular weight estimates of 65,000 for the decarboxylase from rat prostate (10) and 160,000 for one form of ornithine decarboxylase from Physarum (22). These differences may reflect variations between species or cell types. Alternatively, the enzyme may undergo monomer-polymer transitions in vitro and this may account for some of the observed variability in the molecular weights (22). The activity of the Fraction III enzyme

PURIFICATION

OF ORNITHINE

was extremely labile and was found to be stabilized by albumin and other proteins and by polyethylene glycol (Table II). In addition, following prolonged incubation with albumin (or casein), the activity of the enzyme was actually greater than that observed with the addition of these proteins only during the assay interval (Table II). This latter observation, which is difficult to explain by a simple stabilization of the active enzyme by added protein, may suggest the existence of different activity forms of ornithine decarboxylase in vitro. Such an interconversion of active forms of the enzyme with an accompanying change in molecular size has been shown to occur in Physarum (22). ACKNOWLEDGMENTS The authors wish to express their sincere appreciation to Mrs. Janet Covington and Mrs. Sharon Tollefson for their technical assistance throughout the course of these studies. The assistance of Dr. David Puett in the preparation of this manuscript is gratefully acknowledged. REFERENCES 1. RUSSELL, D. H., AND SNYDER, S. H. (1968) Proc. Nat. Acad. Sci. USA 60, 1420-1427. 2. JANNE, J., AND RAINA, A. (1968) Acta Chem. Scand. 22, 1349-1351. 3. PEGG, A. E., LOCKWOOD, D. H., AND WILLIAMSASHMAN, H. G. (1970)Biochem. J. 117,17-31. 4. JANNE, J., AND RAINA, A. (1968) Biochim. Biophys. Acta 166, 419-426. 5. MORRIS, D. R., AND FILLINCAME, R. H. (1975) in Annual Review of Biochemistry (Snell, E. S., ed.), Vol. 43, pp. 303-325, Annual Reviews, Inc., Palo Alto, Calif. H. G., COPPOC, G. L., AND 6. WILLIAMS-ASHMAN, WEBER, G. (1972) Cancer Res. 32, 1924-1932.

DECARBOXYLASE

415

K. J. (1974) Biochim. Biophys. Acta 354, 88-100. DON, S., AND BACHRACH, U. (1975) Cancer Res. 35, 3618-3622. FILLINGAME, R. H., JORSTAD, C. M., AND MORRIS, D. R. (1975) Proc. Nat. Acad. Sci. USA 72, 4042-4045. 10. JANNE, J., AND WILLIAMS-ASHMAN, H. G. (1971) J. Biol. Chem. 246, 1725-1732. 11. FRIEDMAN, S. J., HALPERN, K. V., AND CANELBiophys. Acta LAKIS, E. S. (1972) Biochim. 261, 181-187. 12. ONO, M., INOUE, H., SUZUKI, F., AND TAKEDA, Y. (1972) Biochim. Biophys. Acta 284,285-297. 13. HOLTTA, E. (1975) Biochim. Biophys. Acta 399, 420-427. 14. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 15. MAIZEL, J. V., JR. (1971) in Methods of Virology (Maramorosch, K., and Koprowski, H., eds.), Vol. 5, pp. 179-246, Academic Press, New York. 16. LAZARIDES, E., AND WEBER, K. (1974)Proc. Nat. Acad. Sci. USA 71, 2268-2272. 17. LAZARIDES, E., AND LINDBERG, U. (1974) Proc. Nat. Acad. Sci. USA 71, 4742-4746. 18. LINDBERG, U., AND ERIKSSON, S. (1971) Eur. J. Biochem. 18, 474-479. P., AND THOMP19. MILLER, J. V., CUATRECASAS, SON, E. B. (19721 Biochim. Biophys. Acta 276, 407-415. 20. PEGG, A. E., AND WILLIAMS-ASHMAN, H. G. (1968) Biochem. J. 108, 533-539. 21. HARIK, S. I., AND SNYDER, S. H. (1973) Biochim. Biophys. Acta 327, 501-509. 22. MITCHELL, J. L. A., CAMPBELL, H. A., AND CARTER, D. D. (1976) FEBS Lett. 62, 33-37. 23. FONG, W. F., HELLER, J. S., AND CANELLAKIS, E. S. (1976) Biochim. Biophys. Acta 428, 456465. 24. THEOHARIDES, T. C., AND CANELLAKIS, Z. N. (1976) J. Biol. Chem. 251, 1781-1784. LEMBACH,