Fur. J. Biochem. 81. 545-5550 (1977)

Ornithine Decarboxylase Antizyme Is a Normal Component of Uninduced H-35 Cells and Rat Liver John S HELLER, Dimitri KYRTAKIDIS, Wang F FONG, and E S CANFLLAKIS Department of Pharmacology, Yale University School of Medicine, New Haten, Coniiecticut (Received Junc 27, 1077)

Ornithine decarboxylase antizyme (a non-competitive protein inhibitor of ornithine decarboxylase induced by the addition of putrescine, spermidine, spermine to a variety of cell lines and rat liver) has been detected in unstimulated H-35 cells and in normal rat liver. The ornithine decarboxylase antizyme is bound to subcellular components in a nonreactive form from which it can be released as the free antizyme by treatment with high concentrations of salts or low concentrations of diamines or polyamines. Of the latter, putrescine is the most effective; 1 pM putrescine will release free ornithine decarboxylase antizyme from subcellular particles. The molecular weights and general physical properties of the bound ornithine decarboxylase antizyme are the same as those of the induced ornithine decarboxylase antizyme. In €1-35 cells, 80% of the bound form of the ornithine decarboxylase antizyme is localized in the nucleus whereas only 35 01;of the bound form of the ornithine decarboxylase antizyme is found in the rat liver nucleus. The remaining activity is not associated with ribosomes or polysomes but is localized in a particulate fraction that sediments through a 5-40% sucrose gradient. This bound form of ornithine decarboxylase antizyme also does not react with ornithine decarboxylase unless it is first liberated by polyamines. The presence of ornithine decarboxylase antizyme bound to particles in unstimulated cells and its liberation from the particles by low levels of polyamines as well as its induction in the intact cell by polyamines suggests that it may play a role in modulating ornithine decarboxylase activity in-vivo. Ornithine decarboxylase catalyzes the conversion of ornithine to putrescine and is reported to be the rate-limiting enzyme in the biosynthesis of the polyamines [l,21. Putrescine, a weak competitive inhibitor of ornithine decarboxylase in the assay system in vitro [2,3], has been reported to dramatically inhibit the increase in ornithine decarboxylase activity in rat liver after stimulation by growth hormone [4], partial hepatectomy [4], dexamethasone (J. S. Heller, unpublished result), in mitogen-stimulated human lymphocytes [ 5 ] and in hormone-stimulated mouse 3T3 cells [6]. Evidence from our laboratory indicates that putrescine, when added to cultures of L-I 210, H-35, and N-18 neuroblastoma cells or injected into the intact rat, promotes the synthesis of a protein inhibitor to ornithine decarboxylase [7,8]. This inhibitor has a molecular weight of about 26500 .t 2000, is heatlabile, and its activity is destroyed by treatment with chymotrypsin or trypsin. The ornithine decarboxylase inhibitor is found in the 100000 x g supernatant fraction after induction with putrescine, spermidine or Enzyme. Ornithine decarboxylase (EC 4.1.1.17).

spermine. We have suggested the name antizyme for this type of protein inhibitor of an enzyme whose synthesis is induced by the product of the reaction it inhibits [8]. The ornithine decarboxylase antizyme displays non-competitive kinetics with respect to substrate [7] and binds tightly to ornithine decarboxylase. The assay for ornithine decarboxylase antizyme involves the addition of supernatants or extracts to a known quantity of partially purified rat liver ornithine decarboxylase and measuring the amount of inhibition of the activity of ornithine decarboxylase. It should perhaps be emphasized that, because of the nature of the reaction, ornithine decarboxylase antizyme can be detected by direct assay, only in cell extracts in which there is no ornithine decarboxylase activity (i.e. where ornithine decarboxylase antizyme is present in excess). We have looked for the presence of ornithine decarboxylase antizyme in normal cells that have not been induced for its synthesis. In this fashion we have attempted to establish whether ornithine decarboxylase antizyme is a normal component of cells or whether

546

Ornilhine Decarboxylase Antizymc

it is an artifact of the stress imposed upon the cell by high levels of polyamines. Our results indicate that ornithine decarboxylase antizyme exists in cells, uninduced for the synthesis of ornithine decarboxylase antizyme. In these cases it exists in a particle-bound form, and can be identified in subcellular fractions of H-35 cells and of rat liver. This bound form of ornithine decarboxylase antizyme does not react with ornithine decarboxylase unless it is extracted in free form, most specifically with low concentrations of putrescine. The molecular weight and the general properties of the bound ornithine decarboxylase antizyme are identical to those of the ornithine decarboxylase antizyme isolated from cells which have been induced by polyamines. MATERIALS AND METHODS Materials

~ ~ - [ l - ' ~ C ] O r n i t h i(32.2 n e Ciimol) was obtained from New England Nuclear and diluted with unlabeled DL-ornithine to give a final specific activity of 7.55 Ci/ mol. Putrescine, spermidine, and spermine were from Aldrich and all other chemicals were of reagent grade obtained from Sigma. Female rats (1 50- 180 g) were purchased from Charles River Laboratories (Wilmington, Mass.) The medium and serum were from Gibco. Preparation of Cell Extracts

H-35 Cells. H-35 cells (obtained from Dr. F. T. Kenney, Oak Ridge Lab.) were cultured on 47.5 x 11-cm Bellco roller bottles (Vineland, N. J.) in minimum Eagles medium [Y] (cat, F12) fortified with 10 fetal calf serum in a 5 7; COziair environment at 37 "C. The cells were harvested at confluency by scraping the sides of the bottle with a rubber policeman and washed twice with NaCIlPi. The final cell pellet (approximately 5 x 10' cells) was resuspended in 15ml of Tris buffer A (50 mM Tris pH 7.3 at 23 "C, 50 mM KCl and 1 mM MgCL), and homogenized in a Dounce homogenizer. Nuclei were isolated by centrifuging the homogenate at 600 x g for 10 min. The nuclear pellet was washed twice in Tris buffer A by centrifugation, resuspended in Tris buffer A with 0 . 5 x NP-40 [lo], washed twice by centrifugation at 600 x g and the nuclei resuspended in 5 ml of Tris buffer. The nuclear supernatant fraction was combined with the first two washes; this was centrifuged at 100000 x g for 1 h to provide the supernatant fraction and the pellet; the pellet was resuspended in 15ml Tris buffer A. Examination of the nuclear preparation by phase microscopy showed the nuclei to be relatively free of cytoplasmic tabs. There was a contamination of unbroken cells which varied from 1 */, to 5

x

x.

in

Unstimulated Cells

Rat Liver. Female rats were decapitated, the livers quickly removed and placed in approximately 4 vol. of 0.33 M sucrose and 0.004 M CaC12. The livers were homogenized in a smooth glass PotterElvehjem homogenizer by 9- 10 strokes of a motordriven loose-fitting Teflon pestle. The homogenate was fractionated by differential centrifugation. The nuclei were isolated according to the method of Chauveau et al. [I I ] as modified by Wang [12]. The sucrose concentration of the nuclear supernatant was diluted to 0.25 M with Tris buffer A and the mitochondria were isolated according to Sedgwick and Hubscher [13]. The smooth endoplasinic reticulum, rough endoplasmic reticulum, and polysomes were isolated on discontinuous sucrose gradients according to Aldelmdn et al. [14], The nucleoli and the nucleoplasm were isolated from sonicated nuclei as described by Busch [15]. Morphological criteria as determined by light, phase and electronmicroscopy of these fractions correspond to those provided in the respective references [14,15]. The 100000 x g supernatant fraction and the buffer washes of the particulate fractions were assayed for ornithine decarboxylase antizyme activity after extraction and none was detectable. The routine extraction of all subcellular fractions from rat liver and H-35 cells was performed with 1 0 mM putrescine, spermidine, and spermine in Tris buffer A and the mixtures stirred for 10min at room temperature. EDTA was then added to a final concentration of 2 mM, the mixtures stirred for an additional 10 min, 0.77 mg/ml dithiothreitol added and the samples centrifuged at I00000 x g for 1 h. The supernatants were dialyzed for 15 h at 4 "C against 100 vol. of Tris buffer B, (10 mM Tris pH 7.2-7.3 at 4 " C , 50 mM KCI, 1 mM EDTA, 0.05 mM pqridoxdl-P, 2.5 m M dithiothreitol) and then for 4 h in the same buffer minus KCI. Portions of the dialyzed preparations were assayed for ornilhine decarboxylase antizyme activity using approximately 0.5 unit of partially purified ornithine decarboxylase [16]. Microassay for Ornithine Decarboxylase and for Omithine Decarboxylase Antizyme

The incubation mixture contained in a final volume of 0.05 ml: 2.5 pmol Tris buffer (pH 7.1 at 37 "C), 0.05 pmol EDTA, 0.002 ymol pyridoxal-P, 0.25 pmol dithiothreitol, 0.028 pmol ~ ~ - [ l - ' ~ C ] o r n i thine, approximately 0.5 unit of ornithine decarboxylase activity (62 unitslmg); portions of the dialyzed extracts ranging from 10 p1 to 50 p1 were then added. The reactions were performed in 12 x 75-mm Falcon tubes (2058) each fitted with a 0.25-in (6.35-mm) paper disc impregnated with 0.025 ml of NCS/toluene (lil, vlv) transfixed to a disposable syringe needle which pierced the plastic cap. (NCS was obtained from

547

.I. S. Heller, D. Kyriakidis, W. F. Fong, and E. S. Canellakis

Amersham/Searle.) The reactions were stopped after a 1-h incubation by injecting 0.1 ml of 10 trichloroacetic acid (wlv) through the syringe needle into the incubation mixture, thc syringe needle was stoppered, incubation continued fur an additional hour to ensure complete release of radioactive COZand the paper discs counted as previously described [16]. All ornithine decarboxylase assays were performed in duplicate and ornithine decarboxylase antizyme assayed at three different concentrations of extract. All experiments were performed at least twice and the averages of the normalized values are presented. One unit of enzyme activity is defined as that catalysing the release of 1 nmol of COZ. 1 unit of inhibitor is defined as the amount of inhibitor that will inhibit 1 unit of enzyme activity. Protein concentration was measured by the method of Ross and Schatz using bovine serum albumin as the standard ~71. RESULTS

Table 1. Extraciion qf' ornithine decarbox~vlu,wantizyme from the total particulate fi.action of rat liver The livcrs from 2 rats were homogenized in Tris buffer A (2 ml/g), divided into 7 portions and centrifuged at l 0 0 0 0 0 x g for 1 h. The pellets were resuspended in 5 ml of Tris buffer A, recentrifuged at 100000 x g for 1 h, the indicated extractants added and ornithine decarboxylase antizyme extracted and assayed as described in Methods. The values reported are the means f S.E. for 3 separate experiments except for the KCl and urea extractions which were single experiments Fxtractant

Concn

Ornithine decarboxylase antizyme extracted

activity

specific activity

M

units

units :mg

0.01 0.01 0.01 0.01 0.01 0.01 0.3 1.0 6.0

0.20

total

Putrescine + spermidine + spermine Putrescine Spermine Spermidine KCl KC1 Urea

+ 0.04

0.30 f 0.06

0.15 k 0.02 0.18 & 0.03 0.02 0.15 0.20 0.21 0.30

0.20 & 0.03 0.11 0.02 0.07 0.01 0.07 0.07 0.08

*

**

Properties of the Antizyme

The molecular weights of the extractable ornithine decarboxylase inhibitor activity were determined using Sephadex chromatography. The H-35 and rat liver proteins had molecular weights of 26000 + 2000 and 27000 + 2000, respectively (data not shown) as compared to 26500 2000 reported previously for the induced ornithine decarboxylase antizyine [7]. The other properties, such as heat lability and sensitivity to proteases, were comparable to those previously described for the induced ornithine decarboxylase antizyme [7], i.e. t l i 2 of approximately 8 min at 55 " C ; the activity was unaffected by treatment with RNAse or DNAse while substantial loss of activity occurred after incubation with carboxymethylcellulose-bound chymotrypsin or trypsin. The molecular weights and properties of the extracted antizyme were constant throughout the fractionation studies reported bel ow.

+

Table 2. Treatment of the post-mitochondria/ pellet with puirescine, spcrmidinr and spermine The post-mitochondria1 supernatant from 8 rat livers was centrifuged at 100000xg for 1 h, the pellet resuspended in 100ml of Tris buffer A and recentrifuged. The pellet was resuspended in 30 ml of Tris buffer A. divided into 7 portions. the indicated concentrations of putrescine, spermidine and spermine added and ornithine decarboxylase antizyme extracted and assayed as described in Methods. The values reported are the mean S.E. for 3 separate experiments

*

Polyamine

Putrescine Spermidine

Release of the Antizyme

Sperrnine

Table 1 summarizes the results obtained when the total particulate fraction from rat liver was treated with a variety of agents (urea, KCl, putrescine, spermidine, and spermine). Urea released the largest total amount of ornithine decarboxylase antizyme ; however, this extract had a low specific activity. Treatment of the pellet with a solution containing 10 mM putrescine, spermidine, and spermine released over 70 % of the activity as compared with urea extraction; however, the specific activity was 3.4 fold greater. The total amount of extractable ornithine decarboxylase antizyme from non-induced H-35 cells and

Buffer

Concn

Ornithine decarboxylase antizyme extracted total activity

specific activity

PM

units

units/mg

10 1 10 1 10 1

2.6 0.40 0.6 k 0.10 1.0 0.20 0.3 _+ 0.07 1.0 f 0.20

*

2.5 f 0.40 0.6 f 0.10 1.0 k 0.20 0.2 k 0.04 0.9 f 0.20

k 0.02

0.4

-

0.4

~

* 0.02

rat liver was 4- 8 of that found in the fully induced H-35 cells and rat liver. Table 2 summarizes the data obtained when the post-mitochondria1 pellet was treated with low levels of putrescine, spermidine and spermine. Putrescine at 10 pM was as effective as the higher combined levels of putrescine, spermidine and sperrnine in releasing ornithine decarboxylase antizyme whereas

Ornithine Decarboxylase Antizyine in Unstimulatcd Cells

548 Tablc 3. Distributiori u ] vrnithine decarhoxylase antizymr activity in rai liver and 11-35 cells (A) Subcellular distribution in rat liver and H-35 cells. The aubcelM a r fractions were isolated as described in Methods and the ornithine decarboxylase antizymc extractcd with 10 inM each of putrescine, spermidine and spermine in Tris buffer A. The assays were performed as described in Methods. (B) Relative distribution in rat liver nucleus and endoplasmic reticulum

(A) Tissue

8h

Ornithine decarboxylase antizyme activity in nucleus

mitochondria

100000 x g postmitosupernatant chondrial pellet

-

14 5s

y,, lotal 10

H-35 cells Rat liver

80 38

(B) Fraction

Ornithine decarboxylase antizyme

6

0 0

uU, :

Nucleus Nucleoplasm Endoplasmic rcticulum : smooth rough Polysomcs

so 50

30 70 0

spermidine and spermine at 10 pM each were less effective. Treatment of the post-mitochondria1 pellet with buffer released less than 20% of the ornithine decarboxylase antizyme as compared to that released by putrescine. The higher specific activity in Table 2 as compared to Table 1 reflects the difference in bulk proteins present in the total particulate fraction and the post-mitochondria1 pellet. The higher combined levels of polyamines were used in the subsequent fractionation studies to ensure better recovery of the ornithine decarboxylase antizyme.

Distribution of the Antizynze Table 3 summarizes the subcellular distribution of ornithine decarboxylase antizyme in H-35 cells and in rat liver. Most of the ornithine decarboxylase antizyme activity in H-35 cells was found in the nucleus, whilc only 38 of the activity was in the rat liver nuclei. The ornithine decarboxylase antizyme activity of the rat liver nucleus was found to be distributed equally between the nucleolus and nucleoplasm. The bulk of the remaining activity in the rat liver was found in the post-mitochondria1 pellet. The post-mitochondria1 pellet was further separated into the smooth endoplasmic reticulum, the rough endoplasmic reticulum and the free polysomes. The