'We thank Drs. Jay Degen and David Morris of the Department of Biochemistry, University of Washington, Seattle, for providing us with the procedure for washing ...
THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 256. No. 23. Issue of December 10, pp. 12156-12163, 1981 Printed in U.S.A.
Ornithine Decarboxylase from Saccharomyces cerevisiae PURIFICATION, PROPERTIES, AND REGULATION OF ACTIVITY * (Received for publication, May 29, 1981)
Ani1 K. ”yagi, Celia White Tabor, and Herbert Tabor From the Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,Maryland 20205
Ornithine decarboxylasehas been purified 1,500-fold (11, 12). We have found, however, that spe2 mutants of S. to homogeneity from a spe2 mutant of Saccharomyces cereuisiae that lack S-adenosylmethionine decarboxylase (12) cerevisiae which lacks S-adenosylmethionine decarboxare derepressed for ornithine decarboxylase activity.’ When ylase and is derepressed for ornithine decarboxylase. grown in a defined medium, the spe2-4 mutant had 4 to 6 This ornithine decarboxylase is a single polypeptide times higher ornithine decarboxylase activity than the parent (Mr = 68,000) and requires a thioland pyridoxal phos- wild type strain AN33. This spe2-4 mutnnt of S. cerevisiae phatefor activity. Additionof M spermidine and was used as theenzyme source for the purification and other M spermine to the growth medium reduces the studies. activity of the enzyme by 90%in 4 h. However, immuThe enzyme has been purified 1500-fold to a specific activity noprecipitation studies showed that the extracts of polyamine-treated cells contain as much enzyme protein of 31 pnol/h/mg (Table I in Miniprint)and showed only one as normal cell extracts. Thisloss of ornithine decarbox- band on polyacrylamide gel electrophoresis both in the absence (data not shown)and presence of sodium dodecyl sulfate ylase activity is probably due to a post-translational modification of enzyme protein because we found no (Fig. 7 in Miniprint). The purified ornithine decarboxylase evidence for any inhibitor of activity in the polyamine- can be stored at -80 “C afteraddition of 5 mM dithiothreitol and 0.1 mM EDTA without loss of activity for at least 10 treated cells. weeks. Thus thepresent schemeof purifying Ornithine decarboxylase produced, with a 38% yield, a homogeneous preparation with a higher specific activity and greater stabilitythan Ornithine decarboxylase (EC 4.1.1.17), the first enzyme in reported for several other eukaryotic sources (7-9, 13). Ornithe biosynthesis of polyamines, is potentially important for a thine decarboxylase followed Michaelis-Menten kinetics from wide variety of metabolic processes (1-3). Elevated levels of pH 6.5 to pH9.0. Although V,,, was highest at pH 8.0, the K, ornithine decarboxylase and polyamines have been closely for ornithine was about twice its minimum value at that pH correlated with the increased growth rate of various eukar- (Table I1 in Miniprint). Pyridoxal phosphate was necessary yotic cells after stimulation by a variety of agents and have for catalytic activity ( K , = 0.6 p ~ (Fig. ) 8 in Miniprint). The been observed in tissues actively engaged in protein synthesis purified enzyme showed only 20% of the activity without the or during rapid proliferation (4-6). Ornithine decarboxylase addition of pyridoxal phosphate. This residual activity was has been purified from a varietyof sources, including rat liver, lost upon dialysis of the enzyme against 100 m~ cysteine or rat ventral prostate, and Escherichia coli (7-10). 20mM hydroxylamine. The activity could be restored (95%) In this paper we report the purification and properties of by addition of 50 p~ pyridoxal phosphate. ornithine decarboxylase from the eukaryote Saccharomyces Molecular Weight and Amino Acid Composition of the cereuisiae and the role of polyamines in the regulation of its Enzyme-The molecular weight of yeast ornithine decarboxactivity. Immunochemical studies indicate that polyamine- ylase was 68,000 (determined on a Sephacryl S-200 column). induced inactivation of ornithine decarboxylase in yeast re- Sodium dodecyl sulfate gel electrophoresis in the presence of sults from modification of existing enzyme protein. 5 mM dithiothreitol showed a single band with M, = 68,000, indicating that the native enzyme is a monomer (Fig. 7 in EXPERIMENTAL PROCEDURES’ Miniprint). The amino acid composition of the enzyme is given in Table I11 of the Miniprint. RESULTS Half-life of Ornithine Decarboxylase-Ornithine decarboxActiuity and Purity of the Isolated Enzyme-Previous stud- ylase in various eukaryotic systems is known to have a very ies have shown that wild type S. cereuisiae has a low level of short half-life (8-30 min) when determined by the decay of ornithine decarboxylase when grown to high optical densities activity after cycloheximide (14-17). The activity in yeastwas * The costs of publication of this article were defrayed in part by measured after addition of cycloheximide or trichodermin3 to the medium. Both inhibitors atthe concentrations used the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 blocked protein synthesis by more than 95% in less than 10 solely to indicate this fact. (data not shown). From these data, the half-life of orni’ Portions of this paper (including “Experimental Procedures,” part min thine decarboxylase in yeast was about 6 h (Fig. l),which is of the “Results,” Figs. 7-10, and Tables I-IV) are presented in much longer than in other eukaryotes (14-17). miniprint at the end of this paper. Miniprint is easily read with the Loss of Ornithine Decarboxylase Activity upon Addition aid of a standard magnifying glass.Full size photocopies are available from TheJournal of Biological Chemistry, 9650 Rockville Pike, of Polyamines-The addition of spermidine and spermine to Bethesda, MD 20814. Request Document No. 81M-1289, cite authors, and include a check or money order for $8.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
M. S. Cohn, C. W. Tabor, and H. Tabor, unpublished data. Trichodermin was a generous gift from Dr. W. 0. Godtfredsen of Leo Pharmaceutical Products, Bellerup, Denmark.
12156
Decarboxylase Ornithine from
s. cerevisiae
12157
nmol/h/107 cells, respectively. The K , values for the transport were 4.0 PM for spermidine and4.3 p~ for spermine(Fig. 10 in Miniprint). Preparation of Antiserum and Quantitation of Immunologically Reacting EnzymeProtein-Loss of enzyme activity following polyamine addition could be the result of inhibition, of inactivation, orof actual degradationof enzyme. Hence, we
40
30
20
10
0
4 0
2.0
4.0
6.0
8.0
TIME fhrl
FIG. 1. Decay of ornithine decarboxylase activity following treatment of growing cells with cycloheximide or trichodermin. Cells were grown to an Assoof 1.5, and cycloheximide (200 pg/ m l ) (M or) trichodermin (20 pg/ml) (M was )added to the culture (zero time on the figure). The culture was shaken at 28 "C, and at different time periods samples were removed.The activity of ornithine decarboxylase was measured as detailedin the text and expressed as micromoles of COz/h/mg of protein.
0
2.0
4.0
6.0
6.0
the culture medium effected a rapid loss of ornithine decarboxylase activity. In 4 h >90% of its activity was lost (Fig. 5.0 2 A ) . The doubling time for these cells was 5 h in the absence of polyamines and 4.5 h after the addition of polyamine^.^ Addition of cycloheximide to the medium at the same as time 4.0 polyamines or earlier prevented the rapid loss of ornithine decarboxylase activity. However, if cycloheximide was added to the medium 1 h after the polyamines, the same loss of 3.0 activity wasobserved as with polyamines alone (Fig. 2 A ) . This indicates that the loss of ornithine decarboxylase activity caused by polyamines is dependent on protein synthesis. 2.0 Wheneitherspermidineorsperminewasaddedtothe , decarboxylase activity culture separately (100 p ~ ) ornithine 1.o decreased; spermine was approximately twice as effective as spermidine. Together the effect of these polyamines was additive. No loss of activity was observed when putrescine was 0 added to the medium even a t a concentration of 2.5 mM. In 6.0 0 2.0 4.0 this connection it shouldbe noted that since the spe2 mutant lacksS-adenosylmethionine decarboxylase itcannotmake TIME Ihr) spermidine or spermine from putrescine. FIG. 2. Loss of ornithine decarboxylase activity upon polyfor amine addition to the growth medium and the As the spe2 mutant used in these studies is derepressed effect of cycloornithine decarboxylase, we also studied the effect of polya- heximide. A , yeast mutant spe2 was grown to an A m of 1.5. At this mines on the activity of ornithine decarboxylase in the wild stage the culture was divided into 5 equal portions. To the 1st sample and ) 2ndsample (Lk-EI), a mixture of spermine and typestrain of S. cerevisiae. Polyamines causeda similar (H . the 3rd decrease in ornithinedecarboxylase activity inwild type yeast spermidine was added to a final concentration of 100 p ~ To sample (A-A), the same concentrations of spermine and spermi(Fig. 2 B ) . dine and 200 pgof cycloheximide/ml were added.To the 4th sample Transport of Polyamines-Since the differential inhibition (M only ), cycloheximide (200 pg/ml) was added. To the 5th of ornithine decarboxylase activity by polyamines in the pres- sample (U which ), served as a control, nothingwas added. One ence and absence of cycloheximide might have been the resulthour after these additions,200 pg of cycloheximide/ml were added to Portions wereremoved at designated of a depressed rate of polyamine transport in the presence of the 2ndsample (M). cycloheximide, transport of spermidine and spermine in the intervals, and the activity of ornithine decarboxylase was measured as described under "Experimental Procedures" (Miniprint).B, wild yeast was studied in the presence and absence of cyclohexi- type cells were grown to an A m of 1.5 and the culture divided into 4 mide. Transport of polyamines was a saturable process under equal portions. To the 1st sample (o"-o), 100 p~ of spermine and the conditions used and was not affected by the addition of 100 p~ spermidine(finalconcentration)wereadded;to the 2nd 200 pg of cycloheximide/ml; and to the 3rd sample cycloheximide (Fig. 9 in Miniprint). The V,,, values for the sample (A-A), both polyamines and cycloheximide in the same concentransport of spermidine and sperminewere 4.8 nmol and 0.48 (M), trations. To the 4th sample (U nothing ), was added. Portions These cells were not completely deprived of intracellular polya- were removedat designated time periods, and the activity of ornithine mines. After12 generations inthe absence of polyamines, the doubling decarboxylase was estimated as given in the text and expressed as time is 12 h (12). micromoles of COz/h/mg of protein.
12158
Decarboxylase Ornithine
FIG.3. Immunodiffusion precipitin reaction of rabbit serum with ornithine decarboxylase. T h e center dl ( E ) contained 10 pI of purified enzyme (300pg/ml). The outer u d l s contained 10 p1 of antiserum in well 1 and 10 PI of preimmune serum in u~ell2;the other wells were empty.
from S. cerevisiae (1060 cpm and 1095 cpm, respectively), although the activity of ornithine decarboxylase in the polyamine-grown cells was only 2% of the activity in the control cells. In comparable experiments we showed that the amount of antiserum used was sufficientfor complete precipitation. These studies establish that polyamine-induced loss of enz-yme activity does not result from the degradation of enzyme but that presumably modification of the enzyme proteinresponsible is forrendering it catalyticallyinactive. Search for Dissociable Inhibitor orAntizyme-Since Canellakis and his associatesandothershave described the presence of an “antizyme” in several types of cells (Le. a noncompetitive protein inhibitor which is induced by polyamines and which noncovalently interacts with ornithine decarboxylase) (19-22), we searched for such an antizyme in yeast with the techniques described by Kyriakidis et at. (19) and McCann et al. (22). When extractsof cells (spe2 mutant) grown in the standard medium were mixed with extracts of cells grown in the same medium containing 100 p~ spermidine and 100 p~ spermine, no evidence foran inhibitor was found. Extracts of cells grown in the presence of 100 p~ spermidine and 100 p~ spermine were fractionated with (NH4)*S04; the various fractions were dialyzed extensively to remove (NH4)2S04and then lyophi-
0.5 0.4
assayed the amount of enz-yme protein following polyamine addition by immunoprecipitation.Antiserumagainstyeast 0.3 ornithinedecarboxylasewas raisedin rabbits by injecting them with the purified antigen as described under “ExperimentalProcedures” in Miniprint.Double immunodiffusion 0.2 Ouchterlonyanalysis of immuneandcontrolseraagainst ornithine decarboxylase showed only a single band with im0.1 mune serum (Fig. 3). The antiserum quantitatively precipitated the enzyme (Fig. 4 ) . 0 The results of double immunodiffusion analysis using purified enzyme and extracts of cells grown in the absence or presence of polyamines (2.5 mM spermidine and2.5 mM spermine and containing virtually no ornithine decarboxylase activity) are presentedin Fig. 5. The immune serumgave only one continuous band, and the intensityof bands from polyaminetreated or untreated cells was the same, indicating that enzyme protein was present in approximately equal amountsin the extracts from polyamine-treated and untreatedcells. For quantitation of enzyme protein in these cell extracts, cells labeled with [:’%]methionine were used, and ornithine decarboxylase was immunoprecipitated from labeled extracts of cultures grown with or without polyamines and containing equal amounts of protein. The resulting immunoprecipitates were washed as described5 under “Experimental Procedures” in Miniprintandseparated by electrophoresisonsodium 1.0 3.0 5.0 7.0 9.0 11.0 dodecyl sulfate gels. The gels were then subjected to autoradiography. The immunoprecipitates from both control and ANTIGEN ADDED (pg) polyamine-grown cells showedonly onebandcomigrating FIG. 4. Quantitative precipitation reaction of rabbit serum with the purified enzyme. The intensity of this band was the with purified ornithine decarboxylase. A, the reaction mixture same in the control extract asin the extract frompolyamine- contained 50 p1 of antiserum (M or) 50 pl of preimmune serum treated cells (Fig. 6). These enzyme bandswere then cut from (A-A) and indicated amounts of purified enzyme in a total volume the gels, and the radioactivity was measured by scintillation of 250 pl. After incubation for 60 min at 37 “C followed by overnight spectrometry. The same amount of enzyme protein was pres- incubation at 4 “C, the precipitates were sedimented by centrifuging at 10,OOO X g for 15 min. The precipitates were washed twice with 0.3 ent in the extracts from control and polyamine-grown cells
P
‘We thank Drs. Jay Degen and David Morris of the Department of Biochemistry, University of Washington, Seattle, for providing us with the procedure for washing the immunoprecipitates, in advance of publication.
ml of 0.15 M NaCI, 0.02 M sodium phosphate, pH 7.3, and protein in the precipitate was measured using a modified Lowry-Fohn method. B,the activity of ornithine decarboxylase in the supernatants of the above reaction after removing the immunoprecipitate. With antisewith preimmune serum, M. rum, H;
Decarboxylase Ornithine
FIG.5. Immunodiffusion precipitin reaction of immune serum with purified enzyme, crude cell extract, and extracts of cells grown on polyamines. Thecentral well (S) contained 15 pl of immune serum. The outer toefls ( I and 4 ) contained 7 pl of purified enzyme (200 pg/ml). U‘elfs 2 and 5 contained 20 pl of crude cell extract (15 rng of protein/ml), and wells 3 and 6 contained 20 pI of crude cell extract (15 mg of protein/ml) from cells grown with added polyamines.
S
N
P
116K
68K 43K 30K
”
21K FIG. 6. Autoradiography of sodium dodecyl sulfate gels of immunoprecipitates from labeled cultures grown with and without polyamines. Cells were grown in the medium containing 2 pCi/ml of L-[.”SSJmethionineand 10 pg/ml of L-methionine. At A,,.*,of 1.5, the culture was divided into 2 equal portions, and a mixture of spermidine and spermine (final concentration 100 p ~ was ) added to one of them. Six hours after the addition of polyamines, the cells were harvested and washed 3 times withdistilled water. Extracts were prepared by a single pass through a French press a t 18,000 p s i . and centrifuging the resulting suspension for 30 min a t 20,000 X g.Equal amounts of cell extract protein (400 p g ) were subjected to immunoprecipitation. The immunoprecipitates were collected by using excess of protein A covalently coupled toSepharose CL-4B (39). These immunoprecipitates were washed as described under “Experimental Procedures”(Miniprint)andseparated on sodium dodecyl sulfate gels, and the gels were subjected to autoradiography. S, molecular weight standards; N,immunoprecipitate from control cell extract; P , immunoprecipitate from extracts of cells grown with polyamines.
from S. cerevisiae
12159
lized. The residues were dissolved ina small amountof 25 mM Tris-HC1 buffer, pH 8.0, and checked for inhibitory activity (19). Also, concentrated extracts of the cells grown with polyamines were separated on a Sephadex G-75 column (2.5 cm X 50 cm), and the fractions were tested for any activity of ornithine decarboxylase or inhibitorof ornithine decarboxylase. This chromatography was also performed using 250 mM NaCl to facilitate the dissociation of any enzyme-antizyme complex (22). Extracts were also prepared fromcells grown in the medium containing 10 mM spermine and 10 mM spermidine, or in medium supplemented with all the amino acids plus 100 p~ spermidine and 1 0 0 p~ spermine and chromatographed ona Sephadex G-75 column in a high salt concentration. However, all attempts were unsuccessful in demonstrating the presence of any inhibitor of ornithine decarboxylase activity in these extracts. Alteration in the Affinity for Pyridoxal Phosphate-Sedory and Mitchell have reported two forms of ornithine decarboxylase in Physarum polycephalum which are reversible modifications of the same enzyme protein anddiffer in their affinities for pyridoxal phosphate (23). However, the loss of activity of the yeast enzyme causedby polyamines could not be reversedby pyridoxalphosphate in the concentration range of 0.1 to 200 p~ in the assay system. DISCUSSION
The regulation of ornithine decarboxylase has been studied in a variety of systems, and several models have been suggested. These include feedback inhibition, enzyme degradation, repression of synthesis, and the formationof an inhibitory protein (“antizyme”)(14,15,19-22,24-26). None of these models, however, seems to be directly applicable to the yeast system describedin this paper. Post-translational modification has also beensuggested (23,26-29), but theonly experimental evidence presented for such a change in vivo in the enzyme was an alteration in the affinity for pyridoxal phosphate (23, 29). Very recently, Russell (30) has reported that the activity of ornithine decarboxylase is decreased when the purified enzyme is modified by the in vitro incorporation of putrescine, catalyzed by added transglutaminase. To study the control of ornithine decarboxylase in yeast, we investigated changesin the enzyme activity after spermine and spermidinewere added to thegrowth medium. We found that the activity fell by 90% within 4 h. Immunochemical studies showed that there was no loss in enzyme protein, indicating that proteinmodification, rather than proteindegradation, accountedfor the loss in activity. Noloss of activity was observed when putrescine was added. Feedback inhibition was excluded by measuring the ornithine decarboxylase activity of the inhibited extracts after dialysis and by mixing inhibited and noninhibited cell extracts; also, spermine and spermidine at 100 p~ concentrations did notinhibittheenzymewhendirectlyaddedtotheassay mixture. Repression could not account for the results, since theamount of immunologically reactiveprotein did not change; also, the loss of activity after polyamine addition was much more rapid than the loss of activity aftercycloheximide or trichodermin treatment. A change in the affinity for pyridoxal phosphate was excluded by measuring the activity a t different pyridoxal phosphate concentrations (see “Results”). Wewere particularlyinterested in testingwhetherthe antizyme model applied to the yeast system, sincewith such a model there would be no decrease in the amount of immunologically reactive material. IndeedKallio et al. (31) reported that, although the activity of rat liver ornithine decarboxylase decreases after the administrationof diaminopropane in vivo to rats, therewas a gradual increase in the relative amountof
Decarboxylase Ornithine
12160
immunoreactive ornithine decarboxylase. They showed that their results could be explained by the formation of an inhibitory proteinfollowing the administration of diaminopropane. As described under “Results,” we have not found any evidence for the presence of an antizyme in yeast, although we were able to c o n f i i the findings of Canellakis and his coworkers of an antizyme in E. coli (19). Interestingly, this antizyme preparation from E. coli inhibited the purified preparation of yeast ornithine decarboxylase. It should be noted that even though an antizyme to ornithine decarboxylase has been reported in several systems (19-22), such an inhibitory protein could not be detected in several other tissues and cells after diamine treatment, even thoughthe ornithine decarboxylase activity was completely inhibited (32,33). Thus, yeast appears to have a novel system for the control of ornithine decarboxylase by post-translational modification, and further work is in progress to investigate what modification of the protein results in this inactivation. REFERENCES 1. Tabor, C. W., and Tabor, H. (1976) Annu. Rev.Biochem. 45,285306 2. Williams-Ashman, H. G., and Canellakis, Z. N. (1979) Perspect. Biol. Med. 22,421-453 3. Janne, J., Poso, H., and Raina, A. (1978) Biochim. Biophys. Acta 473,241-293 4. Morris, D. R., and Fillingame, R. H. (1974) Annu. Rev. Biochem. 43, 303-325 5. Pegg, A. E., and Williams-Ashman,H. G. (1968) Biochern. J. 108, 533-537 6. Raina, A., and Janne, J . (1975) Med. Biol. 53, 121-147 7. Friedman, S. J., Halpern, K. V., and Canellakis, E. S. (1972) Biochim. Biophys. Acta 261, 181-187 8. Pegg, A. E., and McGill, S. (1979) Biochim. Biophys. Acta 568, 416-427 9. Janne, J., and Williams-Ashman,H. G . (1971) J. Biol. Chem. 246, 1725-1732 10. Applebaum, D. M., Dunlap, J. C., and Morris, D.R. (1977) Biochemistry 16,1580-1584 11. Kay, D. G., Singer, R. A,, and Johnston, G. C. (1980) J. Bacteriol. 141, 1041-1046 12. Cohn, M. S., Tabor, C.W., and Tabor, H. (1980) J. Bacteriol. 134, 208-213 13. Ono, M., Inoue, H., Suzuki, F., and Takeda, Y. (1972) Biochim.
from S. cereuisiae Biophys. Acta 284,285-297 and Snyder, S. H. (1969) Mol. Pharmacol. 5,25314. Russell, D. H., 262 15. Obenrader, M. F., and Prouty, W.F. (1977) J. Biol. Chem. 252, 2866-2872 16. McCann, P. P., Tardif, C., and Mamont, P. S. (1977) Biochem. Biophys. Res. Commun. 75,948-954 17. Inderlied, C. B., Cihlar, R. L., and Sypherd,P. (1980) J. Bacteriol. 141,699-706 18. Cohn, M. S., Tabor, C. W., and Tabor, H. (1980) J. Bacteriol. 142,791-799 19. Kyriakidis, D. A., Heller, J. S., and Canellakis, E. S. (1978) Proc. Natl. Acad. Sci. U. S. A . 75,4699-4703 20. Heller, J. S., Chen, K. Y., Kyriakidis, D. A., Fong, W. F., and Canellakis, E. S. (1978) J . Cell. Physiol.96,225-234 21. Fong, W. F.,Heller, J. S., and Canellakis, E. S. (1976) Biochim. Biophys. Acta 428,456-465 22. McCann, P. P., Tardif, C., Hornsperger, J. M., and Bohlen, P. (1979) J. Cell. Physiol. 99, 183-190 23. Sedory, M. J., and Mitchell, J. L. A. (1977) Exp. Cell Res. 107, 105-110 24. Clark, J . L. (1974) Biochemistry 13,4668-4674 25. Kay, J . E., and Lindsay, V. J. (1973) Biochem. J., 132, 791-796 26. J h n e , J., and Holth, E. (1974) Biochem. Biophys. Res. Commun. 61,449-456 27. Clark, J. L., and Fuller, J. L. (1975) Biochemistry 14, 4403-4409 28. Kallio, A. K., Poso, H., Scalabrino, G., and Janne, J. (1977) FFBS Lett. 73,229-234 29. Clark, J . L., and Fuller, J . L. (1976) Eur. J. Biochem. 67,303-314 30. Russell, D. H. (1981) Biochem. Biophys. Res. Commun. 99,11671172 31. Kallio, A., Lofmann, M., Poso, H., and Janne, J. (1979) Biochem. J. 177,63-69 32. Pegg, A. E., Conover, C., and Wrona, A. (1978) Biochem. J. 170, 651-660 33. Clark, J . L., and Fuller, J. L. (1976) Biochem. Biophys. Res. Commun. 73, 785-790 34. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 35. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 36. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412 37. Harboe, N., and Ingild, A. (1973) Scand. J . Zmmunol. 2,161-164 38. Mage, R., and Dray, S. (1965) J. Zmmunol. 95,525-535 39. Kessler, S. W. (1976) J. Immunol. 117, 1482-1490 40. Holtta, E., Janne, J., and Pispa, J. (1972) Biochem. Biophys. Res. Comrnun. 47, 1165-1171 41. Edelhoch, H. (1967) Biochemistry 6,1948-1954 42. Means, G . E., and Feeney, R. E. (1971) Chemical Modification of Proteins, Holden-Day, Inc., San Francisco
12161
Ornithine Decarboxylasefrom S. cerevisiae
RESULTS G r o w tH h e d 1aunPdl e p a r a t l oonCf e E l lx t r a c t S t r a i n was grown a t 28OC 1" S D m e d r u m (121 c o n t a m i n g2 %d e x t r o s ea n d 0.7% DlfCO y e a s t n l t r O g e n base without mlno a c l d s .A d e n l n e and threonine Were added t o a f l n a l concent r a t i o n Of 0.005% a n d0 . 0 2 5 % .r e s p e c t i v e l y ,a n dt h e pH was a d 3 u r t e d to 7.0 W l t hd l b a b l cp o t a 6 8 1 mp h o s p h a t e . for cmparative studies. adenine
When t h ep a r e n ts t r a i n
A N 3 3 (121 was w e d
~n t h e above medium was replacedwlrh0.025% were m d i n t l l n e d by grarlng them on YPAD p l a t e s ( a rlch medxum containmng sufficient amountsofpolyamines t o allow noma1 growth Of m u t a n t sd e f i c i e n t i n p o l y a m i n eb i o s y n t h e s i s ) (121. For p u r i f ~ c s t ~ o n , t h e cells were g r o w n i n a 1 0 0 - l L t e r f e r m e n t o r at 28'C and t h e culture was h i g h l y a e r a t e dd u r i n gt h eg r o w t h .T h e cells were h a r v e s t e d a t A650 lun of5.0. For a l l o t h e r Studies, t h e cells were g r o m I" 1-liter f l a s k s c o n t a l n l n g 500 m l medium a t 18'C o n a r o t a r ys h a k e r ( - 150 rpnl. In all Cases. t h e C e l l s were
a r g ~ n l n e . T h e s ee t r a l n s
s u s p e n d e d ln 0.025 H T r i s - c h l o r i d e , pH 7 . 6 , containing 5 mn d l t h m t h r e l t o l , 1 mH llgCI2, and 0 . 1 mM EDTA ( b u f f e r AI and were brokenby a s i n g l ep a s s t h r o u g h a F r e n c h p r e s s a t 18,000 PSI. The r e s u l t i n g suspension Uae Centrzf u g e d for 30 m l n u t e s a t 20,000 x g . P u r i f 1 C a t l O n Of O r n i t h i n e Decarboxylage - P U r l f x a t m n was c a r r l e d o u t With 300 g of cells ( w e t w e i g h t ) . A l l s t e p s d u r l n g p u r i f i c a t i o n were c a r r l e d o u t a t 0-4'c and pH values were measured a t room t e m p e r a t u r e . AmmOnlm S v l f a t e F r a C t l O n a t l O n - F o r e a c h 100 m l o f s u p e r n a t a n t . 10.8 4 Of "enzyme grade' a m m a n i ws u l f a t e was a d d e dw l t hs t i r r l n g . The p r e c i p l r a t e additional animonlm s u l f a t e ( 2 1 . 4 g/loo "1) t h es u p e r n a t a n t .T h er e s u l t i n gp r e c r p i t a t e ,w h i c hc o n t a i n e dm o s t
was d i s c a r d e d ,a n d
was added to
a c t l v l t y , was dissolved i n b u f f e r A, a n dd l a l y r e dO v e r n l q h ta g a l n s t1 5 A wxth 2 c h a n g e s Of b u f f e r .
Of t h e liters
of b u f f e r
-
DE-52 Chromatography T h e d i a l y z e d enzyme wan a p p l i e d to a OE-52 column ( 3 . 9 cm X 22 on) equilibrated w i t h 0.05 M NaCl m b u f f e r A. The column was washed w i t h 1 l i t e r o f e q u i l i b z a t x n g b u f f e r , and D r n l t h i n e d e c a r b o x y l a s e was t h e ne l u t e dW l t h a 550 m l llnear g r a d i e n to f0 . 0 5 M t o 0.35 M ~ a c l . The a . C t l v e f r a c t i o n s weze pooled, and 60116 amnonlvm s u l f a t e wa8 added t o 55% Saturation. A f t e rS t i T r i n qf o r 30 m i n u t e s .t h ep r e c i p i t a t e was collected by c e n t r l f u g a t i o n a t 2 5 , 0 0 0 x g f o r 1 5 m m u t e s an8 d i s s o l v e d m 8 m l o f b u f f e r A.
-
S e p h m r y l 5-200 T h ec o n c e n t r a t e d enzyme was l o a d e d o n t o a S e p h a c r y l 5-200 C O l W (2.5 C m x 90 Cml, whzch had been e q u i l l b r a t e d w l t h b u f f e r A. O r n l t h l n e d e c a r b o x y l a s e was e l u t e d a t d f l o w r a t e of 16ml/hr.Thepeakfract L O n 5 c o n t a l n i n g t h e enzyme were p o o l e da n dS u b l e c t e dt o (NH412S04 p r e c l p i t a t i o n 155% s a t u r a t i o n l . The p e l l e to b t a i n e da f t e rt h e centrifugation was d l = s o l v e d I n b u f f e r A c o n t a m i n g0 . 5 M (NH4l2SO4.
-
PhenylSepharoseChromatoqraphy The f i n a l p u r l f l c a t m n was achzeved on L column o fp h e n y l Sepharose (1.5 cm x 10 _I, Which had b e e n e q u i l l b r a t e d w l r h b u f f e r A c o n t a m i n g0 . 5 M ( N H 4 1 2 S 0 4 .A f t e rl o a d i n g .t h e column vas washedwith 100 m l Of b u f f e r A c o n t a l n l n g 0 . 5 H (NHq)2S0,, f o l l o w e d by 500 m l o f b u f f e r A c o n t a l n i n g0 . 2 5 M (NH4l2SO4. D r n r t h i n ed e c a r b o x y l a s e was t h e ne l u t e dw i t h a llnear reverse g r a d l e n to f0 . 2 5 H t o 0 . 0 M ammOnlUm S u l f a t e ~n 500 m l o f b u f f e r A. The column w a 5 washedWlth an a d d l t l o n a l 200 m l Of b u f f e r A c o n t a l n r n g no INH412S04. F r a c t l O n S near t h e end O f t h eg r a d i e n t , w h i c hc o n t a l n e dO r n i t h i n e d e c a r b o x y l a s e . were p o o l e d ,c o n c e n t r a t e d in an Amcon p r e s s u r e cell w l t h an M - 5 0 memDrane, a n dS t o r e d at - B O T .
Ornithine Decarboxylasefrom S. cerevisiae Enlnne Activity and EffectOf pH
- Under
the assay conditions used, the
reaction rate was proportional to enzyme concentration and was linear with time oyer a period of 20 minutes a t p~ values between 6 . 5 and 9 . 0 . Maximal r a t e s O f ornithine decarboxylation occurred at pH 8 . 0 . The \ O f ornithine at this pH was 91 YM, and the specific activity of the enzyme was 42 uml/hr/ng. Hence the experiments with purified enzyme were carried out at p~ 8 . 0 .
0.16
1
-
Inhibition by Substrate Analogs All Of the compounds listed in Table IV were inhibitors of ornithine decarboxylase activity. Among the amines tested, putrescine showed the highest affinity for theenzyme. Arginine and lyslne were rather weak inhibitors,whereas 0-methyl ornithine was the most potent of a11 Cmpounda. I - . 5-. a d 6-carbon amino acids that lack the 0 ammo group showed weaker affinity for the enzyme, indicating that o-amino groups (whish probably interact wlth bound pyridoxal phosphate during the enzyme reaction1 might be essential for high affinity.
-
Thiol c m p u n d s have been Effects Of Thiol C m p u n d s on the Activity reported to be necessary for the activity of ornithine decarboxylase in the mammalian system 1 9 ) . I D order to test this effect Of thiolcmpounds, the purified enzyme was dialyzed against large volumes Of buffer A containing no dithiothreitol. Removal o f thiols by dialysis caused total lossof activity, and there was Virtually no production Of C02 in the absenceof any sulfhydryl Canpound. Reactivation Of about 751 activity was achieved by the addition o f 1 nvl dithiothreitol, a d preincubating at 37.C for 5 minutesi the maximal dithiothreitol. activity 1- 9511 could bc recovered in the presence Of 5 Considerable reactiVation of enzyme activity was also observed with 5 nvl 2.3dimercapto-1-propanol I 8 5 8 of the activation obtained in the presence of 5 mn dithiothreitol), and lese with 5 m glutathione 1 1 9 8 ) , 5 nvl reduced coenzyme A (ll%), and 5 nvl 2-m.rcaptoethm01 In]. Oxidized dithiothreitol was totally
inactive. Effect
Of
Nucleotideson Ornithine
Decarboxylase
- HIlttl
Activity
5 toll by nucleoside triphosphates, especially by guanosine triphosphate. We tested the following nucleotides for theireffect on Yeast ornithine decarboxylase aCtiVity: w , cyclic M P , ADP, ATP, GMP, GDP, GTP, THP, TDP. TTP, UHP, UDP, VPP, CUP, CDP, and LTP. All of these were tested at nonsaturatinq concentratione of ornithine. At a low concentration 1100 Y W these nucleotides, with the exception of cyclic AMP, ATP, and TUP. activated the enzyme slightly 115-508). At high Concentrations 12.0 Wl the effect Was even leas prminent, and in sane cases slight inhibition Of enzyme activity instead was observed. NUCleOaide triphosphates have been reported to have no effect On mammalian ornithine decarboxylase ( 9 , 19). &
( 4 0 ) have reported activation of Ornithine decarboxylase of
A
116K-
94K68K-
-
I 0
1
I
1
I
1.o
2.0
3.0
4.0
[Pyridoxal ph~sphatel-~ &
I
'
-
F I G . 8. Lineweaver-Burk plots of ornithine decarboxylation as a functlon of pyridoxal phosphate concentration at v a c i o u ~concentrations of ornithlne. Purified e n r y l ~ awas dialyzed against 0.025 n Tris-HCI buffer, pH 8.0. contsininq 5 w dithiothreitol, 1.0 mn MgC12r 0.1 mn EDTA, and 20 nvl hydroxylamine for 6 hours with 1 change Of buffer. followed by dialysis against the s a w This preparation contained 21 o f its buffer containing no hydroxylamine. Original activity when assayedin the absence of added cofactor. while958 of the original activitywas recovered by brief incubation prior toassay with 50 YM pyridoxal phosphate. me symbols represent: 345 214 ornithine (-1, 260 vu Ornithine IA-1, and 112 VU ornithine 1-1. Reactions were carried out at 31-C in 0.025 n Tris-HC1 buffer. pH 8.0. containing 5 mn dithiothrsitol, 1.0 mn MgC12, 0.1 mn E m A , and v a r ~ o u sConcentrations Of pyridoxal phoaphate.
B
-l,;
pkhk-kG TIME Iminl I
30K- 0
21K"
0.3 0.1
Polyacrylamide gel electrophoresla of purified Ornithine decairboxylase from yeast. samples were denatured in 1% sodium dodecyl sulfate, 0 . 6 8 mercaptoethanol, and 5 mn dithiothreitol for 5 minutes at 100*c and loaded onto 10% polyacrylamide g e l s containing 0 . 2 % sodium dodecyl sulfate. I&)Standard marker proteins. Is1 Purified ornithine decarboxylase. FIG.
1.
J
u-
90 30
150
210
270
TIME lmml FIG. 9 . Influence O f cyclohexlrnlde on the transport Of aperrmdlneand spermme. Cella were grown l n SD medium C o n t u n l n g 28 dextrose, 0 . 1 8 D i f m yeast nitrogen base without m l n o aclds, 0 . 0 0 5 8 a d e n m e , and 0 . 0 2 5 % threonine. At an A650 Of 1 . 5 . the cells were diluted Wxth fresh m d l u m to a density of 10' cells/ml. 141 These cell suspensllons were Incubated at 28'C.Il'CIspermzdine was added to one flask to a final concentration of 100 YU 1-1; the second flask received 200 bg Of cyclohexmxde/ml in addition to spermidine I-). The flasks were shaken. and samples 11.0 m l l were removed at desrgnated t l m e periods. RadlODCtiVlty in the cells was measured after collecting the cells on membrane filters (pore slze 0.45 .:ul and washxng rapldly 3 rzmes wlth the medium contalnmg 1.0 nvl spermidine. IBI The transport of spermlne. Studxes were carried Out exactly as ~n (51 except that ll'Clspermlne was added to the medium (final concentration 100 .HI 1-( and 2 0 0 ;1g cyclohexlmlde/ ml, ~n addition to eperrnlne l S - " a l , were used
from S. cerevisiae
Decarboxylase Ornithine
12163 TABLE I1
Effect
3
r
-
Of
pH On Km and Vmx
Of o r n i t h i n e d e c a r b o x y l a s e
A s s a y s were performed as d e s c r i b e di n' E x p e r i m e n t a lP r o c e d n r e s "( m i n i p r l n t ) . H, P h o s p h a t e b u f f e r , 0.025 H, from pH 6 . 5 and 7.5, and Tris-HC1buffer,0.025
1
from pH 8.0 t o 9 . 0 , were used.
R l n e t i cc o n s t a n t s
were d e r i v e d frcm L ~ n e w e a v e ~ -
Burk p l o t s . PH
Km
"max
!E
'
ISperrnldlnel uM
'
vrnol/hr/mg
6.5 1.2 8.0
161 143
18
91
8.5 9.0
57
42 32
80
21
Amino a c i d a n a l y s i s Calculatedfor
TABLE I11 Of o r n i t h i n e d e c a r b o x y l a s e
nearest i n t e g e rv a l u ep e rm o l e moles
Realdue
25
moles
Resldue
of enzyme. 5 Residue
. . 6 1 a l a n i n e . . . . 17 P h e n y l a l a n i n e . . . GlntPmlC a c i d . . 60 Vallne . . . . . 41 Lysrne . . . . . . . Serine . . . . . 45 H192xdlne . . . . . 30 Isolevclne . . . Threonine . . . . 64 Arginine . . . . . . 33 Leuclne . . . . P r o l i n e . . . . . Methionine 23 . . . Tryptophan 10 a.... Glyclne . . . . . 24 TyrOSlne . . . . C y s1t8e i n e .....
A s p a r t l ca c i d
1.0
0
1 1
I
1
I
I
0.05
0.1
0.15
0.2
0.25
[Spermine!
I
0.3
2
' FM '
We
thank B a r b a r a F. T o r a i n f o r p e r f o r m i n g t h e
oeterminadbythemethod D e t e r m i n e db yr e a c t i o nw i t h g u a n i d i n eh y d r o c h l o r i d e( 4 2 ) .
Of
-no
moles 31 66
a 29 4
6
acid analyses
Edelhoch(411.
5,5'-dith~0-bi~~2-n~troben.oic acld) i n 6 H
TABLE IV I n h i b i t i o n of o r n i t h i n e deFaiTboXylase by s u b e t r a t e analogs Assay c o n d i t i o n s were 18 described ~n t h e t e x t e x c e p t t h a t enzyme was i n c u b a t e dw i t hi n h i b i t o r sf o r1 0m l n l l t e sb e f o r ea d d i n gt h eB U b s t r a t Ea n dt h e concent r a t i o no fo r n i t h i n e was 90 V4 (h a, p p r o x m a t e l y i t s X,,,'. Reeactlons were performed a t pH 8.0.
TABLE I
Purification Of ornithine decarboxylase The d a t a r e f e r t o 300 g w e t w e l g h t o f y e a s t [ s t r a i n s p e 2 1 as t h e S t a r t i n g material. A c t L v l t y Of o r n i t h i n e d e c a r b o x y l a s e was measured a t pH 1 . 6 , ln t h e presence Of 1 1 5 un O r n l t h m e and 100 V U p y r i d o x a lp h o s p h a t e a t 37'c. ktails o ft h ep r o c e d u r e are d e s c r i b e d i n " R e 8 U l t s '15 ( m m i p r l n tt e x t ) . acid STpoo eY t cfaiileflidc
alcf tiaiccvatititam vycint iyPnrtoyt e i ns t e p
3 7,900 21 41
umol/hr
urml/hr/mg
8
204 134
0.05
120
2
101
2.50
60 38
120 1,180
e x t r a cCte l l - f r e e
AmnoDIum s u l f a t e DE-52 79 c~llulose 5-200Sephacryl PhenylSepharose
-fold 1
4.240
0.45300 2
64
31
S u b sabntaaeC tlseoot g endc e n t r ar tei 5 inqo0hufn% liobrrei tdi o n
........
6-Amim-n-~dlericacxd L - Z , 4 - D i m i n o - n - h t y r i ca c i d e-Amino-n-caproic
..... ........
............. n-Hexylamine Putrescine . . . . . . . . . . . . . . Spermidine . . . . . . . . . . . . . . Spermine . . . . . . . . . . . . . . . L-Argmine .............. I-Lysine ............... Cadaverme . . . . . . . . . . . . . . 1.3-Diaminopropane o-Hathylomithine
.......... ...........
mn 12 50 20
0.1 10
5 58 40
2 20 0.05
