NOTES & TIPS
a substrate for all the experiments involving apoptotic
Purification of the Death Substrate
Damien D'Amours,* Patrick J . Duriez,* Kim Orth,²
Rashmi G. Shah,* Vishva M. Dixit,²
William C. Earnshaw,³ Emad S. Alnemri,§
and Guy G. Poirier*,2
*Poly(ADP-ribose) Metabolism Group, Health and
Environment Unit, CHUL Research Center, Room 9700,
2705 Laurier Blvd., Ste-Foy (Que Â bec), Canada, G1V 4G2;
²Department of Pathology, University of Michigan Medical
School, 1301 Catherine St., Box 0602, Ann Arbor, Michigan
48109; ³Institute of Cell and Molecular Biology, University
of Edinburgh, Edinburgh EH0 3J R, Scotland, United
Kingdom; and §Department of Pharmacology and the
J efferson Cancer Institute, Thomas J efferson University,
Philadelphia, Pennsylvania 19107
Materials and Methods
Preparation of the thymus extract for dsDNA±cellu-
lose af® nity chromatography.
cation was performed according to Yoshihara et al. (7)
as modi® ed in Shah et al. (8). Brie¯y, the calf thymus
was homogenized in buffer A (50 mM Tris±HCl, pH
8.0; 1 mM EDTA, pH 8.0; 0.3 M NaCl, 25 mM NaHSO3;
10 mM b-ME; 0.1 mM phenylmethylsulfonyl ¯uoride;
0.5 mM DTT) and centrifuged for 15 min at 7500g. The
supernatant (crude extract) was then fractionated by
two sequential precipitations using (NH4)2SO4 at 30
and 70% saturation. The 30±70% saturated extract
was centrifuged (10,000g, 40 min) and the pellet was
suspended in buffer B (same as buffer A except 0.2 M
NaCl) and desalted on Sephadex G-25 (medium; Phar-
macia) gel by elution with buffer B.
Double-stranded DNA±cellulose af® nity chromatog-
raphy of PARP.Puri® cation of PARP on dsDNA ±cel-
lulose was performed according to Zahradka and Ebi-
suzaki (9). The desalted preparation was applied on
a dsDNA ±cellulose (Sigma Chemical Co.) column and
washed with buffer C (50 mM Tris±HCl, pH 8.0; 1 mM
EDTA, pH 8.0; 0.2 M NaCl, 50 mM NaHSO3; 1 mM DTT;
10% glycerol). The enzyme was eluted with a linear
gradient of 0.2 to1.2 M NaCl in buffer C. PARP elution
was monitored by a poly(ADP-ribosyl)ation assay and
by determination of protein concentration (8). Active
fractions were pooled, concentrated ® ve times by bury-
ing in sucrose granules, and stored at 080?C until fur-
ther puri® cation.
3-Aminobenzamide af® nity chromatography of PARP.
PARP puri® cation through 3-AB Af® -Gel chromatogra-
phy was performed according to Ushiro et al. (10) with
modi® cations. Ten percent of the PARP puri® ed
through dsDNA ±cellulose was applied on a 3-AB Af® -
Gel (Bio-Rad) column (12 1 60 mm) and was washed
with 10 ml of buffer D (50 mM Tris±HCl, pH 7.4; 1 M
NaCl; 10 mM b-ME). The enzyme was eluted with a
linear gradient of 1 to 10 mM 3-MB in 50 ml of buffer
D. Fractions (0.5 ml/each) containing PARP, as moni-
tored by silver staining of SDS ±polyacrylamide gels
(11) and by determination of protein content, were
pooled and concentrated ® ve times using Amicon Cen-
tri¯o CF25 centrifugation cones.
Apoptotic fragment analysis.
was digested using bacterially expressed CPP32 and
ICE-LAP3 as described previously (12). Western blot-
ting and Activity±Western blotting wereperformed us-
ing antibodies CII-10 (13) and 10H (14), respectively,
as described (8, 15).
This part of the puri® -
Received October 24, 1996
Poly(ADP-ribose) polymerase (PARP3) (EC 220.127.116.11)
is a nick-sensing enzyme which covalently modi® es a
variety of chromatin proteins toform poly(ADP-ribose)
from NAD/(reviewed in Ref. 1). During the execution
phase of apoptosis, PARP appears tobe among the ear-
liest death substrates to be cleaved (2, 3) and its cleav-
age has been con® rmed in virtually all cases of
apoptosis whenever studied. In previous studies, re-
combinant PARP was widely used as a substrate for
the apoptotic proteinases due to its easy availability.
However, in vivo, PARP displays various levels of post-
translational modi® cation by ADP-ribose polymers (1)
which are potent inhibitors of at least one proteolytic
activity (4). Theinhibitory potential of pADPr on protein-
ases is further underscored by the observation that sev-
eral poly(ADP-ribosyl)ated proteins, including PARP,
have shown a marked resistance tothe proteolytic activ-
ity of various proteinases (5, 6). Consequently, recombi-
nant PARP appears to be an inappropriate substrate to
study apoptosis since its posttranslational status is most
probably different from that of the native enzyme.
We have developed a simple and rapid method to
purify undegraded PARP from calf thymus toapparent
homogeneity. Despite the fact that this puri® cation in-
volves potent inhibitors of PARP, the puri® ed enzyme
still displays substantial activity and it can be used as
1This work was supported by the Cancer Research Society and
the Medical Research Council of Canada.
2To whom correspondence should be addressed. Fax: (418) 654-
3Abbreviations used: 3-AB, 3-aminobenzamide; b-ME, b-mercap-
toethanol; dsDNA ±cellulose, double-stranded DNA ±cellulose; DTT,
dithiothreitol; ICE, interleukin-1-b-converting enzyme; 3-MB, 3-me-
thoxybenzamide; pADPr, poly(ADP-ribose); PARP, poly(ADP-ribose)
3-AB-puri® ed PARP
ANALYTICAL BIOCHE MISTRY 249, 106±108 (1997)
ARTICLE NO. AB972153
Copyright ? 1997 by Academic Press
All rights of reproduction in any form reserved.
NOTES & TIPS
Puri® cation of Calf Thymus Poly(ADP-ribose) Polymerase
Speci® c activity
3-AB Af® -Gel
aUnderestimation, see text.
Results and Discussion
This new puri® cation procedure of PARP was de-
signed for rapid puri® cation of large quantities of
PARP uncontaminated by any of the Ç45- to 80-kDa
degradation fragments often found in some prepara-
tions (16, 17). Since these degradation fragments lack
a substantial portion of the enzyme, we reasoned that
the complete enzyme could be puri® ed from its shorter
fragments by a procedurethat speci® cally selects mole-
cules with complete C- and N-termini in two distinct
steps. Thus, the new puri® cation procedure was based
on two key steps of af® nity chromatography using as
ligands 3-AB, a potent inhibitor of theenzyme(18) that
interacts with the C-terminal catalytic domain, and
DNA, the cofactor of the enzyme that interacts with
the N-terminal DNA binding domain (1).
The summary of a typical puri® cation starting with
500 g of calf thymus is shown in Table 1. The initial
step of this puri® cation procedureinvolves thefraction-
ation of the crude extract by selective precipitation
with ammonium sulfate. Although this step is not
highly selective for PARP, it substantially eliminates
many cellular contaminants of the crude extract and
stabilizes PARP (7). Ammoniumsulfatewas eliminated
from the fraction by exclusion chromatography on
Sephadex G-25 (9). As shown in Table 1 and Fig. 1
(lane 3), most of the cellular proteins are eliminated
after the dsDNA ±cellulose step. This PARP was puri-
® ed 298-fold and had a speci® c activity of 360 U/mg
(Table 1). At this step of the puri® cation, PARP corre-
sponds to approximately 50% of the total protein con-
tent of the sample as evaluated from Fig. 1 (compare
lanes 3 and 4). The sample was then further puri® ed
by 3-AB Af® -Gel af® nity chromatography. 3-AB is a
potent inhibitor of PARP (IC50of 33 mM) (18) and is
believed tointeract with the catalytic domain at the C-
terminus of the enzyme. Although this procedure does
not immobilizePARP on thecolumn becauseof thelow-
af® nity constant of 3-AB for PARP (18), it suf® ciently
delays the enzyme's elution to separate it from other
proteins of thesample(10). Hence, sincethemain crite-
rion in this study was to increase the ef® ciency of the
elution, we eluted the enzyme with a 1 to10 mM gradi-
ent of 3-MB which is a stronger inhibitor of PARP (IC50
of 17 mM) than 3-AB. This consistently resulted in
sharper elution pro® les andhigher yields of PARP com-
pared to results obtained under the previously de-
scribed conditions (10, 19).
Surprisingly, 3-AB-puri® ed PARP displayed a fairly
high speci® c activity (397 U/mg) without further elimi-
nation of the inhibitors (Table 1). Most interestingly,
full catalytic activity of the enzyme or its apoptotic
fragments may be recovered after SDS ±PAGE and
transfer ontonitrocellulose membrane (Fig. 2, compare
lanes 5 and 6). This enables one to use Activity±West-
ern blotting tomapthecleavagesites of theproteinases
along the enzyme (Fig. 2, lane 7). Puri® cation of PARP
on 3-AB Af® -Gel was very ef® cient sinceapproximately
50% of the dsDNA ±cellulose-puri® ed PARP was recov-
ered after 3-AB Af® -Gel chromatography. Further-
more, theenzymepreparation was devoid of any degra-
dation fragments of PARP and displayed apparent
homogeneity as con® rmed by silver staining of SDS ±
polyacrylamidegel (Fig. 1, lane4) andWestern blotting
(Fig. 2, lane 1). Figure 2 shows that 3-AB-puri® ed
PARP can be cleaved by the apoptotic proteinases
F IG. 1.
different steps of the puri® cation procedure. Lane 1, crude extract
(6 mg); lane 2, 30±70% ammonium sulfate fraction (6 mg); lane 3,
dsDNA ±cellulose pool (300 ng); lane 4, 3-AB Af® -Gel 10 pool (165
ng). Other bands were not detected when more 3-AB-puri® ed PARP
was loaded. Silver staining was performed according to Desnoyers
et al. (11).
Silver-stained 10% SDS ±polyacrylamide gel showing the
NOTES & TIPS Download full-text
11. Desnoyers, S., Shah, G. M., Bourassa, S., and Poirier, G. G.
(1995) Anal. Biochem. 232, 138±140.
12. Orth, K., Chinnaiyan, A. M., Garg, M., Froelich, C. J ., and Dixit,
V. M. (1996) J . Biol. Chem. 271, 16443±16446.
13. Lamarre, D., Talbot, B., de Murcia, G., Laplante, C., Leduc, Y.,
Mazen, A., and Poirier, G. G. (1988) Biochim. Biophys. Acta 950,
14. Kawamitsu, H., Hoshino, H., Okada, H., Miwa, M., Momoi, H.,
and Sugimura, T. (1984) Biochemistry 23, 3771±3777.
15. Shah, G. M., Kaufmann, S. H., and Poirier, G. G. (1995) Anal.
Biochem. 232, 251±254.
16. Holtlund, J ., J emtland, R., and Kristensen, T. (1983) Eur. J .
Biochem. 190, 309±314.
17. Kameshita, I., Yamamoto, H., Fujimoto, S., and Shizuta, Y.
(1985) FEBS Lett. 182, 393±397.
18. Banasik, M., Komura, H., Shimoyama, M., and Ueda, K. (1992)
J . Biol. Chem. 267, 1569±1575.
19. Burtscher, H. J ., Auer, B., Klocker, H., Schweiger, M., and
Hirsch-Kauffmann, M. (1986) Anal. Biochem. 152, 285±290.
F IG. 2.
calf thymus PARP and its apoptotic fragments. Detection of PARP or
its 89-kDa apoptotic fragments using CII-10 antibody. 3-AB-puri® ed
PARP (lane 1); 75,000 apoptotic HL-60 cells (lane 2); 3-AB-puri® ed
PARP cleaved by CPP32 (lane 3); and ICE-LAP3 (lane 4). Detection
of catalytically active PARP or its apoptotic fragments using anti-
pADPr antibody 10H. 3-AB-puri® ed PARP (lane 5); PARP puri® ed
by the conventional procedure (9) (lane 6); and 3-AB-puri® ed PARP
cleaved by ICE-LAP3 (lane 7). One hundred and ® fty nanograms of
PARP was analyzed in each lane.
Western and Activity±Western blotting of af® nity-puri® ed
A Radioactive Assay for Sialyltransferase
Activity Using 96-Well Multiscreen
CPP32 and ICE-LAP3 to generate the signature 89-
kDa apoptotic fragment.
We have thus established a rapid and very speci® c
proceduretopurify milligram quantities of undegraded
PARP from calf thymus. This procedure will be ex-
tremely valuable to study the speci® cities and enzy-
matic activities of the ICE-related proteinases toward
a genuine death substrate.
Wouter Laroy, Marleen Maras, Walter Fiers,
and Roland Contreras1
Department of Molecular Biology, Flanders Interuniversity
Institute for Biotechnology and University of Ghent,
B-9000 Ghent, Belgium
ssa for expert technical advice and Manish Garg for technical assis-
tance. Anti-poly(ADP-ribose) antibody 10H was a generous gift of A.
We thank Danie Ále Poirier and Sylvie Boura-
Received December 24, 1996
Sialic acid is a complex sugar commonly found as a
terminal residue on glycoconjugates (1, 2). It appears
in animal cells, where it has a broad range of functions
(3). To synthesize all sialylated oligosaccharides ob-
served, a large number of ST2are needed. Thirteen
enzymes with distinct characteristics have been cloned
so far (4). A number of them are highly cell-speci® c,
while others are generated by alternative splicing or
alternative promoter utilization in a tissue-dependent
way (5±7). The enzyme is normally located in trans-
Golgi but can alsobereleasedin serum after proteolytic
The need for easy and sensitive ST assays is steadily
growing. The enzymatic activity in different animal
1. Lautier, D., Lagueux, J ., Thibodeau, J ., Me Ânard, L., and Poirier,
G. G. (1993) Mol. Cell. Biochem. 122, 171±193.
2. Takahashi, A., and Earnshaw, W. C. (1996) Curr. Opin. Genet.
Dev. 6, 50±55.
3. Casiano, C. A., Martin, S. J ., Green, D. R., and Tan, E. M. (1996)
J . Exp. Med. 184, 765±770.
4. Inagaki, T., Miura, K., and Murachi, T. (1980) J . Biol. Chem.
5. Boulikas, T., and Poirier, G. G. (1992) Biochem. Cell. Biol. 70,
6. Desmarais, Y., Me Ânard, L., Lagueux, J ., and Poirier, G. G. (1991)
Biochim. Biophys. Acta 1078, 179±186.
7. Yoshihara, K., Hashida, T., Tanaka, Y., Ohgushi, H., Yoshihara,
H., and Kamiya, T. (1978) J . Biol. Chem. 253, 6459±6466.
8. Shah, G. M., Poirier, D., DuchaiÃne, C., Brochu, G., Desnoyers,
S., Lagueux, J ., Verreault, A., Ho¯ack, J .-C., Kirkland, J . B., and
Poirier, G. G. (1995) Anal. Biochem. 227, 1±13.
9. Zahradka, P., and Ebisuzaki, K. (1984) Eur. J . Biochem. 142,
10. Ushiro, H., Yokoyama, Y., and Shizuta, Y. (1987) J . Biol. Chem.
1To whom correspondence should be addressed at Laboratory of
Molecular Biology, K. L. Ledeganckstraat 35, B-9000 Ghent, Bel-
gium. Fax: (32 9) 264-53-48.
2Abbreviations used: CMP-NeuAc, cytidine 5?-monophospho-N-
acetylneuraminic acid; ST, sialyltransferase; a-2,3-ST, CMPNeuAc:
Galb1-3/4GlcNAc-R a-2,3-sialyltransferase; a-2,6-ST, CMPNeuAc:
ANALYTICAL BIOCHE MISTRY 249, 108±111 (1997)
ARTICLE NO. AB972168
Copyright ? 1997 by Academic Press
All rights of reproduction in any form reserved.