Worm Breeder's Gazette 14(4): 28 (October 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Medical Chemistry, University Medical School of Debrecen, H-4026 Debrecen, Bem ter 18/B, Hungary, e-mail: dombradi@jaguar.dote.hu
Protein phosphatases (PP) are significant regulatory enzymes
of all eukaryotes. They must be present in C.elegans as witnessed
by DNA-deduced PP-like amino acid sequences unraveledin the
framework of the genom project. About half of them belong to
Ser/Thr PP, while the rest is Tyr PP. For the assay of the main
Ser/Thr PP classes, namely PP1, PP2A, PP2B and PP2C Cohen
and coworkers suggested a straightforward and relatively simple
procedure (1) that was adopted for the analysis of the worm.
Rabbit muscle phosphorylase a phosphorylated by phospho-
rylase kinase, rabbit muscle inhibitor-1 and casein from bovine milk
phosphorylated by bovine heart cAMP-dependent protein kinase
were used as substrates. C.elegans Var. Bristol (N2) wild type
strain were cultured, harvested, rinsed with the extraction buffer,
frozen in liquid nitrogen and homogenized in four volumes of the ice
cold extraction buffer (50 mM Tris-HCl pH=7.4; 0.1 mM EDTA;
10 mM DTT; 0.5% Triton X-100; 2mM PMSF; 5mM benzamidine
and 1 mM o-phenantroline).The homogenates were cleared by cent-
rifugation at 14000 g, 4 oC for 10 min and than were kept frozen
in liquid nitrogen till the assays.
Rabbit skeletal muscle inhibitor-2 (unphosphorylated) was used
as a specific inhibitor ofPP1 with 32P-phosphorylase a substrate in
the presence ofEDTA,a metal ion chelator(Fig.1).The titration curve
reveals that about 75%of the total activity was supplied by PP1 and
5 nM of inhibitor-2 caused about 50% inhibition. Okadaic acid, a
potent marine toxin and tumor promoter, was used to separate PP1
and PP2A activities with 32P-phosphorylase a substrate in the pre-
sence of EDTA (Fig.2). The titration curve with okadaic acid is bi-
phasic, in the first step about 30% of the total activity is inhibited
with an IC50 of ~0.04 nM and the rest of activity was inhibited in a
second step with an IC50 of ~70 nM. Complete inhibition was
achieved by 1 micromol/liter okadaic acid concentration. It is known
that PP2A is more sensitive to okadaic acid than PP1, thus the first
step of the titration can be explained by the presence of PP2A while
the second step can be attributed to PP1 inhibition.
For the detection of PP2B we used 32P-inhibitor-1 substrate
and Ca-calmodulin as an activator. Due to the interference of prote-
ases the 32Pi liberated in the assays was estimated after ammonium
molybdate extraction(2). About 50 nmol substrate was converted in
1 min by the extract. ~70% of the activity was inhibited by 4 mM
EDTA or 1.5 mM trifluoperazine and ~30% was inhibited by 2 nM
okadaic acid (not shown). Thus 70% of the total activity was due
to PP2B and 30% to PP2A.
PP2C was assayed with 32P-casein substrate in the presence
of Mg2+-activator. ~10%of the activity was inhibited by inhibitor-2
(PP1) ~40% of the activity was inhibited by 2 nM okadaic acid
(PP2A) and ~50% was stimulated by Mg2+ (PP2C). Half maximal
activation was achieved at 2 mM Mg2+ concentration, maximal
activation was measured at 20 mM (Fig.3.).
Acknowledgements.This work was supported by the grants
OTKA 6005 and 12840.
(1) Cohen, P., Klump, S., Schelling, D.L. (1989) FEBS Lett. 250,
596-600.
(2) Shenolikar, S., Ingebritsen, T.S. (1984) Meth. Enyzmol. 107,
103-129.
Figure 1.
Inhibition of phosphorylase
phosphatase activity by rabbit
muscle inhibitor-2 in a crude
C.elegans extract.The activity
measured in the absence of
inhibitor-2 was 0.8 mU/mg,
and was taken as 100%.
Figure 2.
Inhibition of phosphorylase
phosphatase activity by okadaic
acid in a crude C.elegans extract.
100% is the same as in Fig.1.
Figure 3.
Stimulation of casein phosphatase
activity by magnesium ions in a
crude C.elegans extract. The
maximal PP2C activity was 0.29
mU/mg and was taken as 100%.