Protein Phosphatase Protocols

314 Pages·1998·20.445 MB·English

by  John W. Ludlow

1
Prokaryotic Protein-SerineiThreonine Phosphatases
Peter J. Kennelly
1. Introduction
1.1. Prokaryotic Protein-Serine/Threonine Phosphatases:
A Brief Review
1.1.1. Why Study Protein Phosphorylation Events in Prokaryotes?
As this chapter deals with the protein-serine/threonine phosphatases of
prokaryotic organisms, some comments on the role of prokaryotes in the study
of these important enzymes would appear to be in order. Prokaryottc organ-
isms dominate the living world. They represent by the largest source of biomass
on the planet, forming the indtspensable foundation of the food cham upon
which all other living organisms depend. They are the exclusive agents for
carrying out biological nitrogen fixation, and are responsible for the majority
of the photosynthetic activtty that generates the oxygen we breath. In absolute
numbers, in number of species, in range of habitat, and in the spectrum of their
metabolic activities, the prokaryotes far outpace their eukaryotic brethren.
More immediately, in humans prokaryotes perform essential functions in the
digestion and asstmilation of nutrients, whereas infection by bacterial patho-
gens can lead to illness or death.
The intrinsic biological importance of prokaryotic organisms m the bio-
sphere renders them important and interesting objects of study (1). Be that as it
may, the question remams as to why protein phosphorylation in prokaryotes
should be of interest to “mainstream” signal transduction researchers whose
attention has long been fixed on humans and other higher eukaryotes. At least
part of the answer lies in the recent realization that prokaryotes and eukaryotes
employ many of the same molecular themes for the construction and operatton
of their protein phosphorylation networks (2,3). Virtually every major family
From Methods IR Molecular Biology, Vol 93 Protern Phosphatase Protocols
Edlted by J W Ludlow 0 Humana Press Inc , Totowa, NJ
1
2 Kennel/y
of protem kinases or protein phosphatases identified in eukaryotlc organisms
possessesa prokaryotic homolog(s), and vice versa. Consequently, the prokary-
otes represent a volummous library of fundamentally important, universally
applicable information concerning the structure, function, origins, and evolu-
tion of protein kmases, protein phosphatases, and their target phosphoprotems.
In addition, prokaryotes offer significant advantages as venues for the study of
protem kmases and protein phosphatases, particularly with regard to dlssectmg
thetr physiological functions and the factors that Influence them. Prokaryotes
carry out their life functions and the regulation thereof utlhzmg a many-fold
smaller suite of genes and gene products than does the typical eukaryote.
Although they employ molecular mechanisms as subtle and sophlstlcated as
any found in “higher” orgamsms, the fewer “moving parts” in the prokaryotes
materially faclhtates the design, execution, and analysis of molecular genetic
experiments. In addition, their robustness m the face of a wide range of nutn-
tional and environmental challenges greatly facilitates the ldentlficatlon and
analysis of resulting phenotypes. The prokaryotes thus represent a rich and
presently underutilized tool for understanding the fundamental prmclples gov-
erning the form and function of protein phosphorylatlon networks.
1.1.2. Not All Prokaryotes Are Created Equal:
A Brief Outline of Phylogeny
Most readers of this chapter were taught that all living organisms could be
grouped mto two phylogenetlc domains whose names were often given as the
eukaryotes and the prokaryotes (4), However, these latter terms actually refer
to a morphological classlficatlon, not a genetic/hereditary one (5). The term
eukaryote describes those organisms whose cells manifest internal compart-
mentation, more precisely the presence of a nuclear membrane. The prokary-
otes include all organisms lacking such mtracellular orgamzatlon, m other
words everythmg that 1s not a eukaryote. Early studies of phylogeny based on
the first protein sequences, the gross structural and functional characteristics
of key macromolecules, the architecture of common metabolic pathways, and
so forth, suggested that this morphological classificatron of hvmg organisms
paralleled their hereditary relationships. However, as researchers gamed facll-
ity with the isolation, sequencmg, and analysis of DNA, a truly genetic out-
line of phylogeny has emerged, one that groups living organisms mto three
distinct phylogenetlc domains-the Eucarya, Bacteria, and Archaea (Archae-
bacteria) (6).
Whereas the prior supposition that the eukaryote morphological phenotype
characterized members of a coherent phylogenetic domain-the Eucarya-
proved correct, molecular genetic analysis revealed that the prokaryotes segre-
gated into two distinct and very different domains: the Bacteria and the
Prokaryotlc Phosphatases 3
Archaea. The domain Bacteria includes essentially all of the prokaryotrc
organisms one encounters m a typical mrcrobrology course E. co& Salmo-
nella, Pseudomonas, Rhizobium, Clostridia, Staphylococcus, Bacillus, Ana-
baena, and so on. The domain Archaea, on the other hand, IS populated largely
by extremophrles that occupy habitats whose heat, acidity, salmtty, or oxygen
tension render them hostile, rf not deadly to other hvmg orgamsms. However,
rt would be wrong to suppose that the Archaea are simply a set of unusual bacte-
ria. Examination of the genes encoding their most fundamentally important
macromolecules, ranging from DNA polymerase to ribosomal RNAs, make it
clear that the Archaea have much more in common with the Eucarya than they
do with the superficially-similar Bacteria (6,7). The earliest detectable branch
point in the evolutionary time line resulted m the segregation of the Bacterza
away from the organism that eventually gave rise to both the Eucarya and the
Archaea. The common progemtor of these latter domains then evolved for a con-
siderable period followmg this first btfurcation. As a consequence,m any mvesti-
gators believe that present day archaeonss till possessn umerous features reflective
of ancient proto-eukaryotes (7). This combmatron of prokaryotrc “srmplrcny”
with high relatedness to medically relevant eukaryotes render the Archaea a par-
ticularly mtngumg target for the study of protein phosphorylatton phenomena.
1. I. 3. Prokaryotic Protein-Serine/Threonine
Phosphatases ldentlfied to Date
When one considers that the modification of prokaryotrc proteins by phos-
phorylation-dephosphorylatron first was reported nearly 20 yr ago (a-lo),
surprisingly little is known about the enzymes responsible for the hydrolyses of
phosphoserine and phosphothreonme residues m these organisms. The first
prokaryotrc protein-serine/threonme phosphatase to be rdentrfied and charac-
terized was the product of the aceK gene in E coli (II). This gene encodes a
polypeptide that contains both the protem kmase and protein phosphatase
activities responsible for the phosphorylation-dephosphorylation of isocitrate
dehydrogenase. Today, AceK remains an anachronism by virtue of its hermaph-
roditic structure, and because the sequences of its protein kinase and protein
phosphatase domains are unique, exhibiting no srgmticant resemblance to other
protein kinases or protein phosphatases (12).
The next prokaryote-associated protein-serine/threonine phosphatase to be
discovered was ORF 221 encoded by bacteriophage h (13,14). This enzyme,
and a potential protein encoded by an open reading frame m bacteriophage
$80, exhibit significant sequence homology with the members of the PP1/2A/2B
superfamily, one of the two major families of eukaryotic protem-serme/threo-
nme phosphatases (15). Whereas this represented the first discovery of a
eukaryote-like protein phosphorylatron network component having any asso-
4 Kennel/y
ciation with a prokaryotic organism, the mobility and malleability of viral vec-
tors begged the question of whether the genes for these protein phosphatases
were bacterial in origin. Moreover, it remains unclear to what degree a protein
phosphatase from a pathogen can shed light on how bacterial proteins are
dephosphorylated under normal physiological cncumstances.
More recently, two unambiguously bacterial enzymes have been described
that possessp rotein-serine/threonine phosphatase activity. The first, IphP from
the cyanobactermm Nostoc commune (16), is a dual-specificity protein phos-
phatase that acts on phosphoseryl, phosphothreonyl, and phosphotyrosyl pro-
teins in vitro (17). Like other dual-specific protein phosphatases, IphP contams
the characteristic HAT (His-Cys-Xaa@g, or His-kg-Thiolate) active site
signature motif characteristic of protein phosphatases capable of hydrolyzing
phosphotyrosine (18). The second 1s SpoIIE from BaczlZuss ubtilu, a bacterial
homolog of the second major family of “eukaryotic” protein-serme/threomne
phosphatases, the PP2C family (19,20).
“Eukaryotic” protein-serine/threonine phosphatases have been uncovered m
the Archaea as well. In the author’s laboratory a protein-serine/threonine phos-
phatase, PPl -arch, has been purified, characterized, cloned, and expressed from
the extreme acidothermophilic archaeon Sulfolobus solfataricus (21,22). This
protein 1s a member of the PP1/2A/2B superfamily, with whose eukaryotic
members it shares nearly 30% sequence identity (22). Surveys of two other
archaeons, which are phylogenetically and phenotypically distinct from S.
solfutaricus, the halophile Haloferax volcanii and the methanogen Methano-
sarcina thermophda TM- 1, indicate that PP 1- arch from S. solfataricus 1s the
first representative of what may prove to be a widely distributed family of
archaeal protein-serine/threonine phosphatases (23,24). This recently has been
confirmed at the sequence level through the cloning of a second form of PPl-
arch from A4. thermophilu via the polymerase cham reaction (PCR).
1.1.4. Limited Applicability of Cohen’s Scheme
to the Classification Prokatyotic Protein-Serine/Threonine Phosphatases
Recent experience with prokaryotic protein phosphatases has revealed that
Cohen’s criteria for classifying the protem-serme/threonine phosphatases can-
not be extrapolated with confidence to prokaryotic enzymes. To briefly review,
in the early 198Os,C ohen and coworkers compiled a set of functional attributes
characteristic of each of the major protein-serme/threonine phosphatases found
in eukaryotes (25). These attributes mcluded their preference for dephosphory-
lating the a- vs the P-subunit of phosphorylase kinase, their sensitivity to the
heat-stable inhibitor proteins I-l and I-2, and the (m)dependence of their cata-
lytic activity on the presence of divalent metal ions such as Mg2+, Mn2+, or
Ca2+.I n later years sensmvity to potent microbial toxins-such as microcystm-
Prokaryotic Phosphatases 5
LR, okadaic acid, and tautomycin-that inhibited the activity of PPl and PP2A
were added to the list (26). While this scheme soon was adopted as standard for
the classification of eukaryotic protein-serine/threomne phosphatases, attempts
to apply it to prokaryotic enzymes have met with mixed success.F or example,
PPl-arch from S. solfataricus is okadaic acid-insensitive and requires exog-
enous divalent metal ions for activity (21). Under Cohen’s scheme, this would
classify it as a member of the PP2C family. However, the amino acid sequence of
PP 1- arch clearly places it in the PP 1/2A/2B superfamily (22). The same holds
true for another divalent metal ion-dependent, okadaic acid-insensitive PP 1/2A
homolog, ORF 22 1 from bacteriophage k (14).
7.2. An Overview of Methods
for Assaying, Purifying, and Identifying Clones
of a Prokaryotic Protein-Serine/Threonine Phosphatase, PPI-Arch
We use [32P]phosphocasein that has been phosphorylated using the catalytic
subunit of the CAMP-dependent protein kinase (27) as a general-purpose sub-
strate for the assay of protein-serine/threonine phosphatase activity in pro-
karyotic organisms. Although it is a eukaryotic phosphoprotein, all of the
prokaryotic protein-serine/threonine phosphatases that have been studied
(16,17,21-24), as well as the ORF 221 protein-serine/threomne phosphatase
from bacteriophage h (14), hydrolyze phosphocasem at a usefully high rate in
vitro. Its major virtue resides m the fact that it is readily prepared in quantity by
procedures that are simple and economrcal with regard to both effort and
expense. The major drawback of phosphocasein is the very high quantity of
unlabeled phosphate that is already bound to it, which renders it unsuitable for
determining kinetic parameters. However, for routine applications-those
requiring knowledge of the relative protein phosphatase activity present in a
sample such as surveying cell homogenates or column fractions, screening
potential activators or inhibitors, and so on-phosphocasem is entirely suitable.
For the assay of PP 1- arch, a sample of protein phosphatase is incubated with
[32P]phosphocasein in the presence of a divalent metal ion cofactor and a pro-
tein carrier, bovine serum albumin (BSA). Inclusion of the divalent metal ion
cofactor is very important. Every PP1/2A homolog characterized to date in
both the Archaea (21,23,24) and bacteriophage h (14) requires divalent metal
ions for activity, as does the bacterial PP2C homolog SpoIIE (20). (Eukaryotic
PPl is a metalloenzyme (28), but it normally binds divalent metal ions in a
sufficiently tenacious manner to render the addition of exogenous cofactors
unnecessary.) In the author’s experience, Mn2’ has proven the most efficacious
and general cofactor. However, activation by Co*+, N?+, or Mg2+ has been
observed on occasion (21,23,24). The assay is terminated by adding trichloro-
acetic acid (TCA) and centrifuging. With the assistanceo f the BSA carrier, the
6 Kennel/y
TCA quantitatively precipitates the unreacted [32P]phosphocasemw hereas the
inorganic [32P]phosphate that was released by the action of the protein phos-
phatase remains in the supernatant ltquid. An aliquot of the supernatant is then
removed and analyzed for 32Pc ontent by liquid scintillation counting. (Meth-
ods for verifying that the radioactivity detected is derived from morgamc phos-
phate and not small, TCA-soluble phosphopeptides produced by the action of
proteolytic enzymes can be found in ref. 21)
Purification of PP 1- arch from S solfaturzcus is a relatively straightforward
process mvolvmg ion-exchange chromatography, gel filtration chromatography,
and absorption onto and elution from hydroxylapatite. As with many prokary-
otic organisms, breaking the cells themselves is a much more arduous task than
is typical for most animal cells. In the case of S. solfaturicus, repeated sonica-
tion is sufficient, but other organisms may require repeated passage through a
French Press or similarly severe methods. Advantage is taken of the fact that S.
solfataricus releases a soluble, pea-green pigment upon cell rupture By moni-
toring the release of pigment at 400 nm after each somcation cycle, the pomt at
which the majority of the cells have been broken open can readily be determmed.
The PPl-arch obtamed by the procedure described herein 1s x1000-fold
purified over the Soluble Extract. Although this preparation falls somewhat
short of absolute homogeneity, the major protein species is PPl-arch, which
constitutes 40-70% of the total protem present. The unambiguous identifica-
tion of the PP 1- arch polypeptide, and subsequent determination of its relative
abundance, was accomplished by assaymg its catalytic activity m gel slices
following polyacrylamide gel electrophoresis in the presence of sodium
dodecyl sulfate (SDS-PAGE) (22). The key to recovering at least a portion of
the PPl-arch m an active state followmg electrophoresis is the selection of a
much lower temperature, 65°C vs the usual lOO”C, for incubatmg of the pro-
tein with SDS Sample Buffer.
In addition to identifying and characterizing archaeal protein phosphatases
by classic purification, sequencing, and cloning techniques (22) the gene
encoding a second member of the PPl-arch family from Ad, thermophdu has
been identified using PCR. This was accomplished using primers modeled after
regions of PPl-arch from S. solfatarzcus, that are highly conserved with
homologous eukaryotic protein phosphatases (see Fig. 1). Included m these
primers are 5’ extensions containing nucleotide sequences suitable for anneal-
ing the ends of the primers to sites cut by the endonucleases EcoRI or BamHI.
This permits the direct cloning of the PCR product(s) into a variety of plasmid
vectors. The selectivity of PCR amplification is enhanced by usmg the “touch-
down” method (29). The touchdown method is essentially a PCR titration m
which the annealing temperature is lowered by one degree every few, usually
three, cycles. Under these conditions, the region of DNA that most tightly binds
Prokaryo t/c Phospha tases 7
PPl s. solf. QDYVDREPQTGVENLSLIL-KKLIESDENKGKTKIVVLRGNRE
PPl rabbit GDYVDRGKQS-LETICLLLAYKI-KYPEN-----FFLLRONRE
PPZA yeast GDYVDRGYYS-VETVSYLVAMKV-RYPHR-----ITILRGNHE
PPZB rat GDYVDRGYFS-IECVLYLWALKIL-YPKT-----LFLLRGNHE
Pruner 1 5’$+ZAAlTCCGGNGA(T/C)TA(T/C)GTNGA(T/C)(A/C)G 3’
EcoRI
Prmr 2 5’CGGGATCCG(T/C)TC(A/G)TG(A/G)TTNCCNG(T/G)NA 3’
Fig. 1. Design of degenerate oligonucleotide primers cloning of PP 1- arch homologs by
PCR At top 1ss hown the sequence of ammo acids 63-l 04 of PP l-arch from S solfatarzcus
aligned with the correspondmg regions of a rabbit PPl, a yeast PP2A, and a rat PP2B
(Adapted from ref. 22). The areas of lughly conserved ammo acid sequence used to design
the primers are designated with bold lettering. The conserved GDYVDR sequence was
used to design pruner 1 and the conserved LRGNHE! sequence was used to design primer
2. Below these protein sequences are gtven the nucleotide sequences of each primer. The
underlined portions represent the extensrons added to enable primer 1 to anneal to restnc-
tion sites for EcoRI and primer 2 to anneal to restriction sites for BamHI Positions where
two bases are enclosed in parentheses indicate that both of the indicated nucleotlde bases
were incorporated at that posrtion in the oltgonucleotide sequence, whereas N indicates
positions where all four possible nucleotide bases were included.
the primers 1s amplified first, and, therefore, constitutes the predommant end
product because rt is amplified through several-fold more cycles than the next
best match. If three cycles are performed at each temperature and two
sequences differ by 2OC in annealing temperature, the higher annealing prod-
uct will be amplified (2)6-, or 64-, fold more than the lower annealing product.
By scanning through a range of temperatures, the experimentalist gains the
selectivity of using the hrghest possible annealing temperature without the risk
of overshootmg it completely. It should be noted, however, that PCR is not a
panacea. Despite biochemical evidence for the existence of a PPl-arch
homolog in the archaeon H volcanii (23), PCR reactions have failed to yield
an oligonucleotrde product derived from its gene.
2. Materials
2.1. Assay of PPl-Arch
2.1.1. Preparation of f2P]Phosphoseryl Casein
1. Catalytic subunit of CAMP-dependent protein kinase: 1000 U, from Sigma
(St. Louis, MO, cat no P 2645).
8 Kennel/y
2 Casein solution. Autoclaved, hydrolyzed, and partially dephosphorylated casem
(5% w/v) from bovine milk from Sigma (cat no C 4765)
3. ATP, 10 mM, pH 7 5
4. [y-32P]ATP, 0 8 mC1 (see Note 1).
5 Buffer A: 50 n&f Tris-HCl, pH 7.0, 1 mA4 dithiothreltol (DTT), 0.1 r&Y EGTA
(see Note 2).
6. Buffer B. 60 mA4 magnesium acetate in Buffer A
7. Buffer C: 5% (v/v) glycerol m Buffer A.
8 Stop solution: 100 mA4 sodium pyrophosphate, pH 7 0, 100 WEDTA.
9. A 1.0 x 17 cm column of Sephadex G-25 fine (Pharmacla, Uppsala, Sweden)
equilibrated in Buffer C (see Note 3)
2.1.2. Assay of Phosphocasein Phosphatase Activity
in Soluble Samples of Protein Phosphatase
1. Buffer D: 50 mMMES, pH 6.5
2. Buffer E: 120 mMMnC12 in Buffer D.
3. Buffer F: 2 mg/mL BSA m Buffer D.
4 TCA, 20% (w/v)
2.1.3. Assay of Phosphocasein Phosphatase Activity
in Slices from SDS- Polyacrylamide Gels
1 SDS Sample Buffer 5% (w/v) SDS, 40% (v/v) glycerol, 0 1% (w/v) bromo-
phenol blue.
2 Buffer D. 50 mA4 MES, pH 6.5.
3 Buffer G* 0.5 mA4 EDTA in Buffer D.
4 Buffer H: 100 mMMES, pH 6.5,0.66 mg/mL BSA, 40 mMMnC1,.
5. Buffer I: 100 mA4 MES, pH 6 5,0.66 mg/mL BSA, 10 mM EDTA.
2.2. Purification of PP7-Arch from Sulfolobus Solfataricus
1. Buffer J. 20 mMMES, pH 6.5,lOO mA4NaC1,l WEDTA, 1 WEGTA, 1 mA4
DTT, 5 pg/rnL leupeptm, 5 pg/mL soybean trypsin inhibitor, 0 5 mM pheny-
lmethylsulfonyl fluoride (PMSF), 0.5 mA4 tosyllysyl chloromethylketone
(TLCK), 0.5 mM tosylphenylalanyl chloromethylketone (TPCK) (see Note 4)
2. Buffer K: 10 mMMES, pH 6.5,O 5 mMEDTA, 0 5 pg/mL leupeptin, 0 2 mMPMSF
3. 150 mMNaC1 m Buffer K
4. 400 mMNaC1 m Buffer K.
5. Buffer L* 1 Wsodium phosphate, pH 6.5,0.5 mMEDTA, 0.5 pg/mL leupeptin,
0.2 mM PMSF.
6. Buffer M: 400 mM sodium phosphate, pH 6.5, 0.5 mkf EDTA, 0 5 pg/mL
leupeptin, 0.2 mA4 PMSF.
7 Buffer N: 20 mM MES, pH 6 5, 10 mM NaCl, 0.5 mA4 EDTA, 0.5 pg/mL
leupeptm, 0.2 mM PMSF.
8. A 10 x 4 cm column (see Note 3) of CM-Trisacryl (Sepracor, Marlborogh, MA)
equilibrated in Buffer K.
Prokaryotic Phosphatases 9
9 A 6 25 x 30 cm column of DE-52 cellulose (Whatman, Clifton, NJ) equrhbrated
in Buffer K
10. A 2.5 x 40 cm column of DE-52 cellulose equilibrated in Buffer K.
11. A 2.5 x 12 cm column of Hydroxylapatrte HT (Bio-Rad, Richmond, CA) equili-
brated m Buffer L
12. A 5.0 x 100 cm column of Sephadex G-100 fine (Pharmacra) equrhbrated m
Buffer M.
13 An FPLC system (Pharmacra) equipped with a 0 5 x 7 cm column of Mono Q that
has been equilibrated in Buffer K.
2.3. Cloning of Phosphatase Genes by PCR
1. The enzymes and buffers of the Perkin-Elmer Cetus GenAmpTM PCR system were
used, although PCR reagents from other commerctal sources presumably can be
substituted without prejudice to the ultimate results.
2 Oligonucleotide primers 1 and 2 as shown m Fig. 1.
3. Methods
3. I. Preparation of [32P]Phosphocasein
1. Combine the following in a 1.5 mL Eppendorff tube: 100 pL of 5% (w/v) casein
(see Note 5), 85 & of buffer B, 10 )..tLo f 10 mMATP, and z 0.8 mC!r of [y-32P]ATP.
The precise volume of [Y-~~P]ATP added will depend on the concentration of the
solution as supplied by the manufacturer as well as the age of the preparation,
since 32P has a relatively short halfdlife of 13 d (see Note 6). Make up the total
volume to 325 pL with distilled water.
2. Take a vial containing 1000 U of lyophilized catalytic subunit of the CAMP-depen-
dent protein kinase. Remove the septum cap. Add 87.5 pL of Buffer A. Agitate
gently by hand to dissolve the solid. Let stand for a moment to permit the liquid to
drain and collect in the bottom Transfer to the Eppendorff tube from step 1.
3. Rinse residual catalytic subunit from its container by addmg another 87.5 pL of
Buffer A and repeating step 2 Securely cap the Eppendorff tube and mix briefly
on a Vortex mixer.
4. Incubate for 8-12 h in a 30°C water bath.
5. At the conclusion of the incubation penod, add 50 & of Stop Solution Mix
briefly on a Vortex mixer You can store at -2O’C or proceed immediately with
the remaining steps.
6. Remove 5 & of the incubation mixture and add to 995 & of distilled water m a
1.5 mL Eppendorff tube. Mix vigorously on a Vortex mixer. Remove three 5 pL
portions of the 1:200 diluted incubation mixture, place in individual scintillation
vials, then add 1 mL of a water-compatible liquid scintillation fluid such as Eco-
Lume (WestChem, Irvine, CA) or Econo-Safe @PI, Mount Prospect, IL). Mea-
sure the radioactivity present in a liquid scintillation counter (see Note 7) Thts
information is then used to calculate the specific radioactivity of the ATP used to
phosphorylate the casein (Assume the cold ATP you added completely accounts
for the concentration of total ATP.) Typical specific activities range from l-3 x
10 Kennelly
1016 cpmlmole Please note that it not necessary to try and convert cpm to dpm as
long as you use the same scintlllatlon counter and sclntlllatlon fluid for all mea-
surements of radioactivity. Under these circumstances, efficiency 1s a constant that
cancels itself m all subsequent calculations of moles of product produced, percent sub-
strate turnover, and so on
7. Apply the mcubatlon mixture to a 1 0 x 17 cm column of Sephadex G-25 fine
that has been equilibrated in Buffer C
8. Develop the column with Buffer C Collect 1 .O mL fractions m numbered 1 5 mL
Eppendorff tubes
9 Remove 5 pL ahquots from each fraction, place each m a separate, numbered
scmtlllatlon vial, add 1 .O mL of scmtlllatton fluid, and count for radloactivlty.
10 Graph the radloactlvlty present in the aliquots as a function of fraction number.
Two peaks of [32P]radloactlvlty should be apparent on the chromatogram. The
first peak 1s the [32P]phosphocasem (see Note 8) and the second 1s the unreacted
[y-32P]ATP.
11 Store the two or three peak fractions of [32P]phosphocasem at -2O’C The con-
centration of casem-bound [32P]phosphate in peak fractions generally ranges from
5-25 w Discard the remaining fractions as radioactive waste. Store the column
m a shielded location until needed again (see Note 9).
3.2 Assay of PPl-Arch Activity
1. Thaw a tube of [32P]phosphocasein solution. Mix the contents using a Vortex
mixer. Spin briefly m a microcentrifuge to centrifuge the contents into the bot-
tom of the tube. This represents an important precaution deslgned to mmimlze
the chances of inadvertently contactmg radioactive material that might otherwise
be clmgmg to the bottomslde of the cap, or scattering it about the lab while open-
ing the tube (see Note 10). Remove 10% of the volume of [32P]phosphocasem
required to perform the planned number of assays, and place m an Eppendorff
tube. Return the rest of the [32P]phosphocasem stock to the freezer
2 For each assay, combme 5 pL of Buffer E and 5 p.L of Buffer F m a 1 5 mL
Eppendorff tube.
3 Add the protein phosphatase sample to be assayed, plus any additional compounds
(activators, inhibitors, and so on) you might wish to test, to the Eppendorff tube The
volume of the sample plus other addltlons should be 5 15 pL. Make up any unutlhzed
portion of tis 15 pL volume, if necessary,w ith Buffer D. Control assayss hould substl-
tute an equal volume of a suitable buffer m place of the protein phosphatase sample
4 Imtlate the assay by adding 5 pL of [32P]phosphocasem solution, mlxmg briefly
on a Vortex mixer, then place in a 25’C water bath (see Notes 11 and 12) This
quantity of phosphocasem solution generally yields a final concentration of
casein-bound [32P]phosphate of l-4 w.
5. Terminate reaction, generally after a period of 10-90 mm, by addmg 100 pL of
20% (w/v) TCA and mlxmg briefly on a Vortex mixer
6 Pellet precipitated protein by centrifuging at 12,000g for 3 mm m a micro-
centrifuge (see Note 10).