Download Disulphide-bond formation in protein folding catalysed by highly

592nd MEETING, LONDON
79
Disulphide-bond formation in protein folding catalysed by highly-purified protein disulphideisomerase
ROBERT B. FREEDMAN,* DAVID A. HILLSON*$ and
THOMAS E. CREIGHTONT
*BiologicalLaboratory, University of Kent, Canterbury
CT2 7NJ,Kent, U.K., and tMRC Laboratory of Molecular
Biology, Hills Road, Cambridge CB2 2QH, U.K.
For many proteins, disulphide-bond formation is a significant
post-translational event involved in the acquisition of the native
tertiary structure. Little is known about how this occurs in cells.
The classic work on the refolding of reduced ribonuclease (see
Anfinsen, 1973) showed that the fully reduced unfolded protein
can regain the correctly disulphide-paired active conformation
without the supply of additional information; however, rapid
refolding requires the presence of a thiol-disulphide redox
system and an enzyme known as protein disulphide-isomerase
(EC 5.3.4.1). Many properties of this enzyme suggest that it
may be involved in catalysing the formation of native disulphide
bonds during protein biosynthesis, but its physiological role has
not been definitely established [see Freedman & Hawkins (1977)
and Freedman & Hillson (1980) for reviews]. In the period
1963-1967, the enzyme from ox liver was shown to contain an
essential thiol group and to be a catalyst of thiol-disulphide
interchange (see Anfinsen, 1973), but its mechanism of action
has not been further explored.
We have recently described a modified method for purification of the enzyme from ox liver that gives material effectively
homogeneous by sodium dodecyl sulphatelpolyacrylamide-gel
electrophoresis and 560-fold purified in protein disulphideisomerase activity (Hillson & Freedman, 1980). We show here
that this preparation is an effective catalyst of the oxidation and
folding of reduced bovine pancreatic trypsin inhibitor by
oxidized dithiothreitol, and we identify the steps catalysed. The
folding pathway of this protein in uitro has been established in
considerable detail (Creighton, 1978). More extensive studies on
the unfolding and refolding of bovine pancreatic trypsin inhibitor
and ribonuclease, catalysed by a cruder preparation of protein
disulphide-isomerase, have been published elsewhere (Creighton
et al., 1980).
The enzyme preparation used here was that described by
Hillson & Freedman (1980). The techniques used for preparation of reduced bovine pancreatic trypsin inhibitor and for
trapping, resolving and determining the intermediates in the
refolding pathway were as described by Creighton (1978) and in
references cited therein.
Fig. 1 shows that highly-purified protein disulphide-isomerase
significantly accelerates folding and oxidation when reduced
bovine pancreatic trypsin inhibitor is oxidised by oxidized
~
bovine
dithiothreitol. Reaction conditions were: 3 0 p reduced
pancreatic trypsin inhibitor, 20 mhf-oxidized dithiothreitol, 0.1 MTris/HCl, pH 7.5, containing 0.2 M-KCI and 1 mM-EDTA, at
25OC in the presence of 10pg of highly purified isomerasefml
(200-fold molar excess of bovine pancreatic trypsin inhibitor
over isomerase). The intermediates that accumulate to significant extents are the same in the absence or presence of the
enzyme. The enzyme does not alter the pathway of protein
folding, but catalyses the rate-determining steps, which in this
system are steps in which a thiol group in the substrate protein
attacks an unstable protein-SS-dithiothreitol-SH mixed disulphide to form a new protein disulphide bond. The slowest step
in the whole pathway is the conversion,of the intermediates I1
(see the legend to Fig. 1) to the two-disulphide intermediate with
the bonds (30-51, 5-55), which then rapidly forms the native
protein. This step involves attack by cysteine-55 on a bond
between either residues 5-14 or 5-38 and is an extreme example
$ Present address: Biophysics Laboratory, Portsmouth Polytechnic,
White Swan Road, Portsmouth PO I 2DT, U.K.
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c)
k
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2:
2
k
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20
It'
1
01
40
20
0
20
10
30
Time (min)
Fig. 1. Oxidation of reduced bovine pancreatic trypsin inhibitor
in the presence (0)or absence (0)of 1Opg ofprotein disulphide
isomeraselml
Oxidations were carried out as described in the text. At intervals,
portions were removed, quenched with iodoacetate, and intermediates were separated by polyacrylamide-gel electrophoresis.
Relative amounts of the intermediates were determined by
densitometry of Coomassie Blue-stained gels and measurement
of peak areas. R, reduced protein; I, one-disulphide intermediates containing either the bond between residues 30-5 1 or
that between 5-30; 11, two-disulphide intermediates containing
either the bonds (30-51, 5-14) or (30-51, 5-38); II', twodisulphide intermediate containing the bonds (30-5 1, 14-38);
N, native protein containing the bonds (30-51,5-55, 14-38).
of protein flexibility; it, too, is catalysed by protein disulphideisomerase. The enzyme-catalysed steps therefore involve both
conformational change in the substrate protein and the chemical
process of thiol-disulphide interchange.
A derivative of bovine pancreatic trypsin inhibitor with the
disulphide 14-38 reduced and alkylated unfolds and refolds
much more slowly than the native protein and follows a different
pathway, since residues 14 and 38 cannot participate in
disulphide bonds (Creighton, 1978). Under the conditions of
BIOCHEMICAL SOCIETY TRANSACTIONS
80
Fig. 1, the reduced form of this derivative forms the 3&51
disulphide bond slowly and the 5-55 bond at a negligible rate.
However, both these processes are significantly catalysed by
highly purified protein disulphide-isomerase.
The results confirm that this enzyme is an effective catalyst, at
physiological pH, of the intramolecular thiol-disulphide interchange reactions involving conformational transitions that are
rate-determining in the formation and rearrangement of protein
disulphide bonds.
Anfinsen, C. B. (1973) Science 181,223-230
Creighton, T. E. (1 978) Prog. Biophys. MoI. Biol. 33,23 1-297
Creighton, T. E., Hillson, D. A. & Freedman, R. B. (1980)J. Mol. Biol.
142,4342
Freedman, R. B. & Hawkins, H.C. (1977) Biochem. SOC.Trans. I ,
348-357
Freedman, R. B. & Hillson, D. A. (1980) in The Enzymology of
Post-translational Modfiations of Proteins (Freedman, R. B. &
Hawkins, H. C., eds.), pp. 157-212, Academic Press, London
Hillson, D. A. & Freedman, R. B. (1980) Biochem. J. 191,377-388
Optical measurement of the plasma-membrane potential of mammalian cells grown in
monolayer culture
C. LINDSAY BASHFORD, KEITH A. FOSTER,
KINGSLEY J. MICKLEM and
CHARLES A. PASTERNAK
Department of Biochemistry, St. George’s Hospital Medical
School, Cranmer Terrace, London S. W.17 ORE, UX.
T
I
I
I
Magnetic
Fluorescent dyes, particularly cyanines and oxonols, have been
stirrer
widely used to monitor membrane potential in systems conI
1
sidered too small for the reliable application of microelectrodes
Light guide
(Waggoner, 1976; Cohen & Salzberg, 1978). Cyanines, which
carry a single, delocalized positive charge are particularly useful
for measuring the potential when the enclosed compartment is
negatively charged with respect to the medium. They have
therefore been used to monitor plasma-membrane potential of
erythrocytes (Sims et al., 1974; Hoffman & Laris, 1974) and of
cells in suspension (Philo & Eddy, 1978). In these cases a
‘null-point’ titration procedure was employed to determine the
concentration of external K+ at which the addition of valinomycin causes no change in dye fluorescence. Valinomycin
specifically increases the K+ permeability of membranes (Harris
& Pressman, 1967) and at the ‘null-point’ the internal ([K+l,)
and external ([K+],) activities are in equilibrium with the
membrane potential, E, according to the relationship E =
RT/Fln([K+],/[K+],) (Philo & Eddy, 1978). We have used the
dye, 3,3’-dipropylthiadicarboyanine [WS-C,-(5); Sims et al.,
19741 to monitor the membrane potential of MDBK (Madin
and Darby bovine kidney) cells grown to confluence in
monolayer culture.
Fluorescence measurements were made with a Johnson
Foundation compensated fluorometer/reflectometer (Harbig et
al., 1976) using a two-branched light guide for optical coupling
(Chance et aL, 1975). Fluorescence was excited at 590nm
(Omega optical interference filter plus Coming glass no. 9872) Fig. 1. Apparatus for measuring fluorescence from monolayer
and measured above 620nm (Kodak Wratten filter no. 92).
cultures of mammalian cells
Fluorescence and reflectance were monitored at 180” with
Primary
excitation
filters are placed at the exit slit of the lamp
respect to the excitation and emission fibres, which were
housing.
Secondary
emission filters are placed at the exit slits of
randomly mixed at the common terminal of the light guide. The
the
beam
splitter
(l),
which diverts 10% of the light to the
emitted light was split 9 : 1 for independent measurement of
reflectance photomultiplier (2), and 9096 to the fluorescence
fluorescence and reflectance. The reflectance channel can be
(3).
used to compensate for fluctuations in excitation and alterations photomultiplier
in the field of view (Harbig et al., 1976) or for measuring
changes in dye absorbance. A diagram of the experimental
arrangement is shown in Fig. 1. Optical signals were recorded cence measurements. The cells were then superfused with a
through the base of the culture dish, which was enclosed in a medium containing 155mM-(Na+/K+)CI-, 5 mwglucose, 1mMlight-tight box. The superfusing medium was stirred as CaCl, and lOmM-Na+/Hepes [4-(2-hydroxyethyl)-l-piperindicated and additions were made with syringes through a port azine-ethanesulphonicacid], pH 7.4. When the fluorescence had
in the top of the box. To determine plasma-membrane potential reached a steady value, normally after 1 or 2min, valinomycin
the cells were incubated with 3 p~-3,3’-dipropylthiadicarbo- (0.2pg/ml final concentration) was added. The ‘null-point’
cyanine iodide, oligomycin (150ng/ml), 150n~-antimycinand [K+l, was determined by plotting the percentage fluorescence
150 nwcarbonyl cyanide p-trifluoromethoxyphenylhydrazone change caused by the addition of valinomycin against log [K+l,.
Initial results with control MDBK cells provide ‘null-points’at
for Smin at 37°C in Earle’s minimal essential medium
containing 1% newborn-calf serum. The inhibitors prevented the [K+], in the range 3 - 4 m w which, assuming that [K+],is about
,
a membrane potential between -90 and
mitrochondrial membrane potential from prejudicing the fluores- 1 5 0 m ~ represents
II
1981