Download Signal sequence peptides at an air-water interface

1131
619th MEETING. CAMBRIDGE
controls were incorporated. Methaemoglobin formation
was measured spectrophotometrically (Harley & Mauer,
1960). The binding of haemoglobin to the membrane was
assessed using the method of Morrison (1965) involving the
conversion of haem to fluorescent porphyrin.
Erythrocyte membranes were prepared (Dodge et a[.,
1963) and suspended in phosphate buffer, pH 7.4, at a final
membrane protein concentration of 1.5mg/ml (Lowry et al.,
1951). Membranes were exposed to the same experimental
treatments as described above for the intact erythrocytes.
Lipid peroxidation was assayed by monitoring the thiobarbituric acid-reactive (TBAR) products from the breakdown
of lipid hydroperoxides (Walls et al., 1976) and by the
measurement of the formation of fluorescent chromolipids
(Tappel, 1962).
The results (Table 1) show that exposing intact erythrocytes to oxidative stress by incubation with the iron II/
ascorbate/hydrogen peroxide system causes almost total
oxidation of the haemoglobin to methaemoglobin after 24 h.
N o significant effect was observed after 5 h. The incorporation of desferrioxamine into the system suppresses haemoglobin oxidation to about 40%, whereas the iron-chelated
complex has no effect. The pattern of increased binding of
haemoglobin to the membrane paralleled the haemoglobin
oxidation as shown in the Table 1. On incorporating substrates for maintaining the ATP levels, haemoglobin oxidation was almost totally inhibited on iron stress.
Lipid peroxidation in the membranes of the erythrocytes
exposed to the various treatments was monitored in the
form of TBAR-products (measured on the basis of nmol/
10" cells) and the production of fluorescent chromolipids.
After the 5 h incubation with the iron/ascorbate/hydrogen
peroxide system there is a small increase in lipid peroxidation
which is not affected by the presence of the iron-chelator
desferrioxamine. More extensive lipid peroxidation has
taken place after 24h incubation but a similar trend is
observed. N o chromolipid formation was observed after
incubation for 5 h, but after the prolonged treatment with
the iron/ascorbate/hydrogen peroxide system, and that in
the presence of ferrioxamine, elevated levels of chromolipids
were detected (relative fluorescence 22 units/pg of phospholipid and 17 units/pg of phospholipid) compared with
the control erythrocytes incubated for the same period (rela-
tive fluorescence 9 units/pg of phospholipid). In the ATPmaintained incubation systems, lipid peroxidation was significantly increased in all treated samples at both time
intervals. These observations suggest the potential role
of methaemoglobin in scavenging propagating oxidative
species in the membrane. Maintenance of ATP levels
presumbly suppresses haemoglobin oxidation on prolonged
incubation by sustaining the activity of methaemoglobin
reductase.
To clarify further the role of haemoglobin in the susceptibility to iron-induced oxidative damage haemoglobin-free
membranes were treated by incubation for 5 h in identical
systems. Lipid peroxidation, expressed as nmoles of TBARproducts/mg of membrane protein, was considerably
increased on treatment with the iron/ascorbate/hydrogen
peroxide system, the increase being totally suppressed in the
presence of desferrioxamine. N o chromolipid formation
was observed.
This study demonstrates that by maintaining the energy
requirements of the erythrocyte, methaemoglobin production is minimized under conditions of iron-stress. However,
under these conditions, the membranes of the erythrocytes
become more susceptible to the oxidative damage and
increased lipid peroxidation ensues. Methaemoglobin therefore has a role in decreasing the susceptibility of erythrocyte
membranes to such oxidant stress and this is confirmed by
the studies on haemoglobin-free membranes.
We gratefully acknowledge financial assistance from the Government of the Turkish Republic of Northern Cyprus.
Dodge, J. T., Mitchell, C. & Hanahan, D. J. (1963) Arch. Biochem.
Biophys. 100, 119-128
Harley, J. 0 . & Mauer, S . M. (1960) Blood 16, 1722-1735
Lowry, 0. J., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951)
J . B i d . Chem. 193, 265-275
Lutz, M. U., Liu, S.C. & Palek, J. (1977) J. Cell Biol. 73, 540-560
Morrison, G . R. (1965) Anal. Chem. 37, 1 1 2 4 1 126
Rice-Evans, C., Baysal, E., Kontoghiorghes, G., Flynn, D. M. &
Hoffbrand, A. V. (1985) Free Radical Res. Commun. 1, 5 5 4 2
Tappel, A. L. (1962) Yitam. Horm. 20. 493-510
Walls, R., Kumar, K. S. & Hochstein, P. (1976) Arch. Biochem.
Biophys. 174, 463-468
Received 1 1 June 1986
Signal sequence peptides at an air-water interface
G E R A R D 0 D. FIDELIO,* BRIAN M. AUSTEN,?
DENNIS CHAPMAN* and JACK A. LUCY*
*Department of Biochemistry and Chemistry, Royal Free
Hospital School of Medicine, Rowland Hill Street,
London N W3 2PF, U . K . . and ?Department of Surgery,
St George's Hospital Medical School, Cranmer Terrace,
London SW17 ORE, U . K .
Secreted proteins are synthesized as precursors with an extra
N-terminal extension that is termed the signal sequence
(Blobel & Dobberstein, 1975). The primary sequences of
signal sequence peptides exhibit little homology, but it has
been reported that they share common features which may
be required for the translocation process (Austen, 1979;
Austen & Ridd, 1981; Austen et al., 1984). Although some
exported proteins, e.g. ovalbumin, are produced without a
transient N-terminal peptide (cf. Wickner, 1980), it has been
proposed that a functionally equivalent signal sequence that
is not proteolytically removed during transport is present
within such proteins (Tabe et a/., 1984). As the precise
mechanism by which peptides with 'signal' properties
mediate the translocation process at membranes is not clear,
Vol. 14
we have investigated the surface properties and the interfacial behaviour of three signal sequences.
Two of the signal sequences studied, the pretrypsinogen 2
and the synthetic 'consensus' peptide (which constitutes a
consensus of known signal sequences), were made on solidphase supports, and have previously been shown to inhibit
the processing in vitro of preproteins in a concentrationdependent manner (Austen & Ridd, 1981; Austen et al.,
1984). The other peptide investigated was the putative signal
sequence contained within ovalbumin (Tabe et al., 1984).
This peptide has recently been isolated as a tryptic fragment
from ovalbumin and shown to inhibit completely the processing of preprolactin (Robinson et al., 1986).
The three peptides were observed to form insolublc
monolayers at the air-water interface and they exhibited
well-defined collapse pressures with values ranging from
26.5 to 43mN/m (Table I). These values are higher than
those of polypeptides and proteins previously studied
including the amphipathic melittin (Fidelio et al., 1982,
1984) (see Table I). Indeed the collapse pressure of the
consensus peptide is similar to that of dioleoylphosphatidylcholine (46mN/m).
1 I32
BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. Collapse pressure for the signal sequences and for the
proteins reported by the literature
References: “G. D. Fidelio, B. Austen, D. Chapman & J. A.
Lucy, unpublished work; Fidelio et al. (1984); ‘ Mitchell et al.
( 1970); dQuinn &
Dawson ( 1 969); ‘Colaccico (1 972); ’Phillips
et al. (1975); RAdamset al. (1971).
Protein
Ovalbumin signal sequence
Pretrypsinogen signal sequence
Consensus signal sequence
Ovalbumin
Melittin
Myelin basic protein
Bovine serum albumin
a-Lactoglobulin
a-Lactoalbumin
Cytochrome c
Folch Lees proteolipid
b-Casein
Total high-density apolipoprotein
Lysozyme
Collapse pressure (mN/m)
26.5
33.5
42.5
19.9“
21.2h
14.2h
13.Ih
18’
20
I 3d
21‘
23’
20’
25R
At its collapse pressure, the pretrypsinogen peptide has a
molecular area of about 0.65 nm2, which is consistent with
an extended ,&structure that is oriented perpendicular to the
interface. By contrast, the observed minimum molecular
area for the consensus and the ovalbumin peptides (about
I .6 nm2) are consistent with an a-helical structure perpendicular to the interface or, alternatively, with a ‘loop’ in
which two antiparallel fl-strands are linked by a /I-turn
region (Austen et al., 1984). (These proposals assume an
average length of amino acid side chains of about 0.5 nm, an
inner diameter of 0.5nm for the a-helix and a distance
between the two opposite fl-strands of 0.4 nm.) Our observations thus indicate that the signal sequences have a considerable degree of secondary structure at the interface.
It is suggested that the ability of signal peptides to support
a high lateral surface pressure may facilitate the binding of
the nascent chain-polysome complex to the endoplasmic
reticulum at the beginning of the translocation process. By
contrast, the subsequent insertion of a completed polypeptide chain into the membrane and loss of its signal peptide
by proteolytic cleavage could, as a consequence of the lower
stabilities of mature proteins at high surface pressures
(Table I), result in the mature protein being extruded into
the cisternae of the endoplasmic reticulum, i.e. in protein
translocation.
This work was supported by the Wellcome Trust
Adams, D. J., Evans, M. T. A., Mitchell, J. R., Phillips, M. C . &
Rees, P. M. (1971) J . Polym. Sci. C34. 167 179
Austen, B. M. (1979) FEBS L e f t . 103, 308 313
Austen, B. M. & Ridd. D. H. (1981) Biochem. Sot. Symp. 46, 235 258
Austen, B. M., Hermon-Taylor. J . . Kaderbhai. M. A. & Ridd. D. H .
(1 984) Biochem. J . 224. 3 17-325
Blobel, G . & Dobberstein, B. (1975) J . Cell Biol. 67, 852 862
Colacicco. G . (1972) Ann. N . Y . Acud. Sci. 195. 224 261
Fidelio, G . D., Maggio, B. & Cumar. F. A. (1982) Bioc~hern.J . 203.
7 17-725
Fidelio, G . D., Maggio, B. & Cumar, F. A. (1984) Chem. P/iys. Lipids
35, 23 1-245
Mitchell, J., Irons, L. & Palmer. G . J. (1970) Biochim. Biophyx. A[,tu
200, 138-150
Quinn, P. J. & Dawson. R. M . C. (1969) Bioc~hern.J . 115, 65 75
Phillips, M. C., Hauser, H.. Leslie. R. B. & Oldani. D. (1975) Biochim. Biophvs. Actu 406, 402 4 I4
Robinson, A., Meredith, C. & Austen. B. (1986) Biochem. Soc.. Truns.
14, 867
Tabe, L., Kreig, P., Strachan. R.. Jackson. D.. Wallis. E. & Colman.
A. (1984) J . Mol. Biol. 180. 645466
Wickner, W. (1980) Science 210. 861 868
Received I 1 June 1986
A method to discriminate transport or consumption of orosomucoid and prealbumin in human
cerebrospinal fluid
teins A or B was calculated according to Weissner (1980):
TILMANN 0. KLEINE
Funktionsbereich Neurochemie, Zentrum fur
Nervenheilkunde der Universitat, 0-355Marburgl Lahn,
West Germany
A method is described to discriminate active or passive
transport and consumption of orosomucoid, a modulator of
immune response (Chiu et al., 1977; Bennett & Schmid,
1980), and transport of thyroxine-binding prealbumin in
human cerebrospinal fluid (CSF). Both proteins have
plasma to CSF concentration gradients of
180: 1 and
160 : 1, respectively, maintained by the blood-brain
barrier (Kleine, 1980; Kleine & Merten, 1983). Concentrations of orosomucoid, prealbumin or albumin were
measured in the same samples of lumbar CSF and corresponding serum samples of 190 patients by immunonephelometric or immunoturbidimetric microassays (Kleine & Merten, 1980, 1983). Albumin synthesized by liver cells was
selected as a reference parameter to study the transport
of orosomucoid and prealbumin through the blood-brain
barrier: Passive transfer (barrier-dependent) of both pro-
-
-
Abbreviations used: CSF, cerebrospinal fluid, CNS, central nervous
system.
[CSF albumin]
x [serum protein A or B]
[serum albumin]
(I)
It was increased, with values above the reference range
(Table I).
Active transport (barrier-independent) of orosomucoid
was calculated by combining two equations:
[CSF orosomucoid] -
[CSF albumin]
[serum albumin]
x [serum orosomucoid]
and according to Delpech & Lichtblau (1972):
(2)
[CSF orosomucoid] : [CSF albumin]
(3)
[serum orosomucoid] : [serum albumin]
I t was increased (decreased) with values of eqns. (2) and
(3) above (below) the reference range (Table 1) showing
simultaneously increased (decreased) orosomucoid levels
in lumbar CSF. Consumption of CSF orosomucoid was
increased with elevated values calculated with eqns. (2) and
(3) and CSF contents lying within or below the reference
range (Table 1). It was decreased with values calculated by
1986