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Transcript
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