Cysteine and glutathione secretion in response to protein disulfide bond formation in the ER

Science

Volumn 277, Issue 5332, 1997, Pages 1681-1684

  Carelli, Stephana ^a   Ceriotti, Aldo ^b   Cabibbo, Andrea ^a   Fassina, Giorgio ^c   Ruvo, Menotti ^c   Sitia, Roberto ^a  

^a SAN RAFFAELE HOSPITAL   (Italy)

^b CNR   (Italy)

^c Tecnogen ^*  (Italy)

Author keywords

[No Author keywords available]

Indexed keywords

CYSTEINE; GLUTATHIONE; THIOL DERIVATIVE;

ANIMAL CELL; ARTICLE; CELL ORGANELLE; CONTROLLED STUDY; DISULFIDE BOND; ENDOPLASMIC RETICULUM; NONHUMAN; OXIDATION REDUCTION REACTION; PRIORITY JOURNAL; PROTEIN FOLDING; PROTEIN SYNTHESIS; SIGNAL TRANSDUCTION;

ANIMALS; BENZOPYRANS; BREFELDIN A; CYCLOHEXIMIDE; CYCLOPENTANES; CYSTEINE; CYSTINE; DISULFIDES; ENDOPLASMIC RETICULUM; EXOCYTOSIS; GLUTATHIONE; GLUTATHIONE DISULFIDE; GOLGI APPARATUS; HEMAGGLUTININ GLYCOPROTEINS, INFLUENZA VIRUS; IMMUNOGLOBULIN LAMBDA-CHAINS; IMMUNOGLOBULIN M; OOCYTES; OXIDATION-REDUCTION; PROTEIN SYNTHESIS INHIBITORS; PROTEINS; TEMPERATURE; TUMOR CELLS, CULTURED; XENOPUS LAEVIS;

ANIMALIA;

EID: 0030765707     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5332.1681     Document Type: Article

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31. Oocytes were obtained from large females of Xenopus laevis and maintained in modified Barths' saline as described (19). Animals were kept and oocytes were obtained in accordance with the institutional guidelines. For the determination of secreted thiols and disulfides, groups of five oocytes were cultured for different times in 50 μl of modified Barths' saline lacking nystatin. The basal levels of thiol secretion varied in batches of oocytes isolated from different frogs, possibly corresponding to different levels of synthesis of endogenous proteins.
32. Oocytes were microinjected with 50 nl of mRNA solution as described (19) with the use of a Narishige IM-1 automatic microinjector controlled by an IM-1T timer.
33. 3CN (C). Gradient: 0 min, 100% A; 30 min, 50% A, 50% B; 40 min, 100% C; 45 min, 100% C; 50 min, 100% A. Flow was at 1.25 ml/min at a temperature of 45°C; ultraviolet monitoring was used (wavelength, 263 nm). The concentration of thiols in the samples was determined by comparison with calibrated amounts of standard solutions of cysteine, GSH, GSSG, and the mixed disulfidebetween cysteine and glutathione (Cys-SG). The identity of individual peaks in the chromatograms was further confirmed by time-of-flight matrix-assisted laser desorption ionization mass spectrometry (4) [T. Tanaka et al., Rapid Comm. Mass Spectrom. 2, 151 (1988)].
34. 3CN (C). Gradient: 0 min, 100% A; 30 min, 50% A, 50% B; 40 min, 100% C; 45 min, 100% C; 50 min, 100% A. Flow was at 1.25 ml/min at a temperature of 45°C; ultraviolet monitoring was used (wavelength, 263 nm). The concentration of thiols in the samples was determined by comparison with calibrated amounts of standard solutions of cysteine, GSH, GSSG, and the mixed disulfidebetween cysteine and glutathione (Cys-SG). The identity of individual peaks in the chromatograms was further confirmed by time-of-flight matrix-assisted laser desorption ionization mass spectrometry (4) [T. Tanaka et al., Rapid Comm. Mass Spectrom. 2, 151 (1988)].
35. 35S]methionine-cysteine (1 mCi/ml) (PRO-MIX, Amersham). After the pulse, the medium was removed, and the oocytes were washed thoroughly with modified Barths' saline and incubated in the same medium supplemented with excess cold methionine and cysteine. After the medium was harvested, each oocyte was homogenized in 40 μl of homogenization buffer (19), supplemented with 50 mM iodoacetamide. Media and homogenates were frozen in liquid nitrogen and stored at -80°C.
36. Homogenates and incubation media were diluted to 1 ml with Net-gel buffer (14) and incubated with specific antibodies for 4 hours on ice before addition of 50 μl of a 10% suspension of protein A-Sepharose CL 4B (Pharmacia, Uppsala, Sweden). After overnight incubation at 4°C, the beads were washed three times with NET-gel buffer and eluted by heating at 95°C in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer [0.062 M tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 0.001% bromophenol blue].
37. We thank F. Cozzolino, D. Fesce, I. Haas, A. Helenius, A. Malgaroli, J. Meldolesi, M. Neuberger, A. T. Palamara, R. Pardi, A. Rubartelli, and L. Wrabetz for helpful discussions and suggestions; C. Fagioli for technical help; and S. Trinca for secretarial assistance. This work was supported through grants from the Associazione ltaliana per la Ricerca sul Cancro (AIRC), Consiglio Nazionale delle Ricerche (CNR), and Ministero della Sanità (AIDS Special Project) to R.S. S.C. was supported by a fellowship from the Fondazione San Raffaele, in partial fulfillment of a Ph.D. from the Open University, London. A.Ca. is the recipient of a fellowship from AIRC.