• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Nov 1, 1998; 335(Pt 3): 597–604.
PMCID: PMC1219821

Reversible penetration of alpha-glutathione S-transferase into biological membranes revealed by photosensitized labelling in situ.

Abstract

Fluorescent lipid analogue 3,3'-dioctadecyloxacarbocyanine incorporated into biological membranes was used to induce photoactivation of a hydrophobic probe 5-[125I]iodonaphthyl-1-azide (125INA) by energy transfer and to thereby confine subsequent radiolabelling of proteins to the lipid bilayer. This approach was applied in bovine chromaffin cells to discover cytosolic proteins that reversibly penetrate into membrane domains. alpha-Glutathione S-transferase (alpha-GST) was identified as the only labelled protein in bovine chromaffin-cell cytosol, indicating that it inserts reversibly into the membrane lipid bilayer. The selectivity of the labelling towards the lipid bilayer is demonstrated by showing that influenza virus haemagglutinin becomes labelled by 125INA only after the insertion of this protein into the target membrane. The molar 125INA:protein ratio was used as a quantitative criterion for evaluation of the penetration of proteins into the membrane lipid bilayer. This ratio was calculated for four integral membrane proteins and four soluble proteins that interact with biological membranes. The values for four integral membrane proteins (erythrocyte anion transporter, multidrug transporter gp-170, dopamine transporter and fusion-competent influenza virus haemagglutinin) were 1, 8, 2 and 2, respectively, whereas for soluble proteins (annexin VII, protein kinase C, BSA and influenza virus haemagglutinin) the values were 0.002, 0, 0.002 and 0.02, respectively. The molar ratio for alpha-GST was found to be 1, compatible with the values obtained for integral membrane proteins.

Full Text

The Full Text of this article is available as a PDF (358K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Mátyus L. Fluorescence resonance energy transfer measurements on cell surfaces. A spectroscopic tool for determining protein interactions. J Photochem Photobiol B. 1992 Mar 13;12(4):323–337. [PubMed]
  • Mosmuller EW, Pap EH, Visser AJ, Engbersen JF. Steady-state fluorescence studies on lipase-vesicle interactions. Biochim Biophys Acta. 1994 Jan 3;1189(1):45–51. [PubMed]
  • Marsh D. Lipid-protein interactions in membranes. FEBS Lett. 1990 Aug 1;268(2):371–375. [PubMed]
  • Butterfield DA, Sun B, Bellary S, Arden WA, Anderson KW. Effect of endotoxin on lipid order and motion in erythrocyte membranes. Biochim Biophys Acta. 1994 Jan 11;1225(2):231–234. [PubMed]
  • Benfenati F, Valtorta F, Rossi MC, Onofri F, Sihra T, Greengard P. Interactions of synapsin I with phospholipids: possible role in synaptic vesicle clustering and in the maintenance of bilayer structures. J Cell Biol. 1993 Dec;123(6 Pt 2):1845–1855. [PMC free article] [PubMed]
  • Neher E, Marty A. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6712–6716. [PMC free article] [PubMed]
  • Rojas E, Pollard HB. Membrane capacity measurements suggest a calcium-dependent insertion of synexin into phosphatidylserine bilayers. FEBS Lett. 1987 Jun 8;217(1):25–31. [PubMed]
  • Kado RT. Membrane area and electrical capacitance. Methods Enzymol. 1993;221:273–299. [PubMed]
  • Brunner J. New photolabeling and crosslinking methods. Annu Rev Biochem. 1993;62:483–514. [PubMed]
  • Bercovici T, Gitler C. 5-[125I]Iodonaphthyl azide, a reagent to determine the penetration of proteins into the lipid bilayer of biological membranes. Biochemistry. 1978 Apr 18;17(8):1484–1489. [PubMed]
  • Bayley H, Knowles JR. Photogenerated reagents for membranes: selective labeling of intrinsic membrane proteins in the human erythrocyte membrane. Biochemistry. 1980 Aug 19;19(17):3883–3892. [PubMed]
  • Raviv Y, Salomon Y, Gitler C, Bercovici T. Selective labeling of proteins in biological systems by photosensitization of 5-iodonaphthalene-1-azide. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6103–6107. [PMC free article] [PubMed]
  • Raviv Y, Pollard HB, Bruggemann EP, Pastan I, Gottesman MM. Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells. J Biol Chem. 1990 Mar 5;265(7):3975–3980. [PubMed]
  • Rosenwald AG, Pagano RE, Raviv Y. Activation of 5-[125I]iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids. J Biol Chem. 1991 May 25;266(15):9814–9821. [PubMed]
  • Pak CC, Krumbiegel M, Blumenthal R, Raviv Y. Detection of influenza hemagglutinin interaction with biological membranes by photosensitized activation of [125I]iodonaphthylazide. J Biol Chem. 1994 May 20;269(20):14614–14619. [PubMed]
  • Ketterer B, Christodoulides LG. Enzymology of cytosolic glutathione S-transferases. Adv Pharmacol. 1994;27:37–69. [PubMed]
  • Mannervik B, Danielson UH. Glutathione transferases--structure and catalytic activity. CRC Crit Rev Biochem. 1988;23(3):283–337. [PubMed]
  • Singhal SS, Saxena M, Ahmad H, Awasthi S, Haque AK, Awasthi YC. Glutathione S-transferases of human lung: characterization and evaluation of the protective role of the alpha-class isozymes against lipid peroxidation. Arch Biochem Biophys. 1992 Dec;299(2):232–241. [PubMed]
  • Benson AM, Talalay P, Keen JH, Jakoby WB. Relationship between the soluble glutathione-dependent delta 5-3-ketosteroid isomerase and the glutathione S-transferases of the liver. Proc Natl Acad Sci U S A. 1977 Jan;74(1):158–162. [PMC free article] [PubMed]
  • Ujihara M, Tsuchida S, Satoh K, Sato K, Urade Y. Biochemical and immunological demonstration of prostaglandin D2, E2, and F2 alpha formation from prostaglandin H2 by various rat glutathione S-transferase isozymes. Arch Biochem Biophys. 1988 Aug 1;264(2):428–437. [PubMed]
  • Boyer TD, Vessey DA. Inhibition of human cationic glutathione S-transferase by nonsubstrate ligands. Hepatology. 1987 Sep-Oct;7(5):843–848. [PubMed]
  • Burns AL, Magendzo K, Srivastava M, Rojas E, Parra C, de la Fuente M, Cultraro C, Shirvan A, Vogel T, Heldman J, et al. Human synexin (annexin VII) polymorphisms: tissue specificity and expression in Escherichia coli. Biochem Soc Trans. 1990 Dec;18(6):1118–1121. [PubMed]
  • Cornwell MM, Gottesman MM, Pastan IH. Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells. J Biol Chem. 1986 Jun 15;261(17):7921–7928. [PubMed]
  • Ambudkar SV, Lelong IH, Zhang J, Cardarelli CO, Gottesman MM, Pastan I. Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8472–8476. [PMC free article] [PubMed]
  • Stern-Bach Y, Greenberg-Ofrath N, Flechner I, Schuldiner S. Identification and purification of a functional amine transporter from bovine chromaffin granules. J Biol Chem. 1990 Mar 5;265(7):3961–3966. [PubMed]
  • Raynal P, Pollard HB. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim Biophys Acta. 1994 Apr 5;1197(1):63–93. [PubMed]
  • Bell RM, Burns DJ. Lipid activation of protein kinase C. J Biol Chem. 1991 Mar 15;266(8):4661–4664. [PubMed]
  • Brocklehurst KW, Pollard HB. Interaction of protein kinase C with chromaffin granule membranes: effects of Ca2+, phorbol esters and temperature reveal differences in the properties of the association and dissociation events. Biochim Biophys Acta. 1989 Feb 27;979(2):157–165. [PubMed]
  • Peters T., Jr Serum albumin. Adv Protein Chem. 1985;37:161–245. [PubMed]
  • Habig WH, Jakoby WB. Glutathione S-transferases (rat and human). Methods Enzymol. 1981;77:218–231. [PubMed]
  • Wilson IA, Skehel JJ, Wiley DC. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. [PubMed]
  • Stegmann T, Doms RW, Helenius A. Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem. 1989;18:187–211. [PubMed]
  • White JM. Membrane fusion. Science. 1992 Nov 6;258(5084):917–924. [PubMed]
  • Mosior M, Newton AC. Mechanism of interaction of protein kinase C with phorbol esters. Reversibility and nature of membrane association. J Biol Chem. 1995 Oct 27;270(43):25526–25533. [PubMed]
  • Ballatori N. Glutathione mercaptides as transport forms of metals. Adv Pharmacol. 1994;27:271–298. [PubMed]
  • Kullak-Ublick GA, Hagenbuch B, Stieger B, Wolkoff AW, Meier PJ. Functional characterization of the basolateral rat liver organic anion transporting polypeptide. Hepatology. 1994 Aug;20(2):411–416. [PubMed]
  • Pulaski L, Jedlitschky G, Leier I, Buchholz U, Keppler D. Identification of the multidrug-resistance protein (MRP) as the glutathione-S-conjugate export pump of erythrocytes. Eur J Biochem. 1996 Oct 15;241(2):644–648. [PubMed]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...