Glutathione conjugates recognize the Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase.
Leukotriene (LT) C4 and other glutathione conjugates are synthesized intracellularly and then move to the plasma membrane for export. The intracellular proteins that bind these molecules and the significance of these interactions are poorly understood. To identify the binding sites of membrane-associated proteins that recognize these molecules, we utilized photoaffinity probes to label the inner leaflet of erythrocytes. The predominant molecule labeled with S-(p-nitrobenzyl)glutathione-[125I]4-azidosalicylic acid (PNBG-[125I]ASA) or LTC4-[125I]4-azidosalicylic acid (LTC4-[125I]ASA) was 38 kDa. The protein was labeled with PNBG-[125I]ASA, electroblotted to polyvinylidene difluoride membranes, digested in situ with lysyl endopeptidase, and two radiolabeled peptides isolated by reverse phase-high performance liquid chromatography. These contained an identity of 7/11 with amino acids 119-129, and 11/11 with amino acids 67-77 of human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Photoaffinity labeling with PNBG-[125I]ASA was blocked completely by 100 microM ATP and greater than 50% with 100 microM NAD+. LTC4-[125I]ASA binding to the NAD+ site was confirmed by V8 protease digestion of purified GAPDH labeled with LTC4-[125I]ASA or PNBG-[125I]ASA, with both labels localized to the 6.8-kDa N-terminal fragment. Photoaffinity labeling of HL-60 cells with LTC4-125I-ASA identified GAPDH as the predominant cytoplasmic binding protein in these cells. These data indicate that GAPDH is a membrane-associated and cytoplasmic protein which binds glutathione conjugates including LTC4.
Puder
M
M
Arthritis Unit and the Department of Medicine, Massachusetts General Hospital, Massachusetts General Hospital East, Charlestown, Massachusetts 02129, USA.
Soberman
R J
RJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004910:Erythrocyte Membrane / Q000201:enzymology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D017997:Leukotriene C4 / Q000031:analogs & derivatives* / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D015394:Molecular Structure; D009243:NAD / Q000494:pharmacology; D017510:Protein Folding; D012459:Salicylates / Q000378:metabolism*
D000345:Affinity Labels; D007457:Iodine Radioisotopes; C105470:S-(4-nitrobenzyl)glutathione-iodo-4-azidosalicyclic acid; D012459:Salicylates; C105469:leukotriene C4-iodo-4-azidosalicylic acid; D009243:NAD; D017997:Leukotriene C4; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005978:Glutathione