Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium.
Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D004735:Energy Transfer; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D008081:Liposomes; D008854:Microscopy, Electron; D009132:Muscles / Q000201:enzymology*; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D011817:Rabbits; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
D008081:Liposomes; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D002118:Calcium