DISSECTION OF DISCRETE KINETIC EVENTS IN THE BINDING OF ANTIBIOTICS AND SUBSTRATES TO THE GALACTOSE-H-COLI( SYMPORT PROTEIN, GALP, OF ESCHERICHIA)

GalP is the membrane protein responsible for H+-driven uptake of D-gal actose into Escherichia coli. It is suggested to be the bacterial equi valent of the mammalian glucose transporter, GLUT1, since these protei ns share sequence homology, recognise and transport similar substrates and are both inhibited by cytochalasin B and forskolin. The successfu l over-production of GalP to 35-55% of the total inner membrane protei n of E. coli has allowed direct physical measurements on isolated memb rane preparations. The binding of the antibiotics cytochalasin B and f orskolin could be monitored from changes in the inherent fluorescence of GalP, enabling derivation of a kinetic mechanism describing the int eraction between the ligands and GalP. The binding of sugars to GalP p roduces little or no change in the inherent fluorescence of the transp orter. However, the binding of transported sugars to GalP produces a l arge increase in the fluorescence of 8-anilino-1-naphthalene sulphonat e (ANS) excited via tryptophan residues. This has allowed a binding st ep, in addition to two putative translocation steps, to be measured. F rom all these studies a basic kinetic mechanism for the transport cycl e under non-energised conditions has been derived. The ease of genetic al manipulation of the galP gene in E. coli has been exploited to muta te individual amino acid residues that are predicted to play a critica l role in transport activity and/or the recognition of substrates and antibiotics. Investigation of these mutant proteins using the fluoresc ence measurements should elucidate the role of individual residues in the transport cycle as well as refine the current model.