Bilitranslocase can exist in two metastable forms with different affinities for the substrates--evidence from cysteine and arginine modification.
Bilitranslocase is an organic anion carrier involved in bilirubin and phthalein uptake by the liver. In rat liver plasma membranes, its function is assayed by recording the electrogenic sulfobromophthalein movement. This has been found to be inhibited by both cysteine-specific and arginine-specific reagents. Inhibition is both partial and it occurs to the same extent, i.e. approximately 50%. The effects are not additive. Here we describe the mechanism underlying the above observations. It is concluded that bilitranslocase occurs in two possible states, featured by high and low affinity for the substrates (for sulfobromophthalein, Km = 5 microM and 37 microM, respectively). Cysteine- or arginine-reactive reagents, by reacting selectively with the low-affinity form, entrap it and shift the equilibrium between the two forms, so that, at completion, only the low-affinity form is present. The substrate concentration in the standard transport assay is 39 microM, a value at which the modified low-affinity form operates in the range of half-maximal velocity. This explains both the apparent half-inhibition measured after the chemical treatments and the lack of additivity. In addition, the substrates are shown to enhance the rate of conversion from the low-affinity to the high-affinity form of the translocator, thus favouring its high-affinity form under physiological conditions.
Passamonti
S
S
Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole Università degli Studi di Trieste, Italy. passamon@bbcm.univ.trieste.it
Battiston
L
L
Sottocasa
G L
GL
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001663:Bilirubin / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002570:Ceruloplasmin; D003545:Cysteine / Q000737:chemistry; D006902:Hydroxymercuribenzoates / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008623:Mercaptoethanol / Q000378:metabolism; D051381:Rats; D013448:Sulfobromophthalein / Q000378:metabolism
D006902:Hydroxymercuribenzoates; D008565:Membrane Proteins; D013448:Sulfobromophthalein; C030601:4-hydroxymercuribenzoate; D008623:Mercaptoethanol; D001120:Arginine; D002570:Ceruloplasmin; C033838:bilitranslocase; D003545:Cysteine; D001663:Bilirubin