Direct photoaffinity labeling of leukotriene binding sites

Abstract

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labcling of leukotriene binding sites. Cryofixation eliminates unspecific labeling taking place in solution by photoisomcrs and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock-freezing of the samples, irradiation-induced highly reactive excited states andlor intermediates can form covalent bonds with the respective binding sitc in the frozen state.After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time-dcpendent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation rcvcaled that only one polypeptide with an M , of 67000 was labeled. This polypeptide was identified as albumin.Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent M , of 48 000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hcpatocytes a polypeptide with an apparent M , of 48 000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent M , of 25000, was identified as subunit 1 of glutathione transfcrases by immunoprecipitation.The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene-metabolizing enzymes, and transport systems.Leukotrienes arc proinflammatory mediators active in vivo at nanoniolar concentrations [I -1 I]. They comprise the chemotactic leukotriene B4 (LTB,) as well as the cysteinyl leukotrienes. LTC4, LTD4, LTE, and its metabolites [ S , 61. Leukotrienes exert their biological effects through distinct receptors at the cell membrane [12]. The cysteinyl leukotriencs are rapidly eliminated from the blood circulation [9, 1 I] by a temperature-and ATP-dependent uptake system at the sinusoidal membrane of hepatocytes 113 -151. Moreover, cysteinyl leukotriene uptake has been demonstrated in renal cells and in intestinal mucosal cells [13], whereas AS-30D rat hepatoma cell% are complctely deficient in the uptake of these lipid mediators [I 51. Hepatocellular uptake is followed by partial intracellular metabolism and by excretion of cysteinyl leukotrienes and their degradation products into bile [9, 1 1,

Characterization of Binding of Leukotriene C4 by Human Multidrug Resistance Protein 1

“…Whether the site involves TM helices 16 and 17 has not been established. LTC 4 contains both conjugated and unconjugated double bonds and is intrinsically photolabile (39). It has been used for photolabeling of intact MRP1, but the regions of the protein where cross-linking occurs, or that are required for high affinity binding, have not been identified.…”

“…It has been used for photolabeling of intact MRP1, but the regions of the protein where cross-linking occurs, or that are required for high affinity binding, have not been identified. This is attributable in large part to practical limitations imposed by the low efficiency with which cross-linking occurs and the low specific activity of [ 3 H]LTC 4 (6,10,28,39,40). In this study, we have extended the utility of [ 3 H]LTC 4 as a photolabeling agent by using baculovirus dual expression vectors that permit co-expression of nearstoichiometric amounts of various MRP1 fragments in Sf21 cells.…”

Identification of the Multidrug‐Resistance Protein (MRP) as the Glutathione‐S‐Conjugate Export Pump of Erythrocytes

“…For this purpose, we used the method of photoaffinity labeling of the transporter protein with a photoactivatable substrate taking advantage of the fact that the high-affinity substrate LTC, itself is a photoactivatable compound [34]. This approach has previously been used to characterize the glutathione-S-conjugate transporting proteins in leukotriene-generating cells [ 171 and to radiolabel this transporter in MRP-overexpressing tumor cell lines [21,22,391.…”

Analysis of Human Multidrug Resistance Protein 1 (ABCC1) by Matrix-Assisted Laser Desorption Ionization/Time of Flight Mass Spectrometry: Toward Identification of Leukotriene C4 Binding Sites

“…In addition, UV irradiation of MRP1 can be expected to result in some degree of intra-or intermolecular cross-linking and/or other chemical reactions. However, because our goal was to identify LTC 4 -modified peptides and because the formation of covalent bonds between LTC 4 and amino acid residues in MRP1 involves the opening of one or more of the triene double bonds during irradiation (Falk et al, 1989), those differences in the obtained spectra that did not match an exact mass shift of 625.5 Da from an unlabeled peptide to a LTC 4 -labeled peptide were excluded from further consideration. In addition, the mass shifts were required to occur only for MRP1 irradiated in the presence of LTC 4 and not for the two controls.…”

“…As mentioned previously, LTC 4 is intrinsically photoreactive, a property attributable to its conjugated triene structure (Fig. 4A) (Falk et al, 1989). In general, the efficiency of photochemical reactions is low and consequently, before mass spectrometry, conditions for optimal photolabeling with LTC 4 were determined by photolabeling a constant amount of purified MRP1 in increasing concentrations of [ 3 H]LTC 4 .…”