Interactions of 5-lipoxygenase with membranes: studies on the association of soluble enzyme with membranes and alterations in enzyme activity.

Treatment of rat basophilic leukemia cells (RBL-1) with the calcium ionophore A23187 resulted in activation of 5-lipoxygenase, as indicated by an induction of leukotriene release [Orning, L., Hammarström, S., & Samuelsson, B. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2017]. The enzyme activation was accompanied by a time-dependent association of 5-lipoxygenase to the particular fraction. When cells were lysed in the presence of 0.05-10 microM CaCl2, the soluble 5-lipoxygenase became associated with the particulate fraction. This was demonstrated by a decrease in immunoreactivities and enzymatic activities in the soluble fraction and a parallel increase in particulate-associated immunoreactivities. The particulate-bound enzyme was not active. Ca2+ induced the membrane association of 5-lipoxygenase when added into the incubation mixtures containing the membrane fraction with either the cytosolic fraction or the purified enzyme. 5-Lipoxygenase also bound to the microsomal-enriched fraction in the presence of Ca2+. Maximal membrane binding was obtained after a 1-min incubation at 4 degrees C. When a fixed amount of isolated membranes (0.2 mg of protein) and increasing cytosolic protein (0.5-4 mg) were used, a linear increase in enzyme binding was observed. The binding became saturated at 3 mg of cytosolic protein/mg of membrane protein. 5-Lipoxygenase binding to the membrane fraction was unaffected by pretreatment of the membranes with trypsin but was inhibited by treating with phospholipase A2, suggesting that phospholipids are involved.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification, characterization, and structural properties of a single protein from rat basophilic leukemia (RBL-1) cells possessing 5-lipoxygenase and leukotriene A4 synthetase activities.

Arachidonate 5-lipoxygenase of rat basophilic leukemia (RBL-1) cells was purified more than 1000-fold by gel filtration and anion exchange protein-high performance liquid chromatography (HPLC). Physical properties of the purified 5-lipoxygenase such as molecular weight (74,000-76,000), N-terminal sequence (30 amino acids), and amino acid composition were determined. The purified enzyme converted [14C]arachidonic acid at 20 degrees to [14C] 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and to [14C]dihydroxyeicosatetraenoic acids (diHETEs). Utilizing [14C] 5(S)HPETE as substrate, the purified enzyme also converted the hydroperoxy acid to [14C]diHETES. The [14C]diHETE reaction products were identified primarily (greater than 80% of recovered radioactivity) as the nonenzymatic hydrolysis products of leukotriene A4 (i.e., 6-trans-leukotriene B4 and 12-epi-6-trans-leukotriene B4) by reverse phase HPLC, scanning spectrophotometry, and gas chromatography-mass spectrometry. The bioconversion of [14C] arachidonate and [14C]5(S)HPETE to reaction products by the purified enzyme was dependent on the presence of both Ca2+ and ATP. The enzymatic activities were inhibited in a similar manner by the lipoxygenase inhibitors nordihydroguaiaretic acid, diphenyldisulfide, and SK&F 86002. The data provide evidence that RBL-1 cell 5-lipoxygenase and leukotriene A4 synthetase activities reside on a single monomeric protein with a free N-terminus and that they possess similar biochemical characteristics.

Further comparison of contractions of the smooth muscle of the guinea-pig isolated vas deferens induced by ATP and related analogs.

Some structural requirements which are important for the contractile activity of adenine nucleotides at P2-purinergic receptors in the guinea-pig isolated vas deferens were examined. The consequences of deletions, substitutions and isosteric rearrangements in 1-N, C-2, C-8, N-9 and N6 of adenine, in the ribose hydroxyls, and in the polyphosphate chain were examined. Several kinds of effects on activity were observed, including potentiation of responses, losses in activity, modifications of response duration, and transitions from biphasic concentration-response curves, which are characteristic of ATP, to monophasic curves. Generally, deletions and substitutions to adenine reduced activity. Ribose modifications were better-tolerated. The presence of 5'-phosphate or phosphorothioate moieties was required for maximum activity. A comparison of the present results with previous reports indicates that certain modifications to ATP result in similar effects in both the vas deferens and urinary bladder, which are opposite those which occur in the taenia coli.

Binding of 4(5)-[2-(4-azido-2-nitroanilino)ethyl]imidazole to histamine receptors in guinea pig cerebral cortical membranes.

The interaction of 4(5)-[2-(4-azido-2-nitroanilino)ethyl]imidazole (AAH), a photolabile histamine receptor antagonist, with the binding of histamine, mepyramine, and tiotidine to guinea pig cerebral cortical membranes was examined to evaluate the specificity of AAH for histamine H1 and H2 receptors. Saturable, specific binding of [3H]histamine, [3H]mepyramine, and [3H]tiotidine to the membranes was observed. Competition assays were used to assess the relative affinity of AAH for H1- and H2-receptors. The rank order of IC50 values obtained was (most to least potent) (i) for competing with [3H]histamine binding: histamine greater than AAH much greater than mepyramine approximately equal to tiotidine; (ii) for competing with [3H]mepyramine binding: mepyramine much greater than AAH greater than histamine greater than tiotidine; and (III) for competing with [3H]tiotidine binding: tiotidine much greater than mepyramine greater than histamine approximately equal to AAH. The affinity of AAH for H1 receptors was ca. 14-fold greater than for H2 receptors. These findings support previous evidence obtained in isolated smooth muscle preparations that AAH shows H1-receptor selectivity as an antagonist.

Characterization of [3H]leukotriene D4 binding sites in guinea-pig ventricular myocardium.

[3H]Leukotriene (LT) D4 was used to identify specific LTD4 binding sites in guinea-pig ventricular myocardial membranes. High-performance liquid chromatography analyses indicated that, in the presence of the gamma-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 3% of membrane-bound [3H]LTD4 was converted to [3H]LTC4 or [3H]LTE4 at 30 degrees C. The specific [3H] LTD4 binding, assayed in the presence of 80 mM L-serine-borate, reached a stable steady state within 45 min at 30 degrees C (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded an apparent dissociation constant (Kd) of 3.4 +/- 2.1 nM and a maximum number of binding sites of 850 +/- 91 fmol/mg of protein. Competition binding studies with [3H]LTD4, synthetic 5S, 6R-LTD4 (LTD4) and its diastereoisomer 5R,6S-LTD4, LTE4, LTC4 and the putative LT antagonists FPL 55712, 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid (2-nor-LTD1) and SKF 88046 demonstrated a potency order of LTD4 greater than LTE4 greater than LTC4 greater than 5R,6S-LTD4 much greater than 2-nor-LTD1. FPL 55712 and SKF 88046 were ineffective in displacing the specific [3H]LTD4 binding. Pretreatment of the heart membranes with the sulfhydryl reducing reagent dithiothreitol decreased the specific [3H]LTD4 binding in a concentration-dependent manner. Scatchard analyses of saturation isotherms indicated that 0.3 mM dithiothreitol pretreatment of heart membranes decreased the maximum number of binding sites of the [3H]LTD4 binding to 368 +/- 61 fmol/mg of protein with minimal effects on the apparent Kd.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of guinea pig myocardial leukotriene C4 binding sites. Regulation by cations and sulfhydryl-directed reagents.

Using [3H]leukotriene C4 (LTC4) and radioligand-binding techniques, specific leukotriene C4 binding sites have been identified in membranes derived from guinea pig ventricular myocardium. High performance liquid chromatography analyses indicated that, in the presence of the gamma-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 2% of membrane-bound [3H]LTC4 was converted at 20 degrees to [3H]leukotriene D4 or [3H]leukotriene E4. The specific binding of 4 nM [3H]LTC4, in the presence of 80 mM L-serine-borate, reached a stable steady state within 15 min at 20 degrees (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded a dissociation constant (Kd) of 27.5 +/- 6.0 nM and a maximum number of binding sites (Bmax) of 19.9 +/- 5.2 pmol/mg of membrane protein. Competition binding studies of [3H]LTC4 with synthetic leukotriene C4, leukotriene D4, and leukotriene E4 and the putative peptidoleukotriene antagonists FPL 55712, SKF 88046, and 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid revealed an order of potency of leukotriene C4 much greater than 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid greater than SKF 88046 greater than LTE4 greater than LTD4 greater than FPL 55712. The specific [3H]LTC4 binding was stimulated by the divalent cations Ca2+, Mg2+, and Mn2+ and to a lesser degree by the monovalent cations Na+, K+, Li+, and NH4+. CaCl2 (3 mM) and NaCl (150 mM) stimulated the LTC4 binding by increasing the Bmax to 42.6 +/- 5.9 and 35.0 +/- 2.0 pmol/mg, respectively, but had minimal effects on Kd. Pretreatment of the heart membranes with the sulfhydryl reagent N-ethylmaleimide decreased the specific [3H]LTC4 binding in a concentration-dependent manner. The N-ethylmaleimide-induced inactivation of [3H]LTC4 binding sites was protected by occupation of the binding site with the agonist leukotriene C4, but no protection was observed with the antagonist SKF 88046. Scatchard analyses of saturation isotherms indicated that 30 microM N-ethylmaleimide pretreatment reduced the Bmax of the [3H]LTC4 binding to 8.2 +/- 3.1 pmol/mg with minimal effects on Kd. The data provide direct biochemical evidence for specific [3H]LTC4 binding sites in the guinea pig heart membranes. The [3H]LTC4 binding sites appear to be modulated by divalent and monovalent cations and free sulfhydryl group(s) may be associated with the agonist-binding site. The results suggest that the physiological effects of the leukotrienes on the guinea pig heart may be mediated through membrane-bound receptors.

Leukotriene E4 binds specifically to LTD4 receptors in guinea pig lung membranes.

High affinity, stereoselective binding sites for [3H]leukotriene E4 ([3H]LTE4) have been identified and characterized in guinea pig lung membranes. [3H]LTE4 bound to these membranes with a pharmacological specificity identical to that previously observed for the [3H]LTD4 receptor in guinea pig lung. [3H]LTE4 specific binding was selectively inhibited by Na+, enhanced by Ca2+, Mg2+ and Mn2+ and modulated by guanine nucleotides. Scatchard analysis of saturation binding data showed a single class of high affinity and saturable binding sites, with a dissociation constant (Kd) of 0.4 +/- 0.2 nM and a density (Bmax) of 430 +/- 50 fmol/mg membrane protein, similar to values observed for the LTD4 receptor in guinea pig lung. The rank order potency of agonist binding to the [3H]LTE4 binding sites was LTD4 greater than LTE4 much greater than LTC4. These results indicate that [3H]LTE4 binds to [3H]LTD4 receptors and suggests that induction of smooth muscle contraction by LTD4 and LTE4 may be mediated by identical mechanisms and receptors in the guinea pig lung.

Interaction of [3H]arylazido aminopropionyl ATP ([3H]ANAPP3) with P2-purinergic receptors in the smooth muscle of the isolated guinea-pig vas deferens.

Following its photolysis in the presence of the isolated guinea-pig vas deferens, the ATP photoaffinity label ANAPP3 produces a specific antagonism of adenine nucleotide-induced contractile responses which are mediated by P2-purinergic receptors. To characterize the site of covalent photoincorporation of ANAPP3, intact vasa deferentia were treated with [3H]ANAPP3 and samples of homogenate, cytosol and a crude membrane fraction were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Photolysis of [3H]ANAPP3 (10(-5) M; 3.0 mu Ci/ml) resulted in the incorporation of radioactivity into cellular components with apparent molecular weights of 54-66 and 43-57 kilodaltons. The photoincorporation of [3H]ANAPP3 was associated with the crude membrane fraction and not the cytosol, was reduced in the presence of ATP in an ATP-concentration-dependent manner, was lessened following pretreatment of the tissues with photolyzed nonradiolabeled ANAPP3, and was unaffected by the nucleoside transport inhibitor, dipyridamole. In tension studies on the same tissues the presence of ATP resulted in a concentration-dependent reduction in the initial contractile response to [3H]ANAPP3 the response to 3H was antagonized in tissues which had been pretreated with nonradiolabeled ANAPP3, and dipyridamole had no effect on the contractile response to [3H]ANAPP3. According to several criteria these findings indicate that the antagonism by photolyzed ANAPP3 of adenine nucleotide-induced responses is a direct result of the covalent insertion at or near the recognition site of cell-surface P2-purinergic receptors.

Regulation of ligand binding to leukotriene D4 receptors: effects of cations and guanine nucleotides.

High affinity, stereoselective specific binding sites for [3H]leukotriene D4 [( 3H]LTD4) have been demonstrated in guinea pig lung membranes. Purine nucleotides quantitatively reduced [3H]LTD4 specific binding with a rank order potency of guanosine-5'-O-3-thiotriphosphate (GTP gamma S) = guanyl-5'-yl-imido-diphosphate [Gpp(NH)p] greater than GTP greater than ATP greater than GDP. In the presence of 1 microM Gpp(NH)p, the maximum number (Bmax) of [3H]LTD4 specific binding sites was reduced to 41 +/- 10 percent of the control level (950 +/- 150 fmol/mg membrane protein). In the presence of 3 microM Gpp(NH)p, the rate of association of [3H]LTD4 to the specific sites was estimated to have increased 2.5-fold. The rate of dissociation of [3H]LTD4 from the specific sites was also increased significantly in the presence of 50 microM Gpp(NH)p. The divalent cations, Ca2+ and Mg2+ (10 mM), increased the Bmax 2-fold and had minimal effects on the dissociation constant (Kd) of [3H]LTD4 specific binding. Sodium ions, at a concentration of 50 mM, reduced the Bmax, and had minimal effects on the Kd of [3H]LTD4 specific binding. These data indicate that guanine nucleotides, Na+, Mg2+ and Ca2+ regulate [3H]LTD4 binding to its receptors in guinea pig lung.

Comparison of the effects of apamin, a Ca2+-dependent K+ channel blocker, and arylazido aminopropionyl ATP (ANAPP3), a P2-purinergic receptor antagonist, in the guinea-pig vas deferens.

Apamin, which blocks Ca2+-dependent increases in K+ permeability, antagonizes ATP-induced relaxation of several smooth muscles. The ATP photoaffinity label arylazido aminopropionyl ATP (ANAPP3), following its photolysis in the presence of the guinea-pig vas deferens, antagonizes contractile responses to ATP. This study was conducted to determine whether apamin antagonizes ATP-induced responses in the guinea-pig vas deferens, and also to evaluate whether ANAPP3 antagonizes responses to ATP by interfering with Ca2+-dependent K+ permeability changes. Apamin (10(-6) M) potentiated ATP-induced contractions. This potentiation was nonspecific in that responses to norepinephrine, histamine and acetylcholine also were enhanced; responses to KCl were unaffected. To evaluate the possible interactions between the two agents at the same cellular site, the effect of apamin was examined in ANAPP3-treated tissues. In such tissues apamin did not potentiate the residual responses to ATP; however, apamin was nevertheless able to potentiate responses of ANAPP3-treated tissues to norepinephrine, histamine and acetylcholine, and responses to KCl remained unaffected. These studies provide additional support for the view that ANAPP3 antagonizes ATP-induced responses of the guinea-pig vas deferens by blocking P2-purinergic receptors. The antagonism by ANAPP3 is not attributable to a blockade of Ca2+-dependent K+ permeability changes.

Leukotriene C4 ([3H]-LTC4) binding to membranes isolated from a hamster smooth muscle cell line (DDT1MF2).

Leukotriene C4 (LTC4) has been demonstrated to induce contraction of the smooth muscle cell line DDT1MF2. A partially purified membrane fraction obtained from these cells exhibited a high affinity binding site for LTC4. Binding of [3H]-LTC4 was saturable, specific and reversible with a dissociation constant (Kd) of 21 +/- 4 nM. The maximum number of binding sites (Bmax) was 55 +/- 5 pmol/mg of protein. Specificity was demonstrated in competition studies in which the Ki of LTC4 against specifically bound [3H]-LTC4 was 12 nM whereas Leukotriene D4 (LTD4) and Leukotriene E4 (LTE4) had a Ki of 38 +/- 4 and 4.7 +/- 0.5 nM respectively. A previously described antagonist of leukotriene-induced smooth muscle contraction PFL 55712 had a Ki of 23 +/- 2 nM as determined by competition binding experiments.

Characterization of the leukotriene D4 receptor in guinea-pig lung.

The effects of monovalent, divalent cations, buffere species and pH dependency on [3H]leukotriene D4 binding to the receptor have been characterized in vitro by using a radioligand binding assay. It was found that Ca2+, Mg2+, Co2+ and Mn2+ enhanced the specific binding. High concentrations of NaCl (150-300 mM) inhibited the specific binding to the receptor. The specific binding was also found to be higher in Pipes buffer (pH 6.5) than in Tris, Hepes and phosphate buffer at pH 7.0-8.0. Conversion of [3H]leukotriene D4 was minimized by inclusion of 1 mM cysteine, glycine in the incubation buffer and maintaining the temperature at 22 degrees C. Under the conditions employed, the dissociation constant (KD) and the receptor density (Bmax) were calculated to be 1.8 +/- 0.9 nM and 2100 +/- 375 fmol/mg protein respectively. The leukotriene antagonist FPL 55712, agonist 5R, 6S-LTD4 and LTE4 competed with the [3H]LTD4 binding to the receptor. Prostaglandins, alpha-, beta-adrenergic and dopaminergic receptor agonists and antagonists did not compete significantly.

Identification of specific binding sites for leukotriene C4 in human fetal lung.

Specific leukotriene C4 (LTC4)1 binding sites were identified in membrane preparations from human fetal lung. Specific binding of [3H]-LTC4 represented 95 percent of total binding, reached steady-state within 10 minutes and was rapidly reversible upon addition of excess unlabeled LTC4. Binding assays were performed at 4 degrees C under conditions which prevented metabolism of [3H]-LTC4 (80 mM serine-borate, 10 mM cysteine, 10 mM glycine). Under these conditions, greater than 95 percent of the membrane bound radioactivity, as analyzed by high performance liquid chromatography, co-eluted with the LTC4 standard. Computer-assisted analyses of saturation binding data showed a single class of binding sites with a dissociation constant (Kd) of 26 + 6 nM and a density (Bmax) of 84 + 18 pmol/mg protein. Pharmacological specificity was demonstrated by competition studies in which specific binding of [3H]-LTC4 was displaced by LTC4 and its structural analogs with inhibition constants (Ki) of 10 to 30 nM, whereas LTD4, diastereoisomers of LTD1, LTE4 and the end organ antagonist FPL 55712 were 150 to 700 fold less potent competitors than LTC4. These results provide evidence for specific, reversible, saturable, high affinity binding sites for [3H]-LTC4 in human fetal lung membranes.

Peptidoleukotrienes: distinct receptors for leukotriene C4 and D4 in the guinea-pig lung.

Using [3H]-leukotriene C4 ([3H]-LTC4) and [3H]-leukotriene D4 ([3H]-LTD4), specific peptidoleukotriene receptors have been identified in membranes derived from guinea-pig lung. In the presence of 0.1 mM guanyl-5'-yl-imidodiphosphate, which completely inhibits [3H]-LTD4 binding, [3H]-LTC4 binding was protein- and temperature-dependent, reached equilibrium within 15 minutes at 20 degrees C and was reversible. Guanine nucleotides had no effect on the [3H]-LTC4 binding. Competition studies with [3H]-LTC4, peptidoleukotrienes C4, D4, E4 and the peptidoleukotriene antagonist FPL 55712 revealed an order of potency of leukotriene C4 much greater than E4 greater than D4 greater than FPL 55712. [3H]-LTD4 competition studies indicated an order of potency of LTD4 greater than LTE4 greater than LTC4 much greater than FPL 55712. Bioconversion of [3H]-LTC4, as determined by high performance liquid chromatography, was less than 3 percent. The data suggest the guinea-pig lung may contain biochemically distinct receptors for LTC4 and LTD4.

Photoaffinity labeling of P2-purinergic and H1-histamine receptors in intact smooth muscle.

This paper discusses the general applicability of photoaffinity labels as pharmacological receptor antagonists in functional studies of intact smooth muscle preparations. Guidelines are suggested that take into account the criteria for photoaffinity labeling studies as well as those for use with conventional antagonists.

Evidence for a contribution by purines to the neurogenic response of the guinea-pig urinary bladder.

In order to determine if ATP contributes as an excitatory transmitter in the guinea-pig bladder, experiments were conducted with ANAPP3, a photoaffinity analogue of ATP, which is an antagonist of adenine nucleotides in several other smooth muscles. With or without photoactivation with visible light, ANAPP3 antagonized contractile responses of in vitro strips of bladder to exogenous ATP. The antagonism was specific in that responses to acetylcholine and KCl were not affected by ANAPP3. Responses of strips of bladder to transmural electrical stimulation were not antagonized by ANAPP3 and were relatively insensitive to atropine. However, combined treatment with ANAPP3 and atropine produced a marked antagonism of the neurogenic response. In experiments with bladders obtained from animals pretreated with 6-hydroxydopamine, the ANAPP3-sensitive component of the neurogenic response was absent. These results suggest that acetylcholine, released from cholinergic nerves, and a purine, released from 6-hydroxy-dopamine-sensitive nerves, are both involved in motor transmission in this tissue.

Comparison of photoaffinity labeling of P2-purinergic receptors of isolated guinea-pig vas deferens by arylazido aminopropionyl ATP and by arylazido aminobutyryl ATP.

Two chemically related arylazido photoaffinity analogs of ATP (arylazido aminopropionyl ATP (ANAPP3) and arylazido aminobutyryl ATP (ANABP3)), which have been reported in the literature to differ in their ability to inhibit myosin ATPase, were compared for their ability to antagonize contractile responses of the isolated guinea-pig vas deferens to ATP. During photolysis in organ chambers the photoconversion (delta A260/delta t) of ANAPP3 occurred with greater than first order kinetics or was multiexponential and t1/2 = 7.5 min, while delta A260/delta t for ANABP3 was first order and t1/2 = 2.25 min. After photolysis of these compounds in the presence of the guinea-pig vas deferens, using irradiation periods which caused 80% consumption of the compounds, ANABP3 was 2-3 times more potent than ANAPP3 in antagonizing contractions to ATP, which are mediated by P2-purinergic receptors. A comparison of concentration-response curves obtained for nonphotolyzed ANAPP3 and ANABP3 used as agonists suggested that the greater antagonism produced by photolyzed ANABP3 is not attributable to a greater potency. The results suggest that the longer 3'-hydroxyl-arylazide bridge length of ANABP3 places the arylnitrene intermediate in a position at or near the P2-receptor which is more favorable for covalent insertion.