Jeffrey S. Wiseman

Role of mast cell chymase in the extracellular processing of big-endothelin-1 to endothelin-1 in the perfused rat lung

Previous studies of endothelin-1 (ET) synthesis have shown that some cultured endothelial cells secrete an intermediate product, big-endothelin-1 (bigET), suggesting that the processing of secreted bigET to ET may be physiologically significant. In this study, two pertinent ET converting enzyme activities, mast cell chymase I (EC 3.4.21.39) and a phosphoramidon-sensitive, neutral metalloprotease, were identified in a rat lung particulate fraction. We perfused rat lungs with bigET and chymostatin or phosphoramidon to study the relevance of these two proteases to the processing of extracellular bigET in vivo. Addition of compound 48/80 (a compound which activates mast cells, causing degranulation and release of chymase) to the perfusion buffer greatly increased hydrolysis of exogenously added bigET to ET. ET formation was inhibited completely by 32 microM chymostatin, whereas inhibition by 50 microM phosphoramidon was incomplete and variable. Perfusate histamine levels were used to monitor the extent of mast cell degranulation, and inhibition of ET production by phosphoramidon was attributed to inhibition of degranulation, per se. There was a direct correlation between perfusate ET and histamine levels in both control and phosphoramidon-treated (but not chymostatin-treated) lungs. Our results suggest that chymase from lung mast cells is capable of physiologically relevant extracellular processing by bigET to ET in the perfused rat lung.

5-Lipoxygenase from rat PMN lysate

The products of arachidonic acid metabolism in the 15,000xg supernatant of sonicated rat PMN are described. Only products derived from 5-lipoxygenase are observed. These products are 5-HETE and products derived from hydrolysis of LTA4, particularly LTB4. Some minor products derived from decomposition of 5-HPETE are also observed. The dependence of the activity of 5-lipoxygenase on enzyme and on substrate concentrations is presented and discussed in terms of a kinetic model that includes enzyme inactivation during turnover and substrate inhibition. The 5-lipoxygenase activity is stimulated by Ca++ and ATP.

Glutathione peroxidase is neither required nor kinetically competent for conversion of 5-HPETE to 5-HETE in rat PMN lysates.

In the 5-lipoxygenase pathway for arachidonic acid metabolism, reduction of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to 5-hydroxyeicosatetraenoic acid (5-HETE) is catalyzed by an activity different from glutathione peroxidase. Glutathione peroxidase here refers to the nonspecific peroxidase that catalyzes the reduction by glutathione of cumene hydroperoxide and a variety of other peroxides including 5-HPETE. This enzyme is inhibited by mercaptosuccinic acid. Preparations of the 15,000xg supernatant from lysed rat peritoneal polymorphonuclear leukocytes were the source of these activities. Thus, when glutathione peroxidase is inhibited to less than 0.5% of its normal activity by mercaptosuccinic acid, 5-HPETE is reduced as efficiently as in the absence of mercaptosuccinate. In lysate preparations from which endogenous glutathione has been removed, reduction of 5-HPETE is still observed but only in the presence of added reducing agents, e.g., 0.2 mM glutathione. When endogenous glutathione peroxidase is not inhibited, reduction of 5-HPETE occurs at a rate greater than 15-fold faster than can be accounted for by this activity. We conclude, therefore, that the glutathione peroxidase in rat PMNs is not kinetically competent to account for reduction of 5-HPETE. There is a distinct peroxidase that catalyzes this reaction. The 5-HPETE peroxidase can utilize glutathione as reducing agent but is not inhibited by mercaptosuccinate, and additional results indicate that it is inactivated during turnover.

Ketones as electrophilic substrates of lipoxygenase

The rate-limiting step of the lipoxygenase reaction involves the abstraction of a hydrogen from the methylene carbon of a 1,4-diene. One possibility for the mechanism of the enzyme is the abstraction of this hydrogen as a proton to generate a carbanionic intermediate or transition state. In order to investigate this possibility, 5-, 8-, 12-, and 15-hydroxy-eicosatetraenoic acid were oxidized to the corresponding ketones and these ketones were assayed as substrates of the 5-, 12-, and 15-lipoxygenases from rat neutrophils, rat platelets, and soybeans, respectively. The ketones were in no case better substrates than arachidonic acid and in some cases the hydroxyeicosatetraenoic acids were equally active as the corresponding ketones. Since no increased rate of oxidation for these electrophilic substrates was observed, it is concluded that no transition state with carbanionic character is generated in the rate-determining step of the lipoxygenase reaction.

Determination of endothelin by an immobilized receptor assay utilizing a 96-well format.

Bovine cerebellar membranes immobilized on 96-well microtiter plates provide receptors for 125I-labeled endothelin-1 as the basis for a competitive binding assay. Adsorption of the membranes to a surface does not significantly alter the ligand-receptor interaction and reduces non-specific binding to 3-7% of total binding compared to 10-20% for a filtration technique. Considerable savings in reagents are realized since assays can be performed in 100 microliter volumes with only 10-20 micrograms of membrane protein. The 96-well format allows the rapid quantitation of large numbers of samples, and the assay is especially attractive in that it utilizes readily available reagents and equipment without the need for specific antibodies. The endothelin-receptor-based assay may be used to measure conversion of big endothelin-1 to endothelin-1 in aqueous assays. Since the presence of serum does not affect this method, tissue culture medium may be directly analyzed for endothelin production by cultured cells. All three isoforms of endothelin are detected, and the specificity of the receptor is retained since fragments and precursor forms of endothelin are not recognized. In cases where multiple endothelin isoforms may be present or where specificity of binding is in question, this assay may be used in conjunction with high pressure liquid chromatography to distinguish active peptides.

An assay for dihydroorotase using high-performance liquid chromatography with radioactivity detection

An assay for measuring dihydroorotase activity was devised. Radiolabeled substrate and product were separated by high-performance liquid chromatography using a reverse-phase column with ion-pairing, and the radioactivity was quantitated by flow detection.

A radiometric kynurenine monooxygenase assay

Kynurenine 3-monooxygenase is a flavin-dependent monooxygenase that catalyzes the oxidation of L-kynurenine to 3-hydroxy-L-kynurenine in the kynurenine pathway of tryptophan metabolism. The enzyme requires NADH or NADPH as a cofactor. A discontinuous assay that utilizes L-[3H]kynurenine as substrate is described. The assay offers high precision and a wide range of accessible substrate and cofactor concentrations. The assay was used to measure kinetic isotope effects and the stereospecificity of oxidation of the cofactor. Hydride is transferred from the A-side (pro-R) of NADH and NADPH since primary deuterium isotope effects were observed for both cofactors when they were deuterated on the A-side but not on the B-side. The large isotope effect on Vmax/Km for NADH is sensitive to the concentration of kynurenine, which indicates that NADH can bind before kynurenine.

The specificity of two proteinases that cleave adjacent to arginine, Cl esterase and acrosin, for peptide p-nitroanilide substrates

Relative values of Vmax/Km for hydrolysis of 40 peptide p-nitroanilides catalyzed by human Cl-s and human acrosin are reported. For Cl-s, Ac-Lys(gamma Cbz)-Gly-Arg is the optimum sequence, but 25% of the substrates have (Vmax/Km)rel greater than 0.25 compared to this sequence. The best acrosin substrate tested has the sequence Tos-Gly-Pro-Arg, although (Vmax/Km)rel greater than 0.15 for more than half of the substrates. Proline at P2 is preferred by acrosin. Both enzymes prefer arginine at P1 greater than or equal to 3-fold over lysine and will not accept citrulline. In addition, occupancy of site S3 may yield an increase in Vmax/Km of greater than or equal to 10-fold with either enzyme, but many residues are accepted at S2, S3 and S4. Thus, an acrosin assay using Tos-Gly-Pro-Arg p-nitroanilide as a substrate is more than 20-times as sensitive as existing assays with blocked arginine derivatives.

Specificity of an HPETE peroxidase from rat PMN

The 15,000xg supernatant of sonicated rat PMN contains 5-lipoxygenase that converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A4 and an HPETE peroxidase that catalyzes reduction of the 5-HPETE. The specificity of this HPETE peroxidase for peroxides, reducing agents, and inhibitors has been characterized to distinguish this enzyme from other peroxidase activities. In addition to 5-HPETE, the HPETE peroxidase will catalyze reduction of 15-hydroperoxyeicosatetraenoic acid, 13-hydroperoxyoctadecadienoic acid, and 15-hydroperoxy-8,11,13-eicosatrienoic acid, but not cumene or t-butylhydroperoxides. The HPETE peroxidase accepted 5 of 11 thiols tested as reducing agents. However, glutathione is greater than 15 times more effective than any other thiol tested. Other reducing agents, ascorbate, NADH, NADPH, phenol, p-cresol, and homovanillic acid, were not accepted by HPETE peroxidase. This enzyme is not inhibited by 10 mM KCN, 2 mM aspirin, 2 mM salicylic acid, or 0.5 mM indomethacin. When 5-[14C]HPETE is generated from [14C]arachidonic acid in the presence of unlabeled 5-HPETE and the HPETE peroxidase, the 5-[14C]HETE produced is of much lower specific activity than the [14C]arachidonic acid. This indicates that the 5-[14C]HPETE leaves the active site of 5-lipoxygenase and mixes with the unlabeled 5-HPETE in solution prior to reduction and is a kinetic demonstration that 5-lipoxygenase has no peroxidase activity. Specificity for peroxides, reducing agents, and inhibitors differentiates HPETE peroxidase from glutathione peroxidase, phospholipid-hydroperoxide glutathione peroxidase, a 12-HPETE peroxidase, and heme peroxidases. The HPETE peroxidase could be a glutathione S-transferase selective for fatty acid hydroperoxides.

Evidence against a role for aspartyl proteases in intracellular processing of big endothelin.

CPAE endothelial cells were cultured in the presence of pepstatin, NH4Cl, or chloroquine in order to assess their effects on the secretion of endothelin-1 (ET-1). The first of these is an inhibitor of aspartyl proteases and the last two are known to neutralize acidic intracellular compartments. The pepstatin was encapsulated into liposomes to aid in its uptake, and uptake was confirmed by measuring the residual aspartyl protease activity in washed, lysed cells. Pepstatin had no effect (less than 5%) on the secretion of ET-1, 25 mM NH4Cl decreased secretion by 30-47%, and 25 microM chloroquine increased secretion by 37-79%. In contrast, each of these reagents is known to inhibit lysosomal degradation of intracellular proteins by 75-90%. Additionally, big ET was shown to be a very poor substrate, in terms of kcat/Km values, for aspartyl proteases. The rate constants were less than 10(4) M-1 s-1, which is approximately 1% of the value for the best substrates. The data, therefore, do not support a role for aspartyl proteases in the formation of ET-1. Similar to chloroquine, 0.5 microM monensin increased the secretion of ET-1 by 40-60%. Both of these reagents have previously been shown to increase the rate of constitutive secretion of peptides by affecting their partitioning between packaging into storage granules and constitutive secretion. The results would therefore provide supportive evidence for the existence of a storage form of ET-1 in endothelial cells.