I

XXO:

Fig. 1. Superoxide generated by xanthine and xanthine oxidase inhibits ECE activity. BAEC membrane proteins containing ECE were incubated with big ET-1, and the production of ET-1 was measured by ELISA. Reaction mixtures were incubated with buffer alone, or with 0.1 mM xanthine and xanthine oxidase (10 mU/ml) (XXO), or with XXO and catalase (Cat, 80 U/ml), or with XXO and SOD (lOOU/ml).

specific to superoxide. To confirm the specificity of the effects of superoxide, some reactions are also incubated with superoxide dismutase (SOD, 100 U/ml) or cata-lase (80 U/ml). SOD but not catalase blocks the inhibition of ECE by superoxide donors, confirming that superoxide inhibits ECE (Fig. 1).

It is important to show that ROS do not affect the substrate and products of the assay. For example, if ROS modified the product ET-1 and decreased the sensitivity of the ET-1 ELISA, a false-positive result would be obtained. We therefore have measured a fixed amount of ET-1 that has been treated or not treated with ROS, to confirm that the ET-1 ELISA is not affected by ROS. We also have treated a fixed amount of the substrate big ET-1 with buffer or with ROS, and measured its conversion by BAEC membrane proteins, in order to confirm that ROS do not modify the substrate of ECE. These two important controls have increased our confidence that any effects of ROS on ECE conversion of big ET-1 into ET-1 are indeed true-positive results.

Radicals and Dimerization of Endothelin-Converting Enzyme

We next explored various mechanisms by which superoxide might inhibit ECE. Radicals can disrupt disulfide bridges, altering the tertiary structure of proteins. We therefore sought to examine the effect of superoxide on the homodimerization of ECE. We have treated ECE-containing protein extracts with buffer alone or superoxide as described above, fractionated the proteins on nondenaturing gels, and then determined the relative mobility by immunoblotting with an antibody to ECE. Radical treatment of ECE does not alter its mobility.

Radicals and Zinc: Indirect Measurements of Zinc in Endothelin-Converting Enzyme

ECE is a zinc metalloproteinase, and shares with other zinc metalloproteinases a zinc-binding HEXXH motif. Zinc is required for ECE activity. We hypothesized that superoxide might eject Zinc from ECE.

Therefore, we have indirectly assessed the effect of superoxide on the zinc content of ECE. We first confirmed that ECE requires a divalent cation for activity. We performed the ECE assay as described above in the presence or absence of EDTA (0-100 pM). We found that 1 ¡iM EDTA inhibits ECE activity by more than 60%. To show that zinc is essential for ECE activity, we performed the ECE assay in the presence of 1 \iM EDTA and 10 \iM ZnCl2. The addition of zinc restores ECE activity. To determine whether superoxide ejects zinc from ECE, we treated ECE-containing BAEC extracts with superoxide as described above for 1 hr, and then added increasing amounts of zinc (1-10 ¡jlM) for 10 min. Zinc restores ECE activity after inhibition with superoxide.

FIG. 2. Superoxide ejects zinc ion from ECE. ECE interacts with zinc ion through two histidine residues in an HEXXH motif and through a third zinc ligand, glutamate. Superoxide may eject zinc ion by reducing it.

Active ECE Inactive ECE

FIG. 2. Superoxide ejects zinc ion from ECE. ECE interacts with zinc ion through two histidine residues in an HEXXH motif and through a third zinc ligand, glutamate. Superoxide may eject zinc ion by reducing it.

These experiments suggest that superoxide inhibits ECE by ejecting zinc from the enzyme (Fig. 2). However, these experiments do not directly assess the zinc content of ECE.

Radicals and Zinc: Direct Measurements of Zinc in Endothelin-Converting Enzyme

Direct measurements of the content of zinc in a protein can be challenging. We use instrumental neutron activation analysis (INAA) to quantify zinc in ECE. A full discussion of INAA is outside the scope of this chapter. In brief, INAA involves neutron irradiation of a sample, and the measurement of the decay of various activation products.

We prepare BAEC membrane proteins as described above, and ECE is then im-munoprecipitated with antibody to ECE. Purified ECE is treated with superoxide generators, or not so treated. We place our ECE preparations into small acid-washed polyethylene vials, using an additional 0.2 ml of buffer solution to ensure complete transfer. (Note: It is essential to use double-distilled, deionized water in buffer solutions, to avoid the possible contamination of the samples with zinc from tap water.)

The samples and standards are irradiated together for 12 hr at a neutron flux of 8 x 1012 n/cm2 sec; transferred to irradiated vials, using two additions of 0.5 ml of unirradiated buffer; and then allowed to decay for approximately 4 weeks. The presence of the activation product 65Zn (half-life, 243.8 days; y energy, 1115.5 keV) is measured in the samples and standards by using four high-purity germanium (HPGe) detectors coupled to a dedicated personal computer (all hardware and software are from Canberra Industries, Meriden, CT). The zinc levels in the proteins are calculated on the basis of the 65Zn activities in the protein samples and the reference materials, the reference sample masses, and the certified zinc reference concentrations.

We found that treatment of ECE with 0.1 mM xanthine and xanthine oxidase (1.0 mU/ml) for 1 hr removes all zinc from ECE (Fig. 2). Thus INAA can be used to assess the content of zinc in proteins such as metalloproteinases.

Conclusion

ROS regulate a variety of vascular signaling pathways. Using an ELISA, we have shown that superoxide regulates ECE activity. Indirect measurements and direct measurements show that superoxide inhibits ECE by ejecting zinc from the enzyme. Several pathways in the cardiovascular system involve the activity of converting enzymes, including the renin-angiotensin-aldosterone pathway. Techniques that measure protease activity, and measure the effect of ROS on proteases, may reveal novel mechanisms by which radicals regulate cardiovascular signaling.

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