br Results br Discussion Many assays in

Results
Discussion Many assays in the literature for DBH require the use of catalase to protect the active site from hydrogen peroxide, which is a by-product in the initial step of the reaction. For the non-immobilized enzyme there was a visible increase in the rate with increasing amounts of catalase. However, for both types of immobilized enzyme minimal amounts of catalase are required for maximal activity. The rates for both forms of immobilized enzyme (see Table 1) are lower relative to the non-immobilized DBH. This suggests the enzyme is working slower and therefore the amount of hydrogen peroxide produced as a by-product is minimal and does not affect the enzyme activity. The immobilized enzymes have conformations that do not require large amounts of catalase for protection. Under ideal assay conditions the Km and Vmax for different substrates were determined for all the enzyme forms. In both cases, immobilization onto Glut-P and IAM, the Km and Vmax showed a decrease. This is understandable when one considers the effects of diffusion. The Nutlin-3 of the substrate from the bulk solution to the microenvironment of the immobilized enzyme can limit the rate. This in turn affects the concentration of the substrate/cofactor in the vicinity of the enzyme. However, differences in the kinetic properties of the DBH–Glut-P and DBH–IAM interphases were observed. The DBH–Glut-P interphase similar to the non-immobilized enzyme gave rise to a ping-pong mechanism. The DBH–IAM interphase did not display a ping-pong mechanism, which is consistent with previous findings [21]. The different forms of immobilization explain these findings. When DBH is immobilized on the Glut-P interphase the enzyme is outside the stationary phase, whereas with the IAM interphase the enzyme is embedded within the interphase surroundings. As such the DBH–Glut-P interphase can be utilized to characterize the enzyme found in the cytosol and information concerning the membrane-bound enzyme can be obtained with the DBH–IAM interphase. Fusaric acid, an antibiotic produced by fungus, has been found to be a potent inhibitor of DBH. Inhibition of DBH has been shown to result in decreased sympathetic activity and marked hypotensive effects [19]. The results are consistent with previously reported results demonstrating that fusaric acid is capable of reducing endogenous levels of norepinephrine [19]. Fusaric acid was found to inhibit the DBH-mediated formation of NE from DA at concentrations as low as 10−6 M. Fifty percent inhibition was achieved at similar concentrations for both enzyme forms. Captopril contains a sulfhydryl moiety, which has been shown to be responsible for the attenuation of the vasoconstriction-induced by sympathetic nerve stimulation [20]. Sulfhydryl compounds are known to inhibit DBH in vivo and in vitro. Noncompetitive inhibition with respect to tyramine was viewed with inhibitions achieved at concentrations as low as 150 μM. The inhibitory effect of captopril was shown to be reversed in a dose-dependent manner by cupric ions. The reversal of the inhibition was achieved with 2.5 μM cupric sulphate. Palatini et al. showed reversal of the inhibition by 140 μM captopril at 1.5 μM Cu2+, as CuSO4 [22]. DBH in chromaffin granules of the adrenal medulla occurs in a soluble form and a membrane bound form. The amino acid compositions of these two states of DBH are essentially identical [15]. However, the two states have shown differences in pH stabilities. The non-immobilized enzyme was shown to have a pH optimum at pH 6.0. Consistent with our results the DBH–IAM interphase displayed a shift of 1.5 pH units, pH 7. The observed optimum pH for immobilized DBH mimics physiological conditions for the membrane-bound DBH. The support utilized for immobilization, IAM, mimics the membrane environment that membrane-bound DBH is accustomed to. The IAM support contains covalently bound phospholipids which is an ideal support due to the fact that the membrane-bound DBH has been shown to be linked with lipids of the membrane [21].