Synthesis and characterization of immobilized dopamine beta-hydroxylase in membrane-bound and solubilized formats.
Dopamine beta-hydroxylase (DBH) catalyzes the beta-hydroxylation of dopamine to norepinephrine. The enzyme in chromaffin granules occurs in a soluble form and a form confined to the surrounding membrane. DBH was noncovalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase and the resulting DBH-IAM stationary phase was enzymatically active and was shown to mimic the membrane-bound form of the enzyme. DBH was also covalently immobilized onto a silica-based support containing, glutaraldehyde-P (Glut-P). The resulting DBH-Glut-P interphase was also enzymatically active, reproducible and shown to display characteristics of the solubilized enzyme. The results demonstrate that the different immobilization methods utilized for the enzyme can be used to quantitatively and qualitatively determine the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions for the two distinct forms of the enzyme. These new entities can be used in basic biochemical studies as well as in high throughput screening of substances for DBH substrate/inhibitor properties.
Department of Medicine, Division of Experimental Medicine, McGill University, Quebec, Montreal, Canada.
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Markoglou
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Wainer
D002021:Buffers; D002216:Captopril / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism; D002853:Chromatography, Liquid; D003300:Copper / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism / Q000494:pharmacology; D004299:Dopamine beta-Hydroxylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D005669:Fusaric Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008567:Membranes, Artificial; D012995:Solubility; D013696:Temperature; D014439:Tyramine / Q000378:metabolism / Q000494:pharmacology
D002021:Buffers; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D003300:Copper; D002216:Captopril; D002374:Catalase; D004299:Dopamine beta-Hydroxylase; D005669:Fusaric Acid; D004298:Dopamine; D014439:Tyramine