Premium
Characterization of Membrane‐Bound and Soluble Catechol‐ O ‐Methyltransferase from Human Frontal Cortex
Author(s) -
Jeffery Douglas R.,
Roth Jerome A.
Publication year - 1984
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1984.tb02755.x
Subject(s) - catechol o methyl transferase , membrane , chemistry , enzyme , transferase , tropolone , catechol , biochemistry , methyltransferase , nordihydroguaiaretic acid , membrane protein , chromatography , methylation , lipoxygenase , organic chemistry , allele , gene
Catechol‐ O ‐methyltransferase (COMT; E.C. 2.1.1.6) from human frontal cortex occurs in both a soluble and membrane‐bound form. Attempts to solubilize the membrane‐bound transferase by repeated washing or by extraction into solutions of high ionic strength were unsuccessful. The finding that Triton X‐100 was capable of solubilizing membrane‐bound COMT suggested that the membrane‐bound transferase is an integral membrane protein. The membrane‐bound and soluble enzymes did not differ in their requirements for magnesium ions or in their pH‐activity profiles; both enzymes showed an optimum near pH 8.0 when assayed in phosphate buffer. In addition, the two enzymes did not differ in the degree of inhibition caused by CaCl 2 , both enzymes displaying 65% inhibition at 2.5 m M CaCl 2 . The competitive inhibitors tropolone and nordihydroguaiaretic acid displayed K i values for the membrane‐bound transferase five‐ to 10‐fold lower than those observed for the soluble transferase. Solubilization of membrane‐bound COMT in Triton X‐100 resulted in an increase in the apparent K m value of the membrane‐bound transferase for dopamine. The increase in K m appeared to be due to apparent competitive inhibition by Triton X‐100 and reached a limiting value of approximately 80 μM. These results confirm that membrane‐bound COMT is an integral membrane protein that may be structurally distinct from soluble COMT.