THE VITAMIN-D-3 HYDROXYLASE-ASSOCIATED PROTEIN IS A PROPIONAMIDE-METABOLIZING AMIDASE ENZYME

Previously we isolated a novel protein that coimmunoprecipitates with the 1,25-dihydroxyvitamin D-3-24R-hydroxylase and 25-hydroxyvitamin D-3-1 alpha-hydroxylase. This kidney-specific protein found in the inner membrane of mitochondria is named the vitamin D-3 hydroxylase-associated protein (VDHAP). To determine a putative function for this protein, an extensive computer search of the deduced amino acid sequence of VDHAP was performed, A BLAST homology search identified amino acid residues 133 through 321 in acetamidase from Aspergillus nidulans that exhibit 38% amino acid identity and 65% amino acid similarity to VDHAP. A protein consensus sequence dictionary, MOTIFS, identified an amidase consensus sequence in VDHAP. This sequence, G-G-S-S-G-G-E-G-A-L-I-A-G-G-G-S-L-L-G-I-G-S-D-V-A-G-S-I-R-L-P-S, in VDHAP is located between amino acids 223 and 254. Propionamide, acetamide, and acrylamide were identified as substrates for an amidase activity in soluble chicken kidney mitochondria. Propionamide is the best substrate with a V-max of 16.1 nmol NH4+/min/mg protein and an apparent K-m of 7.9 mM in soluble chicken kidney mitochondria. A VDHAP monoclonal antibody, IVC2G8, immunoprecipitates 78% of the total propionamidase activity in soluble chicken kidney mitochondria. These results suggest that VDHAP is a propionamidase enzyme in soluble chicken kidney mitochondria and a member of the amidase signature gene family. (C) 1995 Academic Press, Inc.