Ettinger R A, DeLuca H F
Department of Biochemistry, University of Wisconsin-Madison, College of Agricultural and Life Sciences 53706.
Arch Biochem Biophys. 1995 Jan 10;316(1):14-9. doi: 10.1006/abbi.1995.1003.
Previously we isolated a novel protein that coimmunoprecipitates with the 1,25-dihydroxyvitamin D3-24R-hydroxylase and 25-hydroxyvitamin D3-1 alpha-hydroxylase. This kidney-specific protein found in the inner membrane of mitochondria is named the vitamin D3 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 identify 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 Vmax of 16.7 nmol NH4+/min/mg protein and an apparent Km 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.
此前，我们分离出了一种新型蛋白质，它能与1,25 - 二羟基维生素D3 - 24R - 羟化酶和25 - 羟基维生素D3 - 1α - 羟化酶共同免疫沉淀。这种存在于线粒体内膜的肾脏特异性蛋白质被命名为维生素D3羟化酶相关蛋白（VDHAP）。为了确定该蛋白质的假定功能，我们对VDHAP推导的氨基酸序列进行了广泛的计算机搜索。BLAST同源性搜索在构巢曲霉的乙酰胺酶中鉴定出了133至321位的氨基酸残基，其与VDHAP的氨基酸一致性为38%，氨基酸相似性为65%。一个蛋白质共有序列字典MOTIFS在VDHAP中鉴定出了一个乙酰胺酶共有序列。VDHAP中的这个序列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位于223至254位氨基酸之间。丙酰胺、乙酰胺和丙烯酰胺被鉴定为可溶性鸡肾线粒体中乙酰胺酶活性的底物。丙酰胺是最佳底物，在可溶性鸡肾线粒体中的Vmax为16.7 nmol NH4+/min/mg蛋白质，表观Km为7.9 mM。一种VDHAP单克隆抗体IVC2G8免疫沉淀了可溶性鸡肾线粒体中78%的总丙酰胺酶活性。这些结果表明，VDHAP是可溶性鸡肾线粒体中的一种丙酰胺酶，是乙酰胺酶特征基因家族的成员。
