ATP-binding and -hydrolysis activities of ALDP (ABCD1) and ALDRP (ABCD2), human peroxisomal ABC proteins, overexpressed in Sf21 cells.
The peroxisomal ATP-binding cassette (ABC) proteins, adrenoleukodystrophy protein (ALDP, ABCD1) and ALD-related protein (ALDRP, ABCD2), were expressed in Spodoptera frugiperda 21 (Sf21) insect cells using a baculovirus-mediated expression system. Immunoelectron microscopy and subcellular fractionation revealed that the overexpressed ALDP was distributed in various subcellular organelles including mitochondria, nucleus and peroxisomes. The ALDP was not extractable with Na(2)CO(3) treatment, suggesting that it integrated into membranes. ATPase activity was detected in the membrane fraction expressing ALDP. The nucleotide-binding capacities of the expressed ALDP were estimated by the binding to ATP- or ADP-agarose. ALDP exhibited an affinity to both ADP and ATP. In contrast, ALDRP exhibited an affinity to ADP but scarcely to ATP. The ALDP in the Sf21 membrane fraction was extracted with n-dodecyl-beta-maltoside and successively purified with a chelate column. The nucleotide-binding and ATPase activities of the purified ALDP were, however, not detected. It may be that certain membranous components are required for the activity. We demonstrate for the first time that the peroxisomal ABC proteins can be expressed in Sf21 membranes maintaining their nucleotide-binding abilities and ATPase activities, and the expressed proteins will be of use for further characterization.
Morita
Masashi
M
Department of Biological Chemistry, Graduate School of Medicine & Pharmaceutical Sciences, University of Toyama, Sugitani, Toyoma, Japan. masa@pha.u-toyoma.ac.jp
Kurisu
Mikinori
M
Kashiwayama
Yoshinori
Y
Yokota
Sadaki
S
Imanaka
Tsuneo
T
D000074061:ATP Binding Cassette Transporter, Subfamily D; D000074063:ATP Binding Cassette Transporter, Subfamily D, Member 1; D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D006868:Hydrolysis; D009711:Nucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018411:Spodoptera
C115572:ABCD1 protein, human; C431694:ABCD2 protein, human; D000074061:ATP Binding Cassette Transporter, Subfamily D; D000074063:ATP Binding Cassette Transporter, Subfamily D, Member 1; D018528:ATP-Binding Cassette Transporters; D009711:Nucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate