Cloning, purification, and nucleotide-binding traits of the catalytic subunit A of the V1VO ATPase from Aedes albopictus.
The Asian tiger mosquito, Aedes albopictus, is commonly infected by the gregarine parasite Ascogregarina taiwanensis, which develops extracellularly in the midgut of infected larvae. The intracellular trophozoites are usually confined within a parasitophorous vacuole, whose acidification is generated and controlled by the V(1)V(O) ATPase. This proton pump is driven by ATP hydrolysis, catalyzed inside the major subunit A. The subunit A encoding gene of the Aedes albopictus V(1)V(O) ATPase was cloned in pET9d1-His(3) and the recombinant protein, expressed in the Escherichia coli Rosetta 2 (DE3) strain, purified by immobilized metal affinity- and ion-exchange chromatography. The purified protein was soluble and properly folded. Analysis of secondary structure by circular dichroism spectroscopy showed that subunit A comprises 43% alpha-helix, 25% beta-sheet and 40% random coil content. The ability of subunit A of eukaryotic V-ATPases to bind ATP and/or ADP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the FCS data indicates that the ADP-analogues bind slightly weaker to subunit A than the ATP-analogues. Tryptophan fluorescence quenching of subunit A after binding of different nucleotides provides evidence for secondary structural alterations in this subunit caused by nucleotide-binding.
Hunke
Cornelia
C
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
Chen
Wei-June
WJ
Schäfer
Hans-Jochen
HJ
Grüber
Gerhard
G
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000330:Aedes / Q000201:enzymology* / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D017344:Genes, Insect; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D021122:Protein Subunits; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D017931:DNA Primers; D019476:Insect Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases