期刊
BIO-PROTOCOL
卷 13, 期 10, 页码 -出版社
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.4676
关键词
ABC transporter; ATPase; ATP hydrolysis; Radioactive assay; 32P-ATP; P-type ATPase
类别
ATPase assays are commonly used for the characterization of purified ATPases. This radioactive [γ32P]-ATP-based assay, utilizing complex formation with molybdate, offers high sensitivity and can be used for proteins with low ATPase activity or low purification yields. It has various applications including substrate identification, determination of the effect of mutations on ATPase activity, and testing of ATPase inhibitors.
ATPase assays are a common tool for the characterization of purified ATPases. Here, we describe a radioactive [gamma 32P]-ATP-based approach, utilizing complex formation with molybdate for phase separation of the free phosphate from non-hydrolyzed, intact ATP. The high sensitivity of this assay, compared to common assays such as the Malachite green or NADH-coupled assay, enables the examination of proteins with low ATPase activity or low purification yields. This assay can be used on purified proteins for several applications including the identification of substrates, determination of the effect of mutations on ATPase activity, and testing specific ATPase inhibitors. Furthermore, the protocol outlined here can be adapted to measure the activity of reconstituted ATPases.
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