Structural and Functional Characterization of the Putative Pineal ATPase PINA
Copper plays a crucial role in human metabolism as a cofactor of enzymes responsible for respiration, iron metabolism, free radical disposal, and many other functions. Copper ATPases (Cu-ATPases) are a family of enzymes facilitating copper transport in the body. ATP7B is one of them, and it belongs to a P-type class of ATPases. In 1999, an mRNA of a splice vari-ant (PINA) has been discovered in rat pineal gland and retina. Transcription levels of the mRNA increase during the night and decrease during the day. Homologous mRNA (ATP7B.K) was also reported in humans. The goals of this study were to confirm the expression of PINA in rat pineal gland, as well as to verify the temporal expression pattern. We also aimed to express and investigate the enzymatic activity of a recombinant putative PINA homologue from human, and compare its properties to the full-length ATP7B. We have successfully expressed the full-length ATP7B and two putative isoforms of hu-man PINA homologue in K. lactis expression system. We have established a purification proce-dure that enabled us to get active enzymes and study their ATPase activity. This is the first suc-cessful purification of the active full-length human ATP7B reported. The activity of the full-length protein produced according to our procedure is comparable to previously reported by other groups, who studied either the full-length protein in the membrane or its purified truncat-ed variants. The putative PINA homologues showed much higher ATPase activity than the full-length enzyme. We could not detect the putative PINA protein in pineal gland extracts and membrane preparations due to low sensitivity of the available antibodies. Further investigations are needed for establishing copper transport rates of the full-length ATP7B and the PINA homologues, as our new protocol for copper transport quantification needs further optimizations.
PINA, pineal gland, ATP7B, copper, Wilson's disease, circadian rhythm
Master of Science (M.Sc.)