title: Golgi membranes from liver express an ATPase with femtomolar copper affinity, inhibited by cAMP-dependent protein kinase.
abstract: Copper-stimulated P-type ATPases are essential in the fine-tuning of intracellular copper. In the present work we characterized a copper-dependent ATPase hydrolysis in a native Golgi-enriched preparation from liver and investigated its modulation by cyclic AMP-dependent protein kinase (PKA). The very high-affinity Atp7b copper pump presented here shows a K(0.5) for free copper of 2.5×10(-17) M in bathocuproine disulfonate/copper buffer and ATP hydrolysis was inhibited 50% upon stimulation of PKA pathway, using forskolin, cAMP or cholera toxin. Incubation with PKA inhibitor (PKAi(5-24) peptide) raises Cu(I)-ATPase activity by 50%. Addition of purified PKA α-catalytic subunit increases K(0.5) for free copper (6.2×10(-17) M) without modification in the affinity for ATP in the low-affinity range of the substrate curve (∼1 mM). The Hill coefficient for free copper activation also remains unchanged if exogenous PKA is added (2.7 and 2.3 in the absence and presence of PKA, respectively). The results demonstrate that this high-affinity copper pump in its natural environment is a target of the liver PKA pathway, being regulatory phosphorylation able to influence both turnover rate and ion affinity.
authors.affiliation: Laboratório de Físico-Química Biológica Aída Hassón-Voloch, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal de Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil.
authors.forename: Elaine
authors.initials: E
authors.lastname: Hilário-Souza
authors.forename: Rafael H F
authors.initials: RH
authors.lastname: Valverde
authors.forename: Thiago
authors.initials: T
authors.lastname: Britto-Borges
authors.forename: Adalberto
authors.initials: A
authors.lastname: Vieyra
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lowe
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D055162:Biocatalysis; D027682:Cation Transport Proteins / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D010766:Phosphorylation; D034421:Sus scrofa; D013997:Time Factors
chemical_list: D027682:Cation Transport Proteins; D003300:Copper; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases