The regulation of Na, K-ATPase by PLMS, the phospholemman-like protein from shark. Molecular cloning, sequence, expression, cellular distribution and functional effects of PLMS

In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase α-subunit and PLMS showed the presence of α and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E1∼P → E2-P reaction, which is partially rate-limiting in shark, and the K+ deocclusion reaction, E2(K) → E1, are accelerated. The latter may explain the finding that the apparent Na+ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase α-subunit is important for the modulation of shark Na,K-ATPase activity

Kinetic and mesoscopic non-equilibrium description of the Ca2+ pump: A comparison

We analyse the operation of the Ca2?-ATPase ion pump using a kinetic cycle diagram. Using the methodology of Hill, we obtain the cycle fluxes, entropy production and efficiency of the pump. We compare these results with a mesoscopic non-equilibrium description of the pump and show that the kinetic and mesoscopic pictures are in accordance with each other. This gives further support to the mesoscopic theory, which is less restricted and also can include the heat flux as a variable. We also show how motors can be characterised in terms of unidirectional backward fluxes. We proceed to show how the mesoscopic approach can be used to identify fast and slow steps of the model in terms of activation energies, and how this can be used to simplify the kinetic diagram.Process and Energy LaboratoryMechanical, Maritime and Materials Engineerin