No abstract

Incorporation of 32P into telokin, a smooth muscle-specific, 17-18-kDa, acidic (pI 4.2-4.4) protein, was increased by forskolin (20 microM) in intact rabbit ileum smooth muscle (ileum) and by 8-bromo-cyclic GMP (100 microM) in alpha-toxin-permeabilized ileum. Native telokin (5-20 microM), purified from turkey gizzard, and recombinant rabbit telokin, expressed in Escherichia coli and purified to >90% purity, induced dose-dependent relaxation, associated with a significant decrease in regulatory myosin light chain phosphorylation, without affecting the rate of thiophosphorylation of regulatory myosin light chain of ileum permeabilized with 0.1% Triton X-100. Endogenous telokin was lost from ileum during prolonged permeabilization (>20 min) with 0.1% Triton X-100, and the time course of loss was correlated with the loss of 8-bromo-cyclic GMP-induced calcium desensitization. Recombinant and native gizzard telokins were phosphorylated, in vitro, by the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and p42/44 mitogen-activated protein kinase; the recombinant protein was also phosphorylated by calmodulin-dependent protein kinase II. Exogenous cGMP-dependent protein kinase (0.5 microM) activated by 8-bromo-cyclic GMP (50 microM) phosphorylated recombinant telokin (10 microM) when added concurrently to ileum depleted of its endogenous telokin, and their relaxant effects were mutually potentiated. Forskolin (20 microM) also increased phosphorylation of telokin in intact ileum. We conclude that telokin induces calcium desensitization in smooth muscle by enhancing myosin light chain phosphatase activity, and cGMP- and/or cAMP-dependent phosphorylation of telokin up-regulates its relaxant effect.

RhoA, a ubiquitous intracellular GTPase, mediates cytoskeletal responses to extracellular signals. A 2.1 A resolution crystal structure of the human RhoA-GDP complex shows unique stereochemistry in the switch I region, which results in a novel mode of Mg2+ binding.

The ATP-binding cassette subfamily B member 1 (ABCB1) multidrug transporter P-glycoprotein plays a central role in clearance of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. We used double electron electron resonance spectroscopy to uncover the basis of stimulation of P-glycoprotein adenosine 5′-triphosphate (ATP) hydrolysis by multiple substrates and illuminate how substrates and inhibitors differentially affect its transport function. Our results reveal that substrate-induced acceleration of ATP hydrolysis correlates with stabilization of a high-energy, post-ATP hydrolysis state characterized by structurally asymmetric nucleotide-binding sites. By contrast, this state is destabilized in the substrate-free cycle and by high-affinity inhibitors in favor of structurally symmetric nucleotide binding sites. Together with previous data, our findings lead to a general model of substrate and inhibitor coupling to P-glycoprotein.

The F O F 1 ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous F O sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium. KeywordsATP synthase; kinetic mechanism; rotation; transport Like many transporters, the F O F 1 ATP synthase (or F-type ATPase) has been a fascinating subject for the study of a complex membrane-associated process. The ATP synthase is a critically important activity that carries out synthesis of ATP from ADP and Pi driven by a proton motive force, Δµ H+ , or sodium motive force, Δµ Na+ . This final step of oxidative or photo-phosphorylation provides the vast majority of ATP in the cell. The proton or sodium motive force is also needed to power other membrane processes such as secondary transporters or in the case of bacteria, flagellum rotation. In anaerobic conditions, facultative bacteria use the ATP synthase as an ATP-driven H + or Na + pump to generate the Δµ H+ , or Δµ Na+ (see [1] for a textbook review.) The F O F 1 complex is nearly ubiquitous in the cell membranes of eubacteria, in the thylakoid membrane of chloroplasts, and the inner membrane of mitochondria. The transporter has remained structurally and mechanistically conserved, except for a few additional domains or subunits in mitochondria, which may play roles in regulation or assembly.Many years of innovative biochemical, genetic, kinetic, and thermodynamic studies led to the first structural solution of the catalytic F 1 portion of the complex by Walker, Leslie and coworkers [2] in 1994. This landmark structure provided critical information on the catalytic portion of the complex but the subunit arrangement of much of the rest of the complex was still not elucidated. The partial F 1 structure, which at the time was the largest asymmetric unit solved, provided the impetus and the structural information needed to test the notion that the