Evidence of redox-linked signaling for producing a giant signal complex.

Previously we showed that a thiol-reactive heavy metal, HgCl2, crosslinked multiple cell surface receptors through a ligand-independent pathway, which produced massive aggregates of phosphotyrosine (PTYR)-containing proteins beneath plasma membrane [Nakashima et al. (1994): J Immunol 152: 1064-1071]. In this study we characterized these unique aggregates at the molecular level. The lysates in Brij 96 of thymocytes treated with HgCl2 were separated into the supernatant and pellet fractions by simple centrifugation. Selected PTYR-containing proteins and p56lck appeared in the pellet fraction as quickly as 5 s after exposure to HgCl2, and were further increased in amount by 5 min. Although the mechanism of triggering these events was redox-linked, the majority of proteins in the Brij 96-insoluble aggregates were dissociated in SDS-PAGE under nonreducing condition. This suggested that PTYR-containing proteins and p56lck themselves do not form dimer or polymer directly by thiol-mediated bond. The pellet fraction was further found to include some other signal delivery elements, such as GTPase activating protein, phosphatidylinositol 3 kinase, and mitogen-activated protein kinase. Finally, all of these signal elements and selected PTYR-containing proteins were collected in the same fraction by the sucrose density gradient centrifugation. These results suggest a unique redox-linked pathway of formation of a giant signal complex.

Aging: effects on sodium- and potassium-activated adenosine triphosphatase activity and ouabain binding sites in rat brain.

Sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase) activity and [3H]-ouabain binding were examined in homogenates of cerebral cortex, striatum, and hypothalamus of 3-, 8-, and 26-month-old rats to determine if aging-related alterations in energy utilization demonstrated in brain slices and homogenates are potentially associated with alterations in Na,K-ATPase. There were no consistent age-related changes seen in Na,K-ATPase activity, the number of [3H]-ouabain binding sites, or their affinity for ouabain. Moreover, enzyme activities of the two molecular forms of Na,K-ATPase and their inhibition by strophanthidin did not appear to be different in partially purified enzyme preparations obtained from whole brain of 3- and 26-month-old rats. In contrast, the concentration of [3H]-ouabain binding sites was lower in cardiac muscle of senescent rats indicating a reduction in the number of active Na,K-ATPase units. It appears unlikely that aging-related central nervous system changes are associated with alterations in the Na,K-ATPase enzyme system.