The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b 5 , and NADH/FADdependent cytochrome b 5 reductase. mARC proteins share a significant degree of homology to the molybdenum cofactorbinding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/ MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo-or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.In eukaryotes the trace element molybdenum is essential for a number of enzymes where the molybdenum atom is part of the so-called molybdenum cofactor (Moco) 2 in the active site of these enzymes (1). Moco is a pterin-based cofactor with a C6-substituted pyrano ring, a terminal phosphate, and a unique dithiolate group that binds the molybdenum atom. Moco-containing enzymes (Mo-enzymes) catalyze important reactions in the global carbon, sulfur, and nitrogen cycles that are characterized by transfer of an oxygen atom to or from a substrate. In mammals, one Mo-enzyme is sulfite oxidase (SO), which catalyzes the last step in the degradation of sulfur-containing amino acids and sulfatides (2). The active SO protein is a homodimer with each monomer of ϳ52 kDa consisting of a N-terminal cytochrome b 5 (cyt b 5 )/heme-binding domain and a C-terminal Moco-binding domain, the latter also harboring the dimerization interface. Both the Moco-and the heme-binding domain of mammalian SO are similar to the respective domains of nitrate reductase (NR), which catalyzes the first and rate-limiting step in nitrate assimilation in autotrophic organisms like plants, algae, and fungi (3). In addition to its N-terminal Mocobinding domain and the cytb 5 /heme-binding domain, each NR monomer possesses a C-terminal FAD-binding domain. Xanthine oxidoreductase (XOR) is another mammalian Mo-enzyme, and it is active as a homodimer with each ϳ145-kDa monomer consisting of several distinct domains: an N-terminal domain...

The vasodilator-stimulated phosphoprotein (VASP) colocalizes with the ends of stress fibers in cell-matrix and cellcell contacts. We report here that bacterially expressed murine VASP directly interacts with skeletal muscle actin in several test systems including cosedimentation, viscometry and polymerization assays. It nucleates actin polymerization and tightly bundles actin filaments. The interaction with actin is salt-sensitive, indicating that the complex formation is primarily based on electrostatic interactions. Actin binding is confined to the Cterminal domain of VASP (EVH2). This domain, when expressed as a fusion protein with EGFP, associates with stress fibers in transiently transfected cells.z 1999 Federation of European Biochemical Societies.

The morphology of astrocytic processes determines their close structural association with synapses referred to as the ‘tripartite synapse’. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes.Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as isoform-specific functions.