The disassembly and withdrawal of vimentin intermediate filaments (VIF) from the plasma membrane induces membrane ruffling and the formation of a lamellipodium. Conversely, lamellipodium formation is inhibited when VIF are present.

Giant axonal neuropathy (GAN) is an early-onset neurological disorder caused by mutations in the GAN

Although the mechanism of neuronal death

Complex formation between the human erythrocyte transglutaminase (protein-glutamine:amine -glutamyltransferase, EC 2.3.2.13) and fibronectin or its fragments was examined by immunoanalytical procedures and by fluorescence polarization. A 42-kDa gelatin-binding structure, obtained from human plasma fibronectin by thermolytic digestion, showed as high an affinity for the cytosolic enzyme as the parent fibronectin chains themselves. A 21-kDa fragment comprising type I modules 8 and 9, the last two modules in the 42-kDa fragment, bound with an affinity 100-fold less than the 42-kDa fragment. Binding was remarkably specific and could be exploited for the affinity purification of transglutaminase directly from the hemoglobin-depleted erythrocyte lysate. In spite of the high affinity, it was possible to elute active enzyme from the 42-kDa fragment column with 0.25% monochloroacetic acid. This solvent might have general applicability in other systems involving separation of tightly bound ligands.Plasma fibronectin is thought to play an important homeostatic role by acting as a scavenger for cytosolic transglutaminases (TGs; protein-glutamine:amine -glutamyltransferase, EC 2.3.2.13). Such enzymes occur in many different cell types and could pose the danger of polymerizing proteins if discharged freely into the circulation (1-3). We have focused on the interaction of human plasma fibronectin with the human erythrocyte (RBC) enzyme (4). Binding between the two proteins is instantaneous and very tight and occurs even in the absence of Ca2 , which indicates that it does not depend on the unmasking of the active center of TG. Studies with chymotryptic fragments of fibronectin showed that the gelatin (collagen)-binding domain of the molecule was involved also in the binding of TG (5). However, the two sites seem to act independently because attachment to TG and gelatin can take place simultaneously in a ternary complex. Electron microscopic examination demonstrates that, along the contour lengths of the constituent chains of fibronectin, human RBC TG was bound at a distance of 5-10 nm from the N termini, frequently forming ring-like structures (6).By using well-characterized thermolytic fragments of human fibronectin (7), it is now possible to further define the TG-associating domains of the plasma protein. It will be shown in the present paper that an -42-kDa gelatin-binding structure, which is sequentially composed of a type I, followed by two type II and three type I motifs , displays a full strength of binding for the human RBC enzyme. The high specificity for binding was exploited for the affinity chromatographic purification of TG directly from the hemoglobin-depleted RBC lysate. MATERIALS AND METHODSProtein Preparations. The 42-kDa and 30-kDa gelatinbinding fragments of human plasma fibronectin were obtained by digestion with thermolysin as described by Borsi et al. (8). Further treatment of the 42-kDa fragment with pepsin was employed for producing a yet smaller 21-kDa gelatinbinding fragment (6). For use in i...

The inositol phosphate hydrolyzing activity of human phospholipase C␦1 (PLC␦1) is markedly inhibited when the enzyme is coexpressed with the human heart Gh͞transglutaminase (TG) in human embryonic kidney cells. Because the cotransfection does not affect the amount of PLC␦1 in the cells, the depression of phospholipase activity probably is a result of a direct interaction between the two proteins. An ELISA procedure was employed to document the associations of purified TG preparations from a variety of tissues (human red cells, rabbit lens, guinea pig liver) with PLC␦1. Nucleotides (GTP > GDP > ATP > GMP ‫؍‬ ADP, in order of decreasing efficiency) interfered with the formation of the PLC␦1:TG complex. A conformational change in the TG partner, occurring with nucleotide binding, is thought to be responsible for dissociating the two proteins. The structural rearrangement produces a remarkable shift in the anodic mobility of TG in electrophoresis: TGslow ؉ GTP u [TG:GTP]fast. Altogether, our findings indicate that GTP controls PLC␦1 activity by releasing this protein from an inhibitory association with G h͞transglutaminase.