Group-specific component (vitamin D binding protein) prevents the interaction between G-actin and profilin

Abstract

Profilin purified from human platelets formed a 1:1 molar ratio complex with rabbit skeletal muscle G-actin but was displaced by purified serum Gc (vitamin D binding protein) in a dose-dependent fashion as assessed by chromatography and ultrafiltration. This suggested that Gc and profilin competed for the same binding area on G-actin, with Gc-G-actin complexes being more stable than profilin-G-actin complexes in vitro. The binding domain for Gc on G-actin was localized to a 16,000-Da C-terminal fragment of G-actin generated by Staphylococcus aureus V8 protease, as judged by comigration on two-dimensional electrophoresis and also by overlaying electrophoresis gels with 125I-Gc. Previous studies have reported that residues 374 and 375 of G-actin are essential for binding of profilin. In this study, experiments involving tryptic removal of Cys-374 labeled with the fluorescent probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)-ethylenediamine showed that these C-terminal amino acids were not necessary for interaction with Gc.

Role of group-specific component (vitamin D binding protein) in clearance of actin from the circulation in the rabbit.

“…Similarly, 125i-F-actin (> 90% pelletable counts), '25I-G-actin (< 10% pelletable counts), and '25I-albumin were > 95% homogeneous and migrated on SDS-PAGE to positions indistinguishable from those of the native proteins (Fig. 1); '25I-G-actin preparations also retained the potential for polymerization (39).…”

“…Thus, Gc can bind monomers of actin even in the presence of an excess of plasma gelsolin (56). Moreover, although much cellular actin released in the monomeric G-form is likely to be complexed with the major intracellular G-actin-sequestering protein, profilin (29), Gc displays higher apparent affin-ity for G-actin under defined conditions than does profilin, and can displace profilin from actin (39). Furthermore, Gc apparently binds certain fragments ofactin in vitro and in vivo (37, 39 and Fig.…”

“…Most G-actin released is likely to be complexed with profilin, the major intracellular actin-sequestering protein (29,63). However, profilin is probably displaced by Gc (39), resulting again in complexing of actin with Gc. Such complexes are then removed from the circulation by cellular uptake.…”

“…After 30 min incubation, the samples were centrifuged for 60 min at 100,000 g at 22°C. Pelleting assays (39) showed that > 90% 251-actin was in the F-form. '25I-G-actin was prepared in one of two ways.…”

Angiopathic consequences of saturating the plasma scavenger system for actin.

“…Gelsolin has been identified to be a product of several tissues with a tissue and plasma form, and its activities on F-actin are enhanced in the presence ofDBP (13,46). The actin-binding activity ofDBP most closely resembles that of the intracellular protein, profilin, but at 1000 times higher affinity (47,48).…”