<b>IMMUNOASSAY OF GC GLOBULIN (VITAMIN D-BINDING PROTEIN) IN COMPLEXED FORM WITH </b><b>ACTIN </b>

Abstract

A quantitative immunoassay to determine Gc globulin in complexed form with actin is described. This assay involves the monoclonal antibody E12 of which binding to Gc is inhibited by actin, and is designed as follows. Microtiter plates were coated with the monoclonal antibody #44 of which Gc binding was not inhibited by actin. The plates were incubated with Gc in the presence of actin, then with biotinylated E12 followed by streptavidin-peroxidase conjugate. The absorbance was linearly reduced by increasing amounts of actin. This assay system can be applied to human serum. The levels of complexed Gc in serum samples from normal individuals and patients with hepatitis were 10.9 i 4.3% and 24.7 i 13.3%, respectively. Finally, when actin was added to the sera, Gand F-actin were recovered in complexes at increasing levels of 88.9 i 11.3% and 51.5 i 4.8%, respectively, indicating the different binding mechanisms produced by two major binding proteins, Gc and gelsolin, in plasma. Actin is a ubiquitous cytoplasmic protein in eukaryotic cells that participates in cellular motility and structural support. The functions of actin are modulated by several actin binding proteins, which interact with actin in various ways. On the other hand, actin is not considered to be a normal component within the extracellular space, but plasma has potent actin-depolymerizing activity (3, 25). Since actin can be released from cells under a variety of circumstances, particularly during tissue necrosis, a mechanism is necessary to depolymerize and sequester it in monomeric form, then remove it from the extracellular space. This actin scavenging function is attributed to two proteins that bind actin with high afflnity. Gc globulin (also called vitamin D-binding protein) binds only monomeric actin (G-actin) at a ratio of 1: 1 in the absence of Ca2+ and slowly depolymerizes actin filaments (F-actin) by sequestering the pool of actin monomer present at equilibrium (31). In contrast, plasma gelsolin has two or three distinct binding sites for actin monomers and binds both Gand F-actin. The actions of gelsolin on actin are complex, but it rapidly depolymerizes filaments by severing them and blocking the barbed ends in the presence of Ca2+ (2, 11, 23, 33). Both Gc and gelsolin are abundant in plasma (Gc, 6-11 ,uM; gelsolin, 2-4 ,uM) and it appears that they play an important cooperative role in the clearance of actin from the extracellular space (9, 10, 21). Current knowledge of the extracellular actin scavenger mechanism indicates that G-actin binds Gc preferentially. For F-actin, this process is enhanced by gelsolin which severs actin filaments and thereby increases the pool of free G-actin. Further, gelsolin rapidly depolymerizes F-actin and one actin monomer is transferred from the calcium-sensitive site of gelsolin to Gc. This regenerates the actin-binding activity of gelsolin, and frees up gelsolin to interact with additional F-actin (10, 13, 15, 17, 19, 26). It also results in the majority of actin monomers eventually becoming complexed to Gc. Abnormalities in the circulating levels of Gc and the percentage of the complexes have been recently noted in several disorders (6, 12, 16, 18, 22).