Isolation of a calcium‐sensitive, 35,000‐dalton microfilament‐ and liposome‐binding protein from ascites tumor cell microvilli: Identification as monomeric calpactin

Abstract

Microvilli isolated from the MAT-C1 ascites subline of the 13762 rat mammary adenocarcinoma contain a major calcium-sensitive microfilament-binding protein, AMV-p35 (ascites microvillar p35). Association of AMV-p35 with microfilament cores during Triton X-100 extraction of the microvilli is half-maximal at 0.1-0.2 mM calcium. The protein, which comprises 6% of the total microvillar protein, can be isolated from microfilament cores prepared in the presence of calcium by extraction with EGTA and purification by ion-exchange chromatography. Alternatively, the protein can be isolated from Triton extracts of microvilli prepared in the absence of calcium by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on an ion-exchange column. AMV-p35 binds to phosphatidylserine liposomes and F-actin with half-maximal calcium concentrations of about 10 microM and 0.2 mM, respectively. Treatment of AMV-p35 with chymotrypsin yields a 33,000-dalton fragment, behavior similar to the tyrosine kinase substrates calpactins I and II and lipocortins I and II. Immunoblot analyses using antibodies directed against calpactin I, lipocortin I, and lipocortin II showed strong reactivity of AMV-p35 with anti-calpactin I and anti-lipocortin II, but little reactivity toward anti-lipocortin I. The close relationship between AMV-p35 and calpactin I was verified by amino acid sequence analyses of peptides isolated from cyanogen bromide digests of AMV-p35. By gel filtration and velocity sedimentation analyses purified AMV-p35 is a 35,000-dalton monomer. Moreover, AMV-p35 extracted directly from microvilli in Triton/EGTA also behaves as a 35,000-dalton menomer. These findings indicate that AMV-p35 is closely related to the pp60src kinase substrate calpactin I (p36). However, AMV-p35 occurs in the microvilli as a monomer rather than as the heterotetrameric calpactin found in several other cell types.

Cytovillin and other microvillar proteins of human choriocarcinoma cells

“…In its relative molecular weight and calcium-dependent association with the cytoskeleton, it resembles AMV-p35, a calcium-sensitive membranebinding protein of the MAT-Cl microvilli. AMV-p35 is closely related to calpactin I [34], which belongs to a large family of calcium-sensitive membrane-binding proteins [38]. Calpactin I is a peripheral membrane protein, which is expressed in high concentrations in the mammalian intestine and is localized primarily in the cytoskeletal region of the brush border beneath the microvillar array [ 391.…”

Calcium-dependent phospholipid- (and membrane-) binding proteins

“…The Ca2+-dependent membrane-binding proteins coexist in a wide variety of murine, porcine, bovine, and human tissues (Geisow et al, 1984;Davies & Crumpton 1985;Shadle et al, 1985). Western blots of tissue homogenates with antibodies to calpactins I and II showed their uneven distribution ; Hayashi et al, 1987; Khanna et al, 1987;Huang et al, 1986;De, 1986;Liu et al, 1987). Calpactin I is the predominant form in most tissues.…”