Title

Microfilament and membrane associated proteins of 13762 rat mammary adenocarcinoma ascites cell microvilli

Date of Award

1988

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Coralie A. Carothers Carraway, Committee Chair

Abstract

Microvilli isolated from MAT-C1 ascites tumor cell subline of 13762 rat mammary adenocarcinoma contain a number of microfilament- and membrane-associated proteins. Three of them, tropomyosin, AMV-p35 (ascites microvillar p35) and 58K, have been purified and partially characterized.The microvillar tropomyosin is a 60 kD dimer of 31 kD and/or 29 kD subunits. It binds to F-actin in vitro with a 1:6 molar ratio of tropomyosin to actin. While the 31 kD isoform exhibits high Mg$\sp{++}$ requirement ($>$4 mM) for its association with actin, which resembles most of the non-muscle tropomyosins, the 29 kD isoform binds more strongly to actin and the binding reaches saturation at $<$1 mM Mg$\sp{++}$.AMV-p35 comprises approximately 6% of the total microvillar protein and binds to F-actin and phosphatidylserine liposomes in vitro in a Ca$\sp{++}$-dependent manner with a half maximal concentration of 0.2 mM and 10 uM Ca$\sp{++}$, respectively. Immunoblot analysis has shown that AMV-p35 reacts strongly with antibodies against calpactin I and lipocortin II. Further comparison of the partial amino acid sequences suggested that AMV-p35 is highly homologous to calpactin I and is probably a form of calpactin I in MAT-C1 cells. However, AMV-p35 exists as a 35 kD monomer rather than a heterotetramer in which calpactin I was originally found.58K is a major component present in the microvillar core prepared by Triton X-100 extraction. It has been purified by extracting microfilaments with 1 M NaCl in the presence of Triton X-100, and fractionation on Sephadex G-150 and hydroxylapatite column. The purified 58K has a Stokes radius of 4.8 nm and a sedimentation coefficient of 2.9 S. It is an elongated monomeric molecule (frictional ratio = 1.90) with a native molecular weight of 57 kD. In vitro binding studies have shown that 58K interacts with F-actin and phospholipid liposomes. The protein is also phosphorylated both in vitro and in vivo. These properties of 58K support the previous hypothesis that it may play an important role in stabilizing the membrane-microfilament interactions of MAT-C1 cell microvilli.

Keywords

Chemistry, Biochemistry

Link to Full Text

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