Characterization and purification of a GTP hydrolase and its possible function in vitamin A-inhibited membrane transport in rat liver
Abstract
A complex of proteins isolated from endoplasmic reticulum of rat liver showed an in vitro response to retinol by a procedure involving DEAE-cellulose column chromatography, gel filtration chromatography, high performance liquid chromatography and native gel electrophoresis. The complex from sephadex G-200 consisted of dominant peptides at 68 and 55 kDa with minor bands at 93 and 50 kDa and exhibited GTP hydrolase activity that was inhibited by retinol and a protein disulfide isomerase (PDI) activity that was stimulated by retinol. The complex bound retinol with a K$\sb{\rm d}$ of 0.1 nM and with a stoichiometry of 1 mole retinol bound per mole of purified protein. The complex had an optimum pH of 7.5 for GTP hydrolysis, a K$\sb{\rm i}$ for retinol of 0.03 mM and a pI of 5.3 by isoelectric focusing. An antibody raised from rabbit against the complex immunoprecipitated a 55 kDa protein from rat liver that lacked GTPase activity. The common peptide among fractions exhibiting retinol-inhibited GTPase activity was at approximately 68 kDa. The retinol-inhibited GTPase reaction of the native gels was identified using an activity stain. Two major peptides were obtained. Cleavage of the individual peptides by cyanogen bromide (CNBr) at methionine residues produced twelve peptides from the 55 kDa. One of the two peptides from the 55 kDa protein had partial sequence identity with the calreticulin precursor and another with PDI. A 72 kDa peptide had sequence identity with a PDI-related protein. Using an antibody to the G-$\alpha$ common consensus GTP binding sequence, the 55 kDa peptide bound antibody only weakly but the 68 kDa bound strongly. Both may bind retinol based on the stoichiometry of binding and response to enzymatic activities to retinol. The findings point to the isolation and characterization of a novel retinol-responsive activity complex of the transitional endoplasmic reticulum that may combine both retinol and guanine nucleotide binding elements with protein disulfide isomerase activity in the regulation of secretion and/or protein transport.
Degree
Ph.D.
Advisors
Morre, Purdue University.
Subject Area
Biochemistry|Biology|Nutrition
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