Partial purification of fatty-acid binding protein by ammonium sulphate fractionation

Instituto Multidisciplinario de Biología Celular, Buenos Aires, Buenos Aires F.D., Argentina
Archives internationales de physiologie et de biochimie 08/1983; 91(2):103-8. DOI: 10.3109/13813458309078583
Source: PubMed


By fractionation of rat liver cytosol with 70% saturation ammonium sulphate, a soluble fraction showing high affinity for oleic acid was obtained. The binding of oleic acid to this fraction was inhibited by flavaspidic acid. The molecular weight of the main protein present in this fraction was 12 000 as determined by SDS-poly-acrylamide-gel electrophoresis. This soluble fraction stimulated the transfer of oleic acid from microsomes to phosphatidylcholine liposomes as demonstrated by a transfer assay in vitro. The behaviour of this fraction is similar to that described for fatty-acid binding protein.

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    • "Liver cytosol from 6-d-old poults was mixed with 70% ammonium sulfate at 4 C (Avanzati and Catala, 1983) and centrifuged at 4,000 × g; the supernatant was desalted on a PD-10 column. 4 The fraction thought to contain FABP was subsequently applied to a Sephadex G-75 column (1.75 ×X 75 cm), and 3-mL fractions were collected and analyzed for protein and FABP activity using a ligandbinding assay as described below. "
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    ABSTRACT: Turkey (Meleagridis gallopavo) liver cytosolic fatty acid binding protein (FABP) was purified and used as a standard for quantification. An immunoblotting procedure was developed to study the ontogeny of liver cytosolic FABP during embryonic and early posthatch development in turkey poults. Liver FABP activity was also determined indirectly through the use of gel filtration chromatography followed by a ligand-binding assay. The specific activity of liver FABP (ng/mg of cytosolic protein) increased with length of incubation, peaking initially at Day 22, declining between Days 22 and 25, and increasing again from hatch (Day 28) to 6 d posthatch. The specific activity of liver FABP increased 12-fold between Day 13 of incubation and 6 d posthatch compared with total activity, which increased from 946 to 1.01 x 10(6) ng/liver during the same period, a 1,067-fold increase. The results from both analytical procedures were similar, suggesting that the immunoblot method could be used to quantify liver FABP concentrations. The observed increases in FABP activity throughout the embryonic period and first days after hatching paralleled increases in liver lipid concentration. Therefore, liver FABP may be associated with hepatocyte fatty acid transport and metabolism during the latter stages of incubation and early posthatch period.
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    ABSTRACT: Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.
    No preview · Article · Nov 1984 · Archives internationales de physiologie et de biochimie

  • No preview · Article · Feb 1985 · International Journal of Biochemistry
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