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Studies on fatty-acid-binding proteins. The purification of rat liver fatty-acid-binding protein and the role of cysteine-69 in fatty acid binding.

ABSTRACT: 1. A new, simple and high-yield procedure is described for the purification of hepatic fatty-acid-binding protein from rat liver using naphthylaminodecyl-agarose as an affinity column. 2. Cysteine-69 is shown to react slowly, but quantitatively, with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), indicating that the thiol group is free, but may be buried within the protein. 3. Fatty acids do not affect the DTNB reactivity of this cysteine residue; however, cysteine reactivity is enhanced in the presence of haem and oleoyl-CoA. 4. Fatty-acid-binding protein that has been modified with DTNB is still able to bind the fluorescent fatty acid 11-(dansylamino)undecanoic acid, indicating that cysteine-69 may be remote from the fatty-acid-binding site.

SUBMITTER: Wilton DC

PROVIDER: S-EPMC1138812 | biostudies-other | 1989 Jul

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Renal cell carcinoma (RCC) is the most common renal neoplasm. Cancer tissue is often characterized by altered energy regulation. Fatty acid-binding proteins (FABP) are involved in the intracellular transport of fatty acids (FA). We examined the level of brain-type (B) and liver-type (L) FABP mRNA and the protein expression profiles of both FABPs in renal cell carcinoma. METHODS: Paired tissue samples of cancerous and noncancerous kidney parts were investigated. Quantitative RT-PCR, immunohistochemistry and western blotting were used to determine B- and L-FABP in tumor and normal tissues. The tissue microarray (TMA) contained 272 clinico-pathologically characterized renal cell carcinomas of the clear cell, papillary and chromophobe subtype. SPSS 17.0 was used to apply crosstables (chi2-test), correlations and survival analyses. RESULTS: B-FABP mRNA was significantly up-regulated in renal cell carcinoma. In normal tissue B-FABP mRNA was very low or often not detectable. RCC with a high tumor grading (G3 + G4) showed significantly lower B-FABP mRNA compared with those with a low grading (G1 + G2). Western blotting analysis detected B-FABP in 78% of the cases with a very strong band but in the corresponding normal tissue it was weak or not detectable. L-FABP showed an inverse relationship for mRNA quantification and western blotting. A strong B-FABP staining was present in 52% of the tumor tissues contained in the TMA. In normal renal tissue, L-FABP showed a moderate to strong immunoreactivity in proximal tubuli. L-FABP was expressed at lower rates compared with the normal tissues in 30.5% of all tumors. There was no correlation between patient survival times and the staining intensity of both FABPs. CONCLUSION: While B-FABP is over expressed in renal cell carcinoma in comparison to normal renal tissues L-FABP appears to be reduced in tumor tissue. Although the expression behavior was not related to the survival outcome of the RCC patients, it can be assumed that these changes indicate fundamental alterations in the fatty metabolism in the RCC carcinogenesis. Further studies should identify the role of both FABPs in carcinogenesis, progression and with regard to a potential target in RCC.

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Studies on fatty-acid-binding proteins. The purification of rat liver fatty-acid-binding protein and the role of cysteine-69 in fatty acid binding.

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