Graduate Thesis Or Dissertation
 

Alteration in levels and synthesis of proteins in trout hepatocytes due to dietary cyclopropenoid fatty acid[s]

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  • Cyclopropenoid fatty acids (CPFA) are unique compounds that contain a highly strained and reactive cyclopropene ring structure. These compounds have been shown to cause a number of toxic effects in a variety of animals. Rainbow trout (Salmo gairdneri) have proven to be particularly sensitive to CPFA. Studies have revealed that CPFA are both carcinogenic and cocarcinogenic in rainbow trout. However, the mechanism(s) of these adverse biological effects are not understood. In the present report a series of studies were performed in order to determine the effect of CPFA on the levels and synthesis rates of trout hepatocyte proteins. In the first study, the influence of dietary CPFA on protein synthesis was measured via the use of amino acid double labeling experiments in isolated hepatocytes. Both the microsomal and cytosolic subcellular fractions were examined in these studies after separation by lithium dodecyl sulfate polyacrylamide gel electrophoresis. In the cytosolic fraction, the synthesis of proteins with apparent molecular weights in the range of 68,000 to 74,000 were significantly decreased. A marked depression in both the level and synthesis rate of microsomal proteins was observed for proteins that migrate in the 200,000 to 240,000 relative molecular mass region in polyacrylamide gels. These high molecular weight proteins do not appear to be membrane proteins and one of them has biotin associated with it. Using avidin-peroxidase staining, it was shown that the mass of this protein was reduced in CPFA-fed trout by 80%. The possible identity of these proteins is discussed. In the second study, initial attempts were made to use two dimensional gel electrophoresis to study alterations in individual liver microsomal polypeptides from trout fed CPFA. In order to effectively resolve membrane proteins in the first dimension (isoelectric focusing) changes in the standard techniques were needed. Replacement of the detergent nonidet-40 with 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPS) in isoelectric focusing of trout liver microsomes have greatly increased resolution. These results have allowed effective resolution of complex polypeptide patterns for comparative purposes. In the third study, antibodies against β-napthoflavone-fed rainbow trout cytochrome P-450 (LM₂) were employed to localize the corresponding polypeptide(s) via protein blotting and immunochemical staining. Microsomes isolated from β-napthoflavone-fed trout contained only a single polypeptide. In contrast, control microsomes contained two distinct polypeptides differing only in their isoelectric points. Thus, an additional P-450 isozyme in rainbow trout was tentatively identified. CPFA treatment caused a preferential decrease in only one of the isozymes found in the control samples. The presence of concanavalin A binding glycopolypeptides was determined. The two P-450 isozymes localized on control microsomal gels were found to bind concanavalin A, suggesting that these isozymes are giycoproteins. Another result of CPFA treatment was a shift in a closely related group of membrane glycopolypeptides, labeled gp80, gp82, gp80₁, and gp82₁. A decrease in the mass of gp80 and gp82, and a corresponding increase in mass of gp80₁ and gp82₁ was observed.
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