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. / Graduation date: 1985
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27241 |
| Date | 25 June 1984 |
| Creators | Perdew, Gary H. |
| Contributors | Selivonchick, Daniel P. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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