The aim of this study was to investigate the possibility of improving the composition of goat meat, in terms of the fatty acid composition of the different fat deposits. For this purpose, we used two groups of 12 female goats each of which had recently undergone a double birth. The animals were maintained under semi-extensive conditions and trough-fed with a concentrate that was either non-supplemented or supplemented with 50 g/kg of polyunsaturated fatty acids (PUFA)-rich fat protected against ruminant metabolism. The kid goats born to each group were suckled by their dams and a representative sample of each was slaughtered at 45 days after birth. The milk produced by the dams receiving the fat-supplemented diet contained fat with a lower content of saturated fatty acids and a higher content of n-3 PUFA, trans-C18: 1 and CLA. The kid goats suckled by these dams grew faster and the legs of the carcasses presented greater muscular development compared with the non-fat-supplemented diet group. The cover, intermuscular and intramuscular fat presented a different fatty acid composition, with a higher proportion of n-3 PUFA, trans C18: 1 and CLA, while that of n-6 PUFA remained unchanged. The change in the lipid metabolism of the kid goats was made evident by the blood levels of certain biochemical parameters. We discuss the improvement in the quality of the meat obtained, taking into account the feeding strategy provided and the class of animal in question.
A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in lambda gt11. The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative processing peptide. An additional 115 base pairs corresponding to a 3'-untranslated region coding for an mRNA poly(A)+ tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular mass 39,253 daltons (Da), showing 82-88% absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase (kb) KpnI-SacI fragment of the lambda gt11 derivative was subcloned between the KpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates of Escherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase. When assayed under excess Mg2+ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry. In contrast to the native pea chloroplast FBPase, the E. coli-expressed enzyme did not react with the monoclonal antibody GR-PB5.(ABSTRACT TRUNCATED AT 250 WORDS)
A cDNA clone encoding pea (%um sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library. Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach. After subcloning in the Ndel-BamHI sites of pET-lZa, the recombinant supplied 20 mg Trx m/2. Escherichia coli culture. This protein had 108 amino acids and was 12,000 D, which is identical t o the pea leaf native protein. Chloroplast FBPase (EC 3.1.3.11) is a highly regulated enzyme of the Benson-Calvin pathway (Bassham and Krause, 1969). It becomes active in the dark-light transition by reduction of an essential -S-S-bridge of the enzyme molecule, which promotes a conformational change to an active form (Jacquot, 1984). This reduction occurs via the Trx system, by which reduction equivalents from photosynthetic electron transport are channeled to Trx in a process catalyzed by the enzyme Fd-Trx reductase; the reduced Trx, in turn, transfers the reduction equivalents to FBPase by thiol-disulfide exchange (Buchanan, 1980;Scheibe, 1990 parts, both chloroplast Trxs show the same active cluster (-W-C-G-P-C-) and similar molecular size and folding (Eklund et al., 1991). However, their primary structures are quite different; the m form is closer to prokaryotic Trxs, and thef form is more similar to that from mammals (Hartman et al., 1990). Spinach Trxf is functional in the activation of FBPase and other chloroplast enzymes, whereas Trx m has been described as specific in NADP-malate dehydrogenase activation (Jacquot et al., 1978;Schiirmann et al., 1981). However, in the presence of the modulators Ca2' and Fru-1,6-bisphosphate, FBPase is also activated by Trx m (Schiirmann et al., 1985). A similar feature has been de- MATERIALS AND METHODS Chemicals and Biological MaterialRestriction enzymes, PCR reagents, isopropyl-P-Dthiogalactoside, substrates, and auxiliary enzymes for determination of FBPase activity were provided by Promega and Boehringer Mannheim. Chromatographic standards and prepacked Sephadex G-50 columns were from Pharmacia. Reagents and DEAE-cellulose membranes for western blotting were purchased from Sigma, Millipore, and Schleicher & Schuell. Other chemicals, including antibiotics and electrophoretic reagents, were of a molecular biology grade and were obtained from Sigma and Pharmacia.Chloroplast FBPase was obtained from pea leaves according to the method of ; pea Trxs m and f were purified from leaves, as described by Prado et al.
An immunological method for quantitative determination of photosynthetic fructose-1,6-bisphosphatase in crude extracts of leaves is proposed. It is based on the ELISA technique, and offers two modifications. A non-competitive technique has a higher sensitivity and is the right option for samples of low fructose-1,6-bisphosphatase content. However, this method is not sufficiently specific when the total protein is higher than 5 μg/cm(3); so, despite its lower sensitivity, in these circumstances a competitive technique is more suitable. Thus photosynthetic fructose-1,6-bisphosphatase can be measured without interferences from the gluconeogenic cytosolic enzyme of the photosynthetic cell or from a non-specific phosphatase present in the chloroplast.
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