Concentrations of long-chain acyl-acyl carrier proteins during fatty acid synthesis by chloroplasts isolated from pea (Pisum sativum) , safflower (Carthamus tinctoris) , and amaranthus (Amaranthus lividus) leaves

Roughan, G.; Nishida, I.

Archives of Biochemistry and Biophysics 276(1): 38-46

1990


ISSN/ISBN: 0003-9861
PMID: 2297229
DOI: 10.1016/0003-9861(90)90007-l
Accession: 015345889

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Abstract
Fatty acid synthesis from [1-14C]acetate by chloroplasts isolated from peas and amaranthus was linear for at least 15 min, whereas incorporation of the tracer into long-chain acyl-acyl carrier protein (ACP) did not increase after 2-3 min. When reactions were transferred to the dark after 3-5 min, long-chain acyl-ACPs lost about 90% of their radioactivity and total fatty acids retained all of theirs. Half-lives of the long-chain acyl-ACPs were estimated to be 10-15 s. Concentrations of palmitoyl-, stearoyl-, and oleoyl-ACP as indicated by equilibrium labeling during steady-state fatty acid synthesis, ranged from 0.6-1.1, 0.2-0.7, and 0.4-1.6 microM, respectively, for peas and from 1.6-1.9, 1.3-2.6, and 0.6-1.4 microM, respectively, for amaranthus. These values are based on a chloroplast volume of 47 microliters/mg chlorophyll and varied according to the mode of the incubation. A slow increase in activity of the fatty acid synthetase in safflower chloroplasts resulted in long-chain acyl-ACPs continuing to incorporate labeled acetate for 10 min. Upon re-illumination following a dark break, however, both fatty acid synthetase activity and acyl-ACP concentrations increased very rapidly. Palmitoyl-ACP was present at concentrations up to 2.5 microM in safflower chloroplasts, whereas those of stearoyl- and oleoyl-ACPs were in the lower ranges measured for peas. Acyl-ACPs were routinely separated from extracts of chloroplasts that had been synthesising long-chain fatty acids from labeled acetate by a minor modification of the method of Mancha et al. (Anal. Biochem., 1975, 68, 600-608). The results compared favorably with those obtained using alternative analytical methods such as adsorption to filter paper and partition chromatography on silicic acid columns. The acyl-ACP which coprecipitated with ammonium sulfate was not affected by treatments with neutral hydroxylamine or borohydride, whereas that eluted from silicic acid was relatively easily derivatized. A single radioactive polypeptide of Mr 11,500 from pea and amaranthus chloroplasts was revealed by autoradiography of gels from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the silicic acid eluates.