Graduate Thesis Or Dissertation
 

The biosynthesis and metabolism of (-)-kaurene in cell-free extracts of immature pea seeds

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  • Procedures were developed for assaying the biosynthesis of (-)- kaurene-¹⁴C and other intermediates in gibberellin (GA) synthesis from mevalonic acid-¹⁴C in cell-free enzyme extracts of immature pea (Pisum sativum L. cv. Alaska) seeds. This system was utilized to investigate three aspects of GA biosynthesis: (1) apparent capacities for (-)-kaurene biosynthesis in extracts of pea seeds at various stages of development; (2) localization of the enzymes which catalyze (-)- kaurene biosynthesis in immature pea seeds; and (3) further metabolism of (-)-kaurene in extracts of immature pea seeds. It was readily demonstrated that mevalonic acid was incorporated into (-)-kaurene and also into squalene (identification tentative) in extracts of immature seeds. The synthesis of (-)-kaurene was shown to be approximately linear with time through 75 minutes at 30°C, to vary directly with enzyme concentration, and to be dependent on ATP. Both Mg² ⁺and Mn²⁺ stimulated the reaction, but Mn²⁺ was a much better activator than Mg²⁺. The optimum pH was 7.1. The capacity to convert mevalonic acid to (-)-kaurene, as measured in cell-free extracts, was found to vary markedly with the stage of seed development. Using enzyme extracts prepared from seeds at different stages of development, it was observed that the activity increased from a very low initial level to a maximum at about 13 days after anthesis, or when the seeds had attained about halfmaximum fresh weight. Comparative assays of (-)-kaurene biosynthesis and accumulation in cell-free extracts of excised seed coats, cotyledons and embryonic shoot-root axes revealed that the enzymes responsible for (-)-kaurene biosynthesis apparently are localized exclusively in the cotyledons. The rates of (-)-kaurene biosynthesis in extracts of isolated cotyledons were higher than those observed in extracts of whole seeds. The enzymes responsible for the synthesis of squalene were present in both the seed coats and the cotyledons. The enzymes catalyzing the synthesis of (-)-kaurene from meva- Ionic acid were present in the soluble fraction of these extracts, whereas one preliminary experiment indicated that the enzymes catalyzing (-)-kaurene oxidation were localized in a microsomal fraction. The rates of (-)-kaurene synthesis and accumulation were greater in the 100,000 x g supernatant fraction than in the 40,000 x g supernatant fraction of extracts of excised cotyledons, whereas the opposite relationship was observed with extracts of whole seeds. Repeated efforts to demonstrate cell-free metabolism of exogenous (-)-kaurene resulted in uniformly negative results. However, when (-)-kaurene was formed in situ from mevalonic acid in cell-free mixtures containing 10, 000 x g supernatant from cotyledon extracts, its oxidation was observed. The metabolism of (-)-kaurene formed in situ was enzymic, or at least heat labile, and was markedly sensitive to inhibition by carbon monoxide. The difference in (-)-kaurene accumulation between carbon monoxide-inhibited preparations and noninhibited preparations was greater in extracts which were prepared by homogenizing the tissues in the presence of insoluble polyvinylpyrrolidone (PVP) than in extracts prepared without PVP. Although these results are not considered to provide direct evidence, they are interpreted to mean that (-)-kaurene metabolism in these extracts is protected from inhibition by insoluble PVP. The inability of the enzyme extracts to metabolize exogenous (-)-kaurene remains incompletely understood. Two possible reasons for this failure are: (a) binding of the (-)-kaurene to non-catalytic protein; and (b) a possible requirement in this system for some intimate functional association between the enzyme systems catalyzing (-)-kaurene biosynthesis and the enzyme systems catalyzing (-)- kaurene oxidation. Particular significance is ascribed to the latter possibility on the basis of limited indirect evidence. The products formed from mevalonic acid in the experiments designed to investigate the metabolism of (-)-kaurene formed in situ were isolated and tentatively identified as geranylgeraniol, (-)-kaurenol, (-)-kaurenal, and (-)-kaurenoic acid. The identifity of (-)- kaurenol was confirmed by co-crystallization of the radioactive product with authentic kaurenol to constant specific radioactivity. While (-)-kaurenol, (-)-kaurenal, and (-)-kaurenoic acid were synthesized from mevalonic acid-¹⁴C as the exogenous substrate, it is assumed, of course, that (-)-kaurene was a direct precursor of all three products.
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