Graduate Thesis Or Dissertation
 

Changes in enzyme activities of the isoprenoid pathway in germinating Pisum sativum

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  • The nature of the isoprenoid pathway and diversity of biologically significant products suggests that the pathway may be highly regulated, particularly under stress conditions, where the availability of high energy intermediates is limited. It was postulated that germinating seeds suffer from a limited supply of available energy and must therefore conserve energy wherever possible. This assumption should be reflected through enzymic changes in specific isoprenoid enzymes of the pathway, correlated with the developmental requirements for growth and differentiation. An in vivo analysis of nonsaponifiable products formed from mevalonate-2-¹⁴C versus germination in Pisum sativum confirmed that β-amyrin (triterpene) and β-sitosterol (sterol) are sequentially synthesized at different rates, depending upon the germination stage. Kinetic transitions between squalene, squalene-triterpene, and squalene-triterpene/squalene-sterol formation are discussed in relation to the metabolic steps required for either triterpene or sterol biosynthesis. Metabolically, either product must arise from 2, 3-oxidosqualene. Changes in triterpene and sterol formation as a function of germination, however, suggest that the enzymes responsible for such synthesis do not compete for oxidosqualene, but for squalene. A regulatory model based on these observations is proposed. Squalene epoxidase is postulated to combine with specific cyclases as an enzyme aggregate. As an aggregate, the enzyme complex competes for squalene to make the appropriate cyclized product. Regulation and selection of product formation is controlled through association and dissociation of the aggregate. Methods for the biosynthesis and isolation of phosphomevalonate-2-¹⁴C, isopentenyl-4-¹⁴C pyrophosphate and farnesyl-4, 8, 12-¹⁴C pyrophosphate from mevalonate-2-¹⁴C in yeast autolysates, prepared from commercial dry baker's yeast, are described in detail. Comparisons between various biosynthetic methods have been made. Methods of identification, isolation technics and criteria of purity are also discussed; those methods most suitable for the isolation and identification of each isoprenoid intermediate have been emphasized. Individual assays of mevalonic kinase, phosphomevalonic kinase, pyrophosphomevalonic decarboxylase, isopentenyl pyrophosphate isomerase, isopentenyl pyrophosphate/dimethylallyl pyrophosphate prenyltransferase and squalene synthetase in 40,000 g supernatants of Pisum sativum, prepared from germinating seeds at selected intervals after the onset of imbibition, suggest that the enzymes are regulated in such a manner as to always favor isopentenyl pyrophosphate and dimethylallyl pyrophosphate formation. The enzymes are activated in such proportions as to favor nonequilibrium conditions between mevalonic acid and isopentenyl pyrophosphate, and equilibrium conditions between isopentenyl pyrophosphate and dimethylallyl pyrophosphate, thereby insuring the most efficient synthesis of higher isoprenoid products at all times. A reversal of carbon flow from dimethylallyl pyrophosphate to isopentenyl pyrophosphate appears highly probable as a means of maintaining a sufficient supply of isopentenyl pyrophosphate to meet the new biosynthetic requirements induced by development. Dimethylallyl pyrophosphate may serve as a storage pool, to meet biosynthetic demands in times of stress, where the endogenous supply of mevalonic acid is limited. Isopentenyl pyrophosphate/dimethylallyl pyrophosphate prenyltransferase was assayed indirectly in 40,000 g supernatants with mevalonate-2-¹⁴C and more directly with isopentenyl-4-¹⁴C pyrophosphate. Under identical conditions in the preparation of 40,000 g supernatants, the former assay yielded an estimated activity some 15-20 fold greater than by isopentenyl-4-¹⁴C pyrophosphate. These results suggest that the prenyltransferase is allosterically activated by one, or more, of the first three isoprenoid intermediates (i.e., mevalonate, phosphomevalonate, and/or pyrophosphomevalonate). Indirect evidence was also obtained for the presence of two distinct isopentenyl pyrophosphate/dimethylallyl pyorphosphate prenyltransferases, tentatively identified as enzymes I and II. Detection of prenyltransferase I is independent of sucrose, while detection of prenyltransferase II is highly dependent upon the inclusion of sucrose (0.45 M) in the homogenizing buffer. The two prenyltransferases are activated sequentially during the course of germination. Prenyltransferase I is postulated to be associated with the cytoplasm, while prenyltransferase II may be associated with an organelle, possibly with immature chloroplasts (protoplasts) or mitochondria. Enzymic changes in the activities of prenyltransferases I and II appear closely correlated with developmental changes in the seed initiated with the breaking of dormancy. A thermolabile, large molecular weight factor has been tentatively identified as a specific inhibitor of isopentenyl pyrophosphate/dimethylallyl pyrophosphate prenyltransferase. This factor is temporal, appearing at later stages of germination. The action of this factor as a means of diverting the flow of carbon between cytoplasmic and organelle associated pathways is discussed. Suppression of prenyltransferase I activity by this factor, coincident with the rise in prenyltransferase II activity, may suggest that compartmentalized isoprenoid pathways are not completely autonomous, but are suppressed or activated to meet the needs of their respective environments, thereby conserving considerable energy and substrate.
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