Pea root plastids were isolated by differential centrifugation and resulting crude plastid fraction was purified by centrifugation through 10%(v/v) Percoll. Marker enzymes indicated that greater than 50% of the plastids were recovered essentially free from mitochondrial and endoplasmic reticulum contamination. The optimum in vitro conditions for glycerolipid biosynthesis from (U-$ sp{14}$C) glycerol-3-phosphate have been determined. Total glycerolipid biosynthesis was approximately 15 nmole/hr/mg protein in the presence of 200 $ mu$M glycerol-3-phosphate, 0.5 mM each of NADH and NADPH, 15 mM KH$ sb2$CO$ sb3$, 0.05 mM CoA, and 2 mM each of ATP and MgCl$ sb2$, 100 mM Bis Tris Propane (pH 7.5) and incubated at the standard temperature of 25$ sp circ$C. ATP, Coenzyme A and a divalent cation are absolutely required for glycerolipid biosynthesis, whereas reduced nucleotides and bicarbonate improve the synthesis to varying degrees. Dihydroxyacetone phosphate had little effect, while dithiothreitol, detergent and Mn$ sp{2+}$ inhibited activity. Under the optimum conditions, isolated pea root plastids mainly synthesized approximately 15% phosphatidic acid, 16% phosphatidylcholine, 13% phosphatidylglycerol, 32% triacylglycerol. Galactolipid synthesis occurred only when UDP-galactose was supplied. Different concentrations of some cofactors resulted in alterations of glycerolipid distribution. Phospholipase A$ sb2$ and Rhizopus lipase digestions of phospholipids and neutral lipids revealed that radioactive fatty acids were preferentially esterified to position sn 2 of each glycerolipid with generally 2-4 times as much radioactivity as position sn 1. Pea root plastids are composed of approximately 62% phospholipid, 24% neutral lipid and 14% glycolipid. Within these classes PG, TAG, and the galactolipids are the major components representing 24, 12, and 12% of the total plastid lipids.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.26186 |
| Date | January 1993 |
| Creators | Xue, Lingru |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Plant Science.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001385072, proquestno: MM94546, Theses scanned by UMI/ProQuest. |
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