The metabolism of sucrose to long chain fatty acids in the endosperm of developing castor bean (Ricinus communis L.) seeds requires a combination of cytosolic and proplastid enzymes. The total activity and the subcellular distribution of the intermediate enzymic steps responsible for the conversion of sucrose to pyruvate have been determined. Hexose phosphate synthesis from sucrose occurs in the cytosol along with the first oxidative step in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase. The proplastids contain the necessary complement of glycolytic enzymes to account for the in vivo rates of acetate synthesis from glucose 6-phosphate. These organelUes also contain the majority of the cellular 6-phosphogluconate dehydrogenase, transketolase, and transaldolase activities.The consequence of these enzyme distributions is that glucose 6-phosphate or 6-phosphogluconate produced in the cytosol must be transported into the proplastids where conversion to pyruvate occurs. The unique segregation of the two oxidative steps in the pentose phosphate pathway may be required to meet the metabolic needs of these fatstoring seeds. Compartmentation of glucose-6-phosphate dehydrogenase in the cytosol and 6-phosphogluconate dehydrogenase in the proplastids is discussed in light of the NADPH requirements for fatty acid synthesis in these subceflular locations.In photosynthetic tissues (9) and germinating seeds (1) some aspects of metabolism are regulated by means of subcellular compartmentation. Similarly, developing seeds show metabolic control by compartmentation. In the endosperm of developing castor bean seeds, oleic acid is synthesized from acetyl-CoA in proplastids (6,20). Phosphofructokinase and pyruvate kinase (5), the key enzymes of glycolysis as well as the pyruvate dehydrogenase complex (13) are also present in this organelle, suggesting that there is a particulate pathway for the conversion of hexose-P to long chain fatty acids (LFA).2 Furthermore, Yamada and his co-workers (11,18,19) have demonstrated the incorporation of sucrose, hexose, glucose 1-P, glucose 6-P, pyruvate, and acetate into fatty acids by purified proplastids. They suggest that sucrose synthase and UDPG synthase are involved in the initial conversion of sucrose to hexose-P within the organelle. The objectives of the research reported here were: to determine the level of the enzymes of the glycolytic and pentose phosphate pathway present in the cytosol and proplastid fraction; to determine which enzyme activities were adequate to account for the in vivo rate of long chain fatty acid biosynthesis; to determine the relationship between the pentose-P and glycolytic pathways in the supply of NADPH and carbon intermediates. MATERIALS AND METHODSPreparation of Proplastids. Thirty to 40-day-old developing castor bean seeds (Ricinus communis L., Baker 296 Dwarf Inbred) were harvested from plants grown in the greenhouse. The endosperm was extracted and homogenized as described previously (13) and cell debris was removed by centrifugati...

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The pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria was purified 23-fold by high speed centrifugation and glycerol gradient fractionation. The complex had a s20,,0, of 47.5S but this is a minimal value since the complex is unstable. The complex is specific for NAD+ and pyruvate; NADP+ and other keto acids give no reaction.Mg2+, thiamine pyrophosphate, and cysteine are also required for maximal activity. The pH optimum for the complex was between 6.5 and 7.5.Continuous sucrose density gradients were used to separate castor bean (Ricinus communis L.) endosperm proplastids from mitochondria.Pyruvate dehydrogenase complex activity was found to be coincident with the proplastid peak on all of the gradients. Some separation of proplastids and mitochondria could be achieved by differential centrifugation and the ratios of the activities of the pyruvate dehydrogenase complex to succinic dehydrogenase and acetyl-CoA carboxylase to succinic dehydrogenase were consistent with both the pyruvate dehydrogenase complex and acetyl-CoA carboxylase being present in the proplastid. The proplastid fraction has to be treated with a detergent, Triton X-100, before maximal activity of the pyruvate dehydrogenase complex activity is expressed, indicating that it is bound in the organelle. The complex had a sharp pH optimum of 7.5. The complex required added Mg2+, cysteine, and thiamine pyrophosphate for maximal activity but thiamine pyrophosphate was inhibitory at higher concentrations. The sequence of reactions catalyzed by these enzymes is: