Isoenzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinis communis L.

Abstract

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, transketo-lase, 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 fat-storing 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 dehy-drogenase 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, Ya-mada and his co-workers (11, 18, 19) have demonstrated the incorporation of sucrose, hexose, glucose 1-P, glucose 6-P, pyru-vate, 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 organ-elle. 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 glyco-lytic pathways in the supply of NADPH and carbon intermedi-ates. MATERIALS AND METHODS Preparation 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 centrifugation at 500g for 10 min. The homogenate was centrifuged at 10,000g for 30 min to give soluble (S,0) and particulate (P,o) fractions. The Plo fraction was resuspended in homogenizing medium (1 ml/4 g of endosperm) by means of a Teflon homogenizer. In some experiments the Plo was centrifuged at 10,000g and the resulting particulate fraction (washed P10-WP10) was resus-pended as described for the Plo fraction. Proplastids were purified by layering 1 ml of the Plo on a continuous sucrose density gradient buffered with 10 mM TES * NaOH (pH 7.5). The gradient consisted of 30 ml of linear 35 to 55% (w/w) sucrose which also had a linear gradient of 0 to 0.5 mM MgCl2. The gradient rested on a cushion of 5 ml of 60% sucrose. The gradients were centrifuged at 24,000 rpm for 20 min in an SW 27 rotor (15). During this period of time the organelles did not reach equilibrium, therefore the gradient was a combination of velocity and equilibrium density centrifugation. Fractions of 1.1 ml were collected dropwise by puncturing the bottom of the tube. Enzyme Assays. All enzyme assays were performed at 26 C and were linear with respect to time and enzyme concentration. Spectrophotometric assays were performed in a Gilford modified Beckman DU spectrophotometer. When NADH was present in the assay the rates were corrected for NADH oxidase activity. The assay for glucose-6-P dehydrogenase (D-glucose-6-P:NADP+ oxidoreductase, EC 1.1.1.49) (G6PDH) contained 10 mM MgCl2, 0.1% Triton X-100, 0.17 mm NADP+, 0.33 mm glucose-6-P (G6P), 20 mm TES*NaOH (pH 7.5) in a final volume of 3 ml. The reduction of NADP+ was measured by monitoring the A at 340 nm. The assays for 6-P-gluconate dehydrogenase (6-P-D-glucon-ate:NADP+ oxidoreductase [decarboxylating], EC 1.1.1.44), hexokinase (ATP:D-hexose-6-P-transferase, EC 2.7.1.1), hex-ose-P isomerase (D-glucose-6-P-ketol-isomerase, EC 5.3.1.9), P-glucomutase (a-D-glucose-1 ,6-diP: aD glucose -1-P phospho-transferase, EC 2.7.5.1) were identical with that for glucose-6-P 1128