Subceilular Distribution of Gluconeogenetic Enzymes in Germinating Castor Bean Endosperm 1

The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase, sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO2 occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in adtion to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development. There is a massive conversion from fat to sucrose in the endosperm of young castor bean seedlings. It has been demonstrated (see Fig. 7) that the fl-oxidation of fatty acids and the subsequent conversion of acetyl-CoA to succinate occur in glyoxysomes and that succinate is converted to malate or oxaloacetate in the mitochondria 44). However, the intracellular site of the reactions from oxaloacetate to sucrose is still not known. Kobr and Beevers (18) have shown that the concentrations of glycolytic intermediates and coenzymes in castor bean endosperm do not differ strikingly from those in nongluconeogenetic tissues such as pea and buckwheat seedlings. They suggested (17) that gluconeogenesis may occur in a separate intracellular compartment, the plastid, since several glycolytic enzyme activities were found there. In a previous paper we described a method for preparing intact plastids in high yield from protoplasts from castor bean endosperm (24) which allows a quantitative approach to this problem. We report here the intracellular localization of gluconeogenetic and related enzymes using this method, and conclude that the production of sucrose from oxaloacetate occurs in the cytosol. 'Supported by DOE Contract EY-76-03-0034. 2 Recipient of a travel grant from the Japan Society for Promotion of Science and a grant from Matsunaga Foundation (Tokyo). 3 Present address: Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan. MATERIALS AND METHODS Seeds of castor bean (Ricinus communis cv. Hale) were soaked in running tap water for 1 day and germinated in moist Vermiculite at 30 C. The methods for preparation of protoplasts from the endosperm of 4-day-old castor bean seedlings and disruption of the protoplasts in 150 mm Tricine-KOH (pH 7.5)-25% sucrose-l mm EDTA were described previously (24). The ruptured protoplasts (about 10 mg protein in 2 ml) were layered on a gradient composed of: (a) a 1-ml cushion of 60%1o (w/w) sucrose; (b) 13 ml sucrose solution increasing linearly from 30 to 60%o sucrose; and (c) I ml 30%1o sucrose in an 18-ml tube. All sucrose solutions were prepared in 0.1 nmm EDTA (pH 7.5). The gradients were centrifuged at 21,000 rpm for 3 h in a Beckman model L2-65B centrifuge in a Spinco SW 27-1 rotor at 4 C, and sequential fractions (0.4 ml) were collected. Enzyme Assays. All assays except for those of sucrose-P synthetase (37 C), sucrose synthetase (37 C), RuP2 carboxylase (25 C), and fructose diphosphatase (25 C) were carried out at room temperature (about 23 C). Assays for sucrose-P synthetase and sucrose synthetase were carried out by measuring the incorporation of labeled UDP-glucose4 into sucrose-P or sucrose at 37 C for 30 min by a modification of Hawker's methods (14). The reaction mixtures contained, in ,tmol, K-phosphate buffer (pH 7.5), 5.0; EDTA, 2.5; NaF, 1.25; fructose-6-P, 2.5; UDP-['4C]glucose, 0.5 (1 ,uCi); in a total volume of 75 ,ul for sucrose-P synthetase, and Tris-HCl (pH 8.0), 5.0; fructose, 2.5; UDP-[`4C]glucose, 0.5 (0.1 ,uCi); in a total volume of 75 ,lI for sucrose synthetase. The reactions were stopped by heating in boiling water for 2 min. Twenty p1 of reaction mixture were applied to a paper chromatogram. The radioactivity incorporated into sucrose was measured by cutting the chromatogram into 1-cm-wide strips which were assayed for 14C in scintillator solution (0.5%, w/v, PPO in toluene) after chromatography in ethyl acetate-pyridine-water (8:2:1, v/v/ v) for 20 h. For the sucrose-P synthetase assay the remainder of the reaction mixture (50 ,ul) was added to 0.1 ml of 50 mm glycineNaOH buffer (pH 10.6) containing MgCl2 (5 ytmol), 2,l alkaline phosphatase (Sigma type III), and toluene (10 ,ul). After incubation at 30 C for 15 h, 20-,ld samples were chromatographed as above. The differences between the 14C in sucrose before and after phosphatase treatment represent incorporation of 14C into sucroseP by sucrose-P synthetase. It was proved that the product was sucrose by invertase treatment (data not shown). The pH of the assay mixture is the optimum pH determined for these enzyme activities in crude homogenates. Hexokinase, hexose-P isomerase, glucose-6-P dehydrogenase, 6-P-gluconate dehydrogenase, phosphoglucomutase, UDP-glucose pyrophosphorylase, 3-P-glycerate kinase, P-glycerate mutase, pyruvate kinase, transaldolase, transketolase, phosphofructokinase, enolase, NAD and NADP triose4Abbreviations: UDP-glucose: uridine diphosphate glucose; PEP: phosphoenolpyruvate; RuP2: ribulose 1,5-bisphosphate. 31 www.plantphysiol. org on May 1, 2019 Published by Downloaded from Copyright © 1979 American Society of Plant Biologists. All rights reserved. NISHIMURA AND BEEVERS P dehydrogenase were assayed according to Simcox et al. (32). Other assays were those described in the literature as follows, catalase (22), fumarase (28), RuP2 carboxylase (26), PEP carboxylase (8), PEP carboxykinase (8), pyruvate dehydrogenase (29), aldolase (15), fructose diphosphatase (7), NAD malate dehydrogenase (3), triose-P isomerase (23). Coupling enzymes were obtained from Sigma. RESULTS In a previous paper we reported methods for preparing intact plastids in high yield from protoplasts from castor bean endosperm. The enzyme profile of the broken protoplast preparations after sucrose density gradient centrifugations is shown in Figure 1. The prominent protein peak coincides with fumarase activity which is the marker enzyme of mitochondria. Less than 5% of the total fumarase activity was detected in the supernatant fractions. Catalase, the marker enzyme of glyoxysomes, is localized in a fraction at peak density 1.25 g/cm3 (see below). Only 7% of the