Chloroplast and Cytoplasmic Enzymes

Thus, the energy charge is a measure of the extent to which the total pool of 5'-adenylates is present in the form of components containing phosphoanhydride bonds. It has been demonstrated that regulatory enzymes in many biosynthetic (energy-utilizing) sequences show a sharp increase in rate as the energy charge is increased through the physiologically important range of about 0.80 to 0.95 (2). Although this has been shown to be the case for many animal and microbial systems which have been studied, relatively little is known about adenylate energy charge regulation of higher plant systems.The results presented in this paper indicate that the rate of biosynthesis of kaurene from mevalonate in M. macrocarpus endosperm shows a response to variation in energy charge which is typical of a biosynthetic sequence. The rate-determining step in the sequence subject to this regulation appears to be the reaction catalyzed by pyrophosphomevalonate decarboxylase (EC 4.1.1.33) (step 3, Fig. 1). EXPERIMENTAL PROCEDUREMaterials. Immature seeds of M. macrocarpus (wild cucumber) were collected in the Santa Monica Mountains, California, during the spring of 1975 and frozen at -20 C for approximately 1 year before use. Pooled endosperm (100 ml) was then gently disrupted in a Thomas Teflon to glass homogenizer and centrifuged for 10 min at 10,000g to remove cell debris and larger particulate matter. The supematant fraction was further centrifuged at 150,000g for 90 min to remove particulate enzymes which catalyze the oxidative metabolism of kaurene and much of the phosphatase activity. This supernatant solution was then dialyzed against four 1-liter volumes of tris chloride (0.05 M, pH 7) in a rotating rapid-dialyzer over a period of 2 hr. The dialyzed preparation (55 ml, 1.7 mg protein/ml) served as the source of enzymes for all of the experiments reported. All enzyme preparation procedures were done at 0 to 4 C, and the enzyme solution was stored frozen in a liquid N2 refrigerator until ready for use.[2-"4C]MVA lactone (10.9 mCi/mmol) was purchased from

Section: Discussionmentioning

confidence: 99%

Regulation of the Biosynthesis of Ent-Kaurene from Mevalonate in the Endosperm of Immature Marah macrocarpus Seeds by Adenylate Energy Charge

Knotz¹,

Coolbaugh²,

West³

1977

“…5), CO2 fixation was extremely sensitive to the ADP concentration and was already inhibited by 0.5 mm ADP. Low concentrations of AMP (<I mM) caused considerable inhibition which may reflect the reported competition of AMP with ATP (29). Concentrations of ATP up to 5 mm (Fig.…”

Section: Resultsmentioning

confidence: 88%

Characterization of a Photosynthesizing Reconstituted Spinach Chloroplast Preparation

Kow¹,

Gibbs²

1982

Regulation of the photosynthetic carbon reduction cycle with reconstituted chloroplast systems has been studied in several laboratories. These studies have established the reconstituted chloroplast system as a useful research tool to elucidate questions not readily resolved with the intact chloroplast due to a permeability barrier imposed by its envelope (6,16,22,23,26,32,34). Reconstituted systems have been prepared by lysing isolated whole chloroplasts followed by recombining the separated membranes with the soluble components. Prepared in this way, the reconstituted preparations contained low levels of primers (reductive pentose-P cycle intermediates) and cofactors depending upon the amount of soluble material added back to the membranal fraction.Preparations of this kind were reported to fix CO2 at rates approaching 20o of that of a system to which a primer such as PGA had been added (6).We report here a method of preparing a reconstituted chloroplast system capable of photosynthetically fixing CO2 in which the carry-over ofsoluble components is minimal and fixation is strictly dependent on primer. Depending upon the MgC12 concentration in the rupturing fluid and the added primer, a particulate fraction termed the MgC12-broken chloroplast particle is obtained which, in the absence of added stromal protein, can assimilate CO2 yielding the intermediates of the photosynthetic carbon reduction cycle. This light-dependent system capable of fixing CO2 in the order of 1 to 20 ,umol/mg Chl-h, depending upon primer and cofactors may be a useful adjunct to the reconstituted systems previously studied. We have characterized light-dependent CO2 fixation in this particulate preparation with respect to products formed, primers, pyridine nucleotides, adenylates, and ferredoxin.MATERIALS AND METHODS Plant Material. Spinacia oleracea (var. America) leaf tissue was obtained from plants grown in a vermiculite-soil mixture in a growth chamber maintained at 20°C during the light period (12 h) and 16°C during the dark period. Plants were also purchased from local markets.Preparation of MgP' and MgS. Whole chloroplasts were prepared using a modification of the method of Gibbs and Robinson (14). Spinach leaves (12-15 g), deveined and diced, were homogenized in a VirTis blender with 50 to 60 ml chilled medium containing 50 mm Hepes-NaOH (pH 6.8), 5 mm disodium EDTA, 1 mM MgCl2, 1 mm MnCl2, 1 mm Na4P207, and 0.33 M sorbitol. Homogenization (including acceleration time) was carried out for 6 s at 50%o full-line voltage, and the homogenate was filtered through two layers of Miracloth. The fitrate was centrifuged at 755g for 50 s. The pellet (whole chloroplasts) was resuspended in 50 ml of a medium containing 50 mm Hepes-NaOH (pH 7.0) and 0.33 M sorbitol. The washing step was included to minimize the carry over of cytoplasmic enzymes and inorganic ions, especially Mg +. This suspension was recentrifuged at 755g for 50 s, and the resulting pellet was osmotically disrupted with 5 ml medium containing 50 mM Tricine-NaOH (pH 8.5) and varyi...

“…Staining Assays. Prior to assaying, approximately 0.2 ml of each extract was thawed at 4°C and assayed within 4 h. P-glycerate kinase (25), P-fructokinase (10), triose-P isomerase (1 1), pyruvate kinase (15), glucose 6-P dehydrogenase (21), and 6-P-gluconate dehydrogenase (26) were assayed by spectrophotometric assays essentially as described previously.…”

Section: Methodsmentioning

confidence: 99%

Enzymes of Glucose Oxidation in Leaf Tissues

Wurtele

Nikolau²

1986

ABSTRACIThe distribution of the glycolytic enzymes, phosphofructokinase, aldolase, triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, and the oxidative pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, was determined in the leaf tissues of two C3-plants, pea and leek, and two C4-plants, maize and sorghum. All enzymes examined were found in epidermal tissue. In pea, maize, and sorghum leaves, the specific activities of these enzymes were usually higher in the nonphotosynthetic epidermal tissue than in the photosynthetic tissues of the leaves. In leek leaves, which were etiolated, specific activities were similar in both epidermal and mesophyll tissue. The distribution of the rate limiting enzymes of glycolysis and the oxidative pentose phosphate pathways probably reflects the capacity of each tissue to generate NADH, NADPH, and ATP from the oxidation of glucose. This capacity appears to be greater in leaf tissues unable to generate reducing equivalents and ATP by photosynthesis, that is, in epidermal tissues and etiolated mesophyll tissue.Microscopic examination of leaves readily demonstrates the diversity of cell types in this organ (9,16 cells (14, 17-19, 23, 28, 30).Glycolysis and the oxidative pentose phosphate pathway are the major routes of glucose oxidation in plants, providing ATP, NADH, and NADPH to nonphotosynthetic cells in the light,