T. ap Rees

Pathway of starch breakdown in photosynthetic tissues of Pisum sativum

1. The aim of this work was to discover the pathway of starch breakdown in the photosynthetic tissues of Pisum sativum. 2. Measurements of the starch in the leaves of plants grown in photoperiods of 12 or 18 h showed that starch, synthesized in the light, was rapidly metabolized in the dark at rates of 0.04--0.06 mumol glucose/min per g fresh weight. 3. The maximum catalytic activities of alpha-amylase, beta-amylase, hexokinase, alpha-glucan phosphorylase and phosphoglucomutase in extracts of leaves showed no diurnal variation in either photoperiod, and exceeded estimates of the rate of net starch breakdown in the dark. 4. Studies with intact chloroplasts, isolated from young shoots and from leaves, indicated that pea chloroplasts do not contain significant activities of alpha-amylase, beta-amylase and hexokinase, although some of the latter may be attached to the outside of the chloroplast envelope. These studies also showed that pea chloroplasts contained sufficient alpha-glucan phosphorylase and phosphoglucomutase to mediate the observed rates of starch breakdown. 5. It is proposed that starch breakdown in pea chloroplasts is phosphorolytic.

Invertase activity during the development of carrot roots

Abstract Evidence was obtained that roots of carrot ( Daucus carota L.) contain an acid invertase (optimum pH 4·5) and an alkaline invertase (optimum pH 7·4). The acid and alkaline invertases were partially purified and K mm for sucrose found to be 5·6 × 10 −2 M and 2·4 × 10 −2 M respectively. The acid invertase was distributed between the cell wall and supernatant fractions of root homogenates. This distribution was readily and reversibly altered by varying the pH of the extraction medium. Almost all of the alkaline invertase activity was found in the supernatant fraction of the homogenates. Sugar content and the activities of both invertases were shown to vary widely during the development of carrot roots. High acid invertase activity was found in rapidly growing tissue in which the sucrose content was low or was declining rapidly. With the exception of young seedlings, alkaline invertase activity was most marked in tissues that were storing sucrose. Possible roles for the two invertases in the control of sucrose metabolism are proposed.

Alkaline inorganic pyrophosphatase and starch synthesis in amyloplasts

The aim of this work was to see if amyloplasts contained inorganic pyrophosphatase. Alkaline pyrophosphatase activity, largely dependant upon MgCl2 but not affected by 100 μM ammonium molybdate or 60–100 mM KCl, was demonstrated in exracts of developing and mature clubs of the spadix of Arum maculatum L. and of suspension cultures of Glycine max L., but not in extracts of the developing bulb of Allium cepa L. The maximum catalytic activity of alkaline pyrophosphatase in the above tissues showed a positive correlation with starch synthesis, and in the first two tissues was shown to exceed the activity of ADPglucose pyrophosphorylase. Of the alkaline pyrophosphatase activity in lysates of protoplasts of suspension cultures of Glycine max, 57% was latent. Density-gradient centrifugation of these lysates showed a close correlation between the distribution of alkaline pyrophosphatase and the plastid marker, nitrite reductase. It is suggested that much, if not all, of the alkaline pyrophosphatase in suspension cultures of Glycine max is located in the plastids.

Starch synthesis by isolated amyloplasts from wheat endosperm

The aim of this work was to discover which compound(s) cross the amyloplast envelope to supply the carbon for starch synthesis in grains of Triticum aestivum L. Amyloplasts were isolated, on a continuous gradient of Nycodenz, from lysates of protoplasts of endosperm of developing grains, and then incubated in solutions of 14C-labelled: glucose, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glycerol 3-phosphate. Only glucose 1-phosphate gave appreciable labelling of starch that was dependent upon the integrity of the amyloplasts. Incorporation into starch was linear with respect to time for 2 h. At the end of the incubations, 98% of the 14C in the soluble fraction of the incubation mixture was recovered as [14C]glucose 1-phosphate. Thus it is unlikely that the added [14C glucose 1-phosphate was extensively metabolized prior to uptake by the amyloplasts. It is argued that the behaviour of the isolated amyloplasts, and previously published data on the labelling of starch by [13C]glucose, are consistent with the view that in wheat grains it is a C-6, not a C-3, compound that enters the amyloplast to provide the carbon for starch synthesis.

Enzymic properties of amyloplasts form suspension cultures of soybean

Abstract The aim of this work was to determine which enzymes of carbohydrate metabolism are present in amyloplasts. Protoplasts from 4- to 5-day-old suspension cultures of soybean, Glycine max, were lysed and fractionated on a sucrose gradient. This gave an amyloplast fraction that contained stromal enzymes and was not seriously contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. Studies of this fraction provide evidence that, in soybean cells, starch synthase and ADPglucose pyrophosphorylase are confined to amyloplasts; invertase, sucrose synthetase and UDPglucose pyrophosphorylase are absent from the amyloplast and probably confined to the cytosol; the following enzymes, though predominantly cytosolic, are present in the amyloplasts in activities high enough to mediate the rate of starch synthesis observed in vivo: glyceraldehyde-phosphate dehydrogenase (NAD), triosephosphate isomerase, fructose-1, 6-bisphosphate aldolase, fructose-bisphosphatase, glucosephosphate isomerase and phosphoglucomutase. The pathway from sucrose to starch in non-photosynthetic cells is discussed; particularly the possibility that sucrose is converted to triose phosphate for entry into the amyloplast.

Invertase and sugar content during differentiation of roots of Pisum sativum

Abstract Acid and alkaline invertases (pH optima pH 5·1 and pH 7·3; K m for sucrose 5·3 × 10 −3 M and 13·1 × 10 −3 M, respectively) were demonstrated in roots of pea ( Pisum sativum L.). Their activities and the contents of sucrose, glucose, and fructose were related to protein content and cell number in successive 3 mm segments of the apical 24 mm of the root, and to protein content in stele and cortex isolated from the root 6–24 mm from the apex. Alkaline invertase varied little during differentiation but was most active in the stele where the sucrose concentration was highest. Acid invertase activity was low in the apical 3 mm, rose to a high peak 3–9 mm from the apex and then declined to a steady level 15–24 mm from the apex. Nearly all of the acid invertase 6–24 mm from the apex was in the cortex. Acid invertase activity was inversely related to sucrose content. Measurements of invertase activity in cell fractions, and of hexose formation from sucrose supplied to untreated tissues and to tissues pretreated with ethyl acetate, indicated that all the alkaline invertase and at least half of the acid invertase was intracellular.

Phosphoenolpyruvate carboxykinase and gluconeogenesis in cotyledons of Cucurbita pepo

1. The aim of this work was to investigate the role of phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) in the conversion of fat to sugar by the cotyledons of seedlings of Cucurbita pepo. 2. The enzyme was partially purified from the cotyledons of 5-day-old seedlings. The Michaelis constants for oxaloacetate and ATP were 56 and 119 micron, respectively. The decarboxylation reaction was optimum at pH 7.4. A range of intermediary metabolites did not affect the activity of the enzyme, but 3-mercaptopicolinic acid at micron concentrations was an effective inhibitor. 3. Centrifugation of extracts of 5-day-old cotyledons sedimented appreciable proportions of the ribuloseibisphosphate carboxylase, isocitrate lyase and fumarate hydratase present but very little of the phosphoenolpyruvate carboxykinase. 4. Measurements of phosphoenolpyruvate carboxykinase of cotyledons during germination showed that the maximum catalytic activity exceeded, and changed coincidently with, the rate of gluconeogenesis. 5. 3-Mercaptopicolinic acid inhibited gluconeogenesis from [1-14C]- and [2-14C]acetate supplied to excised cotyledons. The detailed distribution of 14C indicated inhibition of the conversion of oxaloacetate to phosphoenolpyruvate. 6. It is concluded that in marrow cotyledons phosphoenolpyruvate carboxykinase is in the soluble phase of the cytoplasm and catalyses a component reaction of gluconeogenesis.

Carbohydrate oxidation during differentiation in roots of Pisum sativum.

Abstract 1. 1. The aim of this work was to see whether the relative activities of glycolysis and the pentose phosphate pathway varied during the differentiation of mature cells from apical cells in roots of Pisum sativum. 2. 2. The detailed distribution of 14C from [I-14C]- and [6-14C]glucose supplied to segments of the apical 26 mm of the root was determined. The distribution in the apical 3 mm indicated a predominance of glycolysis with a relatively small contribution from the pentose phosphate pathway. The distribution in segments from the region 6–26 mm from the apex indicated substantial contributions from both pathways. 3. 3. The activities of four glycolytic enzymes and five enzymes of the pentose phosphate pathway were measured in extracts of root segments at different stages of differentiation. The activities of the glycolytic enzymes predominated in extracts of the apical 5 mm of the root. This predominance was not found in extracts of segments from the region 5–20 mm from the apex. 4. 4. These results were held to support the view that carbohydrate oxidation in the relatively undifferentiated cells of the root apex was mainly via glycolysis and that differentiation involved an increase in the capacity and activity of the pentose phosphate pathway relative to glycolysis.

Control of the pentose-phosphate pathway in yeast

Abstract 1. 1. The aim of this work was to investigate whether variation in the amounts of the enzyme of the glycolytic and pentose-phosphate pathways controlled carbohydrate oxidation in Candida utilis . 2. 2. The activities of glucose-6-phosphate dehydrogenase ( d -glucose-6-phosphate: NADP + oxidoreductase, EC 1.1.1.49) and transketolase ( d -sedoheptulose-7-phosphate: d -glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) in extracts of yeast grown on nitrate were 2.5 times greater than in extracts of yeast grown on a complex medium that contained amino acids. The activities of a number of other enzymes of carbohydrate oxidation showed little variation with the source of nitrogen. 3. 3. Yeast, grown on complex medium, was transferred to a medium that contained nitrate but which would not support cell division. This transfer altered the pattern of enzyme activities in the extracts from that characteristic of yeast grown on complex medium to that characteristic of nitrate-grown yeast. Cycloheximide prevented this alteration. 4. 4. All treatments that resulted in higher activities of glucose-6-phosphate dehydrogenase and transketolase increased 14 CO 2 production from [1- 14 C]glucose far more than from [6- 14 C]glucose. 5. 5. It is suggested that the capacity of the pentose-phosphate pathway in yeast may be controlled by variation in the amounts of glucose-6-phosphate dehydrogenase.

Pathways of carbohydrate oxidation during thermogenesis by the spadix of Arum maculatum

1. The aims of this work were to discover the pathways of carbohydrate oxidation prior to and during thermogenesis by the club of the spadix of Arum maculatum, and whether there was coarse control of these pathways. 2. 14C02 production from [1-14C]-, [3,4-14C]-, and [6-14C]glucose, the detailed distribution of 14C from [1-14C]- and [6-14C]glucose, and the maximum catalytic activities of phosphofructokinase, fructose-1,6-diphosphate aldolase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase were determined at different stages in the development of the spadix. The results indicate that in the early stages carbohydrate is oxidized via both the pentose phosphate pathway and glycolysis, and that a shift to glycolysis occurs during development so that just before and during thermogenesis glycolysis predominates almost exclusively. 3. During development the activities of phosphofructokinase and glucose-6-phosphate dehydrogenase per club increased 100- ans during spadix development, and indicated that the onset of rapid glycolysis at thermogenesis is regulated by fine control or availability of substrate.