Enzymes of starch metabolism in the developing rice grain

The levels of starch, soluble sugars, protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and starch synthetase —were assayed in dehulled developing rice grains (Oryzasativa L., variety IR8). Phosphorylase, Q-enzyme, and R-enzyme had peak activities 10 days after flowering, whereas α- and β-amylases had maximal activities 14 days after flowering. Starch synthetase bound to the starch granule increased in activity up to 21 days after flowering. These enzymes (except the starch synthetases) were also detected by polyacrylamide gel electrophoresis. Their activity in grains at the midmilky stage (8-10 days after flowering) was determined in five pairs of lines with low and high amylose content from different crosses. The samples had similar levels of amylases, phosphorylase, R-enzyme, and Q-enzyme. The samples consistently differed in their levels of starch synthetase bound to the starch granule, which was proportional to amylose content. Granule-bound starch synthetase may be responsible for the integrity of amylose in the developing starch granule.     

Enzymes of carbohydrate metabolism in developing Hordeum distichum grain

Variations in activity of several enzymes associated with carbohydrate metabolism were recorded during the development of barley endosperm. The enzymes investigated were: sucrose-UDP (ADP) glucosyl transferase; invertase; UDPG (ADPG) pyrophosphorylase; hexokinase; glucose-6-phosphate ketoisomerase; phosphoglucomutase, and nucleosidediphosphokinase.     

Enzymes of carbohydrate metabolism in the developing endosperm of maize

A number of enzymes presumably implicated in starch synthesis were assayed at various stages of endosperm development ranging from 8 days to 28 days after pollination. Activity for invertase, hexokinase, the glucose phosphate isomerases, the phosphoglucomutases, phosphorylase I, uridine diphosphate glucose pyrophosphorylase, and the starch granule-bound nucleoside diphosphate glucose-starch glucosyltransferase was present at the earliest stage of development (8 days) studied. Activity was detectable for phosphorylase III, the soluble adenosine diphosphate glucose-starch glucosyltransferase, adenosine diphosphate glucose pyrophosphorylase, and sucrose-uridine diphosphate glucosyltransferase at 12 days. For phosphorylase II and cytidine diphosphate glucose pyrophosphorylase, activity was first detectable at the 14- and 16-day stages, respectively. Rapid increases in starch content are observed prior to detectable activity for adenosine diphosphate glucose pyrophosphorylase, the soluble adenosine diphosphate glucose-starch glucosyltransferase and phosphorylases II and III. For all enzymes, except invertase, activity per endosperm rises to a peak at 22 or 28 days. Greatest activity for invertase is found at 12 days with a steady decline thereafter. The pattern of invertase activity in comparison with that of sucrose-uridine diphosphate glucosyltransferase supports previous suggestions, that the latter plays a key role in the conversion of sucrose to starch. In addition to phosphorylases I, II, and III, multiple forms of glucosephosphate isomerase and phosphoglucomutase were detected.     

Enzymes of carbohydrate metabolism in developing grains of high lysine barley mutant

Activities of UDP(ADP)-sucrose synthetase, hexokinase, phosphoglucoisomerase and phosphoglucomutase have been studied in both a high lysine mutant barley, Notch-2 and its parent NP 113 during development. The Notch-2 mutant had higher average activities of UDP(ADP)-sucrose synthetase, hexokinase and phosphoglucomutase and lower activity on a grain basis of phosphoglucoisomerase than NP 113. This reflected the decreased dry matter in the mutant grain. In general, the average activities of hexokinase and phosphoglucomutase per grain did not differ significantly between Notch-2 and NP 113. It is suggested that the lower level of phosphoglucoisomerase in Notch-2 compared with NP 113 would limit the synthesis of glucose 6-phosphate, which in turn would result in reduced starch synthesis.     

Enzymes of glutamine metabolism in testa-pericarp and endosperm of developing wheat grain

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate puruvate transaminase and glutamate oxaloacetate transaminase have been assayed in developing testa-pericarp and endosperm of two wheat varieties, namely Shera (11.6% protein) and C-306 (9.8% protein). On per organ basis, activities of all the enzymes studied, except glutamine synthetase, increased during development. Glutamine synthetase activity decreased during development in the testa-pericarp, whereas, no glutamine synthetase activity could be detected in endosperm of either variety at any stage of development. Compared to testa-pericarp, endosperm had higher activities of glutamate synthase and glutamate pyruvate transaminase. On the whole, enzyme activities in Shera were higher, as compared to C-306. Developmental patterns and relative levels of enzyme activities in the two varieties were more or less the same, when expressed on dry weight basis or as specific activities. The results suggest that ammonia assimilation in developing wheat grain takes place by the glutamate dehydrogenase pathway in the endosperm; and both by the glutamate dehydrogenase and glutamine synthetase—glutamate synthase pathways in the testa-pericarp.     

Enzymes of carbohydrate and amino acid metabolism in developing and mature nodules of white clover

The enzymic potential for the metabolism of carbohydrate (photosyntheticproducts) and amino acids (the assimilation of was determined by enzyme assay and by immunodetectionin developing and mature nodules of white clover. White nodulesappeared within 6 d of placing stolon tip cuttings in wet sand.The protein content and enzyme activities of these young nodulesincreased by 10-fold within 9 d. The expression of leghaemoglobinand nitrogenase components 1 and 2 were just detectable in nodulesat 7 d. All other enzymes measured were found in young rootsand white nodules as well as in mature red nodules. However,the expression of sucrose synthase and glutamine synthetase(the key enzymes of sucrose metabolism and assimilation, respectively) were greatly enhancedin nodules compared with roots. Nodule protein content, leghaemoglobinlevels and enzyme activities peaked at approximately 50 d aftertaking stolon cuttings, and then declined by about 50% by 80d. The results are discussed in the context of carbon and nitrogenfluxes in legume nodules. Key words: White clover, legume nodules, carbohydrate metabolism, amino acid metabolism, enzymes     

Enzymes of carbohydrate metabolism in Leishmania donovani amastigotes

A method for the isolation of Leishmania donovani amastigotes from infected hamster spleen and liver tissues is described. Over 85% of the isolated amastigotes were viable as judged by acridine orange-ethidium bromide staining and in vitro transformation to the promastigote form. A comprehensive survey of the enzymes of carbohydrate metabolism in L. donovani amastigotes and promastigotes was conducted. Amastigotes and promastigotes possess all of the enzymes of the Embden-Meyerhof pathway, hexose monophosphate shunt, and tricarboxylic acid cycle. Cell-free extracts of both forms demonstrate an active glutamate dehydrogenase, thus linking activity which permits entry of pyruvate into the tricarboxylic acid cycle. Both forms demonstrate an active glutamate dehydrogenase, thus linking amino acid metabolism with carbohydrate metabolism. Pyruvate carboxylase, the enzyme responsible for replenishment of C4 acids by heterotrophic CO2 fixation into pyruvate, was also demonstrable in the tissue and insect forms. In general, activities of promastigote enzymes are higher than the amastigote enzymes. Differences between the vertebrate (amastigote) and invertebrate (promastigote) forms in their potential to utilize carbohydrates as substrates would appear to be quantitative rather than qualitative.     

Lipids in developing and mature rice grain

The neutral fraction of nonstarch lipids in developing brown rice (Oryza sativa L., cv IR42) was accumulated up to 16 days after flowering (DAF), but phospholipids and glycolipids increased only up to 8 DAF. Fatty acids accumulated in nonstarch lipids until 12 DAF. However, the proportion of linolenic acid in the lipid fraction decreased and that of oleic acid increased during this period. Accumulation of fat-by-hydrolysis in the brown rice occurred until 20 DAF and followed closely that of starch. The proportion of linolenic acid decreased and that of linoleic acid increased until 16 DAF. The fatty acid composition of fat-by-hydrolysis and starch lipids were identical and fat-by-hydrolysis accounted for 48% by weight of starch lipids. Nonstarch lipids were mainly composed of triglycerides and were located in the bran and embryo of mature brown rice. Starch lipids were mainly composed of lysophosphatidyl choline, free fatty acids and lysophosphatidyl ethanolamine, and were located in the endosperm.     

Enzymes of carbohydrate metabolism as potential drug targets

The potential for chemotherapeutic exploitation of carbohydrate metabolism in the Trypanosomatidae is reviewed. This review is based largely on discussions held at a meeting of the COST B9 Action, entitled 'Bioenergetics of Protozoan Parasites'. The major questions posed were: which enzymes are the best to target; what further information is required to allow their use for rational drug development; what compounds would constitute the best inhibitors and which of the enzymes of the pentose-phosphate pathway are present inside the glycosomes, as well? Only partial answers could be obtained in many cases, but the interactive discussion between the multidisciplinary group of participants, comprising chemists, biochemists and molecular biologists, provided thought-provoking ideas and will help direct future research.     

Enzymes of orithine metabolism in adult developing rat intestine

The levels of 11 enzymes, most of them involved in the metabolism of orithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, orithine aminotransferase, and orithine transcarbamylase were compared with those in liver. Changes with age (late gestation to adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described.The results suggests that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotraferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue.Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the orithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

Properties of prolamin in mature and developing rice grain

A procedure was developed for preparing lipid- and phenol-free prolamin directly from IR480-5-9 milled rice (Oryza sativa L.).The preparation consisted mainly of one protein band on analytical and SDS-polyacrylamide disc gel electrophoresis with subunit MW of 17000 and a minor fraction with subunit MW 23000. The prolamin eluted as a single peak on SDS-Sephadex G-75 gel filtration and on DEAE-cellulose column chromatography. Prolamin was poor in lysine, histidine, cystine, and methionine but rich in glutamic acid, tyrosine and proline. In dehulled developing grain of two different rices, changes in the aminogram of the prolamin fraction coincided with the start of endosperm protein body synthesis and the appearance from 7 days after flowering of a second prolamin subunit with MW 23 000.     

Robustness of central carbohydrate metabolism in developing maize kernels

The central carbohydrate metabolism provides the precursors for the syntheses of various storage products in seeds. While the underlying biochemical map is well established, little is known about the organization and flexibility of carbohydrate metabolic fluxes in the face of changing biosynthetic demands or other perturbations. This question was addressed in developing kernels of maize (Zea mays L.), a model system for the study of starch and sugar metabolism. ¹³C-labeling experiments were carried out with inbred lines, heterotic hybrids, and starch-deficient mutants that were selected to cover a wide range of performances and kernel phenotypes. In total, 46 labeling experiments were carried out using either [U-¹³C₆]glucose or [U-¹³C₁₂]sucrose and up to three stages of kernel development. Carbohydrate flux distributions were estimated based on glucose isotopologue abundances, which were determined in hydrolysates of starch by using quantitative ¹³C-NMR and GC-MS. Similar labeling patterns in all samples indicated robustness of carbohydrate fluxes in maize endosperm, and fluxes were rather stable in response to glucose or sucrose feeding and during development. A lack of ADP-glucose pyrophosphorylase in the bt2 and sh2 mutants triggered significantly increased hexose cycling. In contrast, other mutations with similar kernel phenotypes had no effect. Thus, the distribution of carbohydrate fluxes is stable and not determined by sink strength in maize kernels.     

Enzymic differentiation in pathways of carbohydrate metabolism in developing brain

The entire sequence of enzymes of the pentose phosphate pathway, the glycolytic pathway and certain enzymes of fatty acid synthesis were measured in developing rat brain. Coordinated increases in enzymes of the glycolytic pathway were observed, with apparent discrimination in that greater changes were found among enzymes of low turnover capability (Vmax: K_m). During development a sequential pattern emerged with early changes in phosphofructokinase and aldolase followed by triosephosphate isomerase, glycerophosphate dehydrogenase and phosphoglyceromutase; enzymes of the pentose phosphate pathway remained relatively constant, while a marked and parallel fall was seen in acetyl CoA car?ylase, fatty acid synthetase and NADP⁺-linked isocitrate dehydrogenase.     

Enzymes of carbohydrate metabolism in fast-growing Rhizobium grown on hexoses or succinate

Enzymatic evidence supports that succinate mediates repression of hexose-catabolising enzymes in fast-growing Rhizobium sp. (Cicer arietinum). Enzymes of the Embden-Myerhof-Parnas, Entner-Doudoroff and pentose phosphate pathways were found present in hexose-grown cells but not in succinate-grown cells. These however could be induced by the presence of hexoses.     

Properties of glutelin from mature and developing rice grain

Aminograms and SDS-polyacrylamide electrophoresis of milled rice glutelin of 12 Oryza sativa samples showed similar composition and ratio of 1 : 1 : 1 for subunits with MW 38 000:25 000: 16 000, indicating little possibility of finding variants of rice glutelins. Fractionation of S-cyanoethyl glutelin of 3 rices on polyacrylamide-agarose gels gave MW subunits differing in amino acid analysis of which the subunits with MW > 38 000 had the highest lysine content. Of the solubility fractions of endosperm glutelin, the fraction extracted by 0.5 M NaCl-0.6 % β-mercapto-ethanol-0.5% SDS was closest to glutelin in properties. In the developing grain of two varieties, appearance of protein bodies and rapid synthesis of glutelin from 7 days after flowering onward coincided with a drop in lysine content and appearance of MW 38 000 and 25 000 of crude glutelin. The MW 38 000 subunit is thus unique to endo-sperm glutelin.