Purification of a beta-galactosidase from cotyledons of Hymenaea courbaril L. (Leguminosae). Enzyme properties and biological function.

Beta-galactosidases are enzymes that can be found in most living beings and in the plant kingdom its activity and genes have been detected in several tissues such as ripening fruits, developing leaves and flowers and storage tissues such as cotyledons. In plants, their activities are usually associated with the secondary metabolism or with oligosaccharide or polysaccharide degradation. Polysaccharide specific beta-galactosidases include beta-galactanases, which attack pectic polymers and beta-galactosidases that attack xyloglucans (XG). In the present work we purified an XG-specific beta-galactosidase (named hcbetagal) from cotyledons of developing seedlings of Hymenaea courbaril, a legume tree from the Neotropical region of the world. The enzyme has a molecular weight of 52-62 kDa and was shown to attack specifically xyloglucan oligosaccharides (XGOs) but not the polymer. It has a pH optimum between 3 and 4 and at this pH range the enzyme increases activity linearly up to 50 degrees C. Kinetic studies showed that hcbetagal is inhibited competitively by free galactose (K(i) = 3.7). The biochemical properties of hcbetagal as a whole suggest that it is involved in storage xyloglucan mobilisation during seedling development. Its high specificity towards XGOs, the low pH optimum and the fact that it is inhibited by its product (galactose) suggest that hcbetagal might be one of the biochemical control points in xyloglucan catabolism in vivo. A possible relationship with functional stability of the wall during cell death as cotyledons undergo senescence is discussed.

Changes in soluble carbohydrates during storage of Caesalpinia echinata LAM. (Brazilwood) seeds, an endangered leguminous tree from the Brazilian Atlantic Forest.

Caesalpinia echinata seeds stored in laboratory environmental conditions lose their viability in one month whilst under low temperatures germination is maintained for 18 months of storage. These seeds are tolerant to desiccation, keeping their viability up to 0.08 gH2O.gDW-1. Since soluble carbohydrates are believed to be involved with desiccation tolerance and seed storability, the aim of this work is to analyze the content and composition of soluble carbohydrates in C. echinata seeds during storage in paper bags (PB) and glass flasks (GF) at laboratory room (RT) and cool (CT) temperatures. In freshly harvested seeds, total soluble carbohydrates comprised approximately 10% of the dry weight, decreasing to ca. 8% over 18 months of storage at RT. In seeds stored at CT, sugars varied differently decreasing initially and being restored at the end of the analysis period. The main neutral sugars in seeds from all treatments were sucrose, fructose and glucose. Raffinose and stachyose were present as traces. Free myo-inositol and other cyclitols were also detected. The main tendency observed was the variation in levels of both glucose and fructose in relation to sucrose, the highest levels of monosaccharides which were found in seeds stored at CT. The values of glucose and fructose were practically constant in seeds stored in paper bags for 18 months at CT, decreasing consistently in the other treatments, mainly at RT. Sucrose contents remained relatively stable. Changes in soluble sugars during storage suggest that the loss of germinability of seeds of C. echinata could be associated with low levels of glucose and fructose in relation to sucrose.

Changes in soluble carbohydrates during storage of Caesalpinia echinata LAM. (Brazilwood) seeds, an endangered leguminous tree from the Brazilian Atlantic Forest

Sementes de Caesalpinia echinata (pau-brasil) perdem a viabilidade em um mês quando armazenadas no ambiente de laboratório, enquanto a capacidade germinativa é mantida quando armazenadas sob temperturas baixas. O presente trabalho teve como objetivos analisar o conteúdo e a composição dos carboidratos de sementes de C. echinata armazenadas em câmara fria (CT) e em temperatura ambiente do laboratório (RT), em duas embalagens distintas (permeável e impermeável), visando a avaliar o envolvimento desses compostos com a capacidade germinativa das sementes. Os resultados mostraram que os carboidratos solúveis são constituídos principalmente de sacarose, glicose, frutose, myo-inositol e traços de rafinose e estaquiose, totalizando cerca de 10% da massa seca das sementes. As variações nos carboidratos solúveis foram semelhantes nos dois tipos de embalagem, mas diferentes quanto à temperatura de armazenamento. Em CT, as proporções dos monossacarídeos encontradas nas sementes recém-colhidas foram mantidas por cerca de 18 meses de armazenamento, coincidindo com alta porcentagem de germinação (80%). Nas armazenadas em RT houve redução expressiva nas proporções de glicose e frutose e perda completa da germinabilidade. O conteúdo de sacarose se manteve relativamente estável durante todo o período de análise. Os resultados indicam que a perda da germinabilidade de sementes de C. echinata está associada à diminuição dos níveis de glicose e frutose em relação aos níveis de sacarose.

Measurement of glomerular filtration rate using inulin prepared from Vernonia herbacea, a Brazilian native species

Vernonia herbacea (Vell.) Rusby (Asteraceae) is a perennial herb native to the cerrado vegetation of tropical areas in Brazil, which accumulates inulin in the underground reserve organs. The aim of this paper was to determine whether the inulin extracted from V. herbacea could replace commercial inulin for the measurement of glomerular filtration rate (GFR). Underground organs of vegetative plants were collected from a preserved area of the Brazilian cerrado. The inulin fraction utilized was obtained by ethanol precipitation after discarding the high molecular mass fructans in the freeze-thawing precipitate. GFR was determined in male Wistar rats anesthetized with inactin (100 mg/kg), which received intravenously commercial inulin obtained from Dahlia sp (Sigma) or Vernonia herbacea inulin (30 mg/100 g) as a priming dose and 0.05 mg min(-1) 100 g(-1) as a sustaining dose in isotonic saline at the rate of 0.055 ml/min. Clearance was determined during 3 periods, with urine collected from the bladder and blood from the carotid artery. There was no significant difference in the GFR measured by clearance of inulin from both sources even when the plasma concentration of inulin from V. herbacea was doubled. The mean arterial pressure did not vary after the application of both inulins, indicating that they do not produce systemic side effects. The filtered load and the excreted amount of inulin from V. herbacea were equal, showing that the substance is not influenced by tubular function. These results demonstrate that the inulin from V. herbacea can substitute for imported inulin for the determination of GFR and in experiments of kidney microperfusion as a marker of tubular water reabsorption.