Tissue and intracellular localization of indican and the purification and characterization of indican synthase from indigo plants

Indican (indoxyl beta-D-glucoside) was found to accumulate only in green leaves of the indigo plant, and not in any other tissues. Comparisons of the indican content of protoplasts and vacuoles showed that indican was stored only in the vacuole of the cell. Indican content appeared and increased with the appearance and growth of leaves. In mature plants, the younger leaves contained larger amounts of indican than the older ones. Cell extracts of young leaves of indigo plant catalyzed the synthesis of indican from UDP-glucose and indoxyl. Indican synthase was extracted and purified from young leaves. The enzyme was separated into two fractions by anion-exchange chromatography. The enzyme in the fraction which was eluted by 0.1 M NaCl had a molecular weight of 53,000 by SDS-PAGE. Optimum pH of the enzyme was at about 10.0, indicating that the enzyme is likely localized in a different intracellular compartment from that of indican storage. The enzyme showed normal Michaelis-Menten kinetics and a K(m) value of 0.13 mM for UDP-glucose.     

Improved preparation and assay and some characteristics of Cl--ATPase activity from Limonium vulgare

We present a revised method for the preparation, storage and assay of the Cl--ATPase activity responsible for salt secretion in Limonium vulgare. Altering the centrifugation step improved the yield and linked-enzyme assay for ADP provided a sensitive method for continuous monitoring of Cl--ATPase activity. The activity of untreated membranes was low but fairly stable. Treatment with detergent gave strong stimulation of Cl--ATPase activity ATP between 0.1 and 0.2 mM. Cl- stimulated the activity up to a maximum at 0.13 M Cl-, and the pH optimum was around 6.3 to 6.4.     

Agar from some Hawaiian red algae

From describing the agars of Gelidiella acerosa Forssk., Gelidium pluma Loomis, G. pusillum (Stackh.) Lejolis, Gracilaria abbottiana Hoyle, G. bursapastoris (Gmelin) Silva, G. canaliculata (Kützing) Sonder, G. coronopifolia J.Ag., G. epihippisora Hoyle, Pterocladia caerulescens (Kützing) Santelices and P. capillacea (Gmelin) Born. & Thur. as found in Hawaiian samples of these species, it is concluded that the species of Gelidium and especially Pterocladia and Gelidiella may merit more consideration for usage due to their agar gel strengths.The nature of the gel from Gracilaria abbottiana suggests the generic status might well be reexamined. The agars from the Gelidiella and the other Gracilaria species should be studied further for their prospective values to the food industry other than gel strength. Mixtures of the agars from G. bursapastoris and G. coronopifolia would merit attention for the taste texture of their mixtures.     

A new HPLC-ELSD method to quantify indican in Polygonum tinctorium L. and to evaluate beta-glucosidase hydrolysis of indican for indigo production

A method to quantify the indigo precursor indican (indoxyl-beta-D-glucoside) in Polygonum tinctorium L. has been developed. Plant material was extracted in deionized water, and indican was identified and quantified using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Results confirmed that with this method it is possible to measure indican content in a short time, obtaining reliable and reproducible data. Using this method, leaf indican content was quantified every 15 days during the growing season (from May to October) in P. tinctorium crops grown in a field experiment in Central Italy. Results showed that indican increased along the growing season until flowering and was positively affected by photosynthetic active radiation (PAR). Indican is naturally hydrolyzed by native beta-glucosidase to indoxyl and glucose, the indoxyl yielding indigo. The activity of two enzymes, sweet almond beta-glucosidase and Novarom G preparation, were compared with P. tinctorium native beta-glucosidase to evaluate indigo production. Results showed that the ability to promote indigo formation increased as follows: almond beta-glucosidase 相似文献   

On the preparation and some properties of closed membrane vesicles from hog duodenal and jejunal brush border

Closed and nearly spherical vesicles were obtained from both hog duodenum and jejunum after mucosa homogenization in the absence of EDTA and a series of fractional centrifugations. The vesicles were found to contain large amounts of two of the characteristic enzyme markers of the brush border membrane (aminopeptidase and alkaline phosphatase). They were seen by electron microscopy on thin sections or after negative staining to be composed of an apparently intact, 90–100 Å-thick membrane overlaid by the fuzzy coat and to be partly filled by a fibrous material tentatively identified with the cross-filaments of the microvilli. This filling was not removed by 5 mM EDTA or/and 1 M Tris unless the structure of the vesicles was largely destroyed. Very few empty vesicles were obtained at the end of these treatments.The vesicles from hog duodenum and jejunum were observed to contain nearly 2 molecules of cholesterol for 1 molecule of phospolipids. Specific differences were noted between both types of vesicles at the level of their sugar composition and associated enzyme activities. For instance, the jejunal vesicles contained no sialic acid and no enterokinase. They contain, respectively, 2 and 4 times as much alkaline phosphatase and aminopeptidase as duodenal vesicles.     

Effect of some nickel compounds on red blood cell characteristics

The effect of certain inorganic and coordinated nickel compounds on the resistance to different destructive substances, rheological properties, and functional activity of healthy human red blood cells (RBC), was investigated. It is shown that nickel compounds affect the erythrocyte membrane lipid bilayer, as well as membrane proteins to various extents, depending on the type of compounds used. In general, the acceleration of erythrocyte aging was observed to be more pronounced in young erythrocytes. The observed results suggest that nickel compounds decrease water permeability across erythrocyte membranes. Almost all the investigated nickel compounds decrease erythrocyte thermostability, deformability, and the rate of O₂ release by erythrocytes.     

Beta-glucosidase-catalyzed hydrolysis of indican from leaves of Polygonum tinctorium

In this article, a HPLC method to identify and quantify the dyes and the indigo precursors produced in Polygonum tinctorium is described. Using this technique, indican has been positively identified in extracts of P. tinctorium. Our work with two cultivars of P. tinctorium has confirmed that the quantity of indican is dependent on the cultivars, harvest period, and age of the leaves. Two enzymes, Novozym 188 (cellobiase) and Novarom G (beta-glucosidase), are compared on the basis of their activities to hydrolyze the indican at several pH values. We observed that Novarom G is more active than Novozym 188 whatever the pH and that optimum pH of both enzymes for indican hydrolysis is 3. Liberated indoxyl can be oxidized in alkaline media and transformed into indigo and indirubin.