Cloning and Overexpression of Strictosidine β-D-Glucosidase GeneShort Sequence from Catharanthus Roseus in Escherichia coli

Purpose: Strictosidine-β-D-glucosidase (SGD) is considered as a key enzyme in the productionof bisindole alkaloids in Catharanthus roseus. The present study illustrated the production of ashort sequence of this enzyme in Escherichia coli without codon optimization.Methods: Strictosidine-β-D-glucosidase (sgd) gene short sequence (1434 bp), which lacksthe conserved sequence KGFFVWS and the localization peptide sequence at the C-terminal,was amplified from cDNA of C. roseus leaves, cloned and expressed in Escherichia coli. Theactivity of the produced protein in cell free lysate was tested using total alkaloid extract of C.roseus leaves.Results: HPLC and LC-MS analysis of the assay mixture revealed the disappearance of thestrictosidine peak.Conclusion: SGD short sequence can be produced in Escherichia coli in active form withoutcodon optimization.<br />

The Single Superoxide Dismutase of Rhodobacter capsulatus Is a Cambialistic, Manganese-Containing Enzyme

ABSTRACT The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SODRc) homologous to iron-containing superoxide dismutase enzymes. Recombinant SODRc, however, displayed higher activity after refolding with Mn2+, especially when the pH of the assay mixture was raised. SODRc isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SODRc behaves as a Mn-containing dismutase with cambialistic properties.

Dihydroneopterin and the generation of superoxide from iron ions

We describe the impact of dihydroneopterin on the formation of superoxide radicals. As radical source ferrous iron in air-saturated aequeous solution was used. Detection of superoxide was achieved using reduction of nitroblue tetrazolium to formazan. Dihydroneopterin decreases formazan formation when high concentrations of ferrous iron are used which induce a rapid superoxide formation within minutes. On the other hand, when low concentrations of ferrous iron are used, addition of dihydroneopterin to the assay mixture leads to an increase ir. formazan formation for several hours of incubation. We therefore conclude that the radical promoting activity of dihydroneopterin is physiologically more relevant compared to its radical scavenging properties.

A Simple, Rapid Extraction and Assay Procedure for NAD+-dependent Sorbitol Dehydrogenase (SDH) in Peach

Sorbitol is the major photosynthetic product in peach [Prunus persica (L.) Batsch.]. In sink tissues, sorbitol is converted to fructose via NAD+-dependent SDH. A new procedure is described that allows rapid, simple quantification of SDH activity in growing tissues. The procedure uses only 0.01 to 5 g of fresh tissue per sample, such that a single shoot tip, a single root tip, or ≈5 g of fruit flesh can be assayed for SDH activity. Storage of samples at 4 or -20 °C overnight resulted in significant loss of enzyme activity. Thus, freshly harvested tissues were ground with sand in buffer at 2 °C in a mortar and pestle, and the homogenate was centrifuged at 3000 gn to remove particulate matter and sand. The supernatant was desalted on a Sephadex G-25 column, and the eluent was assayed for SDH activity immediately. Activity was determined by measuring the production of NADH per minute in the assay mixture using a spectrophotometer (340 nm). Tris buffer at pH 9.0 was the best for extraction of peach SDH. Activity of SDH was strongly inhibited by dithiothreitol (DTT) in the extraction mixture and by DTT, L-cysteine, or SDI-158 in the assay mixture, similar to results reported for SDH from mammalian tissues. Peach SDH has a Km of 37.7 mm for sorbitol and a pH optimum of 9.5, similar to those reported for apple (Malus × domestica Borkh.) SDH. Unlike older protocols for SDH activity in plant tissues, the new procedure features reduced sample size (1/10 to 1/100 of that which was previously used), smaller volumes of buffer, fewer buffer ingredients, greatly reduced time for sample preparation, yet comparable or higher values of SDH specific activity. Following the same procedure, SDH activity was also measured in Prunus fremontii Wats., Prunus ilicifolia (Nutt.) Walp., and Marianna 2624 plum (P. cerasifera Ehrh. × P. munsoniana Wight & Hedr.).

Simple enzymatic determination of total cholesterol in gallstones.

In this simple method for rapid enzymatic determination of total cholesterol in human gallstones, gallstone powder is dissolved in an 80/20 by vol mixture of N,N-dimethylformamide and dimethyl sulfoxide and reacted directly with the aqueous enzymatic reagent, without further treatment. The organic solvents do not interfere with enzymatic activities of cholesterol oxidase, esterase, and peroxidase in the assay mixture. The method is reproducible (CVs 3.7% to 6.6%), analytical recoveries ranged from 98.6% to 102%, and linearity is good. Furthermore, results correlated well with those obtained by infrared spectroscopic measurements.

Interference of coumarin therapy with the "Heptest" owing to declining prothrombin concentrations.

The Heptest kit (Haemachem, Inc., St. Louis, MO) for quantifying heparin in plasma is based on heparin-mediated inhibition of factor Xa, resulting in prolongation of clotting time. In 19 of 55 plasma samples obtained from 32 patients concurrently receiving coumarin and heparin, Heptest results exceeded true heparin values by more than 0.2 int. unit/mL; four samples showed a deviation exceeding 0.4 int. unit/mL. We show here that these deviations are caused by coumarin-induced decreases of plasma prothrombin. This problem can be circumvented by adding purified prothrombin or normal plasma to the assay mixture.

Escape from blockade of interfering heterophile antibodies in a two-site immunoradiometric assay for thyrotropin.

Sandwich-type immunoassays in which mouse monoclonal antibodies are used are subject to positive interference by heterophile antibodies present in human serum. Manufacturers now customarily add nonspecific mouse immunoglobulins to absorb the heterophile antibodies and eliminate such interference. We describe the case of a patient who had spurious increases in thyrotropin concentration in serum despite use of the mouse immunoglobulins included in the assay kit. This resulted in a puzzling clinical picture, a workup, and treatment. We demonstrate that the observed increases in thyrotropin were markedly reduced by including additional amounts of mouse immunoglobulins of the appropriate class, subclass, and fragment type in the assay mixture.

Enzymic dephosphorylation of d-myo-inositol 1,4-bisphosphate in rat brain

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2] phosphatase activities were measured in both 180,000 g (60 min) particulate and supernatant fractions of rat brain homogenates. Although Ins(1,4,5)P3 was mostly hydrolysed by a particulate phosphatase [Erneux, Delvaux, Moreau & Dumont (1986) Biochem. Biophys. Res. Commun. 134, 351-358], Ins(1,4)P2 phosphatase was predominantly soluble. The latter enzyme was Mg2+-dependent and sensitive to thiol-blocking agents (e.g. p-hydroxymercuribenzoate). In contrast with Ins(1,4,5)P3 phosphatase activity measured in the soluble fraction, Ins(1,4)P2 phosphatase was insensitive to 0.001-1 mM-2,3-bisphosphoglycerate. Lithium salts, widely used in psychiatric treatment, inhibited both Ins(1,4)P2 and Ins(1)P1 phosphatase activities of the crude soluble fraction. In particular, 50% inhibition of phosphatase activity, with 2 microM-Ins(1,4)P2 as substrate, was achieved at 3-5 mM-LiCl. At these concentrations, LiCl did not change Ins(1,4,5)P3 phosphatase activity measured in the same fraction with 1-4 microM-Ins(1,4,5)P3 as substrate. Chromatography of the soluble fraction of a rat brain homogenate on DEAE-cellulose resolved three phosphatase activities. These forms, peaks I, II and III, dephosphorylated Ins(1,4,5)P3, Ins(1)P1 and Ins(1,4)P2 respectively. If LiCl (10 mM) was included in the assay mixture, it inhibited both peak-II Ins(1)P1 phosphatase and peak-III Ins(1,4)P2 phosphatase, suggesting the existence of at least two Li+-sensitive phosphatases.

Effect of dexamethasone on the synthesis of dolichol-linked saccharides and glycoproteins in hepatocytes prepared from control and inflamed rats

Hepatocytes were prepared from control and inflamed rats. The incorporation of [14C]mannose into protein was increased in inflamed compared with control hepatocytes. The incorporation of [14C]mannose into protein was also increased when the hepatocytes were cultured in presence of dexamethasone (1 microM), either from control or inflamed rats. At the same time the incorporation of [14C]mannose into dolichol phosphate mannose and dolichol-linked oligosaccharide was increased due to inflammation. The presence of dexamethasone in the hepatocyte culture caused an increased formation of these two products; in particular its effect on oligosaccharide lipid formation was very pronounced. The ratios of activities of formation of [14C]mannose-labelled oligosaccharide lipid in inflamed over control hepatocytes gradually decrease when increasing amounts of exogenous dolichol phosphate was added in cell homogenate assay mixture. These results suggest that the increase of oligosaccharide lipid formation in inflammation could be due to a higher concentration of endogenous dolichol phosphate, as was shown for dolichol phosphate mannose formation in inflammation [Sarkar & Mookerjea (1984) Biochem. J. 219, 429-436]. In contrast, the ratio of activities of [14C]mannose-labelled oligosaccharide lipid between dexamethasone-treated and untreated hepatocytes shows only a slight increase when increasing concentrations of exogenous dolichol phosphate were added to the assays. This suggests that the stimulation of dolichol pyrophosphate oligosaccharide synthesis observed in dexamethasone treatment is probably due to the higher level of enzymes involved in oligosaccharide synthesis rather than higher level of endogenous dolichol phosphate in these cells.