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One-pot process for the production of ginsenoside Rd by coupling enzyme-assisted extraction with selective enzymolysis was explored in this paper. Several detection methods including HPLC-MS were used to identify and quantify the products in the enzymolysis solution of pectinase. Results showed that ginsenoside Rd was the main component in enzymolysis solution, pectinase specifically hydrolyzes protopanaxadiol (PPD)-type ginsenoside and was a selective enzyme to convert ginsenoside Rb1 to Rd in a way. In addition the influencing factors on the yield of ginsenoside Rb1 and Rd were optimized using L9(34) orthogonal design data. The enzymolysis conditions for the higher yield of Rd were 52.5 °C, pH 6 and 1 h with a yield of 0.8314 from 50 mg drug material. The controllable transformation hypothesis of the PPD-type ginsenoside was also explored from the perspective of the molecular steric hindrance. Pectinase could be used as an efficient enzyme for one-pot producing ginsenoside Rd.

Inositol-3-phosphate synthase (INO1) has previously been genetically validated as a drug target againstTrypanosoma brucei, the causative agent of African sleeping sickness. Chemical intervention of this essential enzyme could lead to new therapeutic agents. Unfortunately, no potent inhibitors of INO1 from any organism have been reported, so a screen for potential novel inhibitors ofT. bruceiINO1was undertaken. Detection of inhibition ofT. bruceiINO1 is problematic due to the nature of the reaction. Direct detection requires differentiation between glucose-6-phosphate and inositol-3-phosphate. Coupled enzyme assays could give false positives as potentially they could inhibit the coupling enzyme. Thus, an alternative approach of differential scanning fluorimetry to identify compounds that interact withT. bruceiINO1 was employed to screen ~670 compounds from the MayBridge Rule of 3 Fragment Library. This approach identified 38 compounds, which significantly altered the Tmof TbINO1. Four compounds showed trypanocidal activity with ED50s in the tens of micromolar range, with 2 having a selectivity index in excess of 250. The trypanocidal and general cytotoxicity activities of all of the compounds in the library are also reported, with the best having ED50S of ~20 μM againstT. brucei.

A spectrophotom etric assay for th e determination of branched-chain L-amino acid am inotransferase activ­ ity is described. It is based on the transamination of L-leucine in the presence of 2-oxoglutarate yielding 4ethyl-2-oxopentanoate. The rate of formation of the branched-chain 2-oxo acid is specifically monitored in a coupled enzymatic reaction using NADf -dependent D*2-hydroxyisocaproate dehydrogenase from Lactoba­ cillus casei sap. pseudoplantarum as coupling enzyme by m easuring the decrease in NADH absorbance at 334 nm. Optimized assay conditions are provided as evalu­ ated for the (iso)enzyme from rat heart, c 1995 Academic Press, Inc.

We have developed a systematic approach to optimization of reagent concentrations for assays of alanine aminotransferase and aspartate aminotransferase: (a) Michaelis constants describing the initial-velocity kinetics of the coupled enzyme reactions were evaluated by a nonlinear least-squares fit of the appropriate equation to measured enzyme activities. Activities of more than 50 normal and pathological sera were measured at 30 °C. (b) These kinetic equations are used to calculate the set of reagent amino- and keto-acid concentrations that all yield a selected fraction of the theoretical maximum enzyme velocity. An optimal pair is determined by defining an additional criterion, such as minimal reagent cost or minimal concentration to Km ratio. (c) The optimum amounts of reagent NADH and coupling enzyme, being a function of desired pre-incubation and measurement intervals, maximum aminotransferase activity to be measured, and endogenous keto-acid concentration, are determined by computer simulation. An approximate relationship and an exact method for computing assay lag time are presented, along with experimentally measured endogenous keto-acid concentrations in serum. All procedures may be applied to other enzyme assays if appropriately modified.

2,4-Dinitrophenol fails to stimulate the breakdown of the soluble high energy intermediate of oxidative phosphorylation isolated from Alcaligenes faecalis extracts. Once the intermediate is broken down, by other reactions, the coupling enzyme is prevented from reassociation with the electron transport particles by 2,4-dinitrophenol. Phosphorylating particles washed with 2,4-dinitrophenol lose coupling enzyme and with it the ability to form the energy-rich intermediate and to couple phosphorylation to electron transport. Phosphorylating particles containing bound coupling enzyme are able to form the high energy intermediate even in the presence of 2,4-dinitrophenol. However, after this first cycle of formation, no more can be formed because 2,4-dinitrophenol prevents the reassociation of coupling enzyme with the electron transport particles. 2,4-Dinitrophenol stimulates respiration in the bacterial phosphorylating particles.

The stimulation of energy-linked reactions by low levels of oligomycin varies greatly with the type of submitochondrial particle. Particles made by disruption of combined heavy and light mitochondria by exposure to sonic oscillation and depleted by urea treatment showed no stimulation over a wide range of oligomycin concentration. However, the mitochondrial coupling factor described previously stimulated energy-linked reactions in these particles. Similar particles from heavy mitochondria gave some stimulation with low levels of oligomycin. High stimulation was obtained with alkali-treated particles derived from heavy mitochondria. The magnitude of stimulation appeared to be related to the concentration of endogenous coupling factors. Both severely depleted and fully supplemented particles were not stimulated by oligomycin, but particles with intermediate levels of endogenous factors gave striking effects.