Micromoles Of Substrate Research Articles

Lead Diversification at the Nanomole Scale Using Liver Microsomes and Quantitative Nuclear Magnetic Resonance Spectroscopy: Application to Phosphodiesterase 2 Inhibitors.

In this report, we describe a method whereby lead molecules can be converted into several new analogues each using liver microsomes. Less than one micromole of substrate is incubated with liver microsomes (mouse, rat, hamster, guinea pig, rabbit, dog, monkey, or human) to produce multiple products which are isolated and analyzed by quantitative cryomicroprobe NMR (qNMR) spectroscopy. The solutions from qNMR analysis were then used as stocks that were diluted into biochemical assays. Nine human phosphodiesterase-2 (PDE2) inhibitors yielded 36 new analogues. Products were tested for PDE2 inhibition, intrinsic clearance in human hepatocytes, and membrane permeability. Two of the products (2c and 4b) were 3-10× more potent than their respective parent compounds and also had improved metabolic stability. Others offered insights into structure-activity relationships. Overall, this process of using liver microsomes at a submicromole scale of substrate is a useful approach to rapid and cost-effective late-stage lead diversification.

Synthesis, characterization and catecholase-mimetic activity of mononuclear copper(II) aspirinate complexes

Four mononuclear copper(II) complexes have been prepared by allowing copper(II) aspirinate to react with benzimidazole, 2-methylbenzimidazole, metronidazole or 2-methyl-5-nitrobenzimidazole ligands. Elemental analyses, UV-Vis, IR, EPR and magnet moment data are consistent with mononuclear square planar complexes that contain two aspirinate ligands and two N-containing ligands to give a CuO 2N 2 chromophore. The benzimidazole, 2-methylbenzimidazole and metronidazole complexes catalyze the oxidation of catechol to o-quinone. This reaction was monitored spectrophotometrically by following the increase in intensity of the o-quinone band at 390 nm with time. Enzyme-mimetic activity of these copper complexes was determined and expressed as micromoles of substrate per mg catalyst per min. The activities were found to be 0.314 for the benzimidazole complex, 0.0998 for the 2-methylbenzimidazole complex, and 0.129 for the metronidazole complex. In the case of the 2-methyl- 5-nitrobenzimidazole complex the initial formation of o-quinone is very rapid; however, after 2–3 min the catalyst is poisoned and the concentration of o-quinone drops slightly and remains constant.

Serum enzymes in the diagnosis of disease in man and animals

The clinical pathologist is concerned with aiding the diagnosis of disease by investigating the disease process in the patient. These investigations seek to identify the pathogen (e.g., toxin, virus, bacterium or parasite) or to detect diagnostic pathological changes by haematology, cytology and biochemistry. Clinical enzymology is a branch of clinical biochemistry which has grown rapidly in the last 50 years. This rapid growth has been associated with the development of methods of enzyme assay. The molecular concentrations of enzymes in serum are very much lower than those of most other metabolites of clinical importance. Only special radioimmunoassay techniques are sufficiently sensitive for the direct determination of serum enzyme protein concentration. Fortunately, enzymes can be determined indirectly by measuring their activity, i.e. their ability to accelerate the particular chemical reactions which they catalyse. Thus the activity of an enzyme may be determined by incubation with its substrate and measurement of loss of substrate, formation of product or change in a coenzyme. Activity varies with temperature, pH, substrate concentrations and the presence of activators or inhibitors, so that enzyme assay conditions must be carefully controlled. It is desirable to saturate the enzyme with substrate to ensure zero-order kinetics during the assay. There is considerable variation in the stability and assay requirements of different enzymes. Enzymes which are very unstable or difficult to assay are of limited value in diagnostic investigations. Practical instructions for many enzyme assays are provided by Bergmeyer ( 1983). Early investigators used a variety of units of enzyme activity. In 1961 the Commission on Enzymes of the International Union of Biochemistry (IUB) recommended a standard international unit (IU) as that amount of enzyme which will catalyse the transformation of 1 micromole of substrate per minute under standard conditions. It is convenient to perform serum enzyme assays at 30°C and to express the results in IU per litre. The newer SI System recommends the “katal” (kat) as the unit of enzyme activity. This is the amount of activity which transforms 1 mole of substrate per second. Thus 1 p kat is equivalent to 60 IU. The enzyme names and numbers recommended

Glycosidases of normal human donor cornea.

Although there have been reports of glycosidases in various ocular tissues, there are very few quantitative data concerning human corneal glycosidases. In this study, the pH optima, apparent Km, and Vmax values were determined for seven glycosidases of human donor corneas obtained from an eye bank. In terms of micromoles of substrate cleaved per milligram of protein per hour, the enzymatic activity of the glycosidases was in the following order of decreasing magnitude: β-galactosaminidase, β-glucosaminidase, β-galactosidase, β-glucuronidase, α-mannosidase, α-fucosidase, α-glucosidase. The pH optima of the enzymes ranged from 3.4 to 5.7.

Lysosomal acid hydrolases in primary monolayer cultures derived from neonatal brains exposed transplacentally to ethylnitrosourea.

SummaryThe effect of transplacental exposure of the fetal rat brain to the carcinogen ethylnitrosourea (ENU) on lysosomal acid hydrolase activities in glial cells has been reported. Primary monolayer cultures of brain cells derived from the brains of newborn rats exposed transplacentally to ENU and from unexposed newborn brains were used to provide concentrated samples of glial cells for hydrolase assay. The hydrolases assayed were β-glucuronidase, N-acetyl-β-D-glucosaminidase, arylsulfatase A and B and acid phosphatase and data were expressed as micromoles of substrate hydrolyzed per hour per milligram of protein. Experimental and control cultures were prepared, incubated and assayed contemporaneously and the ratios between treated and control hydrolase activities were used to evaluate the effect of transplacental ENU exposure.Results showed that there was no effect on the activities of the lysosomal acid hydrolases measured in primary monolayer cultures of brain cells derived from newborn rat brains exp...

Oxidative Deamination of S-Adenosyl-L-homocysteine by Rat Kidney L-Amino Acid Oxidase

Cell-free extracts prepared from rat kidney or liver catalyzed the oxidative deamination of S-adenosyl- L -homocysteine to S-adenosyl-γ-thio-α-ketobutyrate. This reaction was found to be catalyzed by L -amino acid oxidase ( L -amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2). In the presence of catalase, 0.48 µmole of oxygen was consumed for each micromole of substrate oxidized, and 1 µmole each of S-adenosyl-γ-thio-α-ketobutyrate and ammonia were formed. In the absence of catalase oxygen consumption was doubled. The pH optimum for S-adenosyl-γ-thio-α-ketobutyrate formation ranged from 8.8 to 9.2 and the Km value for S-adenosyl- L -homocysteine was 2.48 × 10−2 M . At substrate concentrations of 10 m M and at pH 9.0, the ratio of specific activities with S-adenosyl- L -homocysteine to that with L -leucine or with L -methionine was 0.31 and 0.41, respectively. Partially purified L -amino acid oxidase was also found to be active with L -homocysteine and S-adenosyl- L -methionine.

BETA-HYDROXYACYL DEHYDROGENASE IN HUMAN ARTERIAL TISSUE.

SummaryDeterminations were made of the beta-hydroxyacyl dehydrogenase activities exhibited by intima-media layers of various types of human blood vessels. A total of 215 vascular specimens was included in the study. The average activity of the thoracic descending aorta, expressed in units (micro-moles of substrate metabolized per gram tissue per minute) was .214; the corresponding values recorded for the abdominal aorta, pulmonary artery, coronary artery, cerebral arteries, and inferior vena cava were, respectively, .342, .217, .250, .152, and .082. The mean enzyme activity exhibited by atherosclerotic aortic tissue samples was 72.7% of that observed for normal tissue of the same arterial specimens.

Oxidation of octanoic acid by cell-free bacterial extracts.

STUDIES with extracts from the anaerobic organism Clostridium kluyveri have shown that certain fatty acids are oxidized to acetate by processes similar in essentials to those in mammalian systems1 ; but the mechanisms employed by aerobic bacteria have remained obscure. Thus, although results with whole cells of Serratia marcescens2 did not apparently accord with β-oxidation, it has been shown more recently that extracts from Pseudomonas fluorescens3 catalyse the formation of acetate from decanoate with the uptake of one micromole of oxygen per micromole of substrate. In experiments with cell-free extracts prepared from a vibrio4, we have now been able to show that octanoic acid is oxidized by molecular oxygen to 2C units ; that diphosphopyridine nucleotide, coenzyme A and magnesium ions are co-factors in this process ; and that subsequent oxidation is by way of the tricarboxylic acid cycle.