A rapid assay for ribokinase.

A rapid method capable of detecting low levels of ribokinase is given. [γ-³²P]ATP is converted to ribose 5-[³²P]phosphate which is not absorbable onto charcoal. The assay is linear in enzyme concentration to a lower limit of at least 4 × 10^?2 mg of enzyme/ml.     

Liposome-cell interactions. A rapid assay for cells in suspension culture

A method has been developed for the rapid separation of cells in suspension from non-cell associated lipid vesicles in various assays for vesicle-cell interaction. Separation is achieved on a discontinuous Ficoll-Paque gradient. Cells and free vesicles are totally separated, as evidenced by both radiolabelled vesicles, and vesicles containing the fluorescent dye 6-carboxyfluorescein. The main advantages of this method are the rapidity, efficacy, and gentleness of the separation. Viability of the cells remains consistently high (greater than 96%) throughout the separation. Since this method involves a one-step centrifugation, it precludes the necessity for repeated washings of cells which have been incubated with lipid vesicles.     

A microbiological assay for sodium azide

A reproducible and sensitive method is presented for quantitating sodium azide (NaN₃) that exploits the fact that NaN₃ inhibits Escherichia coli RNA synthesis. A linear correlation is observed between incorporation of [³H]uridine into a trichloroacetic acid-precipitable form and NaN₃ concentration over a 31- to 2000-μg range of azide. This technique was used to determine the azide content of a complex enzyme solution where established colorimetric azide determinations proved to be unworkable. This technique when properly controlled should be applicable to a variety of similar solutions.     

A rapid colorimetric assay of killer toxin activity in yeast

Abstract The pale yellow redox indicator 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is reduced to a dark blue end-product, MTT-Formazan, by the mitochondrial dehydrogenases of living cells. MTT reduction can be measured spectrophotometrically at a wavelength of 570 nm and a method is described to assay the cidal activity of Williopsis mrakii killer toxin against sensitive cells of Candida glabrata . The MTT assay is rapid, quantitative and compares favourably with traditional plating techniques for the assessment of sensitive viability.     

A simple and rapid method for screening transformant yeast colonies using PCR.

A simple and rapid procedure for screening transformant yeast colonies is described. In this method, a trace amount of plasmid DNA is isolated from a small amount of yeast cell mass; then, the presence of the exogenous DNA in each yeast colony is detected by PCR amplification.     

Regulation of 86Rb influx during accumulation of Rb+ or K+ in yeast

The influx rate of ⁸⁶Rb decreases during accumulation of K⁺ or Rb⁺ into metabolizing yeast cells under anaerobic conditions with glucose as substrate. Possible causes for the decrease in the influx rate are examined. It is ruled out that the decrease in the influx rate is caused by an increased turgor pressure of the cells or to an impairment of their energization. During the accumulation of K⁺ or Rb⁺, no decrease but an increase in the protonmotive force of the cells is found. The concomitant increase in cell pH accounts only in small part for the decrease in the influx rates. Acidification of the cells on adding butyrate to the suspension causes a transient increase in the influx rates, whereas the cellular monovalent cation content is still increased. Consequently, no direct relationship is found between the influx rate and the cellular content of K⁺ and Rb⁺.     

A rapid and sensitive assay for chitinase using tritiated chitin

Radioactive chitin, prepared by acetylation of chitosan with tritiated acetic anhydride, was used as substrate in a rapid and extremely sensitive assay for chitinase. The procedure is based on the insolubility of chitin and the solubility in water of the reaction product, diacetylchitobiose. The course of the chitinase reaction is nonlinear, a result that cannot be attributed to an artifact of the method, to inhibition by product, or to instability of the enzyme. Some evidence points to structural heterogeneity of the substrate as a cause for this behavior. Reacetylated chitosan was also used as an adsorbent in the purification of chitinase with better results than with the previously used colloidal chitin.     

A rapid assay for drug sensitivity of glioblastoma stem cells

Glioblastoma (GBM) is a highly infiltrating, aggressive brain cancer with no available curative treatment. We developed a rapid assay for assessing the effect of various drugs on GBM stem cells. The assay uses a small number of separated CD133+ cells (20,000 in 0.2 ml) in 96-well plate that form neurospheres within 1-2 days. Various drugs disperse the neurospheres within 24-36 h, which can be quantified microscopically. We used the GBM cell line A-172 to develop the conditions for the assay, utilizing Gleevec, the gamma-secretase inhibitor DAPT, and the anti-bacterial peptide amph1D. The results show dispersion of the neurospheres leading to cell death, at relatively low drugs concentrations (<25 microM). Drug combination showed a synergistic effect and disruption of neurospheres under lower concentrations. We applied this assay to the CD133+ cells of surgical specimens from three patients that showed similar results. This assay facilitates a rapid test of drugs on small amounts of fractionated patient's GBM stem cells.     

A rapid filtration assay for soluble receptors using polyethylenimine-treated filters

Ligand bound to detergent-solubilized or cytosolic receptors can be separated from free ligand by filtration through glass-fiber filters which have been pretreated with polyethylenimine (PEI). Receptors which can be assayed by this technique include detergent-solubilized muscarinic, adenosine A1, alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic, dopamine D2, opiate, bradykinin, and benzodiazepine receptors as well as naturally soluble estradiol receptors. For muscarinic, adenosine, alpha 2, dopamine, and estradiol receptors, specific binding measured by the PEI-filter technique was 84-110% of specific binding measured by gel filtration, demonstrating that the technique gave almost quantitative recovery of bound ligand.     

A rapid colorimetric assay for heparinase activity.

A rapid, sensitive, assay for enzymes that degrade heparin is described. The procedure is based on the interference of heparin with color development during the interaction of protein with the dye Coomassie brilliant blue. The loss of this property when the glycosaminoglycan is degraded by heparinase can be used to quantify activity of the enzyme in pure form, or in complex biological samples such as tissue homogenates or serum. The assay is also suitable for studying dependence of heparinase activity under conditions such as varying pH and temperature.     

A rapid sensitive assay for phosphatidate phosphohydrolase.

For a purified preparation of the soluble form of phosphatidate phosphohydrolase (EC 3.1.3.4) from guinea pig cerebral cortex, 1-O-alkyl-rac-glycerol 3-phosphate was found to be accepted as a substrate. This substrate analog was tritium-labeled in order to serve in a rapid sensitive assay for the enzyme, in which labeled 1-alkyl glycerol is released. Heat denaturation and enzyme activity dependence on pH indicated that 1-O-alkyl-rac-glycerol 3-phosphate phosphohydrolase and phosphatidate phosphohydrolase activities in the preparation are attributable to the same enzyme. 1-O-Alkyl-rac-glycerol 3-phosphate was hydrolyzed with a V_max of 1.7 nmol min^?1 mg^?1 of protein and a K_m of 270 μm.