A simple micromethod for rapid kinetic analyses of yeast enzymes in situ.
A simple approach for rapid assay of enzymes in large numbers of samples of whole cells of yeast is described. The technique involves treatment of yeast cells with toluene dissolved in ethanol (1:4, v/v; TE), rendering them selectively permeable to small molecules, while letting the enzymes remain intracellular and catalytically active but accessible to exogenously added assay reagents. The permeated cell preparations permit rapid assay of nitrate reductase activity in situ [P. V. Choudary and G. Ramananda Rao (1976) Current Sci. 45, 324-327]. Investigation of enzyme kinetic parameters such as pH optima, temperature dependence, and substrate saturation, which involves several samples and needs to be investigated very rapidly and accurately, is greatly facilitated by permeated cells prepared from a very small culture of cells, eliminating the need for elaborate preparations of cell extracts. By using permeabilized cell preparations, the need to supplement the reaction mixture with expensive cofactors can also be obviated to a large extent. Further, the enzyme activity displays a broader range of tolerance in situ to variations in reaction conditions compared to that in vitro. Permeabilized cell preparations thus seem to present the enzymes in the form of relatively less disintegrated functional complexes, making them a valuable tool in biochemical and cell biological investigations, and facilitate the correlation of in vitro data with the in vivo phenomena more confidently.
D002175:Candida / Q000201:enzymology*; D002463:Cell Membrane Permeability; D003067:Coenzymes / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D017680:Potassium Compounds; D013696:Temperature
D003067:Coenzymes; D009566:Nitrates; D017680:Potassium Compounds; D009565:Nitrate Reductases; C023844:potassium nitrate