SpeedScreen is a novel, label-free, in-solution, affinity-based selection methodology for high-throughput screening (HTS) developed at Novartis Pharma. The SpeedScreen protocol comprises in-solution affinity selection, followed by size exclusion chromatography in combination with microbore-liquid-chromatography/electrospray-ionization mass spectrometry (micro-LC/ESI-MS). The authors describe the basic concept behind assay development, HTS, and data analysis with the SpeedScreen technology. Advantages and limitations of SpeedScreen compared to alternative screening technologies are discussed, and an example is given from a SpeedScreen campaign applying this innovative affinity selection concept in HTS.
Pentalenolactone (PL) irreversibly inactivates the enzyme glyceraldehyde-3phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD' oxidoreductase (phosphorylating)] (EC 1.2.1.12) and thus is a potent inhibitor of glycolysis in both procaryotic and eucaryotic cells. We showed that PL-producing strain Streptomyces arenae TU469 contains a PL-insensitive glyceraldehyde-3-phosphate dehydrogenase under conditions of PL production. In complex media no PL production was observed, and a PL-sensitive glyceraldehyde-3-phosphate dehydrogenase, rather than the insensitive enzyme, could be detected. The enzymes had the same substrate specificity but different catalytic and molecular properties. The apparent Km values of the PL-insensitive and PL-sensitive enzymes for glyceraldehyde-3-phosphate were 100 and 250 ,uM, respectively, and the PL-sensitive enzyme was strongly inhibited by PL under conditions in which the PL-insensitive enzyme was not inhibited. The physical properties of the PLinsensitive enzyme suggest that the protein is an octamer, whereas the PLsensitive enzyme, like other glyceraldehyde-3-phosphate dehydrogenases, appears to be a tetramer.
An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity-staining, produced two bands of different molecular masses and chain compositions. The highmolecular-mass band occurred in low quantity and consisted of, probably eight, apparently identical chains of M , = 33 000, as judged from sodium dodecyl sulfate electrophoreses. The low-molecular-mass band contained two types of chains with MF = 63000-65000 and M! = 61000-62000. The N termini of both chains were threonine, whereas their C-terminal sequences were different:Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different.In all properties investigated, the low-molecular-mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess ( c I~)~ structure, the apoenzyme a/?. SH reagents inactivated the enzyme. Binding and fluorescence of 2-p-toluidinonaphthalene-6-sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2-Hydroxy-5-nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor-binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase.Pyruvate decarboxylase (PDC) is known to occur in yeasts, several other fungi, many plant materials (review: [l]) and a few bacteria [2, 31. One of the early sources for its preparation has been wheat germ [4]. When our efforts to achieve a detailed chemical analysis of yeast PDC became hampered by uncontrollable actions of contaminating proteinases [5], a practical alternative seemed to be the investigation of PDC from wheat germ, which was expected to contain fewer proteinases. The original method for the preparation of wheat germ PDC [4] has been applied without major alterations in all instances when this enzyme was hitherto needed for a variety of purposes [6 -111. Re-examination of this procedure with modern electrophoresis methods demonstrated that it yields PDC of insufficient chain homogeneity for the requirements of peptide mapping and sequence analysis. Therefore a more efficient isolation scheme was elaborated.
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