PubMed · 9664367
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
M Page, R Thorpe. 1998. IgG purification.. https://doi.org/10.1007/978-1-59259-257-9_10
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
Explore the source record for details and available documents.
A few organisms, notably some fungi, have the ability to metabolize thymidine to uracil, thus conserving the pyrimidine ring for subsequent metabolic use. Neurospora crassa possesses this pathway, termed the thymidine salvage pathway, and can utilize thymidine as a total pyrimidine source. The enzyme isoorotate decarboxylase (IDCase) completes this pathway via the enzymatic removal of the carboxylate from isoorotate to yield uracil. We describe in this communication two assays for IDCase and their application to determine activity levels, kinetic constants, and inhibitory properties. One uses [carboxy-14C]isoorotate from which the enzymatically generated 14CO2 is collected and quantitated. The second assay utilizes the spectral difference between 2-thioisoorotate and its decarboxylated product, 2-thiouracil. The spectral difference is greatest at 334 nm, out of the range of absorbance of total protein and thus usable for a spectrophotometric assay. The assays are sufficiently sensitive and accurate to be used in the measurement of Km values for both substrates. IDCase activity is found to be significantly higher in N. crassa strains lacking uc-1, a putative regulatory gene, suggesting a degree of metabolic control over this pathway. 5-Nitrouracil is found to inhibit IDCase with an estimated Ki value that is too low for accurate determination.
Significant improvement in the resolution and quality of the X-ray diffraction of crystals of MTCP-1 protein was observed on post-crystallization soaking. The MTCP-1 crystals grown from 1.5 M ammonium sulfate diffracted to only 3.0 A resolution with some disorder in the diffraction. After post-crystallization soaking in a solution containing 2.0 M ammonium sulfate, the disorder was eliminated and diffraction extended to better than 2.0 A resolution. Both native and selenomethionine-enriched crystals demonstrated better diffraction after soaking for several months. This simple technique may be useful to improve the diffraction quality of protein crystals generally.