Crystallization of Arthrobacter sp. strain 1C N-(1-D-carboxyethyl)-L-norvaline dehydrogenase and its complex with NAD+

The novel NAD+-linked opine dehydrogenase from a soil isolate Arthrobacter sp. strain 1C belongs to an enzyme superfamily whose members exhibit quite diverse substrate specificites. Crystals of this opine dehydrogenase, obtained in the presence or absence of co-factor and substrates, have been shown to diffract to beyond 1.8 ? resolution. X-ray precession photographs have established that the crystals belong to space group P21212, with cell parameters a = 104.9, b = 80.0, c = 45.5 ? and a single subunit in the asymmetric unit. The elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this novel class of dehydrogenases to enable a comparison to be made with other enzyme families and also as the basis for mutagenesis experiments directed towards the production of natural and synthetic opine-type compounds containing two chiral centres.     

Effects of solutions used for storage of size-exclusion columns on subsequent chromatography of peptides and proteins

The effects of storage of size-exclusion column packing materials in methanolic or azide-water solutions on subsequent separations were tested. Three commercially available columns were used in these studies; the Toyo-Soda Bio-Sil TSK 125, Bio-Sil TSK 250 and the DuPont Bio-Series GF-250. Upon initial chromatography, all three columns bound up to 760 micrograms of cytochrome c tryptic peptides. Sample binding to packing material is probably a function of the positively charged basic groups on peptides or proteins interacting with silanol groups. The larger the peptide, the less the opportunity for silanol-charged group interaction, hence, less binding. Initial samples introduced to a new column occupy the binding sites. Equilibration with neat methanol removes the bound protein revealing sites which bind sample. After absorption of peptides to binding sites on the packing material, storage in neat methanol regenerates the binding sites. Storage in 10% methanol diminished the binding phenomenon, but storage in azide-water reduced binding to a range below detection at the microgram level. Our recommendation to users of size-exclusion chromatographic columns is that one satisfy the absorption capacity of a new column by injecting a sufficient quantity of a basic peptide standard or other convenient sample to reduce available binding sites before using the column for important separations. Store columns in azide-water or 10% methanol to prevent the regeneration of exposed silanol groups.     

Computable error bounds for pointwise derivatives of a Neumann problem

In this paper we discuss the recovery of derivatives and thecomputation of rigorous and useful upper bounds for the pointwiseerror in the recovered derivatives, for finite element approximationsof the Laplace equation with Neumann boundary conditions, especiallyat points close to or on a smooth, curved boundary. We analyzethe dipole image technique for the case of curved boundaries,and show how to compute reliable recovered derivatives and errorbounds even in the limiting case of points lying on the curvedboundary. Numerical experiments show reasonably tight errorbounds for points both close to and away from a curved boundary.     

High-performance liquid chromatographic quantitation of rhodamines 123 and 110 from tissues and cultured cells

Rhodamine 123 is a fluorescent vital dye which has potential for therapeutic use in cancer treatment. The dye concentrates in mitochondria of normal and neoplastic cells but accumulates in and is toxic to neoplastic cells. When dye-treated cells are irradiated with blue laser light at 514 nm, mitochondrial injury or cell death results. Rhodamine concentration in cultured cells and tumor tissue was quantitated to correlate cell or tumor death with drug dose. A reversed-phase separation of rhodamine 123 was accomplished using a gradient of 0.05 M phosphate buffer pH 2.85 (mobile phase A) and acetonitrile (mobile phase B), 10-80% B in 15 min with a DuPont Golden Series C8 column. Effluent was monitored with a fluorescence detector at 295 nm excitation and 520 nm emission. Stock rhodamine 123 contained approximately 6-8% of rhodamine 110, the parent compound, which eluted at 9.8 min whereas rhodamine 123 eluted at 11.7 min. Structural verification of both compounds by field desorption mass spectrometry was performed. This is the first report of the chemical separation and quantitation of rhodamine 123 from cultured tumor cells or tumor tissue.