RESUMEN
Cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15,000 x g supernatant fraction of porcine corpora lutea. The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg2+, DL-isocitrate, dithiothreitol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48,000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optimal pH for activity from 7.5 to 9.0. The Km for the substrates DL-isocitrate and NADP+ were 13 and 12 microM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnover number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.
Asunto(s)
Cuerpo Lúteo/enzimología , Isocitrato Deshidrogenasa/aislamiento & purificación , NADP/farmacología , Animales , Western Blotting , Cationes Bivalentes , Fenómenos Químicos , Química Física , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Femenino , Glicerol/farmacología , Concentración de Iones de Hidrógeno , Isocitrato Deshidrogenasa/metabolismo , Isocitratos/farmacología , Cinética , Cloruro de Magnesio/farmacología , Peso Molecular , Desnaturalización Proteica , PorcinosRESUMEN
Mouse monoclonal antibodies (mAbs) were raised against the major capsid protein, L1, of human papillomavirus type 16 (HPV16), produced in Escherichia coli with the expression plasmid pTrcL1. Epitope specificity could be assigned to 11 of these 12 antibodies using a series of linear peptides and fusion proteins from HPV16. One mAb (MC53) recognized a novel linear epitope that appears to be unique to the HPV16 genotype. A further 11 mAbs were characterized as recognizing novel and previously defined linear and conformational epitopes shared among more than one HPV genotype. The apparently genotype specific mAb could be useful for the development of diagnostic tests for vegetative virus infection in clinical specimens.