[EBOOK] Purification Of Nicotinamide Adenine Dinucleotide Phosphate Specific Glutamate Dehydrogenase From C L O R E L L A S O R O K I N I A N A And Partial Characterization Of Its Physical Kinetic And Immunological Properties PDF Download

Chlorella

Purification and Partial Kinetic and Physical Characterization of Two NADP specific Glutamate Dehydrogenase Isoenzymes and Their Protein Precursors and Measurement of the Patterns of Accumulation and Rates of Degradation of Their Nonidentical Subunits in Synchronized Cells of Chlorella Cultured in Different Concentrations of Ammonia

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Download or read book Purification and Partial Kinetic and Physical Characterization of Two NADP specific Glutamate Dehydrogenase Isoenzymes and Their Protein Precursors and Measurement of the Patterns of Accumulation and Rates of Degradation of Their Nonidentical Subunits in Synchronized Cells of Chlorella Cultured in Different Concentrations of Ammonia written by Newell F. Bascomb and published by . This book was released on 1985 with total page 290 pages. Available in PDF, EPUB and Kindle. Book excerpt: Two ammonium-inducible, chloroplast-localized, NADP-specif ic glutamate dehydrogenases were purified from Chlorella sorokiniana . They were homopolymers of either alpha or beta subunits with molecular weights of 55,500 and 53,000, respectively. Purification was achieved by gel filtration (Sephacryl S-300), fast protein liquid chromatography (Pharmacia, Mono Q column), and substrate affinity chromatography (Pharmacia, NADP-Sepharose 4B, Type 4). These isoenzymes were separated by their differential binding to the substrate affinity column. Peptide mapping of purified alpha and beta subunits showed them to have a high degree of sequence homology. Because the ammonia K of alpha holoenzyme was sensitive to changes in NADPH concentration, its K ranged from 0.02 mM to 3.5 mM. With m changes in NADPH concentration, the aminonia K^ of beta holoenzyme remained constant at 70 mM. By use of SDS slab-gel electrophoresis and a Western blot/immunodetection procedure, patterns of accumulation of alpha and beta subunits (in their holoenzyme) were measured in cells cultured in media, containing different concentrations of ammonia. Pulse-chase experiments with Ssulfate were performed to measure the rates of degradation of the two isoenzymes. When the culture medium contained 2 mM ammonia or lower, cells accumulated only the alpha holoenzyme. Above 2 mM ammonia, cells contained both isoenzymes; however, their patterns of accumulation and rates of degradation were very different. The physiological role of alpha and beta holoenzymes appears to be ammonia assimilation at low and high external ammonia concentrations, respectively. From in vitro translation studies with total cellular poly(A)"^ RNA, isolated from cells engaged in synthesis of alpha or beta holoenzymes or both, it was concluded that alpha and beta subunits have protein precursor(s) of identical molecular weight (M = 58,500). When the putative protein-precursor (s) were incubated in vitro, with cell-free extracts from Chlorella cells, they were processed to proteins the size of alpha and beta subunits. From in vitro processing experiments, it was concluded that either two different precursors of the same molecular weight are specifically processed to give alpha and beta subunits, or the specificity or type of processing-enzyme changes to give the two subunits from a single precursor -protein.

Purification Physical and Chemical Characterization of the Nicotinamide Adenine Dinucleotide specific Glutamate Dehydrogenase from Chlorella Sorokiniana