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

Thesis/Dissertation · Wed Jan 01 04:00:00 EST 1986

OSTI ID:7065720

Bascomb, N F

Two ammonium-inducible, chloroplast-localized, NADP-specific 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. 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. 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 (/sup 35/S)sulfate were performed to measured 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 enzymes; 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)/sup +/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) or identical molecular weight (M/sub r/ = 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.

• Research Organization: Florida Univ., Gainesville (USA)
• OSTI ID: 7065720
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALGAE
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
BIODEGRADATION
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL CULTURES
CHEMICAL REACTIONS
CHEMISTRY
CHLORELLA
CHLOROPLASTS
COENZYMES
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DRUGS
ELECTROPHORESIS
ENZYMES
EVEN-ODD NUCLEI
GLUTAMIC ACID
HEMIACETAL DEHYDROGENASES
ISOENZYMES
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPOTROPIC FACTORS
METHIONINE
MICROORGANISMS
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
NADP
NUCLEI
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
PLANTS
RADIOISOTOPES
SULFUR 35
SULFUR ISOTOPES
SYNCHRONOUS CULTURES
UNICELLULAR ALGAE