Researching the green fuse: an historical memoir

2002 ◽  
Vol 80 (12) ◽  
pp. 1231-1238
Author(s):  
John King
Keyword(s):  
Tissue Culture ◽  
Genetic Variants ◽  
Somatic Hybridization ◽  
Datura Innoxia ◽  
Purification And Characterization ◽  
Metabolic Intermediates ◽  
Physiology And Biochemistry ◽  
Biochemical Genetic ◽  
Branched Chain

The author outlines the major milestones in his 40-year career in plant research. His research program might be described, broadly, as explorations into the physiology and biochemistry of plants and their cells using physiological, biochemical, genetic, molecular biological and chemical techniques and technologies. The main areas encompassed by the program are the pathways of biosynthesis of branched-chain amino acids, pantothenate, folates and one-carbon units, uptake of organic and inorganic sources of nitrogen by plant cells, and somatic hybridization and genetic transformation of plant protoplasts. Many of these investigations were carried out with the aid of auxotrophic and resistance genetic variants selected either from cultures of Datura innoxia cells or from among mutagenized populations of Arabidopsis thaliana seedlings. Some technologies found to be especially useful were plant cell and tissue culture, detection of 14C-labelled isotopes in metabolic intermediates, isolation, purification and characterization of enzymes, and most recently, 13C nuclear magnetic resonance (NMR) spectroscopy.Key words: Arabidopsis, Datura, biochemical genetic variants, tissue culture, nitrogen metabolism.

scholarly journals Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft

2000 ◽  
Vol 350 (1) ◽  
pp. 321-328 ◽  
Author(s):  
Helen S. TOOGOOD ◽  
Clyde A. SMITH ◽  
Edward N. BAKER ◽  
Roy M. DANIEL
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Fold Increase ◽  
Branched Chain Amino Acids ◽  
Disulphide Bond ◽  
Purification And Characterization ◽  
Active Site Cleft ◽  
Binding Cleft ◽  
Branched Chain

Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P1 site, such as valine, alanine or phenylalanine. The Km and kcat of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca2+ ions. At 70°C, a 10-fold increase in Ca2+ concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca2+ to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca2+-binding site in Ak.1 protease. Other metal ions, such as Sr2+, increase the thermostability of the enzyme, but to a significantly lower degree than does Ca2+. The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.

Purification and Characterization of an NAD-Malic Enzyme from Bradyrhizobium japonicum A1017

1998 ◽  
Vol 64 (10) ◽  
pp. 4073-4075 ◽  
Author(s):  
Fan Chen ◽  
Youichi Okabe ◽  
Kaoru Osano ◽  
Shigeyuki Tajima
Keyword(s):  
Carbon Metabolism ◽  
Malic Enzyme ◽  
Bradyrhizobium Japonicum ◽  
Molecular Characteristics ◽  
Purification And Characterization ◽  
Metabolic Intermediates ◽  
Molecular Masses

ABSTRACT An NAD-malic enzyme was purified to homogeneity fromBradyrhizobium japonicum A1017, and its molecular characteristics were surveyed. The enzyme exhibited native and subunit molecular masses of 388 and 85 kDa, respectively, suggesting that it exists as a homotetramer, and was activated by metabolic intermediates in glycolysis. The role of the enzyme in bacteroids’ carbon metabolism is discussed.

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