Please use this identifier to cite or link to this item:
Multiple forms of bacterial hydrogenases
|uhm_phd_6910593_r.pdf||Version for non-UH users. Copying/Printing is not permitted||3.92 MB||Adobe PDF||View/Open|
|uhm_phd_6910593_uh.pdf||Version for UH users||3.87 MB||Adobe PDF||View/Open|
|Title:||Multiple forms of bacterial hydrogenases|
|Authors:||Asato, Robert Noriyoshi|
|Abstract:||Hydrogenase enzymes from many distinct bacterial species have been shown by disc electrophoresis on polyacrylamide gel to exist in multiple forms. The enzymes of the hydrogenase systems have different electrophoretic mobilities and produce a band pattern that is unique for each species. These studies have demonstrated the utility of this method for the taxonomic identification of hydrogenase containing bacteria. A tentative identification of an isolated bioluminescent bacteria as P. phosphorium was made using this method. This multiplicity of hydrogenase forms was found both in bacteria which contain mostly soluble hydrogenases and in those where the hydrogenase is predominantly associated with particulate material. When solubilization of this particulate material could be effected, at least two solubilized hydrogenases were released, and, of these, one would have the same electrophoretic properties (RF) as one of the soluble hydrogenases already present in small amounts within the cell. Different growth conditions for various types of bacteria, such as the nitrogen source, iron in the medium, the degree of aeration, and photosynthetic versus aerobic growth in the dark, as well as the conditions under which the cells were stored, markedly affected the hydrogenase activity of the cells, but not their hydrogenase band patterns. The specificities of various isoenzymes of certain bacteria were tested with redox dyes of different potentials, and no difference among isoenzymes of a particular organism was observed. Three of the six forms of C. pasteurianum were found to be interrelated with the ∝ and β forms always converting to the more stable y species. The molecular weights of these forms were approximately 50,000. Recently developed techniques such as sucrose density gradient centrifugation, and gel filtration allowed studies on hydrogenase in the presence of other proteins by a direct assay of its biological activity. Comparative studies were done on the size and shape of hydrogenase enzymes of C. butyricum and C. butylicum, with the former possessing isoenzymes of the same weight as those of C. pasteurianum. Kinetic studies on purified preparations of C. pasteurianum and C. butylicum hydrogenases demonstrated that molybdenum was involved in the one-electron transfer to methyl viologen. The participation of iron in the viologen assay of C. butylicum hydrogenase was also shown. A purification scheme for the isolation of C. butylicum hydrogenase is presented. The final extract possessed a specific activity of approximately 4.Z liters HZ evolved per hour per mg protein. This represented a 500 fold purification over the crude preparation and was stable when stored under HZ at 0° for up to several weeks. The estimation of the turnover number of this partially purified form of hydrogenase indicate that it is at least three times more active than catalase, the most active enzyme known. It has been shown that for all hydrogenase enzymes examined there is an abrupt change in the Arrhenius activation energy at 17°. This was true for a variety of hydrogenase enzymes, catalyzing a variety of reactions. This transition temperature corresponds exactly with a similar transition in the electrophoretic mobilities of the various hydrogenases on polyacrylamide gel. It is believed that this phenomenon is the manifestation of a conformational change occurring at this temperature.|
Thesis (Ph. D.)--University of Hawaii, 1968.
Bibliography: leaves -171.
x, 171 l graphs, tables
|Rights:||All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.|
|Appears in Collections:||Ph.D. - Biomedical Sciences (Biochemistry)|
Please contact email@example.com if you need this content in an alternative format.
Items in ScholarSpace are protected by copyright, with all rights reserved, unless otherwise indicated.