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Category: Microbial Genetics and Molecular Biology
Bacterial Respiration, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555817497/9781555812232_Chap21-1.gif /docserver/preview/fulltext/10.1128/9781555817497/9781555812232_Chap21-2.gifAbstract:
This chapter provides a brief introduction to assay the activity of respiratory enzymes. Spot assays can facilitate the rapid detection of enzyme activities in whole or permeabilized cells, in subcellular samples, or in chromatography fractions to follow protein purification. In-gel activity stains, or zymograms, offer an alternative approach to detect oxidoreductase enzymes, especially when the level of enzyme activity is at or below the sensitivity limit of a standardized quantitative enzyme assay. Conversely, if the cellular location of the enzyme is unknown, it can be determined experimentally. Finally, knowledge of the enzyme location can be useful in understanding the physiological role(s) of the enzyme in cell metabolism and in energy conservation. This section outlines experimental approaches to determine the cellular location of a redox enzyme following cell fractionation. Since succinate dehydrogenase may be partially deactivated by tightly bound oxaloacetate at its active site, the enzyme requires activation prior to enzyme assay by incubation with either malate or succinate. Prokaryotes exhibit considerable enzymatic diversity with respect to the number and types of cytochrome oxidase enzymes present for reduction of molecular oxygen to water. A section of the chapter describes a number of commonly used assays for anaerobic respiratory enzymes that act on terminal electron acceptors, including nitrate, nitrite, nitric oxide, nitrous oxide, fumarate, TMAO, DMSO, and metal oxides. The activity of NADH-dependent NirB-type enzymes is assayed by monitoring the oxidation of NADH.
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Location of redox respiratory enzymes in the cell. The active site of a membrane-bound oxidoreductase may face the cytoplasm (A) or the periplasm (B). A soluble-type oxidoreductase may be located either in the periplasmic space (C) or in the cytoplasm (D). The electron acceptor substrate (S) is converted to the reduced product (P).
Location of redox respiratory enzymes in the cell. The active site of a membrane-bound oxidoreductase may face the cytoplasm (A) or the periplasm (B). A soluble-type oxidoreductase may be located either in the periplasmic space (C) or in the cytoplasm (D). The electron acceptor substrate (S) is converted to the reduced product (P).
Anaerobic cuvette gassing arrangement for performing anaerobic dye-dependent enzyme assays.
Anaerobic cuvette gassing arrangement for performing anaerobic dye-dependent enzyme assays.
Reactions of the dissimilatory sulfate reduction pathway.
Reactions of the dissimilatory sulfate reduction pathway.
Standard redox potentials of selected electron donors and acceptors involved in bacterial oxidation-reduction reactions
a Values derived from reference 91.
Standard redox potentials of selected electron donors and acceptors involved in bacterial oxidation-reduction reactions
a Values derived from reference 91.
Standard redox potentials of artificial and physiologicial redox substrates commonly used to assay respiratory enzymes
a Extinction coefficient expressed at the indicated wavelength.
Standard redox potentials of artificial and physiologicial redox substrates commonly used to assay respiratory enzymes
a Extinction coefficient expressed at the indicated wavelength.
Artificial redox dye substrates that are able to permeate to the cytoplasmic membrane
Artificial redox dye substrates that are able to permeate to the cytoplasmic membrane
Artificial redox dye substrates that are unable to permeate to the cytoplasmic membrane.
Artificial redox dye substrates that are unable to permeate to the cytoplasmic membrane.