Cell Extracts Of Bacteria Research Articles

Cytosolic NADP phosphatases I and II from Arthrobacter sp. strain KM: implication in regulation of NAD+/NADP+ balance.

NADP phosphatase (NADPase) is an enzyme that converts NADP+ into NAD+ through dephosphorylation of NADP+, and is considered to be one of the possible candidates for regulation of the NAD+/NADP+ balance in vivo. In order to obtain an intrinsic NADPase, the NADP+-degrading activity in a membrane-free cell extract of a Gram-positive bacterium, Arthrobacter sp. strain KM, was first assessed and demonstrated to be mainly achieved through the NADPase reaction, indicating NADPase is essential for degradation of NADP+ and therefore for regulation of the NAD+/NADP+ balance in cytosol. Then, the isolation of cytosolic NADPase was attempted using NADP+ as a substrate. Two NADPase isozymes, designated as NADPases I and II, were purified from the cell extract of the bacterium, and were indicated to be the sole cytosolic NADPases regulating the balance of NAD+/NADP+. NADPases I and II are homodimers of 32 and 30 kDa subunits, respectively, and most active at pH 7-8. The N-terminal amino acid sequences of the two enzymes are similar to each other. Among the biological substrates tested, both enzymes showed the highest activity toward NADP+ and NADPH. AMP, ADP, and pyridoxal 5'-phosphate were also dephosphorylated, but to lower extents. Comparison of the features of NADPases I and II with those of other acid phosphatases possessing NADPase activity suggested that NADPases I and II are novel enzymes participating in regulation of the NAD+/NADP+ balance in the cytosol.

A colorimetric assay for detecting haloalkane dehalogenase activity

A pH indicator dye-based colorimetric assay was adapted to measure haloalkane dehalogenase activity quantitatively in cell extracts of bacteria using the microplate format. The assay is based on a decrease in the pH of a weakly-buffered medium when protons and chloride ions are released by the enzyme. Our assay is more convenient than other instrumental or chemical assay methods presently used to monitor dechlorination reactions. In addition, our assay is rapid, quantitative, can be automated to handle a large number of samples using microplates, and uses inexpensive and nontoxic reagents. The assay may be adapted to monitor dechlorinating reactions mediated by other dehalogenase enzymes.