0000000000241836
AUTHOR
Juan L Lequerica
Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A
Mutations enhancing the thermostability of β-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed β-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while …
Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.
Background The Na+/H+ exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H+ ion in exchange for extracellular Na+ and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. Methods To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluo…
A halocin acting on Na+/H+ exchanger of Haloarchaea as a new type of inhibitor in NHE of mammals
10 páginas, 3 figuras.