Purification and in vitro complementation of mutant histidinol dehydrogenases. [Salmonella typhimurium]

Journal Article · Tue Sep 01 04:00:00 EDT 1987 · J. Bacteriol.; (United States)

OSTI ID:6030700

Lee, S Y; Grubmeyer, C T

The biochemistry of interallelic complementation within the Salmonella typhimurium hisD gene was investigated by in vitro protein complementation of mutant histidinol dehydrogenases (EC 1.1.1.23). Double-mutant strains were constructed containing the his01242 (constitutive overproducer) attenuator mutations and selected hisDa or hisDb mutations. Extracts from such hisDa986 and hisDb1799 mutant cells failed to show histidinol dehydrogenase activity but complemented to produce active enzyme. Inactive mutant histidinol dehydrogenases were purified from each of the two mutants by ion-exchange chromatography. Complementation by the purified mutant proteins required the presence of 2-mercaptoethanol and MnCl/sub 2/, and protein-protein titrations indicated that heterodimers were strongly preferred in mixtures of the complementary mutant enzymes. Both purified mutant proteins failed to catalyze NAD-NADH exchange reactions reflective of the first catalytic step of the two-step reaction. The inactive enzymes bound /sup 54/Mn/sup 2 +/ weakly or not at all in the presence of 2-mercaptoethanol, in contrast to wild-type enzyme which bound /sup 54/Mn/sup 2 +/ to 0.6 sites per monomer under the same conditions. The mutant proteins, like wild-type histidinol dehydrogenase, behaved as dimers on analytical gel filtration chromatography, but dissociated to form monomers in the presence of 2-mercaptoethanol. This effect of 20-mercaptoethanol was prevented by low levels of MnCl/sub 2/.

• Research Organization: New York Univ., NY
• OSTI ID: 6030700
• Journal Information: J. Bacteriol.; (United States), Journal Name: J. Bacteriol.; (United States) Vol. 169:9; ISSN JOBAA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AZOLES
BACTERIA
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CATALYSIS
CATIONS
CHARGED PARTICLES
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ENZYME ACTIVITY
ENZYMES
GENETIC MAPPING
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HISTIDINE
IMIDAZOLES
INTERMEDIATE MASS NUCLEI
IONS
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MANGANESE 54
MANGANESE COMPOUNDS
MANGANESE ISOTOPES
MAPPING
MICROORGANISMS
MUTANTS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
PROTEIN STRUCTURE
PURIFICATION
RADIOISOTOPES
REACTION KINETICS
SALMONELLA
SALMONELLA TYPHIMURIUM
THIOLS
TRACER TECHNIQUES
TRANSITION ELEMENT COMPOUNDS