Purification and partial characterization of a novel calcium-binding protein from Bacillus cereus T spores and inhibition of germination by calmodulin antagonists

Miscellaneous · Sun Jan 01 00:00:00 EST 1989

OSTI ID:6308300

Shyu, Y

A novel calcium-binding protein has been purified from the dormant spores of Bacillus cereus T. B. cereus T spores were extensively washed, broken, and heated at 90{degree}C for 2 min. Using calcium-dependent hydrophobic interaction chromatography plus DEAE-cellulose and hydroxylapatite columns, a single protein was obtained which possessed calcium-binding capacity and some characteristics of calmodulin. This heat-stable protein was retained by hydrophobic matrices or a calmodulin antagonist in a calcium-dependent manner. The crude spore extract displaced bovine brain calmodulin from its antibody in a radioimmunoassay and the immunoreactive specific activity of the partially purified fraction which eluted from phenyl-Sepharose was ca. 200-fold greater than the crude spore extract. Purity of this protein was verified by sodium dodecyl sulfate-polyarcylamide gel electrophoresis and reversed-phase HPLC. Calcium-binding ability was verified with a competitive calcium binding assay using Chelex-100 resin and {sup 45}Ca autoradiography. SDS-PAGE and amino acid composition indicated the molecular weight of the protein was 24-kDa. The effects of two calmodulin antagonists, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) on L-alanine-induced germination of Bacillus cereus T spores were examined by measuring commitment to germination, loss of heat resistance, release of calcium, decrease in optical density at 660 nm and phase-contrast microscopy.

• Research Organization: North Carolina State Univ., Raleigh, NC (USA)
• OSTI ID: 6308300
• Resource Relation: Other Information: Thesis (Ph. D.)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BACTERIAL SPORES
GERMINATION
CALMODULIN
CHEMICAL COMPOSITION
FRACTIONATION
AUTORADIOGRAPHY
BACILLUS CEREUS
CALCIUM
ELECTROPHORESIS
INHIBITION
LIQUID COLUMN CHROMATOGRAPHY
MOLECULAR WEIGHT
RADIOIMMUNOASSAY
ALKALINE EARTH METALS
BACILLUS
BACTERIA
BIOASSAY
CHROMATOGRAPHY
DIAGNOSTIC TECHNIQUES
ELEMENTS
IMMUNOASSAY
IMMUNOLOGY
ISOTOPE APPLICATIONS
METALS
MICROORGANISMS
ORGANIC COMPOUNDS
PROTEINS
RADIOASSAY
RADIOIMMUNODETECTION
RADIOIMMUNOLOGY
SEPARATION PROCESSES
SPORES
TRACER TECHNIQUES
550201* - Biochemistry- Tracer Techniques