Purification and properties of an intracellular calmodulinlike protein from Bacillus subtilis cells.
Although calcium ions are crucial in a variety of bacterial processes, including spore development, reports of calmodulin in procaryotes have been few. We have purified to homogeneity a calmodulinlike protein (CaLP) from sporulating cells of Bacillus subtilis grown in a chemically defined sporulation medium; purification involved heat treatment, fractionation with ammonium sulfate, affinity chromatography, and gel filtration on high-performance columns. The protein was eluted from a phenothiazine affinity column in a calcium ion-dependent manner, stained poorly with Coomassie blue and silver stain dyes, bound poorly to nitrocellulose filters, and was not an inhibitor of the major intracellular serine proteinase. It stimulated bovine brain phosphodiesterase in a dose- and Ca2(+)-dependent manner and stimulated NAD kinase from peas in a dose-dependent manner. The B. subtilis calmodulin reacted with anti-bovine brain calmodulin antibodies in enzyme-linked immunoabsorbance assays. The amino acid composition data showed it to be distinctly different from eucaryotic calmodulins, having particularly high levels of serine and glycine. The pI of the protein was estimated to be 4.9 to 5.0. The molecular weight was estimated to be 23,000 or 25,000, based on amino acid composition and detergent gel electrophoresis, respectively. The protein reacted with rhodamine isothiocyanate, which blocked its enzyme-activating capacity and greatly increased its electrophoretic mobility and Coomassie dye-binding ability.
Fry
I J
IJ
Department of Chemistry, New Mexico State University, Las Cruces 88003.
Becker-Hapak
M
M
Hageman
J H
JH
D000596:Amino Acids / Q000032:analysis; D001412:Bacillus subtilis / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000737:chemistry* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis; D010770:Phosphotransferases / Q000096:biosynthesis; D017853:Phosphotransferases (Alcohol Group Acceptor); D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D002147:Calmodulin; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; D010727:Phosphoric Diester Hydrolases; D002118:Calcium