Calmodulin-like activity in the soluble fraction of

Iwasa, Yasushi; Yonemitsu, Kosei; Matsui, Kazuo; Fukunaga, Kohji; Miyamoto, Eishichi

doi:10.1016/0006-291x(81)91164-5

Cited by 47 publications

(18 citation statements)

References 13 publications

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“…Several authors have reported finding calmodulinlike proteins (CaLPs) in procaryotes (18,20,22). The analogy drawn between calmodulin and these procaryotic proteins, however, has relied on physical properties characteristic of calmodulin, such as amino acid sequence motifs (18,41,42), Ca2+ binding (27), and binding to anticalmodulin antibodies (22).…”

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confidence: 99%

Purification and properties of an intracellular calmodulinlike protein from Bacillus subtilis cells

1991

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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 iondependent 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 dosedependent manner. The B. subtilis calmodulin reacted with anti-bovine brain calmodulin antibodies in enzymelinked 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 pl 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.

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confidence: 99%

Purification and properties of an intracellular calmodulinlike protein from Bacillus subtilis cells

1991

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“…While calmodulin was initially considered to be absent in bacteria (3, 6), calmodulin-like proteins (CAMLPs) exhibiting either calcium-binding properties or bovine phosphodiesterase (PDE)-stimulating properties were found in some bacteria (8,10,12,13,15,24,34,36) A917, 1993]. These results support the view that mycobacteria contain a protein functionally similar to eukaryotic calmodulin.…”

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confidence: 67%

“…While calmodulin was initially considered to be absent in bacteria (3,6), calmodulin-like proteins (CAMLPs) exhibiting either calcium-binding properties or bovine phosphodiesterase (PDE)-stimulating properties were found in some bacteria (8,10,12,13,15,24,34,36), but their functions were not explored. Fry et al (11) purified a CAMLP from Bacillus subtilis but did not demonstrate any role for the protein.…”

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confidence: 99%

Cloning and Expression of the Gene for a Novel Protein from Mycobacterium smegmatis with Functional Similarity to Eukaryotic Calmodulin

et al. 2003

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A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame. M. smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids (16%) in M. smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M. smegmatis CAMLP. Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M. smegmatis CAMLP.Calmodulin, a highly conserved and ubiquitous protein in eukaryotic cells (16,17), is a small, heat-and acid-stable protein composed of about 155 amino acids; it undergoes a conformational change upon binding calcium and modulates the function of a number of target proteins (20,21,40). While calmodulin was initially considered to be absent in bacteria (3, 6), calmodulin-like proteins (CAMLPs) exhibiting either calcium-binding properties or bovine phosphodiesterase (PDE)-stimulating properties were found in some bacteria (8,10,12,13,15,24,34,36) A917, 1993]. These results support the view that mycobacteria contain a protein functionally similar to eukaryotic calmodulin. As part of our studies on a possible regulatory role of CAMLP in the growth and metabolism of mycobacteria, we report here the purification of CAMLP from M. smegmatis, the nucleotide sequence of the gene encoding CAMLP, and the expression and partial characterization of the protein.Purification of M. smegmatis CAMLP. The activity of M. smegmatis CAMLP was assayed by the method of Fry et al. (11) based on the stimulation of bovine heart PDE activity by eukaryotic calmodulin. M. smegmatis ATCC 14468 was grown from a stab in Brodie and Gray's medium (1.3% BactoCasamino Acids, 0.1% potassium fumarate, 0.2% Tween 80, 0.1% K 2 HPO 4 , 0.03% MgSO 4 , 0.002% FeSO 4 [pH 7.0], with KOH) (2) at 37°C in a rotary shaker for 2 to 3 days. With this used as a fresh inoculum, 12 1-liter cultures were grown overnight. The cells were sedimented at 10,000 ϫ g for 10 min at 4°C and washed with 10 mM Tris-HCl (pH 7.5). About 70 g (wet weight) of cells was obtained.All operations were carried out at 4°C except for the high-

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“…Ca2+ has multiple binding affinities for different envelope constituents [27] such as peptidoglycans, lipopolysaccharides, m em brane proteins, and its action may be mediated in E. coli cells by a calm o dulin-like factor [28]. In this context, C a2+ inhibi tion of polycationic bacteriolytic and lytic free bactericidal effects would be explained by a possible competitivity exerted by bivalent cation action.…”

Section: Factors Suggested To Represent Another Class O F Enzymes -Thmentioning

confidence: 99%

Polycation-Bacterium Interactions and Wall Subunits as Endocytosis Factors. Topoisomeraselike Action of Basic Polypeptides Suggesting a 7th Class of Enzymes: The Stereases

S¹

1982

Zeitschrift Für Naturforschung C

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Calmodulin-like activity in the soluble fraction of

Cited by 47 publications

References 13 publications

Purification and properties of an intracellular calmodulinlike protein from Bacillus subtilis cells

Purification and properties of an intracellular calmodulinlike protein from Bacillus subtilis cells

Cloning and Expression of the Gene for a Novel Protein from Mycobacterium smegmatis with Functional Similarity to Eukaryotic Calmodulin

Polycation-Bacterium Interactions and Wall Subunits as Endocytosis Factors. Topoisomeraselike Action of Basic Polypeptides Suggesting a 7th Class of Enzymes: The Stereases

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