Isolation, purification and characterization of intracellular calmodulin like protein (CALP) from Mycobacterium phlei

Sarma, P

doi:10.1016/s0378-1097(97)00538-7

Cited by 2 publications

(3 citation statements)

References 12 publications

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“…With the current method, the electrophoretic mobilities observed were highly reproducible, and the large mobility shift caused by the chelator suggests a possible dimer to monomer conversion. Sarma and coworkers (46) have found that the circular dichroism spectrum of the calmodulinlike protein from M. phlei suggests the free protein contains 52% ␤-sheet and only 20% ␣-helix, which shifts to 42% ␤-pleated sheet and 46% ␣-helix when 10 mM calcium ion is added. In contrast, eukaryotic calmodulins contain 30 to 45% ␣-helix and 15 to 20% ␤-pleated sheet structure (23).…”

Section: Discussionmentioning

confidence: 99%

“…Onek and Smith (38) thoroughly reviewed the earlier evidence for the existence of calmodulinlike proteins in seven genera of bacteria. In the last 8 years, further evidence for calmodulinlike proteins has appeared in Mycobacterium smegmatis (7), Mycobacterium tuberculosis (16,17), and Mycobacterium phlei (46); in Escherichia coli which has been induced with EGTA (26); in Nostoc sp. strain PCC 6720 (39); in three species of Bordetella, including B. pertussis (33,34); and in Halobacterium salinarium (44).…”

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confidence: 99%

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Immunocytochemical Localization of a Calmodulinlike Protein in Bacillus subtilis Cells

Domı́nguez

Adams²,

Hageman

1999

J Bacteriol

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To determine possible functions of the calmodulinlike protein ofBacillus subtilis, the time course of its expression during sporulation and its cellular localization were studied. The protein was expressed in a constitutive manner from the end of logarithmic growth through 8 h of sporulation as determined by antibody cross-reactivity immunoblots and enzyme-linked immunosorbent assays (ELISAs). In partially purified extracts, the immunopositive protein comigrated upon electrophoresis with a protein which selectively bound [45Ca]CaCl2, ruthenium red, and Stains-all. Previous studies showed increased extractability of the calmodulinlike protein from B. subtilis cells when urea and 2-mercaptoethanol were used in breakage buffers, implying that the protein might be partially associated with the membrane fraction. This was confirmed by demonstrating that isolated membrane vesicles ofB. subtilis also gave positive immunological tests with Western blotting and ELISAs. To more precisely locate the protein in cells, thin sections of late-log-phase cells, sporulating cells, and free spores were reacted first with bovine brain anticalmodulin specific antibodies and then with gold-conjugated secondary antibodies; the thin sections were examined by transmission electron microscopy. The calmodulinlike protein was found almost exclusively associated with the cell envelope of these fixed, sectioned cells. A possible function of the calmodulinlike protein in sensing calcium ions or regulating calcium ion transport is suggested.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Immunocytochemical Localization of a Calmodulinlike Protein in Bacillus subtilis Cells

Domı́nguez

Adams²,

Hageman

1999

J Bacteriol

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“…A 14-kDa protein with calcium-binding property and PDE-stimulating activity was isolated from M. phlei (33). However, the amino acid sequence of this protein was not reported, and hence its homology with calmodulin cannot be determined.…”

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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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Isolation, purification and characterization of intracellular calmodulin like protein (CALP) from Mycobacterium phlei

Cited by 2 publications

References 12 publications

Immunocytochemical Localization of a Calmodulinlike Protein in Bacillus subtilis Cells

Immunocytochemical Localization of a Calmodulinlike Protein in Bacillus subtilis Cells

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

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