Large Scale Preparation of Calmodulin

Newton, Dianne L.; Krinks, Marie; Kaufman, J.; Shiloach, Joseph; Klee, Claude B.

doi:10.1080/00327488808062527

Cited by 20 publications

(13 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ram testis calmodulin was purified as described by Newton et al [13]. FK506 and rapamycin were generous gifts from S. Schreiber (Harvard University) and okadaic acid from A. Takai (Nagoya University).…”

Section: Materandlsmentioning

confidence: 99%

Factors responsible for the Ca²⁺‐dependent inactivation of calcineurin in brain

et al. 1995

View full text Add to dashboard Cite

The Ca2+-dependent protein phosphatase activity of crude rat brain extracts measured in the presence of okadaic acid, exhibits the characteristic properties of the calmodulin-stimnlated protein phosphatase, calcineurin. It is stimulated more than 200-fold by Ca 2+ and inhibited by the calmodnlin-binding peptide, MI3, and by the immunosuppressive drug, FKS06. It is insensitive to rapamycin at concentrations up to 1 pM. Its specific activity, based on calcineurin concentration determined by quantitative analysis of Western blots exposed to anti-bovine brain IgG, is ten to twenty times that of purified rat brain calcineurin assayed under similar conditions. Unlike the purified enzyme it is rapidly and irreversibly inactivated in a time-, temperature-, and Ca2+/ calmodulin-dependent fashion without evidence of extensive proteolytic degradation. The enzyme is converted to a state which does not lose activity by removal of low molecular weight material by gel filtration. Reconstitution of a labile enzyme is achieved by the addition of the low molecular weight-containing fraction elnted from the gel filtration column. These observations indicate that calcineurin in crude brain extracts is under the control of Ca2+/calmodulin-dependent positive and negative regulatory mechanisms which involve unidentified endogenous factor(s).

show abstract

Section: Materandlsmentioning

confidence: 99%

Factors responsible for the Ca²⁺‐dependent inactivation of calcineurin in brain

et al. 1995

View full text Add to dashboard Cite

show abstract

“…protein expression was induced by addition of IPTG to 30 pM followed by incubation at 30 "C for a further 16-20 h. PhenylSepharose chromatography could not be used for the multiple binding site mutants and therefore an alternative purification scheme was devised, based on that of Newton et al (1988). The start point for the purification was a 100,ooO-g supernatant prepared after French press disruption of the bacterial suspensions as described previously (Maune et al, 1992b).…”

Section: P Mukherjea Et Almentioning

confidence: 99%

“…However, no conditions could be found under which calmodulins with more than one mutant binding site would bind to phenyl-Sepharose. A more conventional procedure, based on that of Newton et al (1988) and involving ammonium sulfate fractionation, DEAE-Sephacel chromatography, and HPLC anion exchange chromatography, was therefore developed for purification of these proteins (see the Materials and methods). Drosophila calmodulin contains no tryptophan, nine phenylalanines, and a single tyrosine residue.…”

Section: Protein Purificationmentioning

confidence: 99%

Interlobe communication in multiple calcium‐binding site mutants of Drosophila calmodulin

1996

View full text Add to dashboard Cite

We have generated mutants of Drosophila calmodulin in which pairs of calcium-binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the -Z position glutamate residue with glutamine. Mutants inactivated in both N-terminal sites (B12Q) or both C-terminal sites (B34Q), and two mutants with one N-and one C-terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV-difference spectroscopy and near-UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr-138) of the protein. These studies uncovered four situations in which Tyr-138 in the C-terminal lobe responds to a change in the calcium-binding properties of the N-terminal lobe. Further, they suggest that N-terminal calcium-binding events contribute strongly to the aberrant behavior of Tyr-138 seen in mutants with a single functional C-terminal calcium-binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr-138 produced by mutation of site 3 toward the wild-type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK-MLCK) established that all of the multiple binding site mutants are poor activators of SK-MLCK. Thus, globally, the calcium-induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr-138 within the crystal structure of calmodulin suggests that effects of the N-terminal lobe on this residue may be mediated via changes to the central linker region of the protein.

show abstract

“…Our results led to the suggestion that the integration of Ndomain, C-domain and the linker peptide is essential for activation of enzymes. We propose that the three regions of calmodulin have their own assigned functions in activating enzymes, which is in contrast to the view that the different domains of calmodulin are working for the activation of different enzymes (8). 1 Present address: Institute of Immunological Sciences, Hokkaido University.…”

mentioning

confidence: 62%

“…However, Newton et al (8) have reported that calcineurin and PDE could not be activated by these fragments when purified using HPLC.…”

mentioning

confidence: 97%

Calmodulin Fragments Can Not Activate Target Enzymes

Minowa¹,

Yazawa²,

Sobue³

et al. 1988

The Journal of Biochemistry

View full text Add to dashboard Cite

Under conditions where nM level of calmodulin was able to show full activation of myosin light chain kinase and cyclic-nucleotide phosphodiesterase, the fragments of calmodulin at concentrations as high as 20 microM failed to activate these enzymes in the presence of Ca2+. The fragments tested were Ala1-Lys75 (F12), Ala1-Arg74 (F12'), Lys75-Lys148 (F34'), Met76-Lys148 (F34'), Asp78-Lys148 (F34), Ala1-Arg106 (F123), and His107-Lys148 (F4). Purification of the proteolytic fragments through HPLC was necessary to remove contaminant calmodulin. Among the fragments, that corresponding to the C-terminal half domain inhibited myosin light chain kinase activity with the inhibition constant of 13 microM. The integrated structure of calmodulin consisting of N-terminal half domain, C-terminal half domain, and the linker peptide was indispensable for the enzyme activation. We discuss the functions of the two structural domains (N-domain and C-domain) in the activation of various enzymes.

show abstract

Large Scale Preparation of Calmodulin

Cited by 20 publications

References 26 publications

Factors responsible for the Ca²⁺‐dependent inactivation of calcineurin in brain

Factors responsible for the Ca²⁺‐dependent inactivation of calcineurin in brain

Interlobe communication in multiple calcium‐binding site mutants of Drosophila calmodulin

Calmodulin Fragments Can Not Activate Target Enzymes

Contact Info

Product

Resources

About

Large Scale Preparation of Calmodulin

Cited by 20 publications

References 26 publications

Factors responsible for the Ca2+‐dependent inactivation of calcineurin in brain

Factors responsible for the Ca2+‐dependent inactivation of calcineurin in brain

Interlobe communication in multiple calcium‐binding site mutants of Drosophila calmodulin

Calmodulin Fragments Can Not Activate Target Enzymes

Contact Info

Product

Resources

About

Factors responsible for the Ca²⁺‐dependent inactivation of calcineurin in brain

Factors responsible for the Ca²⁺‐dependent inactivation of calcineurin in brain