Preparation and properties of calcium-dependent resins with increased selectivity for calmodulin

Hart, Russell C.; Hice, Rita E.; Charbonneau, Harry; Putnam-Evans, Cindy; Cormier, Milton J.

doi:10.1016/0003-2697(83)90753-4

Cited by 12 publications

(5 citation statements)

References 17 publications

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“…Bovine brain calmodulin was purified according to Hart et al (1983). The affinity resin was the kind gift of Dr. Russell Hart of the University of Georgia.…”

Section: Methodsmentioning

confidence: 99%

Probable role of amphiphilicity in the binding of mastoparan to calmodulin

McDowell¹,

Sanyal²,

Prendergast³

1985

Biochemistry

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Two-dimensional helical wheel diagrams and calculations of mean hydrophobic moments show mastoparan, mastoparan X, and Polistes mastoparan to have all the properties expected for amphiphilic helices. Circular dichroic properties are consistent with a random form for these peptides in dilute aqueous solution, but greater than 50% helix is apparent when the peptides are dissolved in 70% trifluoroethanol/water mixtures (v/v) or when the peptides are bound to calmodulin. Changes in the fluorescence spectra, anisotropy, and accessibility of tryptophan whose indole side chain is on the apolar surface of the amphiphilic helix imply a significant role for the apolar surface in the binding of the mastoparans and another amphiphilic peptide, melittin, to calmodulin. These data provide a useful model for designing high-affinity synthetic peptide inhibitors of calmodulin.

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“…Bovine brain calmodulin was purified according to Hart et al (1983). The affinity resin was the kind gift of Dr. Russell Hart of the University of Georgia.…”

Section: Methodsmentioning

confidence: 99%

Probable role of amphiphilicity in the binding of mastoparan to calmodulin

McDowell¹,

Sanyal²,

Prendergast³

1985

Biochemistry

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“…Testing the binding capacity of TAPP Sepharose with pure CaM resulted in a 5.5 ± 1.3 mg CaM bound and eluted per mL settled resin, a comparable binding capacity to the previously reported 5.7 mg CaM/mL settled resin. 14 , 15 Testing the purification ability of 10 mL settled TAPP Sepharose resin with CaM-expressing bacterial lysate resulted in 95 mg of CaM at ≥95% purity as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( Supporting Information ). CaM purified by TAPP Sepharose resin was compared to that purified by the more common phenyl Sepharose resin.…”

Section: Resultsmentioning

confidence: 99%

“…When CaM binds the calcium ion, its exposed hydrophobicity is greatly increased. Unfortunately, phenyl Sepharose purification has drawbacks including the necessity of two consecutive columns and frequent additional purification methods such as ion exchange chromatography or heat treatment as phenyl Sepharose alone does not fully purify CaM as determined by gel electrophoresis. − Another method for CaM purification is the use of a 2-trifluoromethyl-10-aminopropylphenothiazine (TAPP) Sepharose affinity resin. − In our experience, TAPP Sepharose requires only one column and fully purifies CaM from bacterial lysate without any additional steps. − Despite its superiority, TAPP Sepharose is rarely used for CaM purification as it is not commercially available. Instead, the TAPP molecule must be synthesized and coupled to epoxide-activated Sepharose.…”

Section: Introductionmentioning

confidence: 99%

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Improved Synthesis of 2-Trifluoromethyl-10-aminopropylphenothiazine: Making 2-Trifluoromethyl-10-aminopropylphenothiazine Readily Available for Calmodulin Purification

et al. 2018

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“…Materials. Soybeans of Amsoy variety were purchased locally; full-fat soybean flour available commercially was obtained from Archer Daniels Midland, Decatur, IL; DEAE cellulose (DE-52) was from Whatman Co.; calmodulin coupled to Sepharose 4B was either purchased from Sigma Chemical Co., St. Louis, MO, or synthesized in our laboratory according to the procedure of Hart et al (1983); 5'-nucleotidase, cAMP, and PDE were obtained from Sigma. All reagents used were of analytical grade.…”

Section: Methodsmentioning

confidence: 99%

Soybean metal-binding proteins: isolation of a phosphatase that inhibits calmodulin activity

Wang¹,

Ling²,

Ho³

1988

J. Agric. Food Chem.

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Soybean seeds and seedlings contain monophosphatase (MP), phosphodiesterase (PDE), and a calmodulin (CAM) that stimulates PDE activity. Interactions of these proteins likely regulate phosphate metabolism in the seed. This paper identifies proteins that interact with CAM and defines the function of CAM in soybeans. We find that an M P enzyme can inhibit CAM activity and is associated with CAM in soybean whey. MP enzyme is separated into a single peak by DEAE chromatography in the presence of a calcium chelator, EGTA, and further chromatography on a calmodulin affinity column. Its binding to the calmodulin affinity column is reversible and depends on the presence of Ca2+ ions. HPLC, gel electrophoresis, and gel filtration analyses of the MP enzyme show that it contains two subunits (24 and 20 kDa), but neither of the subunits alone exhibits phosphatase activity. The 20-kDa subunit retains the ability to inhibit CAM stimulation of PDE. Recombination of the two subunits yields MP activity that can be further enhanced by the addition of CAM. MP activity appears to be less stable than the CAM inhibitor activity.As part of studies on CAM in soybeans, we isolated the protein from soybean whey by chromatographing extracts on DEAE and then subjecting collected fractions to affinity chromatography on W-7 agarose to obtain pure CAM. The purified protein is similar to calmodulin from spinach and bovine brain in terms of ability to stimulate PDE activity and electrophoretic mobility as a 18-kDa protein (unpublished result). Its behavior during purification suggests that it is associated with other calcium-binding proteins in the whey, particularly in the presence of Ca2+ ions. A calcium chelator (EGTA) is required during DEAE chromatography to separate CAM from other whey proteins. We now have evidence that the proteins to which CAM binds include an MP enzyme that hydrolyzes p-nitrophenyl phosphate. This MP enzyme also inhibits the stimulatory action of CAM on PDE. It is a multisubunit

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Preparation and properties of calcium-dependent resins with increased selectivity for calmodulin

Cited by 12 publications

References 17 publications

Probable role of amphiphilicity in the binding of mastoparan to calmodulin

Probable role of amphiphilicity in the binding of mastoparan to calmodulin

Improved Synthesis of 2-Trifluoromethyl-10-aminopropylphenothiazine: Making 2-Trifluoromethyl-10-aminopropylphenothiazine Readily Available for Calmodulin Purification

Soybean metal-binding proteins: isolation of a phosphatase that inhibits calmodulin activity

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