Refolding parameters for the allosteric homodimeric guanylyl cyclase catalytic core from the atrial natriuretic peptide receptor.
Protein folding continues to be an important biophysical topic in molecular biology. We report the parameters for successfully refolding the guanylyl cyclase core of the ANP receptor, an allosteric homodimeric enzyme. Urea was a better chaotropic solvent than guanidine HCl, and physiological salt concentrations and pH were needed for optimal recovery of enzymatic activity. Renaturation was more sensitive to alkaline compared to acidic deviations in solvent conditions. The time course of refolding was sigmoidal producing an enzyme with a specific activity of 16,000 pmol cGMP/min/mg using 60 microM concentration of substrate. Additional factors are described in this unusual case of renaturing an allosteric homodimeric enzyme in vitro.
Thorpe
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DS
Department of Discovery Biology, Selectide Corporation, Tucson, Arizona 85737, USA. davidthorpe@hmri.com
Niu
S
S
Morkin
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D000494:Allosteric Regulation; D002384:Catalysis; D002460:Cell Line; D006162:Guanylate Cyclase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism*; D012492:Salts; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012997:Solvents; D013379:Substrate Specificity
D012492:Salts; D015842:Serine Proteinase Inhibitors; D012997:Solvents; D010664:Phenylmethylsulfonyl Fluoride; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor