Purification and Characterization of Two High-Affinity (Adenosine 3',5'-Monophosphate)-Binding Proteins from Yeast. Identification as Multiple Forms of Glyceraldehyde-3-Phosphate Dehydrogenase

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Abstract
Two high-affinity cAMP-binding proteins (I and II) were purified to homogeneity from baker''s yeast by a procedure avoiding proteolytic damage. These proteins were identified as multiple forms of glyceraldehyde-3-phosphate dehydrogenase The 2 cAMP-binding proteins are similar in affinities for cAMP, have identical elution volumes on gel filtration, and contain one type of subunit (MW 37,500). The form II of glyceraldehyde-3-phosphate dehydrogenase is free of NAD+ and has a Kd of 1.3 .times. 10-6 M with respect of cAMP. A marked concentration-dependent self-association of the subunits of the form-II protein was revealed by Yphantis sedimentation equilibrium studies. Significant monomer concentrations are present at total concentrations less than 0.02 mg/ml. Coventional sedimentation equilibrium analyses indicated a tetramer MW of 170,000. The high-affinity binding of cAMP to glyceraldehyde-3-phosphate dehydrogenase may significantly reduce intracellular cAMP levels and is discussed in relation to the nature of eukaryote cAMP-binding proteins with similar native or subunit MW values which are at present functionally undefined.