Abstract
Abstract Adenosine 5'-monophosphate, an inhibitor of the d-fructose 1,6-diphosphatase isolated from Candida utilis, induces a specific dissociation of this enzyme at pH 9.2. On the basis of sucrose density gradient centrifugation and gel filtration on Sephadex G-100, it may be concluded that the molecule has dissociated into subunits having one-half the molecular weight of the native enzyme. The dissociation occurs only at alkaline pH and in the cold. It is prevented by low concentrations of fructose 1,6-diphosphate (0.01 mm). d-Fructose 1,6-diphosphatase which has been desensitized to the inhibitory action of 5'-AMP is not dissociated. The results suggest that the dissociation induced by 5'-AMP may be an extreme manifestation of more subtle alterations in the quaternary structure of the enzyme which occur in the presence of substrate at physiological pH.
Highlights
Adenosine 5’-monophosphate,an inhibitor of the D-fructose 1,6diphosphatase isolated from Candida utilis, induces a specific dissociationof this enzyme at pH 9.2
We have previously reported that the D-fructose 1,6-diphosphatase isolated from C. utilis dissociates reversibly at pH 4.0 into molecules which have approximately one-half the molecular weight of the native enzyme [5]
EJect of Alkaline plf-At pH 7.0, n-fructose 1,6-diphosphatase was found to sediment in sucrcse density gradients as a symmetrical peak with a molecular weight (estimated according to Martin and Ames [6]) of approximately 100,000 (Fig. 1)
Summary
Adenosine 5’-monophosphate,an inhibitor of the D-fructose 1,6diphosphatase isolated from Candida utilis, induces a specific dissociationof this enzyme at pH 9.2. Uti1i.scan be rendered permanently insensitive to inhibition by AMP (i.e. irreversibly desensitized) by dinitrophenylation or iodination in the presence of fructose-l ,6-di-P [2] When so treated it retains the catalytic properties, pHactivity relationship, and molecular weight of the native enzyme. We have previously reported that the D-fructose 1 ,6-diphosphatase isolated from C. utilis dissociates reversibly at pH 4.0 into molecules which have approximately one-half the molecular weight of the native enzyme [5]. Gradients containing 5’-AMP (1.0 mM) could not be analyzed for protein at wave lengths lower than 220 rnp, and high concentrations of enzyme (100 to 200 pg) were required to demonstrate the absorbance peaks These tracings were frequently of poor quality but s&iced to confirm the presence of a single absorbance peak for the dissociated enzyme. All chemicals were obtained in reagent grade form from commercial sources
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