Abstract
Sodium borohydride-reduced phosphorylase b dissociates completely to a monomeric form in 6.7% formamide, as determined by sedimentation velocity and equilibrium methods. No dissociation of the native enzyme occurs under these same conditions. A slow inactivation of NaBH4-reduced phosphorylase b occurs in formamide, and the degree of inactivation in different formamide concentrations seems related to the extent of dissociation. Removal of formamide by dialysis restores the enzymic activity and subunit structure. Results of several experiments suggest that the monomeric form is catalytically active. Approximately 30% activity remains after 1 hour of incubation in 6.7% formamide. After this stage, little change of enzymic activity occurs even after 7 hours of incubation. After 1 hour, sedimentation velocity experiments show that a single component is present with an s20,w of 5.6 S, indicating that dissociation is complete at this time. No change in s20,w was observed in the presence of substrates, glucose 1-phosphate, amylodextrin, and activator, AMP, indicating that reassociation of the monomer does not occur under conditions that closely approximate those used in the assay. Kinetic studies showed that the affinities of the enzyme with respect to its substrates and activator were reduced markedly by formamide. No homotropic interactions were seen with respect to AMP for the reduced enzyme incubated in formamide, further indicating the presence of an active monomer. Formamide affects the spectral properties of enzyme-bound pyridoxal phosphate in NaBH4-reduced phosphorylase b.
Highlights
Sodium borohydride-reduced phosphorylase b dissociates completely to a monomeric form in 6.7% formamide, as determined by sedimentation velocity and equilibrium methods
Ultracentrifugal studies showed that NaBHtreduced phosphorylase b is sensitive to low concentrations of formamide, whereas native phosphorylase 6 is not
NaBHa-reduced enzyme, upon incubation with 6.7% formamide, shows a time-dependent change of enzymic activity, and after several hours, it has approximately 30% of the activity of enzyme not incubated with formamide
Summary
Crystalline rabbit muscle phosphorylase 6 was prepared according to a procedure of Fischer and Krebs [15] and was recrystallized three times before use. NaBHd-reduced phosphorylase b was obtained by a method described by Graves et al [16]. Dipotassium glucose l-phosphate and AMP were purchased from Calbiochem. Shellfish glycogen was a product of Sigma Chemical Co. The glycogen was further purified according to the procedure of Sutherland and Wosilait [17]. 99%, was purchased from Matheson, Coleman and Bell. Formamide was further purified by distillation at reduced pressure. Amylodextrin, DPs7, was a gift from Dr John Robyt, Iowa State University. All other chemicals were obtained from commercial sources and were reagent grade
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
