Purification and characterization of phosphorylase a from human platelets

Gergely, Pál; Castle, Alan G.; Crawford, N.

doi:10.1016/0020-711x(79)90053-3

Cited by 8 publications

(3 citation statements)

References 30 publications

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“…Gergely and Bot described partially phosphorylated hybrid forms of phosphorylase in rabbit muscle (16,17) and human platelets (18). The activities of these isoenzymes were intermediate between those of phosphorylases b and a. Karpatkin et al (19,20) found that incubation of human platelets with MgATP+ resulted in an increase in total phosphorylase activity and concluded that the data were "consistent with the presence in human platelets of inactive dimer and monomer species of phosphorylase, which require MgATP for activation."…”

supporting

confidence: 48%

“…In mammalian and nonmammalian tissues, forms ofphosphorylase have been described (16)(17)(18)29) that are less sensitive or insensitive to concentrations ofAMP that maximally activate normal human myophosphorylase. In canine liver (30), human platelets (18,19), and human leucocytes (31), there is a phosphorylase isozyme that remains inactive at concentrations of AMP that maximally activate phosphorylase b. Addition of up to 100 mM AMP did not activate McArdle myophosphorylase, suggesting that this enzyme, although it could be activated by phosphorylase b kinase, is not phosphorylase b.…”

Section: Methodsmentioning

confidence: 99%

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Phosphorylation of McArdle phosphorylase induces activity.

Cerri¹,

Willner²

1981

Proc. Natl. Acad. Sci. U.S.A.

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In McArdle disease, myophosphorylase deficiency, enzyme activity is absent but the presence of an altered enzyme protein can frequently be demonstrated. We have found that phosphorylation of this protein in vitro can result in catalytic activity. We studied muscle of four patients; all lacked myophosphorylase activity, but myophosphorylase protein was demonstrated by immunodiffusion or gel electrophoresis. Incubation of muscle homogenate supernatants with cyclic AMP-dependent protein kinase and ATP resulted in phosphorylase activity. The activated enzyme comigrated with normal human myophosphorylase in gel electrophoresis. Incubation with [y-32P]ATP resulted in incorporation of 32P into the band possessing phosphorylase activity. Activation of phosphorylase by cyclic AMP-dependent protein kinase was inhibited by antibodies to normal human myophosphorylase or by inhibitory protein to cyclic AMP-dependent protein kinase. Incubation of muscle homogenates with phosphorylase b kinase and ATP also resulted in phosphorylase activity. After the action of cyclic AMP-dependent protein kinase, the resulting activity was similar to that ofphosphorylase b. However, incubation with phosphorylase kinase resulted in activity similar

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supporting

confidence: 48%

Section: Methodsmentioning

confidence: 99%

Phosphorylation of McArdle phosphorylase induces activity.

Cerri¹,

Willner²

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…The formation of phosphorylase ab was demonstrated both in vitro [4,5] and in vivo [6][7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation-induced conformational changes in the phosphorylaseab hybrid as revealed by resolution of pyridoxal 5′-phosphate with imidazole citrate and cysteine

1992

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The accessibility of pyridoxal 5'-phosphates of the phosphorylase ab hybrid to resolution by imidazole citrate and cysteine was studied and compared with that of the b and a forms. Promotion of resolution of phosphorylated forms by raising the temperature or in the presence of glycogen indicates that the resistance of phosphorylase a and ab to resolution at 0 degrees C is due rather to their tetrameric state than their phosphorylation-related active conformation. The pattern of resolution of the ab hybrid was similar to that of the a and differed from that of the b forms in that it occurred at 30 degrees C and 37 degrees C but not at 0 degrees C, moreover, it did not show first-order kinetics. On the other hand, inhibition of resolution by ligands binding to the nucleotide site of phosphorylase reflected an intermediate sensitivity of the ab form between that of the b and a forms. We conclude that partial phosphorylation of phosphorylase b elicits conformational change(s) in both subunits which influence the monomer-monomer interactions and resolution of pyridoxal 5'-phosphates. Resistance of ab hybrid to monomerizing agents as imidazole citrate, comparable to that of other forms, argues for its stability, ruling out its reshuffling into mixtures of phosphorylase b and a.

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