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PubMed2001

Direct visualization of the calmodulin subunit of phosphorylase kinase via electron microscopy following subunit exchange.

Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme, (alphabetagammadelta)4. To introduce specifically labeled calmodulin into the phosphorylase-b kinase complex for its eventual visualization by electron microscopy, we have developed a method for rapidly exchanging exogenous calmodulin for the intrinsic delta subunit. This method exploits previous findings that low concentrations of urea in the absence of Ca(2+) ions cause the specific dissociation of only the delta subunit from the holoenzyme [Paudel, H. K., and Carlson, G. M. (1990) Biochem. J. 268, 393-399]. In the current study, phosphorylase-b kinase was incubated with excess exogenous calmodulin and a threshold concentration of urea to promote exchange of its delta subunit with the exogenous calmodulin. Size exclusion HPLC was then used to remove the excess calmodulin from the holoenzyme containing exchanged delta subunits. Using metabolically labeled [35S]calmodulin to allow quantification and optimization of exchange conditions, we achieved exchange of approximately 10% of all delta subunits within 1 h, with the exchanged holoenzyme retaining full catalytic activity. Calmodulins derivatized with Nanogold for visualization by scanning transmission electron microscopy were then exchanged for delta, which for the first time allowed localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex. The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzyme's catalytic gamma subunit.

Animals↗
PubMed2002

The calmodulin-binding domain of the catalytic gamma subunit of phosphorylase kinase interacts with its inhibitory alpha subunit: evidence for a Ca2+ sensitive network of quaternary interactions.

Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca(2+) ions, of the (alphabetagammadelta)(4) phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory alpha and catalytic gamma subunits. The gamma subunit is also known to interact with the delta subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca(2+) ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the gamma (residues 343-386) and alpha (residues 1060-1237) subunits interact. The association of this region of alpha with gamma was corroborated by the isolation of a cross-linked fragment of alpha containing residues 1015-1237 from an alpha-gamma dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of gamma that we found to interact with alpha has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163), we tested the influence of Ca(2+) on the conformation of the alpha subunit and found that the region of alpha that interacts with gamma was, in fact, perturbed by Ca(2+). The results herein support the existence of a Ca(2+)-sensitive communication network among the delta, gamma, and alpha subunits, with the regulatory domain of gamma being the primary mediator. The similarity of such a Ca(2+)-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the gamma subunit of PhK.

Animals↗
PubMedDate not supplied

A computational analysis of substrate binding strength by phosphorylase kinase and protein kinase A.

Protein kinases exhibit various degrees of substrate specificity. The large number of different protein kinases in the eukaryotic proteomes makes it impractical to determine the specificity of each enzyme experimentally. To test if it were possible to discriminate potential substrates from non-substrates by simple computational techniques, we analysed the binding enthalpies of modelled enzyme-substrate complexes and attempted to correlate it with experimental enzyme kinetics measurements. The crystal structures of phosphorylase kinase and cAMP-dependent protein kinase were used to generate models of the enzyme with a series of known peptide substrates and non-substrates, and the approximate enthalpy of binding assessed following energy minimization. We show that the computed enthalpies do not correlate closely with kinetic measurements, but the method can distinguish good substrates from weak substrates and non-substrates.

Binding Sites↗
PubMed1994

Elevated phosphorylase kinase activity in psoriatic epidermis: correlation with increased phosphorylation and psoriatic activity.

To determine whether abnormal activity of a calmodulin-containing enzyme which catalyses phosphorylation reactions may play a pathogenetic role in psoriasis, the presence and activity of phosphorylase kinase (PK) in human epidermis were determined in patients with untreated/active psoriasis (n = 10), treated/resolving psoriasis (n = 10), and non-psoriatic controls (n = 10). Biopsies were taken from involved and uninvolved skin for PK, organic phosphorus, and inorganic phosphate estimation, and light and electron microscopy. The enzyme was present in involved and uninvolved skin of every patient in the study. PK activity (units/mg protein) was significantly higher in active psoriasis than in resolving psoriasis and controls. PK activity correlated directly with organic phosphorus levels, and inversely with the extent of cellular glycogenolysis measured by the depletion of glycogen granules within the keratinocytes. The study demonstrates that PK is present in both psoriatic and normal epidermis, with significantly higher levels in active psoriasis. Furthermore, higher levels of PK activity, glycogenolysis and phosphorylation are associated with increased clinical psoriatic activity. We conclude that PK, a calmodulin-containing enzyme, is involved in regulating calcium-dependent phosphorylation events in human epidermis, and disturbance of its activity may play a key role in the clinical manifestations of psoriasis.

Cytoplasmic Granules↗
PubMed1988

Allosteric nucleotide specificity of phosphorylase kinase: correlation of binding, conformational transitions, and activation. Utilization of lin-benzo-ADP to measure the binding of other nucleoside diphosphates, including the phosphorothioates of ADP.

Recent work has shown that ADP is an allosteric activator of nonphosphorylated phosphorylase kinase from rabbit skeletal muscle (Cheng, A., Fitzgerald, T. J., and Carlson, G. M. (1985) J. Biol. Chem. 260, 2535-2542). The specificity of the allosteric site for nucleoside diphosphates is further investigated in this study. Only purine nucleoside diphosphates are capable of causing allosteric activation, and an amino group at position 2 or 6 of the purine ring is required. Comparisons are made of the abilities of 5'-diphosphate analogs of ADP, including phosphorothioates, to activate, to bind, and to induce in the enzyme's beta subunits conformational changes associated with activation. Binding is measured by competition titrations utilizing fluorescence polarization of lin-benzo-ADP, itself an allosteric activator; and conformational changes are measured by partial proteolysis and chemical cross-linking. When measured at an identical percentage of saturation at the allosteric site, the abilities of ADP analogs to induce conformational changes in the beta subunits parallel their abilities to activate the holoenzyme. An unmodified beta-phosphate of ADP, although not necessary for binding at the allosteric site, is needed to fully drive the activating conformational transition. The activating nucleoside diphosphate appears to be the free species, as opposed to its Mg2+ complex.

Adenosine Diphosphate↗
PubMed1989

Skeletal muscle glycogen content, structure, and metabolism are normal in rats with hepatic glycogen phosphorylase kinase deficiency.

Skeletal muscle glycogen content and structure, and the activities of several enzymes of glycogen metabolism are reported for the hepatic glycogen phosphorylase b kinase deficient (gsd/gsd) rat. The skeletal muscle glycogen content of the fed gsd/gsd rat is 0.50 +/- 0.11% tissue wet weight, and after 40 hours of starvation this value is lowered 40% to 0.30 +/- 0.05% tissue wet weight. In contrast the gsd/gsd rat liver has an elevated glycogen content which remains high after starvation. The skeletal muscle phosphorylase b kinase, glycogen phosphorylase, glycogen synthase and acid alpha-glucosidase activities are 17.2 +/- 2.9 units/g tissue, 119.9 +/- 6.4 units/g tissue, 12.2 +/- 0.4 units/g tissue and 1.4 +/- 0.4 milliunits/g tissue, respectively, with approx. 20% of phosphorylase and approx. 24% of synthase in the active form (at rest). These enzyme activities resemble those of Wistar skeletal muscle, and again this contrasts with the situation in the liver where there are marked differences between the Wistar and the gsd/gsd rat. Fine structural analysis of the purified glycogen showed resemblance to other glycogens in branching pattern. Analysis of the molecular weight distribution of the purified glycogen indicated polydispersity with approx. 66% of the glycogen having a molecular weight of less than 250 X 10(6) daltons and approx. 25% greater than 500 X 10(6) daltons. This molecular weight distribution resembles those of purified Wistar liver and skeletal muscle glycogens and differs from that of the gsd/gsd liver glycogen which has an increased proportion of the low molecular weight material.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

X-linked liver glycogenosis type II (XLG II) is caused by mutations in PHKA2, the gene encoding the liver alpha subunit of phosphorylase kinase.

X-linked liver glycogenosis type II (XLG II) is a recently described X-linked liver glycogen storage disease, mainly characterized by enlarged liver and growth retardation. These clinical symptoms are very similar to those of XLG I. In contrast to XLG I patients, however, XLG II patients do not show an in vitro enzymatic deficiency of phosphorylase kinase (PHK). Recently, mutations were identified in the gene encoding the liver alpha subunit of PHK (PHKA2) in XLG I patients. We have now studied the PHKA2 gene of four unrelated XLG II patients and identified four different mutations in the open reading frame, including a deletion of three nucleotides, an insertion of six nucleotides and two missense mutations. These results indicate that XLG II is due to mutations in PHKA2. In contrast to XLG I, XLG II is caused by mutations that lead to minor structural abnormalities in the primary structure of the liver alpha subunit of PHK. These mutations are found in a conserved RXX(X)T motif, resembling known phosphorylation sites that might be involved in the regulation of PHK. These findings might explain why the in vitro PHK enzymatic activity is not deficient in XLG II, whereas it is in XLG I.

Amino Acid Sequence↗
PubMed1981

Interaction of phosphorylase kinase with the 2',3'-dialdehyde derivative of adenosine triphosphate. 2. Differential inactivation measured with various protein substrates.

The 2',3'-dialdehyde derivative of ATP was used as an affinity label to inactivate phsosphorylase kinase in either the presence or absence of Ca2+ and Mg2+ ions. Following inactivation, the residual activity of phorphorylase kinase toward various protein substrates was measured and compared with that retained for conversion of phosphorylase b. Three different classes of substrates were distinguished by this analysis. For the first class (glycogen synthase), inactivation proceeded at the same rate as that measured with phosphorylase conversion, regardless of whether the inactivation was carried out in the presence or absence of the metal ions. For the second class of substrates (troponin I and troponin T), inactivation of the kinase in either the presence or absence of the metals was much more rapid with phosphorylase as substrate. Phosphorylation of the third class of substrates (phosphorylase kinase itself and a synthetic tetradecapeptide) was inactivated in parallel with phosphorylase b when modification was performed in the absence of metals; however, inclusion of Ca2+ and Mg2+ ions in the inactivation mixture caused activity toward phosphorylase b to be lost more rapidly than that toward the alternative substrates. Our results are consistent with a model in which glycogen synthase and phosphorylase b are preferentially phosphorylated at one type of catalytic site in phosphorylase kinase and troponin I and troponin T at another.

Adenosine Triphosphate↗
PubMed1980

Phosphorylation of rabbit cardiac-muscle troponin I by phosphorylase kinase. The effect of adrenaline.

1. Incubation of rabbit cardiac-muscle troponin I with phosphorylase b kinase leads to the incorporation of .07-1.2 mol of Pi/mol. 2. The major site of phosphorylation is a serine residue at position 72. 3. Lesser amounts of phosphate are incorporated into threonine-138, threonine-162 and serine 20. 4. Serine-20 is the only site that contains a significant amount of phosphate before incubation with phosphorylase b kinase. 5. Unlike the situation with serine-20, the extent of phosphorylation of serine-72 and threonine-138 in the perfused rabbit heart does not change when the heart is exposed to adrenaline (4 microM).

Amino Acids↗
PubMed1992

Expression of the phosphorylase kinase gamma subunit catalytic domain in Escherichia coli.

The catalytic subunit of phosphorylase b kinase (gamma) and an engineered truncated form (gamma-trc, residues 1-297) have been expressed in Escherichia coli. The truncated protein included the entire catalytic domain as defined by sequence alignment with other protein kinases but lacked the putative calmodulin binding domain. Full-length protein was produced in insoluble aggregates. Some activity was regenerated by solubilization in urea and dilution into renaturating buffer but the activity was found to be associated with a smaller molecular weight component. Full-length protein could not be refolded successfully. The truncated gamma subunit was produced in the soluble fraction of the cell as well as in inclusion bodies. The insoluble protein was refolded by dilution from urea and purified to homogeneity, in a one step separation on DEAE-Sepharose to give a protein mol. wt 32,000 +/- 2000 with a high sp. act. of 5.3 mumol 32P incorporated into phosphorylase b(PPB)/min/nmol. Kinetic parameters gave Km for ATP 46 +/- 3 microM and Km for PPb 27 +/- 1 microM. The sp. act. and the Km values are comparable to those observed for the activated holoenzyme and indicate that the gamma-trc retains the substrate recognition and catalytic properties. The ratio of activities at pH 6.8/8.2 was 0.84. gamma-trc was inhibited by ADP with a Ki of 52 microM and was sensitive to activation by Mg2+ and inhibition by Mn2+, properties that are characteristic of the holoenzyme and the isolated gamma subunit. Calmodulin which confers calcium sensitivity on the isolated gamma subunit had no effect on the enzymic properties of gamma-trc.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1975

Control of platelet glycogenolysis; Activation of phosphorylase kinase by calcium.

An apparent enigma during platelet aggregation is that increased glycogenolysis occurs despite a fall in cyclic AMP levels; Activation by a classical cascade is therefore unlikely, and an alternative stimulus for phosphorylase a formation was sought. It was found that low levels of Ca-2+ markedly activate phosphorylase b kinase from human platelets, with a Ka of 0i muM Ca-2+, which is similar to that for the skeletal muscle enzyme; The kinase activity is unstable, and on enzyme ageing is a 50% loss in activity with the Ka decreasing to 0.33 muM Ca-2+. In unstilulated platelets, phosphorylase a was 13.3% of toal measured activity, and glycogen synthetase I was 32.3%. Aggregation induced by ADP did not change the percentage of I synthetase, while increasing that for phosphorylase a. Dibutyryl cyclic AMP did, as expected, increase the percentage of both phosphorylated enzymes; These findings suggest that the natural activator of platelet glycogenolysis during aggregation is Ca-2+, which directly stimulates phosphorylase b kinase without altering glycogen synthetase activity. The cyclic AMP-dependent protein kinase does not appear to be involved;

Adenosine Diphosphate↗