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  • calmodulin-binding peptides  (1)
  • phosphatidylinositol turnover  (1)
Materialart
Erscheinungszeitraum
Schlagwörter
  • 1
    Digitale Medien
    Digitale Medien
    Springer
    Molecular and cellular biochemistry 127-128 (1993), S. 19-30 
    ISSN: 1573-4919
    Schlagwort(e): phosphorylase kinase ; calmodulin ; calmodulin-binding peptides ; Ca2+-binding
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Chemie und Pharmazie , Medizin
    Notizen: Abstract Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the α subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the β subunit and a peptide from the α subunit present in a region deleted in the α′ isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca2+ binding to calmodulin in presence of peptides was measured. By this way, the peptides α 542–566, α 547–571, α 660–677 and β 597–614 have been found to bind specifically to calmodulin. Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the α and β subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in γ as well as to two regions in α and β. Exogenous calmodulin can bind to two regions in α and in β. A binding stoichiometry of 0.8mol of calmodulin/αβγδ protomer of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23nM calmodulin which is in the affinity range of calmodulin binding peptides from β to calmodulin. Therefore, binding of exogenous calmodulin to β activates the enzyme. A model for switching endogenous calmodulin between α, β and γ and modulation of ATP binding to α as well as Mg2+/ADP binding to β by calmodulin is presented.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 2
    ISSN: 1573-4919
    Schlagwort(e): phosphatidylinositol turnover ; glycolytic pathway ; skeletal muscle
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Chemie und Pharmazie , Medizin
    Notizen: Abstract Skeletal muscle triads are possessing the whole set of enzymes of the phosphatidylinositol (PI)-linked signal generating pathway, PI-kinase, PI(4)P-kinase, and PI(4,5)P2-phospholipase C (PLC). The activities of these enzymes are comparable to those found in other cell types for which a functional role of the PI-pathway in intracellular signal transduction has been established. For skeletal muscle an unequivocal function and an initiating signal for Ins(1,4,5)P3-liberation is still unknown. However, the observed Ca-dependency of PLC activity suggests that here Ins(1,4,5)P3 production is a consequence rather than a cause of increasing cytosolic Ca2+. Recently, the glycolytic enzyme aldolase, whose activity can be modulated by inositol polyphosphates, has been localized in the triadic structure. The enzyme which has a high affinity to Ins(1,4)P2, Ins(1,4,5)P3 and Ins(1,3,4,5)P4, seems to be compartmentalized to the junctional foot structure from which it is released upon binding of these molecules. This phenomenon could reflect a capability for regulation of the glycolytic flux even for aldolase, especially if a non steady-state situation in the junctional gap is considered. Meanwhile we have accumulated evidence for the operation of a partial glycolytic sequence in the junctional region established by the enzymes aldolase, glyceraldehyde-3-P (GAP) dehydrogenase and phosphoglycerate kinase. This system is able to produce ATP upon oxidation of GAP and could be, because of the inositol polyphosphate-sensing abilities of aldolase, a target for the membrane associated PI-pathway. The ATP production is however transient which indicates the coupling to an ATP hydrolyzing reaction. Thus, it appears that the ATP produced by the membrane associated system is effectively utilized by an ATP consuming membrane localized system like PI-metabolism or protein kinases. There are indications that exogeneously added ATP does not equilibrate with the ATP synthesized in the junctional region which suggests an effective structural or kinetical compartmentalization of this system. Therefore it is hypothesized that the ATP synthesized by the membrane associated glycolytic sequence is utilized in membrane localized reactions.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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