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Calcium‐Stimulated Protein Phosphorylation in Synaptic Membranes
Author(s) -
Sorensen Roger G.,
Mahler Henry R.
Publication year - 1983
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1983.tb13577.x
Subject(s) - phosphorylation , calcium , kinase , biochemistry , chemistry , membrane , substrate (aquarium) , biophysics , protein kinase a , protein phosphorylation , biology , ecology , organic chemistry
Synaptic membranes from rat brain contain several calcium‐requiring protein kinase (PK) activities with different substrate specificities: (a) an activity (Ca H ‐PK) effective at high concentrations of Ca 2+ ion in the absence of Mg 2+ (active on class F substrates); (b) a (Ca + Mg)‐PK activity that is mediated by Ca 2+ ion in the presence of Mg 2+ (active on class B substrates); (c) (Ca‐CaM)‐PK activities that exhibit simultaneous requirements for both Ca 2+ ion and CaM (for class C and D substrates). Also described are three activities (d–f) that do not require Ca 2+ ion: (d) a Mg‐PK activity in which the presence of Ca 2+ causes the inhibition of phosphorylation (active on class A substrates); (e) an activity affecting a diverse group of substrates (class E substrates), the phosphorylation of which occurs in the presence of Mg 2+ ion alone (Mg‐PK activity) and is unaffected by the addition of Ca 2+ ion and CaM, the substrates of which show different responses to several types of inhibitors; and, finally, (f) the previously well characterized cAMP‐dependent PK activities. Several of the substrates of these kinases have been identified in a fairly unambiguous manner: among them are P43 (class A), as the α subunit of pyruvate dehydrogenase; P54 (class B), as the presynaptic protein B50; and the doublet P75–P80, as proteins IA and IB of Ueda and Greengard. The most interesting activity is that requiring both Ca 2+ and CaM. The half‐maximal stimulation (K 0.5 ) for Ca 2+ in the presence of CaM was found to be 1.0 μM Ca 2+ F in untreated membranes. There is little change in this value on prior EGTA extraction of the membranes, which removes the bulk of its Ca 2+ and reduces its residual CaM by ≥ 50%. The apparent K 0.5 for CaM in the presence of excess Ca 2+ ion was found to equal 0.4 μg per reaction mixture (8 μg/ml) or 1.35 μg per reaction mixture (27 μg/ml), for the untreated and EGTA‐treated membranes, respectively.