Yasushi Iwasa

Occurrence of Two Types of Ca2+-Dependent Protein Kinases in the Cytosol Fraction of the Brain

Abstract— Two types of Ca2+‐dependent protein kinases were demonstrated and partially purified from the cytosol fraction of rat brain by DEAE‐cellulose, Sephadex G‐200, and calmodulin‐affinity column chromatography, using endogenous proteins and chicken gizzard myosin light chains as substrates. The molecular weights of the enzymes were 88,000 (peak I) and 120,000 (peak II) on gel filtration. Peak I had no affinity for calmodulin, whereas peak II had a high affinity for it, with a Ka value of 16.7 nm. The Ka values of peaks I and II for Ca2+ were 2.4 and 1.6 μm, respectively.

Demonstration of a high affinity Ca2+-ATPase in rat liver plasma membranes☆

Abstract Rat liver plasma membranes contained a high affinity Ca 2+ -ATPase which had an apparent half saturation constant of 0.2 μM for calcium. The Ca 2+ -ATPase was not stimulated by adding magnesium and/or calmodulin. Conversely, the addition of these substances diminished the calcium-stimulation of the ATPase. Orthovanadate (7 nM-2 mM), mitochondrial ATPase blockers (NaN 3 , KCN, dicyclohexylcarbodiimide), Na + , K + and ouabain had no effect on the ATPase activity. The ATPase was separated from nonspecific divalent cation stimulatable ATPase (Mg 2+ -ATPase) by solubilization with Triton X-100 followed by a Sephadex G-200 column chromatography and showed an apparent molecular weight of 200,000.

Calmodulin-like activity in the soluble fraction of Escherichia coli

Abstract A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca 2+ -dependent cyclic AMP phosphodiesterase of Escherichia coli . The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca 2+ ,Mg 2+ )ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca 2+ -dependent fashion with an apparent K a of 5 × 10 −5 M . The factor and brain calmodulin had no effect on the phosphodiesterase of E. coli . It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes.

Interaction of calmodulin with chromatin associated proteins and myelin basic protien

Abstract Chromatin associated proteins such as histone and protamine and myelin basic protein inhibit the activities of calmodulin-dependent cyclic nucleotide phosphodiesterase and myosin light chain kinase supported by Ca 2+ and calmodulin in a dose-dependent manner. The inhibition of these enzymes induced by the proteins is completely abolished by high concentration of calmodulin but not with that of Ca 2+ . Kinetic analysis of this inhibition reveals that the proteins inhibit these enzyme activities in a competitive fashion with calmodulin. The proteins bind to calmodulin on a calmodulin coupled-agarose affinity column in the presence of Ca 2+ . It is suggested that endogenous basic proteins interact with calmodulin and may modulate intracellular regulation by calmodulin.

Multiple specificities of brain Ca2+- and calmodulin-dependent protein kinase for substrate.

The purified Ca2+- and calmodulin-dependent protein kinase from rat brain, which has a M.W. of 120,000 by gel filtration analysis, showed a broad substrate specificity. In addition to myosin light chain from chicken gizzard, the enzyme phosphorylated myelin basic protein, casein and two endogenous substrates in a Ca2+- and calmodulin-dependent manner. In contrast, chicken gizzard myosin light chain kinase exclusively phosphorylated myosin light chain.

Modulation by phosphorylation of interaction between calmodulin and histones

Ca*+-Sensitive cyclic nucleotide phosphodiesterase (EC 3 .I .4.17) and its Ca*+-dependent modulator protein were first reported in [l-3]. An activator protein of phosphodiesterase in bovine brain was reported independently [4]. This protein, now called calmodulin, is thought to be a ubiquitous protein in eukaryotic cells and to play a pivotal role in regulation of Ca*+-dependent cellular processes through activation of several Ca*+-sensitive enzymes [5-81. Some cellular proteins, other than Ca*‘-sensitive enzymes, which interact with calmodulin in a Ca*+dependent fashion have been demonstrated in various mammalian and avian cells and Escherichia coli [ 9161. However, physiological functions of these proteins are still under exploration. Cellular basic proteins such as histone, protamine and myelin basic protein have been shown to interact with calmodulin [17-211. These basic proteins were suggested to bind to calmodulin in a Ca*+-dependent manner [ 17,181. However, in [ 191 this interaction was postulated to depend mainly on the high charge density between calmodulin and the basic proteins [ 191. Here we show that the interaction of calmodulin with histones is a Ca*+and charge density-dependent reaction and that this interaction is modulated by phosphorylation of histones.

A calcium-dependent cyclic nucleotide phosphodiesterase from Escherichia coli

Ca2+ is considered to have an important role in cellular response to hormonal stimulation; the Ca2+dependent cyclic nucleotide phosphodiesterase (EC 3.1.4.17) present in many eukaryotic cells [I] plays an important role. In prokaryotes, phosphodiesterases have been reported but are Ca’+-independent [2,3]. The enzyme of Escherichia coli requires Fe2+ or an activator protein (M, 90 000) for its activity [2]. Here, we report the occurrence of an additional species of phosphodiesterase in the soluble fraction of E. coli. The enzyme is Ca’+-dependent and hydrolyzes both cyclic AMP and cyclic GMP. The existence of Ca’+-dependent phosphodiesterase suggests that, in prokaryotes as well as in eukaryotes, one of the roles of Ca2+ in hormonal regulation is acceleration of cyclic nucleotide degradation through the activation of the enzyme.

A high affinity Ca2+ -ATPase in C57 black mouse liver plasma membranes.

Plasma membranes of a cell contain 2 kinds of Ca2÷-extruding mechanisms to maintain [Ca 2÷] of cytoplasm at submicromolar levels, ATP-dependent and extracellular NaLdependent mechanisms. In plasma membranes of several tissues, the ATP-dependent Ca2+-pumps associate with (Ca 2+ + Mg2÷)-ATPase activity [ 1-3 ]. In the case of erythrocyte plasma membrane, a reconstitution study has clearly demonstrated the identity of the (Ca 2÷ + Mg2+)-ATPase and the Ca2+-extruding pump [4]. A variant type of (Ca 2÷ + Mg2÷)-ATPase has been shown in adipocyte and corpus luteum plasma membranes [5,6]. This ATPase has a high affinity for, Ca 2÷ with Ko.s of 0.14-0.3/aM and some other characteristics consistent with those of erythrocyte (Ca 2÷ + Mg2+)ATPase. However, this ATPase shows different sensitivity to Mg 2÷, calmodulin and calmodulin-antagonists from that of erythrocyte enzyme. In rat liver plasma membrane, no (Ca 2÷ + Mg2÷)ATPase had been detected although a Ca2÷-extruding activity in rat liver cells was shown to be ATP-dependent and extracellular Na+-independent [7,8]. A (Ca 2+ + Mg~+)-ATPase distinct from (Ca 2÷ + Mg2+)ATPases of erythrocyte, adipocyte and corpus luteum has been reported in rat liver plasma membranes [9]. This enzyme has very high affinities for Ca 2÷ (Ko.s = 13 nM) and Mg 2÷ (Ko.s = <12/aM), and is activated

A heat-stable inhibitor protein for bovine brain cyclic nucleotide phosphodiesterase from Escherichia coli

dependent reactions including activation of several Ca2+-dependent enzymes and demonstrated in various eukaryotes [3-51. The activation mechanism of Ca’+- dependent enzymes by calmodulin involves the bind- ing of Ca2+ to calmodulin followed by the association of the enzyme with the Ca2+ . calmodulin complex [3-51. Inhibitor proteins of Ca2+-dependent phospho- diesterase have been reported in mammalian tissues, and one type of them is heat-stable [6-l 21. The inhib- itor proteins inhibit the Ca2+ . calmodulin-activated phosphodiesterase by forming a complex of inhib- itor . Ca2+ . calmodulin [6-l 71. Namely, the inhibitor proteins are considered to be a kind of calmodulin- binding protein. Furthermore, other kind of calmo- dulin-binding proteins have also been reported, which functions should be resolved, in various mammalian tissues [ 13-171. In other than mammalian tissues, there has been no report concerning a calmodulin- binding protein. We have found calmodulin-like activ- ity and a Ca2+-dependent phosphodiesterase, which is not sensitive to calmodulin, in the soluble fraction