Shiro Kakiuchi

Calcium dependent phosphodiesterase activity and its activating factor (PAF) from brain: Studies on cyclic 3′,5′-nucleotide phosphodiesterase (III)

Abstract Crude extract from rat brains revealed two different activities of cyclic 3′,5′-nucleotides phosphodiesterase, namely the basal activity and the calcium dependent activity, and the total activity was shown as the sum of these two. The calcium dependent enzyme activity required for a heat-stable, nondialyzable factor (PAF) present in the brain extract. Regulation of calcium ion on the enzyme activity, in the presence of PAF, was observed within the range of pCa 5.9 to 4.9 and seems to be the physiological mechanism.

Occurrence of a Ca2+- and modulator protein-activatable ATPase in the synaptic plasma membranes of brain.

Ca2*-dependent modulator protein was discovered as an activator of phosphodiesterase [ 1,2], or a protein factor required for the Ca*+-dependent activation of phosphodiesterase [3,4]. Later, this protein was shown to be structurally similar to troponin-C [5,6] and to cause Ca2+-dependent activation of several enzymes including phosphodiesterase, brain adenylate cyclase [7], myosin light chain kinases from skeletal muscles [g-10] and chicken gizzard muscle [ll], and actomyosin ATPase [ 12,131. Recently, two groups [14,15] have demonstrated that the activator protein [16,17] for the erythrocyte membrane ATPase is identical to this modulator protein. (Ca” t Mg2+)-ATPase activity was also found in brain tissue [ 18,191. However, requirement of brain enzyme for an activator has not been reported. In the present study, we are able to show the dependency of the activity of brain enzyme upon the modulator protein. Although brain (Ca2’ + Mg*‘)-ATPase activity was detected in all particulate fractions upon subcellular fractionation, only enzyme in the synaptic plasma membrane fraction was responsive to modulator protein.

Lack of tissue specificity of calmodulin: A rapid and high-yield purification method

The discovery of Ca2+-activatable phosphodiesterase [l] led to the discovery of the modulator protein which confers the Ca’+-sensitivity upon this enzyme [2,3]. An activator of phosphodiesterase was independently reported. The identity of both proteins was established subsequently [5]. This modulator protein nowadays termed calmodulin exhibits the Ca2+-dependent activation of a number of enzymes and is now regarded as an intracellular mediator of actions of Ca2+ [6]. Its ubiquitous distribution in the animal and plant kingdoms and its structural and functional conservativeness throughout molecular evolution are well established [6,7]. Although there is general agreement concerning the gross similarities among vertebrate calmodulins, minor differences in structure and mobility upon polyacrylamide gel electrophoresis were seen between calmodulin preparations from rat testis [ 81, bovine uterus [9] and bovine brain [ 10,111. It is thought that these differences are rather artifactual or due to mistaken assignments of the amino acid sequence, but this has not yet been proved or disproved. The present study supports the proposal that there is no difference between calmodulins from different tissues. It also describes a convenient purification method calmodulin giving an overall recovery of >70%. for

Control of the cytoskeleton by calmodulin and calmodulin-binding proteins

Abstract Calmodulin controls the contractile apparatus and cytoskeleton through two groups of calmodulin-binding proteins. Spectrin (erythrocyte) and spectrin-like proteins (brain and other tissues), both associated with membranes as the major member of the submembranous cytoskeleton, constitute one group. The other consists of τ protein (brain microtubules), caldesmon (smooth muscle) and a M r 135 000 protein (smooth muscle), collectively referred to as flip-flop switch proteins. These proteins interact with calmodulin and cytoskeletal protein (either tubulin or actin) alternately depending upon the concentration of Ca 2+ .

Effects of phospholamban phosphorylation catalyzed by adenosine 3′:5′-monophosphate- and calmodulin-dependent protein kinases on calcium transport ATPase of cardiac sarcoplasmic reticulum ☆

To elucidate the role of 22000-dalton protein phospholamban, a putative regulator of Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum, we examined the relationship between cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban and their effects on ATPase activity and calcium transport of cardiac sarcoplasmic reticulum. Cardiac microsomes were incubated with [gamma-32P]ATP or unlabeled ATP, catalytic subunit of cyclic AMP-dependent protein kinase and/or exogenous calmodulin, and subsequently assayed for ATPase activity and calcium uptake by cardiac sarcoplasmic reticulum. Cyclic AMP-dependent phosphorylation of phospholamban was independent of Ca2+, whereas calmodulin-dependent phosphorylation of phospholamban was dependent on Ca2+ within a range between 0.2 and 50 microM. Cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban occurred independently; when both kinases were operative, the amounts of phosphorylation were additive. Under these conditions, the phosphoproteins formed by cyclic AMP- and calmodulin-dependent protein kinases electrophoretically migrated as 11000-dalton components when sodium dodecyl sulfate-solubilized phosphoproteins were boiled prior to polyacrylamide gel electrophoresis. The ATPase activity was stimulated by either cyclic AMP- or calmodulin-dependent phosphorylation of phospholamban at Ca2+ concentrations up to 2 microM. The extents of stimulation of ATPase activity were additive when both types of phosphorylation were functional. Calcium uptake was similarly augmented by cyclic AMP- and/or calmodulin-dependent phosphorylation of phospholamban. These results indicate that Ca2+-dependent ATPase and calcium transport of cardiac sarcoplasmic reticulum are regulated by phospholamban phosphorylation catalyzed by cyclic AMP- and calmodulin-dependent protein kinases, thus suggesting a dual role of phospholamban in active calcium transport.

Mechanism of stimulation of Ca2+ plus Mg2+-dependent phosphodiesterase from rat cerebral cortex by the modulator protein and Ca2+

Abstract The activity of phosphodiesterase (“Ca2+ plus Mg2+-dependent” phosphodiesterase) of a preparation from brain was found to depend on the presence of both Ca2+ and a protein factor called modulator. It was shown by gel filtration that the active enzyme-modulator complex (MW, about 200,000) was formed from the modulator (MW, 28,000) and an inactive enzyme (MW, about 150,000) in the presence of Ca2+. When EGTA was added, this active enzyme-modulator complex dissociated into inactive enzyme and modulator. These results, together with the finding of Teo and Wang that Ca2+ binds to the modulator, could explain the stimulatory effect of Ca2+ on this enzyme as follows: The “Ca2+ plus Mg2+-dependent” phosphodiesterase may exist as the inactive free form in equilibrium with the active enzymemodulator (Ca2+) complex, and Ca2+, through binding to the modulator, may shift the equilibrium towards formation of the active enzyme-modulator (Ca2+) complex, thereby increasing the activity of the mixture. On decreasing the concentration of Ca2+, the process is reversible.

The amino acid sequence of the Tetrahymena calmodulin which specifically interacts with guanylate cyclase

Abstract The amino acid sequence of the Tetrahymena calmodulin was determined. The protein is composed of 147 amino acids and the amino-terminal is acetylated. Compared to bovine brain calmodulin, there were eleven substitutions and one deletion of amino acid residues. The substitutions and deletion were concentrated in the carboxyl-terminal half of the molecule. Among the substitutions, those at positions 86 (Arg → Ile), 135 (Gln → His) and 143 (Gln → Arg) may introduce the functional difference. The deletion occurred near the carboxyl-terminal, this region being assumed to be exposed to the surface area ( R.H. Kretsinger and C.D. Barry (1975) ). The change in the sequence at this terminal region may be attributable to the specific activation of guanylate cyclase.

The calmodulin-binding protein in microtubules is tau factor

The discovery of Ca2+-activatable cyclic nucleotide phosphodiesterase [ 1 ] and subsequent demonstration of a protein factor which confers Ca2+-sensitivity upon this enzyme [2,3] coincided with the discovery of a protein activator of brain phosphodiesterase [4]. Since then, this protein factor, nowadays called calmodulin, has been shown to cause CaZ+-dependent activation of a variety of enzymes [5]. Moreover, calmodulin has been shown to interact with a number of proteins apparently devoid of enzyme activities by forming complexes with them [6-10]. Although possibilities are raised that these calmodulin-binding proteins may perform yet unknown functions in the cell, the biological activities of these proteins have not been clarified, in spite of various attempts. We have purified a 150 000-M r calmodulin-binding protein (caldesmon) from chicken gizzard muscle [ 11,12]. Caldesmon associates with F-actin when not interacting with calmodulin. Therefore, in the presence of the 3 protein species, calmodulin, caldesmon and F-actin, the concentration of Ca 2+ acts as a fl ipflop switch toward the formation of the caldesmon calmodulin complex at the increased level (>10 -6 M) and toward the formation of the caldesmon • F-actin complex at the decreased level. This is the first demonstration of the Ca2+-dependent regulation of calmodulin on the cytoskeleton and contractile system of eucaryotic cells other than striated muscles. This communication deals with the interaction of cal-

Cyclic 3′,5′-nucleotide phosphodiesterase, IV. Two enzymes with different properties from brain

Two active peaks of phosphodiesterase, I and II, were resolved by a gel filtration column chromatography of the high speed supernatant of brain extract. The peak II represented Ca++ plus Mg++ dependent phosphodiesterase, the occurence of which in the supernatant of brain extract had been reported (1, 3), while the peak I may be called as “Ca++ independent” and Mg++ dependent phosphodiesterase from its nature. The former decomposed cyclic AMP, cyclic GMP, and cyclic UMP with the stimulatory effect of Ca++ion. The latter, decomposing cyclic GMP at the comparable rate to cyclic AMP, showed negligible activity to cyclic UMP.

Calmodulin-binding protein of erythrocyte cytoskeleton

Abstract Previously we have shown that purified spectrin binds calmodulin in the presence of Ca2+ with a Kd value of 3 μM (Sobue, K. et al. (1980) Biochemistry International 1, 561–566). We now provide evidence that the calmodulin-binding activity found in the human erythrocyte cytoskeleton is indeed due to spectrin and no other binding proteins are involved, i.e. the binding activity was purified from the erythrocyte cytoskeleton quantitatively and the purified peak contained spectrin as the only protein constituent. Moreover, Kd value (2.8 μM) and the maximum binding capacity (160,000 – 200,000 calmodulin per cell) obtained from the kinetic analysis of the binding activity in the crude cytoskeleton agreed with the corresponding values reported for purified spectrin. Since the concentration of calmodulin in the erythrocyte cell, which was 2.5 μM or 1.6 × 105 molecules per cell, is close to both the Kd value and the number of the binding sites in the cell, respectively, free calmodulin in the erythrocyte cell may be in a dynamic equilibrium with the spectrin-bound form in vivo depending upon the intracellular concentration of Ca2+.