Reiko Yamazaki

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

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.

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.

Distribution in rat tissues of modulator-binding protein of particulate nature: Studies with 3H-modulator protein

Studies on Ca”-activatable cyclic nucleotide phosphodiesterase [l] led to the discovery of a protein modulator that is required for the activation of this enzyme by Ca2+ [2-41. Modulator protein is identical to the activator protein originally discovered by Cheung [5,6]. Later, this protein has been shown to cause the Ca”-dependent activation of several enzymes that include phosphodiesterase [l-6], adenylate cyclase [7], a protein kinase from muscles [8-l 11, phosphorylase b kinase [ 121, actomyosin ATPase [ 13,141, membranous ATPase from erythrocytes [ 1.5 ,161 and nerve synapses [ 171. Thus, modulator protein appears to be an intracellular mediator of actions of Ca2+. Besides the above enzymes, modulator protein has been shown to associate, in a Ca2’-dependent fashion, with cellular proteins whose functions are yet to be identified [ 18-251. It is expected that these modulatorbinding proteins may represent Ca”/modulator protein-regulated enzymes(proteins) [ 191, or subunits of them [22]. While these studies [ 18-251 were carried out with the supernatant fraction of tissues, work in our laboratory [26] revealed the presence of modulator-binding protein of particulate nature in brain which, in the presence of Ca’+, can associate -l/3 of the soluble modulator present in this tissue. The present work shows the distribution of this particulate modulator-binding component in rat tissues. This paper also describes the labeling of modulator protein with tritium without deteriorating

Identity of the particulate form of calmodulin with soluble calmodulin

Discovery of Ca2+activatable phosphodiesterase [l] and subsequent demonstration of protein modulator which confers Ca2+-sensitivity upon this enzyme [2,3] coincided with the discovery of a protein activator of brain phosphodiesterase [4]. However, it took several years before these two independent lines of the research merged when the identity of the two proteins as a Ca2+-receptive protein was finally established [S-7]. This protein, now called calmodulin, is considered an intracellular mediator of the stimuluslinked actions of Ca’+in the cell. Although calmodulin is generally regarded as a soluble protein, it was observed [8] that, when calmodulin was prepared from the brain homogenate, inclusion of EDTA in the homogenizing buffer greatly increased the yield of this protein in the soluble fraction. Subsequently, work in our laboratory clarified the existence of calmodulinbinding protein(s) of particulate nature that bind calmodulin in the presence of Ca2+ in the brain and other tissues [9,10]. However, an extensive extraction of the particulate fraction with EGTA could not solubilize all the calmodulin activity, leaving a certain amount of its activity in the insoluble fraction [9]. We shall henceforth refer to this residual activity as the particulate form of calmodulin. The concentration of this particulate form calmodulin decreased in hepatoma tissues when compared with the normal liver [ 111. Since this change was accompanied by an increased level of the soluble