Calcium-dependent cyclic nucleotide phosphodiesterase inhibition of basal activity by heat-stable factors from rat cerebrum

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca²⁺-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca²⁺-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in K_m for cyclic GMP and a decrease in $\text{V}$. In the presence of calcium ions and purified activator protein, the Ca²⁺-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca²⁺-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was mimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca²⁺-independent activation of Ca²⁺-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca²⁺-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca²⁺-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phosphodiesterase activity.     

Calcium-dependent regulation of NAD kinase.

An activator protein of NAD kinase from the pea, $\text{Pisum}\text{satavum}$ L., has been shown to be Ca²⁺-dependent. This plant activator protein also stimulates the activity of modulator protein dependent-cyclic nucleotide phosphodiesterase from porcine brain. This stimulation is similar to that observed with modulator protein isolated from animal sources. Furthermore, Ca²⁺-dependent modulator proteins isolated from porcine brain, bovine brain, and the coelenterate, $\text{Renilla}$, will regulate the NAD kinase activity of peas. Other common properties of the plant activator protein and animal modulator proteins are their acidic nature, heat stabilities, similar Stokes' radii, and their interactions with troponin I.     

Modulation of lyso-platelet-activating factor:acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca²⁺ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca²⁺. The effect of Ca²⁺ was on the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the enzyme for the substrate acetyl-CoA without showing any significant effect on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca²⁺ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca²⁺, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca²⁺ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca²⁺ and decreased approx. 1000-times when the Ca²⁺ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (Ca_iZ) decreases in the order of $\text{Ca}{}_3\text{Z}\text{>}\text{Ca}{}_4\text{Z}\text{>}\text{Ca}{}_2\text{Z}\text{?}\text{CaZ}$. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca²⁺-ATPase in the range of 10^?7–10^?6 M Ca²⁺, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca²⁺-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca²⁺-transport ATPase in erythrocytes at the prevailing Ca²⁺ concentration (probably 10^?7 – 10^?8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca²⁺-ATPase requires that the Ca²⁺ concentration rises to 10^?6 – 10^?5 M.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

K-254-I (Genistein), a New Inhibitor of Ca2+ and Calmodulin- Dependent Cyclic Nucleotide Phosphodiesterase from Streptosporangium vulgare

A new calmodulin antagonist, genistein, was isolated from the culture broth of Strepto-sporangium vulgare K-254. The spectral data of K-254-I indicated that the compound was identical with genistein, 4^/,5,7-trihydroxyisoflavone. Genistein inhibited the Ca²⁺/calmodulin-depen- dent activity of cyclic nucleotide phosphodiesterase from bovine brain (IC₅₀ = 20 μΜ) without appreciably affecting its basal activity. The inhibitory activity of genistein was antagonized by higher concentrations of calmodulin. Although phosphatidylserine did not reverse the inhibition of calmodulin, genistein inhibited the phospholipid-sensitive, Ca^{2 +}-dependent protein kinase (protein kinase C) from bovine brain (IC₅₀ = 35.3 μΜ). The activity of cAMP-dependent protein kinase was not affected by 700 μΜ of genistein.     

Compound 4880 is a selective and powerful inhibitor of calmodulin-regulated functions

Compound $\text{48}\text{80}$, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound $\text{48}\text{80}$ was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca²⁺-transport ATPase, with IC₅₀ values of 0.3 and 0.85 μg/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 μg/ml. Inhibition of Ca²⁺ transport into inside-out red blood cell vesicles by compound $\text{48}\text{80}$ follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca²⁺-transport ATPase induced by calmodulin is inhibited by compound $\text{48}\text{80}$ according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound $\text{48}\text{80}$ bind to calmodulin in a Ca²⁺-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound $\text{48}\text{80}$ is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca²⁺-transport ATPase that has been described hitherto. In addition, compound $\text{48}\text{80}$ was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca²⁺-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound $\text{48}\text{80}$, respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca²⁺-transport ATPase or calf cardiac sarcolemma (Na⁺ + K⁺)-transport ATPase are far less influenced by compound $\text{48}\text{80}$ as compared with trifluoperazine and calmidazolium. Because of its high specificity compound $\text{48}\text{80}$ is proposed to be a promising tool for studying calmodulin-dependent processes.     

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurosproa crassa

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca²⁺-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in rat kidney cytosol

The effect of regucalcin, a novel Ca²⁺-binding protein, on Ca²⁺/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10^-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl₂ and calmodulin. However, the activatory effect of both CaCl₂ (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10^-8 to 10^-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl₂ (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10^-7 M). The inhibitory effect of regucalcin (10^-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl₂ (10 µM) and regucalcin (10^-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.     

Regulatory interaction between calmodulin and ATP on the red cell Ca2+ pump

The interactions between calmodulin, ATP and Ca²⁺ on the red cell Ca²⁺ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ by 5–10-fold and the rate of Ca²⁺-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ along a biphasic concentration curve ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>1</mtext>}}\text{≈ 1.4 μ}\text{M}\text{, K}{}_{\mathrm{<mtext>m</mtext><mtext>2</mtext>}}\text{≈ 330 μ}\text{M}$), but in stripped membranes the curve is essentially hyperbolic ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{≈ 7 μ}\text{M}$). In calmodulin-bound membranes Ca²⁺ activates $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ at low concentrations ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{< 0.28 μ}\text{M}$) in stripped membranes the apparent Ca²⁺ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E₂ and E₁ forms of the Ca²⁺ pump protein.     

Calcium-dependent cyclic nucleotide phosphodiesterase from glial tumor cells

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been identified in homogenates of C-6 glial tumor cells. The Ca²⁺-dependent phosphodiesterase was resolved by ECTEOLA-cellulose chromatography into two fractions. One fraction contained a protein regulator of the enzyme which was identical to a homogeneous Ca²⁺-binding protein (CDR) from porcine brain by the criteria of electrophoretic migration, biological activity, heat stability, and behavior in diverse chromatographic systems. The second fraction contained deactivated enzyme (CDR-dependent phosphodiesterase) which regained full activity upon the readdition of both Ca²⁺ and CDR. In subcellular fractionation experiments both the CDR and the Ca²⁺-dependent phosphodiesterase were predominantly located in the 100,000g supernatant fraction.The apparent K_m values of the phosphodiesterase for cyclic AMP (cAMP) and cyclic GMP (cGMP) were 10 and 1.2 μm, respectively, when CDR was not rate limiting. Minor increases in the apparent K_m for cAMP were observed at rate-limiting concentrations of CDR. At the ratio of CDR to CDR-dependent enzyme present in the C-6 cell homogenate, half-maximal activation was conferred by 4 μm Ca²⁺ for the hydrolysis of 25 μm cGMP and by 8 μm Ca²⁺ for the hydrolysis of 25 μm cAMP. Increased ratios of CDR to CDR-dependent phosphodiesterase increased the sensitivity of the enzyme to Ca²⁺. The enzyme was more sensitive to CDR with cGMP as substrate than with cAMP, and more sensitive at high than at low cyclic nucleotide substrate concentrations. The quantity of enzyme in the assay also influenced the amount of CDR required for half-maximal activation.     

A high affinity,calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells

Although acute alterations in Ca²⁺ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca²⁺-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca²⁺-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca²⁺ of 280 nM, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg²⁺ could replace Ca²⁺ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg²⁺ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca²⁺-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca²⁺-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca²⁺ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.     

Calmodulin-binding proteins in brain

It is now widely accepted that actions of intracellular Ca²⁺ are mediated by a four-domain Ca²⁺-binding protein, calmodulin. Brain is especially rich in calmodulin, containing about 400 mg (24 μmol) of EGTA-extractable calmodulin per kg of brain. However, only a fraction of the above amount is required for the calmodulin-activated enzymes and most of the rest may be assigned to calmodulin-binding proteins, proteins which are apparently devoid of enzyme activities but undergo Ca²⁺-dependent associations with calmodulin. Several of such proteins have been recently discovered in brain. These include a heat-labile 80 K phosphodiesterase inhibitor protein (calcineurin), a heat-stable 70 K phosphodiesterase inhibitor protein, a 50 K protein, myelin basic protein, tubulin, microtubule τ (tau) factor, a spectrin-like doublet protein (240 plus 235 K) (calspectin; fodrin) and a particle-associated 155 K protein.Functions of these calmodulin-binding proteins have not been fully elucidated yet. Some proteins may be calmodulin-regulated enzymes catalyzing yet unknown biochemical reactions, e.g. a protein phosphatase activity was found for calcineurin. Some proteins may interact with contractile elements or cytoskeleton of the cell, e.g. τ factor and calspectin interacted with tubulin and F-actin, respectively and tubulin itself is a calmodulin-binding protein. So, interesting possibilities are the regulation of the functions of cytoskeleton by calmodulin through these calmodulin-binding proteins. Regulation of microtubule assembly by Ca²⁺-dependent binding of calmodulin to tubulin and/or τ factor and possible involvement of calspectin in the mechanism regulating axonal transport of neuronal proteins have been suggested. Thus, the exploration of the regulating functions of Ca²⁺/calmodulin in brain depends largely upon the further study of the properties of these calmodulin-binding proteins.     

Studies On Cyclic Nucleotide Metabolism In Tetrahymena Pyriformis: Partial Characterization of Cyclic Amp- and Cyclic Gmp-Dependent Phosphodiesterases*

SYNOPSIS. Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in Tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction: the activities were optimal at pH 8.0–9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg²⁺ or Mn^2-. A kinetic analysis of the properties of the enzymes yielded 2 apparent K_III values ranging in concentration from 0.5 to 50 μM and from 0.1 to 62 μ M for cyclic AMP and GMP. respectively. A Ca²⁺-dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, Tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca²⁺, although this activator did not stimulate the phosphodiesterase. the results suggested that Tetrahymena might contain 2 types of Ca²⁺-dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Studies on calmodulin isolated from Tetrahymena cilia and its localization within the cilium

Tetrahymena calmodulins from cilia, cell bodies and whole cells were isolated separately and compared. These calmodulins showed just the same properties: they co-migrated in SDS-polyacrylamide gel electrophoresis, had a Ca²⁺-dependent electrophoretic mobility change in alkali gel, held the same antigenic determinants in common, and activated brain cyclic nucleotide phosphodiesterase Ca²⁺-dependently with identical activation curves. Distributions of calmodulin and calmodulin-counterpart in Tetrahymena cilium were investigated by using alkali gel electrophoresis in the presence of Ca²⁺ or EGTA, and by immunoelectron microscopy. Calmodulin was detected in the membrane plus matrix fraction and outer-doublet microtubule fraction, and its Ca²⁺-dependent counterpart existed exclusively in the latter fraction. However, neither calmodulin nor its counterpart was detected in the crude dynein fraction. Immunoelectron microscopy revealed that calmodulin was localized along the longitudinal axis of outer-doublet microtubules at regular intervals of about 90 nm. The calmodulin-binding site in the ciliary axoneme was suggested to be interdoublet links.     

The presence and binding characteristics of calmodulin in microsomal preparations enriched in sarcoplasmic reticulum from rabbit skeletal muscle

ATP-dependent oxalate facilitated calcium transport in sarcoplasmic reticulum (SR) preparations obtained from rabbit vastus lateralis muscle (fast skeletal muscle; F_sr) and soleus (slow skeletal muscle; S_sr) was determined. Addition of exogenous calmodulin did not stimulate calcium transport in either F_sr or S_sr preparations. F_sr and S_sr previously washed in 1 mM EGTA demonstrated a reduced capacity to transport Ca²⁺; the exogenous addition of calmodulin (0.24 μM) under these conditions, did not restore uptake activity but significantly decreased the steady-state level of Ca²⁺ uptake. Extracts of skeletal SR prepared by treatment with 0.2 mM EDTA and boiling produced significantly more stimulation of red cell Ca²⁺ATPase activity than extracts prepared by boiling alone. This stimulation of red cell Ca²⁺-ATPase was inhibited to a significant extent by $\text{48}\text{80}$, a known anti-calmodulin agent. Radioimmunoassay revealed that extracts prepared by boiling or EDTA-treatment followed by boiling contained considerable amounts of calmodulin. Washing with 1 mM EGTA, though, did not release any calmodulin from SR. These studies reveal that calmodulin is present in both F_sr and S_sr and can only be removed by harsh treatments. The role of calmodulin in skeletal muscle Ca²⁺-transport remains to be determined.     

Regulation of the Ca2+-pump by calmodulin in intact cells

ATP-enriched human red cells display high rates of Ca²⁺-dependent ATP hydrolysis (16 mmol·litre cells^?1·h^?1) with a high Ca²⁺ affinity ($\text{K}{}_{0.5}\text{～0.2 μ}\text{M}$). The finding suggests a mechanism for regulation of cell Ca²⁺ levels, involving highly-cooperative stimulation of active Ca²⁺ extrusion following binding of calmodulin to the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$.     

Further characterization of Ca2+-activated phospholipase A2 in cat adrenal cortex

When cat adrenocortical cells were incubated with exogenous phospholipid substrate (autoclaved $\text{E.}$$\text{coli}$) in the presence of corticotropin, there was a Ca²⁺-dependent increase in phospholipid breakdown activity, suggesting that a hormone-stimulated phospholipase is localized to the plasma membrane. Phospholipase activity in a particulate fraction from lysed cells at neutral pH was a function of the Ca²⁺ concentration. The addition of increasing Ca²⁺ concentrations to a subcellular fraction of lysed cells which had been prelabelled with [¹⁴C]arachidonic acid produced graded increases in fatty acid release. A depletion of label from phosphatidylcholine was observed, as well as a marked increase in radioactivity associated with phosphatidylethanolamine. The subcellular fraction of cells prelabelled with [¹⁴C]palmitic acid failed to release fatty acid in response to Ca²⁺, although a loss of label from phosphatidylcholine and a modest gain in label by phosphatidylethanolamine was demonstrable. A Ca²⁺-activated deacylation-reacylation reaction preferentially involving phosphatidylethanolamine was evident in cortical cells prelabelled with archidonic acid; whereas, other Ca²⁺-stimulated lipolytic reactions also appeared to be operative in cells prelabelled with either arachidonic or palmitic acid. The Ca²⁺-dependent mobilization of arachidonic acid from an endogenous phospholipid pool lends additional support to the idea that Ca²⁺-mediated activation of phospholipase A₂ participates in the control of adrenocortical activity. However, since Ca²⁺ also stimulated arachidonic acid liberation from cortical triglycerides, these lipid moieties may also contribute to the observed effects of Ca²⁺ on fatty acid release.     

Multiple forms of cyclic nucleotide phosphodiesterase from bovine carotid artery smooth muscle.

DEAE-cellulose chromatography, in the presence and absence of Ca²⁺, of the 16,000g supernatant from bovine carotid artery smooth muscle has been used to separate four different types of cyclic nucleotide phosphodiesterase (3′:5′-cyclic-nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) activity, designated types A, B, C, and D. Type A is a high affinity, cyclic AMP-specific form of phosphodiesterase (K_m = 1.6 μM) and elutes at relatively high ionic strength. Type B is a high affinity (K_m = 2 μM), cyclic GMP-specific form which elutes at low ionic strength. Type C is a mixed substrate form, displaying anomalous kinetics for the hydrolysis of both cyclic AMP and cyclic GMP. It elutes from DEAE-cellulose at an ionic strength intermediate to that of types A and B. Type D is also a mixed substrate form of phosphodiesterase. However, its elution pattern from DEAE-cellulose differs, depending on whether Ca²⁺ is present or not, suggesting a Ca²⁺-dependent interaction between this enzyme form and the acidic Ca²⁺-dependent regulator protein (CDR). The hydrolytic activity of type D is stimulated by CDR, and activation requires the simultaneous presence of Ca²⁺ and CDR. Kinetic analysis of cyclic AMP hydrolysis by type D gives a linear double reciprocal plot; activation has no effect on the K_m but increases the velocity approximately sixfold. Activation of cyclic GMP hydrolysis apparently affects both the K_m and V. At all concentrations tested, the degree of activation is higher with cyclic AMP than with cyclic GMP. It is suggested that while the activable form of phosphodiesterase may play a relatively minor role in the overall hydrolysis of cyclic nucleotides, Ca²⁺-dependent activation may have a more important role in regulating the level of cyclic AMP than that of cyclic GMP in vascular smooth muscle.