Calcium-dependent Regulator Research Articles

Inability of parvalbumin to function as a calcium-dependent activator of cyclic nucleotide phosphodiesterase activity.

The calcium-sensitive phosphodiesterase-stimulating activity sometimes associated with parvalbumin preparations is due to contaminating (less than 0.1%) amounts of carp muscle CDR (calcium-dependent regulator)-like protein. This protein can be resolved from parvalbumins by Sephadex G-75 chromatography and has many characteristics of the CDR. Parvalbumin itself causes a nonspecific stimulation of phosphodiesterase at all calcium concentrations which, in the presence of CDR, can cause an apparent shift to a lower concentration of the calcium level required for half-maximal stimulation.

The Interactions between Calcium-Dependent Regulator Protein of Cyclic Nucleotide Phosphodiesterase and Microtubule Proteins I. Effect of Calcium-Dependent Regulator Protein on the Calcium Sensitivity of Microtubule Assembly

We examined the effect of porcine brain Ca2+-dependent regulator (CDR) protein on microtubule (MT) assembly from microtubular proteins isolated from porcine brain by temperature-dependent cycles of assembly-disassembly. CDR exhibited a potent inhibitory effect on MT assembly in the presence of Ca2+, whereas it had little or no effect on the extent of MT assembly in the absence of Ca2+. The increase in KCl concentration greatly potentiated the Ca2+-dependent inhibitory effect of CDR. The effect of CDR was reversible in a Ca2+ concentration-dependent manner, and the extent of inhibition by CDR at a fixed concentration of free Ca2+ was roughly proportional to the concentration of CDR. Moreover, the Ca2+ concentration required for the half-maximal inhibition of MT assembly from a fixed concentration of purified microtubular proteins (PMP) decreased with increasing CDR concentration. On the basis of these results, together with data on the Ca2+-dependent association of CDR and tubulin (J. Biochem., accompanying paper), we propose the following model; Ca2+ + CDR in equilibrium Ca2+-CDR Ca2+-CDR + tubulin in equilibrium Ca2+-CDR-tubulin (nonpolymerizable).

The Interactions between Calcium-Dependent Regulator Protein of Cyclic Nucleotide Phosphodiesterase and Microtubule Proteins II. Association of Calcium-Dependent Regulator Protein with Tubulin Dimer

The Ca2+-dependent regulator protein (CDR) of cyclic nucleotide phosphodiesterase (PDE) was reported to be a Ca2+-dependent regulator of microtubule (MT) assembly in the preceding paper. In this paper, the binding of Ca2+-CDR complex to tubulin dimer was investigated in order to elucidate the Ca2+-dependent inhibitory action of CDR on MT assembly. Purified microtubular proteins (PMPs) isolated from porcine brain did not affect the ability of CDR to activate Ca2+-activatable PDE, and did not include any inhibitory protein of Ca2+-activatable PDE. The binding of CDR to the tubulin dimer was observed on Sephadex G-200 gel filtration and ammonium sulfate fractionation in a Ca2+-dependent manner. CDR did not bind to microtubule associated proteins. We now assume that Ca2+-dependent inhibition of MT assembly by CDR is due to the binding of CDR to tubulin dimer in a Ca2+-dependent manner.

Regulation of rat lung adenylate cyclase by cytoplasmic factor(s) during development

Basal adenylate cyclase activity in rat lung homogenate was low prenatally but increased several-fold after birth and remained elevated to maturity. The results also demonstrate the appearance of some factor(s) in the lung cytoplasm at a certain age which markedly activated adenylate cyclase. During late gestation and early neonatal life, when the cytoplasmic factor(s) was low or absent, basal adenylate cyclase activity was low and norepinephrine and NaF produced maximum activation of the enzyme. However, when the cytoplasmic factor(s) appeared in the adult lungs, basal adenylate cyclase activity was elevated and both norepinephrine and NaF produced little or no activation of the enzyme. These data suggest a role for the cytoplasmic factor(s) in regulating rat lung adenylate cyclase. The cytoplasmic factor(s) appeared to be a protein since it was inactivated by trypsin digestion and by heating to 75 degrees C. Activation of adenylate cyclase was not due to small ions or other low molecular weight components of the cytoplasm as dialysis of the supernatant did not alter its activation of adenylate cyclase. The cytoplasmic factor(s) did not appear to be either GTP or calcium-dependent regulator of cyclic AMP phosphodiesterase as these did not activate the rat lung adenylate cyclase.

Stimulation of glycogen synthase phosphorylation by calcium-dependent regulator protein.

Phosphorylation of skeletal muscle glycogen synthase catalyzed by a protein kinase is stimulated up to 10-fold by the calcium-dependent regulator (CDR) protein. Half-maximal stimulation requires about 1 microgram of CDR/ml. Phosphorylation by the CDR-dependent synthase kinase is more rapid at pH 8.6 than at pH 6.8 and is blocked by ethylene glycol bis(beta-aminoethyl-ether)N,N'-tetraacetic acid and trifuloperazine. Approximately 60 to 70% of the phosphate is incorporated into the trypsin-insensitive region of glycogen synthase resulting in conversion of the a form to the b form of the enzyme. The CDR-dependent synthase kinase is not myosin light chain kinase, as this enzyme does not phosphorylate glycogen synthase. Furthermore, synthase phosphorylation by the cAMP-dependent protein kinase catalytic subunit is not affected by CDR. The possibility that CDR-dependent synthase kinase may be phosphorylase kinase is being investigated.

Glycogen synthase kinase-2 from rabbit skeletal muscle is activated by the calcium-dependent regulator protein

Glycogen synthase kinase-2 from rabbit skeletal muscle is activated by the calcium-dependent regulator protein

Calcium-sensitive regulation of actin-myosin interactions in baby hamster kidney (BHK-21) cells.

A fraction has been obtained from baby hamster kidney (BHK-21) cells that will stimulate the actin-moderated ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of both BHK-21 myosin and gizzard smooth muscle myosin. This activation is associated with the specific phosphorylation of the myosin 20,000-dalton light chain. The BHK-21 myosin light chain kinase preparation contains a major protein of approximately 105,000 molecular weight as determined by sodium dodecyl sulfate gel electrophoresis. Both the actin activation and phosphorylation events require the presence of Ca2+ and the so-called modulator or calcium-dependent regulator protein that has been isolated from smooth muscle, brain, and other tissues. On the basis of these results we propose that this kinase system constitutes a Ca2+-dependent regulatory mechanism for myosin-actin interactions in nonmuscle mammalian cells.

Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3‘: 5‘-monophosphate-dependent protein kinase.

Turkey gizzard smooth muscle light chain kinase was purified by affinity chromatography on calcium dependent regulator weight of 125,000 +/- 5,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When myosin light chain kinase is incubated with the catalytic subunit of cyclic AMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of myosin kinase. Brief tryptic digestion of the 32P-labeled myosin kinase liberates a single radioactive peptide with a molecular weight of approximately 22,000. Phosphorylation of myosin kinase results in a 2-fold decrease in the rate at which the enzyme phosphorylates the 20,000-dalton light chain of smooth muscle myosin. These results suggest that cyclic AMP has a direct effect on actin-myosin interaction in smooth muscle.

Ca2+-dependent protein phosphorylation system in membranes from various tissues, and its activation by "calcium-dependent regulator".

Analysis of membranes from a variety of tissues has revealed a widespread distribution of a protein phosphorylation system dependent on the presence of both Ca2+ and "calcium-dependent regulator" (CDR). This protein phosphorylation system has been studied in some detail in nervous tissue. Neuronal membranes contain a protein phosphorylation system that requires Ca2+ and a soluble heat-stable protein [Schulman, H. & Greengard, P. (1978) Nature (London) 271, 478--479]. This protein has been purified to homogeneity from bovine cerebral cortex, with use of an assay based on its ability to stimulate Ca2+-dependent protein phosphorylation in membranes. This protein kinase activator appears to be identical to CDR of cyclic nucleotide phosphodiesterase. Throughout its purification, this single entity was found to activate both Ca2+-dependent protein kinase and cyclic nucleotide phosphodiesterase. The kinase activator purified here and authentic CDR were equally effective in their ability to activate Ca2+-dependent protein kinase.

Specific binding of the calcium-dependent regulator protein to brain membranes from the guinea pig

The interaction of a calcium-dependent regulator protein (CDR) of brain adenylate cyclase (EC 4.6.1.1) with synaptic membranes from guinea pig brain was examined using 125I-CDR as a tracer molecule. 125I-CDR binding was reversible, saturable, and temperature sensitive. The same Ca 2+ and Mg 2+ dependence was observed for 125I-CDR binding and for brain adenylate cyclase activation by CDR.

Calcium-dependent regulator of NAD kinase in higher plants

An activator protein of NAD kinase from the pea, Pisum satavum L., has been shown to be Ca 2+-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, Ca 2+-dependent modulator proteins isolated from porcine brain, bovine brain, and the coelenterate, 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.

Control of microtubule assembly-disassembly by calcium-dependent regulator protein.

The Ca2+-dependent regulator (CDR) protein of cyclic nucleotide phosphodiesterase is a low molecular weight, acidic, Ca2+-binding protein which has been implicated in a number of Ca2+-dependent enzymatic functions. Indirect immunofluorescence has revealed that CDR is specifically associated with the chromosome-to-pole region of the mitotic apparatus during metaphase-anaphase in a pattern distinctly different from that of similar cultured cells stained with antitubulin. This characteristic localization in the mitotic half-spindle suggested a role for CDR in the control of microtubule assembly-disassembly during mitosis. Thus, CDR was examined for its effects on microtubule polymerization in vitro. It was determined that stoichiometric concentrations of CDR and a homologous Ca2+-binding protein, skeletal muscle troponin C, both inhibited and reversed microtubule assembly in a Ca2+-dependent manner. CDR-dependent inhibition of in vitro microtubule assembly occurred at physiological Ca2+ concentrations (approximately 10 micron) that, in the absence of CDR, caused only a slight reduction in polymerization. At Ca2+ concentrations in the low physiological range (less than 1 micron), no inhibition was observed. These biochemical results, together with the immunofluorescent localization of CDR in the mitotic half-spindle, provide evidence that Ca2+ is an endogenous regulator of microtubule disassembly through the activity of CDR.

Hormonal Regulation of Sertoli Cells

The Sertoli cell is the primary target within the testis for both FSH and androgens. FSH binds to cell surface receptors, alters cyclic nucleotide metabolism and affects Ca++ flux. Cyclic AMP and Ca++, via an intracellular receptor protein, the calcium‐dependent regulator (CDR), control the cytoskeletal network which is comprised of microtubules and microfilaments. By affecting these intracellular components FSH regulates specific protein secretion and cell motility. In addition FSH results in an overall stimulation of protein synthesis and specifically modulates the levels of at least two intracellular regulatory proteins. Androgens bind to a specific cytoplasmic receptor which is translocated into the nucleus of interphase cells. This class of steroids is also involved in androgen binding protein metabolism but the precise mechanism of control is unknown. The interrelationships of FSH and androgen in the control of spermatogenesis is discussed. In addition, consideration is given to the possible development of male contraceptive by interfering with various steps in the mechanism of action of peptide and steroid hormones upon the Sertoli cell.

Calcium-dependent regulator protein: Localization in mitotic apparatus of eukaryotic cells

Calcium-dependent regulator protein is a low molecular weight (17,000), thermostable, calcium binding protein which is structurally homologous to skeletal muscle troponin C. This protein is present in all nonmuscle cells and has been shown to decorate stress fibers in interphase cells by indirect immunofluorescence. Using this procedure we have investigated the distribution of the protein during mitosis of eukaryotic cells. As the cells enter prophase, the distinct cytoplasmic localization disappears commensurate with the dissolution of the cytoskeleton. The regulator protein seems to be randomly distributed throughout the prophase cell, including the region around the condensed chromosomes. However, at prometaphase, it is localized in association with the half-spindles of the mitotic apparatus. Through metaphase and most of anaphase, the protein remains localized between the chromosomes and the poles of the spindle. During late anaphase the protein is also found in the interzone region but rapidly condenses into two small regions, one on each side of the midbody that separates the daughter cells. The regulator protein is not localized in the cleavage furrow during telophase, whereas actin is demonstrable in this region. Indeed, placement of the protein during mitosis is distinct from both that of actin and that of tubulin. The localization of calcium-dependent regulator protein during mitosis suggests that it may mediate the calcium effects on the mitotic apparatus and thus play a role in chromosome movement.

Stimulation of brain membrane protein phosphorylation by calcium and an endogenous heat-stable protein.

THE important role of Ca2+ in the physiology of the nervous system is well documented1,2. However, the biochemical mechanisms underlying certain of its physiological effects, such as stimulus–secretion coupling3,4 and synthesis of cate-cholamines5,6, have not yet been elucidated. Calcium has been implicated in several biochemical reactions of potential importance to synaptic function. Thus, calcium and a heat-stable calcium-binding protein activate the cyclic nucleotide phosphodiesterase from mammalian brain7,8. The calcium-dependent regulator (CDR) from porcine brain9, bovine heart10 and bovine brain11 has been purified and characterised. CDR seems to be a calcium receptor as it binds calcium strongly and specifically. There is evidence that a CDR·Ca2+ complex is the true activator of cyclic nucleotide phosphodiesterase12–14. A detergent-solubilised preparation of brain adenylate cyclase can also be activated by this calcium-binding protein15. Calcium stimulates protein phosphorylation in both intact16,17 and lysed18,19 synaptosomes and this cyclic nucleotide-independent mechanism may mediate or modulate some of the intracellular effects of Ca2+ on the function of presynaptic nerve terminals. We report here that calcium-dependent phosphorylation of synaptosomal membrane fractions from rat cerebral cortex requires an endogenous protein factor present in the synaptosomal cytoplasm. Calcium stimulated phosphorylation is lost on purification of synaptic membranes and can be effectively recovered by reconstitution with either the synaptosomal cytoplasm or with a purified preparation of CDR. Thus, regulation by calcium of calcium-dependent protein kinase activity may be mediated physiologically by the calcium-binding protein postulated to regulate cyclic nucleotide phosphodiesterase and adenylate cyclase.

Isolation and Regulation of the Protein Kinase Inhibitor and the Calcium-Dependent Cyclic Nucleotide Phosphodiesterase Regulator in the Sertoli Cell-Enriched Testis1

The cAMP-dependent protein kinase inhibitor (PKI) and calcium-dependent cyclic nucleotide phosphodiesterase regulator (CDR) proteins were examined in the Sertoli cell-enriched (SCE) rat testis. PKI activity was stable at 100 C for 10 min whereas the rate of decline of CDR activity at this temperature was first order (t½ of 7 min). Greater than 90% of both activities were found in the soluble fraction of the cell with the remaining 10% in the participate fraction. PKI and CDR could be separated by DEAE-cellulose chromatography and by polyacrylamide gel electrophoresis. Gel filtration chromatography also resulted in their physical separation, CDR eluting slightly before PKI. Molecular weights estimated by gel filtration were 21,400 and 28,800 daltons for PKI and CDR, respectively. Sufficient PKI was present in the SCE testis to inactivate only 7% of the total cAMP-dependent protein kinase whereas CDR was present in vast excess over the amount required to activate maximally total calciumdependent phosphodies...

Epinephrine- abd glucagon-stimulated cardiac adenylyl cyclase activity: Regulation by endogenous factors

1. 1. Epinephrine and glucagon sensitivity of cardiac adenylyl cyclase activity of crude particulate preparations (10 000 × g pellets) was 30–50% that of homogenates prepared using hypo-, iso-, or hypertonic media. The 10 000 × g supernatnat of the homogenate restored epinephrine- and glucagon-stimulated adenylyl cyclase. 2. 2. A substance or substances with similar activities on heart membrane adenylyl cyclase was found in liver, skeletal muscle, diaphragm, stomach, duodenum, jejunum, ileum, adipose tissue, spleen, and lung, but not in brain or adrenal gland 10 000 × g supernatants. 3. 3. The factor in the 10 000 × g supernatant from heart was sensitive to trypsin treatment, not precitated at 105 000 × g for 2 h, inactivated by boiling, non-dialyzable, bound to Dowex 1-X8 and DEAE-cellulose at pH 7.4, unstable to storage at 4°C, and precipiated by 80% saturated (NH 4) 2SO 4. DEAE-cellulose chromatography of the 10 000 × g supernatant resolved a factor that enhanced epinephrine-stimulated adenylyl cyclase activity from that enhancing glucagon stimulation. The separated factors showed differential stability upon storage at −10°C. 4. 4. Calcium-dependent regulator (CDR) did not restore hormone sensitivity in these cardiac particulate preparations. Therefore, the factor reported in this study is not CDR. These results add to the probability that hormone sensitivity of membrane-bound adenylyl cyclase activity is regulated in part by endogenous factors.

Regulation of adenylate cyclase from glial tumor cells by calcium and a calcium-binding protein.

A biphasic response to changes in Ca2+ concentration was observed for basal and norepinephrine-stimulated adenylate cyclase activity in homogenates of C-6 glioma cells. The enzyme was stimulated approximately 40% by low concentrations of free Ca2+ (less than or equal to 1 muM) and inhibited to successively greater extents as free Ca2+ concentrations were increased to approximately 100 muM. Ca2+ did not alter the concentration of norepinephrine required for enzyme activation. Homogenates of C-6 cells were separated into particulate and supernatant fractions by centrifugation at 27,000 X g for 20 min. The particulate fraction contained nearly all of the adenylate cyclase activity. This activity was stimulated approximately 40% by the addition of untreated supernatant fraction, by boiled or dialyzed supernatant fraction, and by a homogenous Ca2+-binding protein (calcium-dependent regulator (CDR) prepared from brain. Addition of either the supernatant fraction or CDR lowered the Ca2+ concentration required for maximal stimulation of the adenylate cyclase. The factor in the supernatant fraction which activated the particulate enzyme was subsequently identified in acrylamide gel electrophoretic studies to be CDR. The amount of CDR required for maximal activation of the enzyme was found to be lowered as the Ca2+ concentration in the assay was increased. High amounts of added CDR (100 to 1000 ng) were inhibitory. Use of the monionic detergent, Lubrol PX, to prepare dispersed adenylate cyclase from the particulate fraction resulted in large losses of activity. The resultant preparation of enzyme contained some CDR which could not be removed by chromatography of the preparation on anion exchange columns. Addition of homogeneous CDR to the assay activated the enzyme several-fold at low Ca2+ concentrations. At higher Ca2+ concentrations the enzyme was activated fully by the CDR endogenous to the preparation and added Ca2+. CDR was inhibitory.

Adrenal medullary cyclic nucleotide phosphodiesterase: Lack of activation by the calcium-dependent regulator

Calcium-dependent regulator, a calcium-binding protein isolated from brain and adrenal medulla, has been shown to activate a brain calcium-sensitive cyclic nucleotide phosphodiesterase. To determine if this protein has the same role in the adrenal medulla, the cyclic nucleotide phosphodiesterase of adrenal medulla was characterized. Neither crude nor partially purified adrenal medullary phosphodiesterase was inhibited by EGTA or stimulated by calcium and the calcium-dependent regulator, whereas similar brain preparations displayed sensitivity to these agents. As the calcium-dependent regulator does not appear to stimulate adrenal medullary cyclic nucleotide phosphodiesterase activity, alternate roles of this protein in adrenal medulla are suggested.

Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase.

An activating factor of adenylate cyclase (EC 4.6.1.1) HAS BEEN OBTAINED FROM DETERGENT-DISPERSED PREPARATIONS OF PORCINE CEREBRAL CORTEX BY COLUMN CHROMATOGRAPHY ON ECTEOLA-cellulose. The factor was identified by acrylamide gel electrophoresis and by enzyme activation studies as the Ca2+-binding protein that regulates the activity of a brain cyclic nucleotide phosphodiesterase. This Ca2+-binding protein confers a Ca2+-dependent activation upon the adenylate cyclase, which is reversed by the subsequent addition of egta in excess of the free Ca2+. It is proposed that this Ca2+-dependent regulator controls enzymatic activities responsible for the synthesis of adenosine 3':5'-monophosphate and for the hydrolysis of guanosine 3':5'-monophosphate.