Tetrahymena calcium-binding protein is indeed a calmodulin

We previously isolated a Ca2+-binding protein from a ciliate, Tetrahymena, and designated it as TCBP (Tetrahymena Ca2+-binding protein). The present paper reports that TCBP, which has two high affinity Ca2+-binding sites (Kd=4.6 X 10(-6) M), could activate porcine brain cyclic nucleotide phosphodiesterase at a concentration of over 10(-6) M free Ca2+, with the same mode of activation as that of authentic (porcine brain) calmodulin. In addition, the amino acid composition of TCBP was essentially the same as that of brain calmodulin. Therefore, we conclude that TCBP as an activator of Tetrahymena guanylate cyclase is indeed a calmodulin.     

Posttranslational Modification of Calmodulin in Rat Brain and Pituitary

The posttranslational modification of calmodulin has been studied in six brain regions and the anterior pituitary. Carboxylmethylation, calmodulin converting enzyme, and calmodulin (lysine) N-methyltransferase activities were determined. Incubation of calmodulin with cytosolic extracts of these tissues in the presence of the methyl donor [methyl-3H]-S-adenosyl-L-methionine and identification of labeled proteins by gel electrophoresis and fluorography indicated that calmodulin is a substrate for protein carboxylmethyltransferase in all tissues tested. In hippocampus, caudate nucleus, cerebral cortex, and anterior pituitary, but not in cerebellum, superior colliculus, brainstem, or diencephalon, a second methylated protein was found when calmodulin was added to incubation mixtures. This protein was shown to be identical to the previously described product of calmodulin converting enzyme. Converted calmodulin was isolated by fast protein liquid chromatography and shown to be des(Lys)calmodulin, lacking the carboxy terminal lysine residue of calmodulin. The anterior pituitary had by far the highest levels of calmodulin converting enzyme; this enzyme, in turn, was identified as a cobalt-stimulated carboxylpeptidase B. In contrast to the regional differences in these parameters, the levels of calmodulin (lysine) N-methyltransferase did not differ greatly among brain regions, although regional differences in the activity of this enzyme were statistically significant.     

Isolation of calmodulin from the protozoan, Tetrahymena pyriformis, by the use of a tubulin-Sepharose 4B affinity column

We developed a simple new method for the isolation of calmodulin, a ubiquitous Ca2+-binding protein, by using tubulin-Sepharose 4B column chromatography, and succeeded in rapid isolation of calmodulin from Tetrahymena pyriformis. The procedure was also shown to be successfully applicable to the isolation of calmodulins from starfish ovary, porcine brain, and monkey brain and, therefore, may be of general use for the rapid isolation of calmodulin.     

The purification and identification of calmodulin from human placenta

A protein which showed similarity to bovine brain calmodulin in electrophoretic mobilities on polyacrylamide gels in the presence of 40% glycerol (pH 8.6) and 0.1% sodium dodecyl sulfate (pH 7.2) was isolated from human placenta. Its final yield was approx. 4 mg per kg human placenta. The placenta protein was similar to bovine brain calmodulin in stimulating bovine brain calmodulin-deficient cyclic nucleotide phosphodiesterase in the presence of calcium. However, its stimulating activity was eliminated by ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or trifluoperazine. In addition, there is a close resemblance in amino acid composition between the placental protein and bovine brain calmodulin. These results indicate that calmodulin is present in human placenta.     

"Buttonin," a unique button-shaped microtubule-associated protein (75 kD) that decorates spindle microtubule surface hexagonally

A 75-kD protein was purified from sea urchin egg microtubule proteins through gel filtration. It enhanced the polymerization of porcine brain tubulin, but was not heat-stable and did not bind to calmodulin in the presence of calcium as demonstrated by calmodulin affinity column chromatography. Rotary shadowing of the freeze-etched 75-kD protein adsorbed on mica revealed the protein to be a spherical molecule (approximately 9 nm in diameter). Quick-freeze deep-etch electron microscopy revealed that the surface of microtubules polymerized with 75-kD protein was entirely covered with hexagonally packed, round, button-like structures that were quite uniform in shape and size (approximately 9 nm) and similar to the buttons observed on microtubules of mitotic spindles in vivo or microtubules isolated from mitotic spindles. Judging from calibration studies of molecular mass by gel filtration, the 75-kD protein probably exists in a dimeric form (approximately 150 kD) in its native condition. The stoichiometry of tubulin (dimer) versus 75-kD protein (dimer) in the polymerized pellet was 3-3.4:1. Hence, we concluded that the 75-kD protein was a unique microtubule-associated protein that formed the microtubule button in vivo and in vitro. We propose to name this protein "buttonin".     

A high affinity acceptor for phospholipase A2 with neurotoxic activity is a calmodulin

One of the high affinity binding proteins for ammodytoxin C, a snake venom presynaptically neurotoxic phospholipase A(2), has been purified from porcine cerebral cortex and characterized. After extraction from the membranes, the toxin-binding protein was isolated in a homogenous form using wheat germ lectin-Sepharose, Q-Sepharose, and ammodytoxin-CH-Sepharose chromatography. The specific binding of (125)I-ammodytoxin C to the isolated acceptor was inhibited to different extents by some neurotoxic phospholipases A(2), ammodytoxins, bee venom phospholipase A(2), agkistrodotoxin, and crotoxin; but not by nontoxic phospholipases A(2), ammodytin I(2), porcine pancreatic phospholipase A(2), and human type IIA phospholipase A(2); suggesting the significance of the acceptor in the mechanism of phospholipase A(2) neurotoxicity. The isolated acceptor was identified as calmodulin by tandem mass spectrometry. Since calmodulin is generally considered as an intracellular protein, the identity of this acceptor supports the view that secretory phospholipase A(2) neurotoxins have to be internalized to exert their toxic effect. Moreover, since ammodytoxin is known to block synaptic transmission, its interaction with calmodulin as an acceptor may constitute a valuable probe for further investigation of the role of the latter in this Ca(2+)-regulated process.     

The phosphorylation of choline acetyltransferase

Human placental Choline Acetyltransferase (ChAT) has been shown to be phosphorylated in vitro by kinases present in rat brain. Phosphorylation occurs at a single site with the exclusive phosphoamino acid being serine. ChAT phosphorylation was shown to be calcium, and not cyclic nucleotide, dependent and was inhibited by inhibitors of calcium/calmodulin protein kinases including anti-calmodulin anti-sera. ChAT phosphorylation was stimulated by calmodulin (9 fold) and, to a lesser extent, by phosphatidylserine (4 fold). These results indicate the involvement of a calcium/calmodulin and possibly also a calcium/phosopholipid kinase. This finding was confirmed by demonstrating ChAT phosphorylation using both purified multifunctional calcium/calmodulin protein kinase (CaMK) and calcium/phospholipid protein kinase C (PKC) from rat brain. A stoichiometric incorporation of 0.9 mol phosphate/mol ChAT was achieved by CaMK. Phosphorylated ChAT could be isolated from freshly prepared rat brain synaptosomes. The results obtained with this model system support the hypothesis that in vivo a fraction of ChAT exists phosphorylated.     

山莨菪碱对经有限蛋白水解后的脑突触膜Ca~(2+)-ATPase的影响

 从猪脑中提取钙调蛋白和突触质膜,我们研究了山莨菪碱对经有限蛋白水解和磷脂酶A_2处理后的突触膜Ca~(2+)-ATPase活性影响。发现药物对不同预处理后的Ca~(2+)-ATPase表现出不同影响并调节钙调蛋白对它的激活作用。     

The binding of calmodulin to myelin basic protein and histone H2B.

1. A calmodulin-binding protein of apparent mol.wt. 19 000 has been purified from chicken gizzard. Similar proteins have been isolated from bovine uterus, rabbit skeletal muscle and rabbit liver. 2. These proteins migrated as an equimolar complex with bovine brain calmodulin on electroporesis on polyacrylamide gels in the presence of Ca2+ and 6M-urea. The complex was dissociated in the presence of EGTA. 2. The chicken gizzard calmodulin-binding protein has been shown to be identical with chicken erythrocyte histone H2B on the basis of partial amino acid sequence determination. 4. The calmodulin-binding proteins of apparent mol.wt. 22 000 isolated previously from bovine brain [Grand & Perry (1979) Biochem. J. 183, 285-295] has been shown, on the basis of partial amino-acid-sequence determination, to be identical with myelin basic protein. 5. The activation of bovine brain phosphodiesterase by calmodulin is inhibited by excess bovine uterus calmodulin-binding protein (histone H2B). 6. The phosphorylation of myelin basic protein by phosphorylase kinase is partially inhibited, whereas the phosphorylation of uterus calmodulin-binding protein (histone H2B) is unaffected by calmodulin or troponin C. 7. The subcellular distribution of myelin basic protein and calmodulin suggests that the two proteins do not exist as a complex in vivo.     

Further characterization of the membrane-bound (Ca2+ + Mg2+)-ATPase from porcine erythrocytes

1. The kinetic and physicochemical properties of the calcium-pumping protein, (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) were studied in ghost membranes isolated from porcine erythrocytes. 2. The membrane-bound enzyme in situ has a specific activity of 3.12 +/- 0.08 micron/mg protein/hr and a Vmax of 3.47 +/- 0.21 mumol/mg protein/hr in the absence of calmodulin. 3. Its activity was stimulated by calmodulin about 5-fold. The enzyme is also highly sensitive to inhibition by vanadate (Ki = 1.6 +/- 0.2 microM). 4. Calmodulin also affects the pH- and Ca2+-sensitivity of the enzyme. The optimum pH, in the presence of calmodulin, is 7.5 and the optimum temperature is 38 degrees C with an activation energy of 11.9 kcal/mol.     

Identity of a membrane-bound vasoactive intestinal peptide-binding protein with calmodulin.

A vasoactive intestinal peptide (VIP)-binding protein purified from guinea pig lung membranes (p18) was digested with trypsin, and the amino acid sequence of the peptide fragments was determined. The sequence of six tryptic fragments of p18 was identical with subsequences present in mammalian calmodulin. Authentic porcine brain calmodulin and p18 co-migrated on an sodium dodecyl sulfate-electrophoresis gel and displayed identical chromatographic behavior on a reverse phase high performance liquid chromatography column. The VIP-binding properties of p18 and calmodulin were indistinguishable. Both proteins displayed saturable and apparent high affinity binding of VIP, evidenced by potent inhibition of complexation with [Tyr10-125I]VIP by unlabeled VIP (IC50 = 6.0-8.1 nM). Rat growth hormone releasing factor and a C terminally extended form of VIP ([Leu17]VIP-GKR) also displayed potent inhibition of the binding (IC50 = 6.4 and 4 nM, respectively). These neuropeptides are potential modulators of calmodulin function.     

Purification and characterization of calmodulin from porcine renal medulla

A calcium sensitive phosphodiesterase (PDE) activated by an endogenous calmodulin was identified in the cytosolic fraction of porcine renal medulla. The PDE and calmodulin were separated from each other by DEAE-cellulose column chromatography. Calmodulin was purified from a heat-treated supernatant by column chromatography with DEAE-cellulose and hydroxylapatite. The purified renal calmodulin has a molecular weight of 17,500, is heatstable, and has a pI of 4.2. Activation of the renal PDE by calmodulin was immediate and stoichiometric. The renal calmodulin and PDE cross react with bovine brain calmodulin and PDE, indicating a lack of tissue and species specificity. Thus, renal calmodulin is very similar to bovine brain calmodulin. However, renal calmodulin did not affect detergent-solubilized or membrane-bound renal adenylate cyclase or the antidiuretic hormone-stimulated activity of the enzyme. These results suggest that calmodulin may function in the renal medulla to regulate cAMP levels by stimulation of PDE but not adenylate cyclase. However, the ubiquitous distribution of calmodulin in eukaryotic cells and its effects on a number of other enzymes allow the possibility that calmodulin may have a role in renal function other than cAMP metabolism.     

Studies on Limulus amoebocyte. Isolation and identification of a membrane-bound protein activator of cyclic nucleotide phosphodiesterase from Limulus amoebocyte

A protein isolated from Limulus polyphemus amoebocyte activates the hydrolysis of cyclic AMP by phosphodiesterase. The protein activator, like calmodulin, requires Ca2+ for its activity and is antagonized by calmodulin-modulating protein from bovine brain. 2-Chloro-10-(3-aminopropyl)-phenothiazine, a compound known to bind calmodulin, also inhibits the effect of the protein activator. This Limulus protein activator is an acidic protein with high percentage of glutamate and aspartate; it contains trimethyllysine, a characteristic amino acid found in all calmodulin. It is different from calmodulin isolated from other species, however, in its molecular weight (4 to 5 times greater), amino acid composition, antigenicity, and binding ability on 2-chloro-10-(3-aminopropyl)-phenothiazine affinity column chromatography. The amino acid composition, gel electrophoresis pattern, and molecular weight of this protein activator are indistinguishable from endotoxin-binding protein which we isolated previously by other independent methods. Immunologic studies demonstrate that these two proteins are essentially identical. The endotoxin-binding protein thus has the dual functions of binding endotoxin, and showing calmodulin-like activity. It may play an important role in degranulation of Limulus amoebocytes which is induced by minute amounts of gram-negative bacterial endotoxin.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

Comparison of the membrane-bound (Ca2+ + Mg2+)-ATPase in erythrocyte ghosts from some mammalian species

The properties of the membrane-bound calcium-pumping protein, the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) were compared in erythrocyte ghosts isolated from five mammalian species--human (Homo sapiens), bovine (Bos taurus), porcine (Sus scrofa melitensis), ovine (Ovis aries crassicandus) and caprine (Capra hircus syriaca). The specific activity of the enzyme in porcine erythrocytes is one order of magnitude higher than in the other species. It was also stimulated to various extents by the regulator protein, calmodulin, and by phosphatidylinositol in all the species. Analysis of membrane proteins revealed a number of differences which seem to suggest that the molecular architecture of the red cell membrane influences the activity of the enzyme.     

Possible involvement of calmodulin in maturation and activation of Chaetopterus eggs

We report the isolation of calmodulin from oocytes of Chaetopterus pergamentaceus. The identification of this protein is based on (1) activation of beef heart cAMP phosphodiesterase, (2) heat stability, (3) sensitivity to chlorpromazine, and (4) electrophoretic mobility identical to that of porcine brain calmodulin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of either Ca2+ or EGTA. We treated oocytes with chlorpromazine and W-7 to investigate the involvement of calmodulin in meiosis initiation and egg activation. Very low concentrations of chlorpromazine inhibited germinal vesicle breakdown (GVBD). This effect was shown to be dependent upon bright indirect light, since the drug was much less effective at GVBD inhibition under conditions of very low illumination. Higher concentrations of chlorpromazine and W-7 (100 microM) inhibited GVBD and activated eggs with intact germinal vesicles as determined by fertilization envelope formation and the onset of ameboid activity. Neither egg activation nor inhibition of calmodulin stimulation of phosphodiesterase activity in vitro was affected by light. These results are consistent with a role for calmodulin in egg activation and GVBD, but suggest that chlorpromazine in bright light may prevent GVBD by some mechanism other than calmodulin inhibition.     

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.     

Activation of ATPase Activity in the Chromatin Fraction of Pea Nuclei by Calcium and Calmodulin

ATPase, especially the Ca^2$-dependent enzyme, in the chromatinfraction isolated from nuclei of pea seedlings was activatedby bovine brain calmodulin and by protein that was judged tobe calmodulin from its Ca^2$-dependent activation of NAD kinase.This calmodulin-dependent ATPase activity was blocked by calmodulin-specificinhibitors. (Received July 11, 1983; Accepted October 24, 1983)     

African trypanosomes contain calmodulin which is distinct from host calmodulin

Studies were initiated to determine whether African trypanosomes utilize Ca2+ fluxes to coordinate complex morphological and biochemical life cycle changes. We have identified the ubiquitous intracellular Ca2+ receptor, calmodulin, in two developmental stages of Trypanosoma brucei rhodesiense. The transition from rapidly dividing, slender bloodstream trypomastigotes to slow growing procyclics in axenic culture was accompanied by changes in specific calmodulin content (3 micrograms/mg cell protein to 1 microgram/mg cell protein, respectively) and a shift in intracellular calmodulin distribution, Trypanosome calmodulin is physically and functionally distinct from that of host tissues, including bovine brain and rat erythrocytes. It is similar to but distinct from Tetrahymena calmodulin. Comparisons among these proteins isolated from the four sources were made using the following criteria: (1) mobility on sodium dodecyl sulfate discontinuous polyacrylamide gels; (2) Ca2+-induced conformational changes; (3) CNBr-cleavage fragments; (4) activation of bovine brain cyclic nucleotide phosphodiesterase in both a Ca2+-dependent and calmodulin-dependent manner; (5) activation of human erythrocyte (Ca2+ + Mg2+)-ATPase; and (6) inhibition of calmodulin activity by trifluoperazine and penfluridol. Trifluoperazine but not trifluoperazine sulfoxide was cytotoxic to trypanosomes in vitro. Half maximal effect occurred at 15 microM. We conclude that calmodulin is a functional component of Africal trypanosomes and suggest that it plays an important role in mediating the host-parasite relationship.     

P-57 is a neural specific calmodulin-binding protein

P-57 is a novel calmodulin-binding protein which has recently been isolated from bovine cerebral cortex (Andreasen, T. J., Luetje, C. W., Heideman, W., and Storm, D. R. (1983) Biochemistry 22, 4615-4618). In contrast to all other calmodulin-binding proteins characterized thus far, P-57 has equivalent or higher affinity for calmodulin in the absence of free Ca2+ compared to the presence of Ca2+. In this study, the distribution of P-57 in other tissues and within brain was examined using a radioimmune assay and photoaffinity labeling with azido-125I-calmodulin. P-57 was not found in tissues other than brain, retina, and spinal cord. Within brain, P-57 levels varied from 0.1% of the total protein in white matter regions to about 0.5% in cell body-rich fractions. The protein was found in both membrane and soluble fractions. P-57 is the most abundant calmodulin-binding protein in brain and appears to be neural specific. The concentrations of P-57 in brain and its affinity for calmodulin in the absence of Ca2+ are sufficient to complex a significant fraction of the total calmodulin present.