Publisher Summary This chapter outlines chemical crosslinking studies of the interaction of calmodulin (CaM) with its receptors. Klevit and Vanaman developed techniques for specifically derivatizing Tyr 99 in calcium-binding site III or Tyr 138 in site IV (1), both in the C-domain of mammalian CaM. Benzophenone-labeled CaM, which was radio-iodinated at its sole tyrosine (125I-Bz-CaM), and native wheat germ CaM were found to be very similar to bovine CaM in their abilities to stimulate two mammalian enzyme systems—the Ca-ATPase of red blood cell membrane and the CaM-dependent kinase of cardiac Sarcoplasmic Reticulum (SR). The major product of the affinity labeling is a complex with a molecular mass of 40,000 daltons, which has been shown to be composed of CaM and phospholamban. Benzophenone-maleimide probe spans a distance of about 10 A from the sulfhydryl group of CaM to the photoactivatable carbonyl carbon of benzophenone, suggesting that Cys 27 is within 10 A of a binding site on phospholamban. Thus, this derivative will be a useful tool for CaM structure-function studies and studies defining CaM interactions with its receptors.
This chapter describes the calbindin-D28k distribution in chick ultimobrancial and rat thyroid glands. Calbindin-D28k [CaBP], the vitamin D-dependent calcium binding protein, appears as an end-organ marker of the vitamin D endocrine system in several tissues. The protein was first discovered in chick intestine and subsequently in other chick tissues. CaBP is also present in other species and a lower molecular weight form, calbindin-D9K, predominates in mammalian intestine. White Leghorn cockerels were raised on a normal diet and sacrificed from 4 days to 5 weeks of age. Mature Sprague-Dawley rats, raised on a normal diet, were also utilized. The UB glands from chicks and thyroids from rats were rapidly dissected, quick-frozen and fixed by freeze substitution. After fixation, embedding in paraffin, sectioning and deceration, the sections were reacted immunohistochemically with anti-CaBP. The connective tissue elements of the UB gland and the isolated islands of parathyroid tissue did not contain CaBP. It is found that when UB gland sections adjacent to those stained for CaBP were reacted with anti-CT, the co-localization of CaBP and CT was observed.
This chapter discusses the structure and the function with [3H]ryanodine, which is a highly specific radioligand for studying the structure and the function of putative ligand-gated Ca2+ release channels localized at the Terminal Cisternae (TC) of the Sarcoplasmic Reticulum (SR), which may participate in Excitation–Contraction (EC) coupling in striated muscle. Regulation of the [3H]ryanodine binding site in skeletal muscle parallels many of the properties exhibited by Ca2+ induced release of Ca2+ from skinned and intact muscle fibers and TC SR vesicle preparations indicating that [3H]ryanodine binds to an open state of the channels involved. [3H]ryanodine allows direct evaluation of whether these diverse inotropic agents influence unrelated and biochemically distinct sites at the SR or or act on a single-channel gating mechanism. Caffeine and adenine nucleotides in tandem act synergistically to influence cooperativity in Ca2+ activation of [3H]ryanodine binding suggesting different allosteric mechanisms for adenine nucleotides and caffeine perhaps via overlapping domains. Contribution of the Ca2+ ATPase to the integrity of the Ca2+-ryanodine receptor complex is assessed by treating TC SR vesicles with polyoxyethylene-9-laurylether (C12E9), which preferentially solubilizes and separates this protein from the other TC SR proteins.
This chapter discusses the results of NMR studies of five calmodulin (CaM)-binding peptides aimed at determining the existence and the nature of peptide-induced conformational changes in CaM. Titrations were performed at a CaM concentration of 0.5 mM in the NMR tube. In their original characterization of M13 as a CaM-binding domain, Blumenthal et al. noted that the peptide sequence exhibited α-helical probability >1 for residues 1–18. The results indicate that a single high-affinity species of peptide-CaM complex is formed and that the conformation of both the peptide and CaM are markedly altered upon complex formation, thus supporting our earlier conclusions based on the 27-residue peptide M13. The NMR results obtained for the CaM-binding peptides are more complex. The demonstration that different peptides induce different conformational changes in CaM provides the first structural evidence for a model for CaM-target enzyme interactions in which an enzyme-specific conformational change in CaM plays an important role in the enzyme's activation.
This chapter presents an interdisciplinary approach to the molecular mechanisms of calmodulin action. The VU-1 calmodulin has an amino acid sequence that is a hybrid of vertebrate and plant calmodulins. The bacterially expressed protein lacks two of the post-translational modifications common to vertebrate and some other calmodulins. These include the acetylation of the amino terminus and the trimethylation of lysine-115. Apart from the lack of acetylation, the changes in the VU-1 calmodulin structure have been observed in the naturally occurring calmodulins. The study of calmodulin-activated proceses by protein engineering requires structural information about specific target enzymes. The detailed studies have focused on the calmodulin dependent enzyme Myosin Light Chain Kinase (MLCK). The amino acid sequences of potential calmodulin binding sites have been elucidated for skeletal and smooth muscle MLCK. A new calmodulin expression vector that allows for mutagenesis, amplification, characterization and expression of mutant calmodulin genes in E. coli has been constructed. The new expression vector, pVUCH-1, is a hybrid of pVUC-1 and pUC8. The orientation of the gene relative to the tac promoter elements within the plasmid is the same as that found in pVUC-1.
This chapter reviews comparative metal ion binding properties of the α-subunit of S-100a and the parent molecule using fluorescence spectroscopy. Bovine brain S-100 protein is a mixture of two major components, S-100a and S-100b, with a subunit composition of αβ and β2, respectively. S-100 proteins undergo a conformational change upon binding Ca2+ and Zn2+. α-subunit is an ideal system for this purpose, because the protein has only 1 cysteine at the C-terminal end in position 85. The fluorescent probe, Acrylodan, when reacted with the α-subunit in the presence of Ca2+ resulted in labeling 0.31 thiol group/α-chain. The red shift observed upon Ca2+ addition is because of a conformation change induced by Ca2+ binding whereby the probe moves to a more polar microenvironment. Ca2+ and Zn2+ can bind, and perturb to the thiol group in the presence of each other. The small perturbation observed with Zn2+ may be attributed to the fact that histidine residues, which are known to act as ligands for coordinating Zn2+ are only found at the N-terminus of the α-subunit, at positions 16 and 18, well away from cysteine 85 located at the other end.
This chapter discusses the early events of stimulus–secretion coupling in parathyroid cells and that center on the mechanisms by which changes in extracellular Ca2+ are transformed into corresponding changes in [Ca2+]. In dissociated bovine parathyroid cells loaded with fura-2, small increases in the concentration of extracellular Ca2+ evoke rapid and transient increases that are followed by lower yet sustained (steady-state) increases in [Ca2+]i. Receptor-dependent mobilization of cellular Ca2+ by extracellular stimuli generally results from the increased formation of inositol-1,4,5-trisphosphate (IP3), which then acts upon nonmitochondrial pools to release sequestered Ca2+. Fluoride, in the presence of trace amounts of aluminum contaminating most solutions, appears to act by mimicking the terminal phosphate of GTP, thus permanently activating target G-proteins and rendering them insensitive to regulation by receptors. Mobilization of cellular Ca2+ in the parathyroid cell shows many of the features characteristic of receptor-dependent processes in other cells and thus provides evidence for the presence of Ca2+ receptors on the surface of this cell. This putative receptor is presumably a protein that binds Ca2+, although it must certainly differ from intracellular Ca2+-binding proteins such as calmodulin.
This chapter explores metal ion binding properties of nitrated S-100b protein. S-100b protein exists as a dimer of 21,000 molecular weight in native solvents. The S-100b protein polypeptide chain (β-subunit) consists of 91 amino acid residues and contains a typical EF-hand type calcium-binding region located between residues 61 and 72. Tetranitromethane (TNM) under controlled conditions can be used specifically to modify tyrosine residues in proteins. The absorption spectrum of nitrated S-100b was similar to that of native S-100b. Tyrosine CD bands were located at 284 and 278 nm, and the phenylalanine CD bands were positioned at 268.5 and 262 nm. Even though the modified protein binds Ca2+ it differs significantly from the native protein in the manner in which it responds to Ca2+. Ca2+ and Zn2+ ions are believed to bind at different sites on the polypeptide chain based on our earlier spectroscopic studies with the apoprotein. Because the Zn2+ binding site, possibly involving histidine 15 or 25, is very close to tyrosine 17, the nitrated S-100b changes were monitored in the environment of tyrosine as a result of Zn2+ addition.
This chapter discusses the appearance of active calcium absorption at the time of weaning that might reflect a vitamin D-regulated induction of the ATP-dependent calcium pump. In rats prior to weaning, calcium absorption is entirely passive, whereas an active calcium absorption appears at weaning. Recently, vitamin D has been reported to increase the activity of an active, ATP-dependent calcium pump in the basolateral plasma membrane of rat intestine. Basolateral plasma membranes were isolated from washed enterocytes by differential and density gradient centrifugations. Basolateral membranes from preweaned (2-week old) and weaned (5-week old) animals were comparably enriched in marker enzymes including Na, K-ATPase and disaccharide. Calcium uptake by basolateral membranes was unaffected by weaning. The apparent effect of calcitriol on the calcium pump may have reflected a response to increased extracellular calcium. The weanling rat provides a model for study of the intestinal effects of vitamin D. At weaning, despite increases in plasma calcitriol, serum calcium changes little.
This chapter presents results of a study conducted to analyze Mg2+-ion binding to TnC and CaM by means of 25Mg Nuclear Magnetic Resonance (NMR). The eluation experiments with Phenyl-Sepharose columns were made using a reversed salt gradient. All NMR experiments were performed on a Nicolet 360 WB spectrometer at 22.15 MHz for 25Mg and 24.34 MHz for 43Ca using a homemade probe. The results showed that the temperature dependence in the 43Ca NMR line width for a sample containing 1 mM TR1C from TnC and an excess of Ca2+ is typical for intermediate exchange and similar to TnC itself. Adding Mg2+-ions results in a decrease in the 43Ca NMR line width. Considering reported binding constants for Ca2+ and Mg2+-ion to TnC and its fragments, the results are only in agreement with a model where Mg2+-ions can bind to site(s) different from the Ca2+ binding sites and that this ion binding will modulate the dynamics in the binding of the Ca2+-ions.
This chapter discusses how Myotoxin a prevents the formation of decavanadate-induced membrane crystals in a time- and concentration-dependent manner. With increasing incubation time with Myotoxin a at 20°C, the number of Sarcoplasmic Reticulum (SR) membrane crystals remaining decreases. The decrease parallels the decrease in Ca-uptake and the increase in Mg-dependent Ca-ATPase activity. The extraction of some membrane lipids and all proteins but the Ca-ATPase does not alter the susceptibility of the crystals to Myotoxin a treatment. Addition of lipids to increase the lipid content to twice the amount of the native SR membrane has no effect on the Myotoxin a-SR membrane crystal interaction. Myotoxin a appears to interact mainly with the Ca-ATPase. Because the Ca-ATPase seems to be present as a dimer during its catalytic cycle, monomerization of the ATPase because of its interaction with Myotoxin a might lead to the uncoupling of ATP hydrolylis from Ca-translocation.
This chapter elaborates the calcium-dependent functional hydrophobic regions of calcium-modulated proteins. Calcium-modulated proteins including calmodulin, troponin C, S-100 protein, myosin light chain and parvalbumin may have two kinds of function, calcium-binding and interacting with target proteins or drugs. The EF hand structure was found in these proteins and proposed to be responsible for calcium-binding function. It has been proposed the possibility that calcium ion binding produces conformational changes in calmodulin that expose hydrophobic regions on the surface of the molecule and which may act as active sites of interaction with its binding proteins or calmodulin antagonists. The calcium-dependent hydrophobic regions of troponin C and S-100 protein were also demonstrated by using fluorescent hydophobic probes and calcium-dependent hydrophobic chromatography. However, these calcium binding proteins cannot activate calcium-dependent enzymes. These calcium-modulated proteins, except parvalbumin, produced an enhancement in the fluorescence intensity of 1-NPN, 2-NPN, 1,8-ANS, 2,8-ANS and 2,6-ANS. Parvalbumin never be able to produce an enhancement in the fluorescence intensity of hydrophobic probes. These results show that parbalbumin may not have target proteins and its function may be the soluble relaxing factor of muscle by its Ca-binding.
The conventional alternating access model for Ca2+ transport by the sarcoplasmic reticulum Ca2+ pump is modified, partly on the basis of the proposed MacLennan-Green domain structure for the Ca2+-pump protein. The present model divides the uptake state (E1) of the protein into three substates, differing in the condition of the Ca2+-binding domain. The domain is an open cavity in the first substate and can bind only a single Ca2+ ion. A fast "jaw-closing" (or "hinge-bending") step then partially closes the cavity to generate the second substate that has a second Ca2+-binding site. Occupation of this site is followed by another jaw-closing step that closes the binding cavity and occludes the bound ions. The subsequent translocation step (to form E2) remains unchanged from previous models. The modified model predicts a constant transport stoichiometry of two Ca2+ per pump reaction cycle. It suggests a plausible mechanism for coupling between Ca2+ binding and ATP utilization: the model predicts (in agreement with experiment) that Ca2+ binding should be a mandatory requirement for phosphorylation of the pump protein, though ATP binding per se does not require Ca2+. The model is consistent with high cooperativity in equilibrium binding of Ca2+, both in the absence and presence of ATP.
Publisher Summary This chapter analyzes small calcium-binding proteins (CaBP) and 1,25-dihydroxyvitamin D receptor distribution. The presence of 1,25-dihydroxyvitamin D [1,25(OH)203] receptors in a number of new target tissues and the hormone's physiological role are studied. Small CaBPs were visualized by 45 Ca-incubation and autoradiography. In addition to the intestinal mucosa and the kidney, tissues classically known to contain both 1,25(OH) 2 D 3 receptors and vitamin D-related CaBPs, CaBPs were examined in tissues recently shown to contain receptors and tissues without receptors. The CaBP detected with these procedures included unidentified bands at 13 kDa, 23.5 kDa, 29 kDa and 30.5 kDa. All tissues contained calmodulin that appeared as a doublet at 18.5 and 19.5 kDa. A band at 14.5 kDa that co-migrated with parvalbumin was very prominent in prostrate. Immunoblotting demonstrated the cross reactivity of this protein in testis and heart with antibodies to authentic rat renal 28 kDa CaBP D . Prostrate, lung and bladder also contained CaBP band comigrating with the vitamin D-related 10 kDa mucosal CaBP. It is found that there is no apparent correlation between the CaBP patterns and the 1,25(OH) 2 D 3 receptors.
This chapter discusses the localization of calcium and zinc binding sites. Proton NMR titration experiments with bovine and human apo-α lactalbumin suggest that the protein undergoes only minor rearrangement in its overall structure upon binding a variety of di- and tri valent metal ions. Titration with one equivalent of Ca2+ and lanthanides causes changes in some of the upfield shifted methyl resonances, very minor changes in the aromatic resonances and a marked upfield shift of the Met-90 resonance. The existence of separate binding sites for the Ca2+ and Zn2+ classes of metal ions. Because both Zn2+ and Ca2+ have an effect on the Met-90 residue it is likely that the Zn2+ binds relatively close to the Ca2+-site, in agreement with earlier fluorescence measurements which indicated a distance of 12 Å between the sites. Both sites can be occupied simultaneously, but the occupation of the Ca2+ site has a dominant effect on the protein conformation.
This chapter explains chick Calbindin genomic organization by electron microscopy. Two partially overlapping genomic clones (8 and 2) constructed in EMBL3 phage (8 and 2) were necessary to map the complete gene. Hybridization was performed using linearized pWH11 (2.1 kbp cDNA inserted pUC12) and genomic clones 8 or 2. Analysis of the hybrids using clone 8 revealed 7 exons whose length appeared rather homogeneous; introns length was much more variable . Hybridization between clone 8 and clone 2 revealed a overlapping fragment of about 13 kbp bordered by two unhybridized portions corresponding respectively to the 51 end of clone 8 (3270 bp + 200 bp) and to 3′ end of clone 2. Analysis of the hybrids showed an unhybridized DNA fragment of (665 + 80) bp, which is believed to represent an up stream portion of Calbindin gene.
This chapter elaborates the ability of insulin to inhibit calmodulin-dependent activities, such as the plasma membrane Ca2+ extrusion pump could involve the phosphorylation of calmodulin itself. Various lines of evidence suggest that alterations of intracellular Ca2+ may be involved in insulin action. Recent evidence indicates that an important transduction mechanism for the action of insulin may be a tyrosine-specific protein kinase associated with the B-subunit of the insulin receptor. Physiological concentrations of insulin resulted in the phosphorylation of three 17 kDa phosphoproteins, the most acidic of which was calmodulin. The stimulation of adipocytes with insulin is accompanied by the phosphorylation of calmodulin. This phosphorylation is a direct consequence of activation of the insulin receptor kinase by insulin. If the phosphorylated molecule has altered bioactivity, the direct phosphorylation of calmodulin could be an important link between the binding of insulin to its receptor and the final effects on cellular metabolism.
A thyroid particulate fraction contains an NADPH-dependent H2O2-generating enzyme which requires Ca2+ for activity. A Chaps solubilized extract of the thyroid particulate fraction partially purified by DEAE chromatography did not show a dependence on Ca2+ for activity. Preincubation of the particulate fraction with Ca2+ yielded a preparation insensitive to Ca2+. The non-particulate fraction obtained after incubation of the particles in the presence of Ca2+ was able to inhibit, in the presence of EGTA, the Ca2+-desensitized particulate fraction and the enzyme isolated on DEAE. It is concluded that the reversible Ca2+ activation of the NADPH-dependent H2O2 generation was modulated in porcine thyroid tissue by (a) calcium-releasable inhibitor protein(s).
