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The NH 2 -terminal blocking group of the Ca 2+ -binding B-subunit of calcineurin (protein phosphatase-2B) has been identified as myristic acid by fast atom bombardment mass spectrometry and gas chromatography. The sequence, myristyl-GlyAsnGluAla-, is very similar to that of the catalytic subunit of cyclic AMP-dependent protein kinase, the only other protein known to contain this fatty acid. This finding, and the elution of all myristyl peptides at 57% acetonitrile on reverse phase HPLC, may facilitate the identification of other proteins with this blocking group.

Since it had been previously shown that in Paramecium cells exocytosis involves the dephosphorylation of a 65-kD phosphoprotein (PP), we tried to induce exocytotic membrane fusion by exogenous phosphatases (alkaline phosphatase or calcineurin [CaN]). The occurrence of calmodulin (CaM) at preformed exocytosis sites (Momayezi, M., H. Kersken, U. Gras, J. Vilmart-Seuwen, and H. Plattner, 1986, J. Histochem. Cytochem., 34:1621-1638) and the current finding of the presence of the 65-kD PP and of a CaN-like protein in cell surface fragments ("cortices") isolated from Paramecium cells led us to also test the effect of antibodies (Ab) against CaM or CaN on exocytosis performance. Microinjected anti-CaN Ab strongly inhibit exocytosis. (Negative results with microinjected anti-CaM Ab can easily be explained by the abundance of CaM.) Alternatively, microinjection of a Ca2+-CaM-CaN complex triggers exocytosis. The same occurs with alkaline phosphatase. All these effects can also be mimicked in vitro with isolated cortices. In vitro exocytosis triggered by adding Ca2+-CaM-CaN or alkaline phosphatase is paralleled by dephosphorylation of the 65-kD PP. Exocytosis can also be inhibited in cortices by anti-CaM Ab or anti-CaN Ab. In wild-type cells, compounds that inhibit phosphatase activity, but none that inhibit kinases or proteases, are able to inhibit exocytosis. Exocytosis cannot be induced by phosphatase injection in a membrane-fusion-deficient mutant strain (nd9-28 degrees C) characterized by a defective organization of exocytosis sites (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). We conclude that exocytotic membrane fusion requires an adequate assembly of molecular components to allow for the dephosphorylation of a 65-kD PP and that this step is crucial for the induction of exocytotic membrane fusion in Paramecium cells. In vivo this probably involves a Ca2+-CaM-stimulated CaN-like PP phosphatase.

Occupancy of one of the two phenothiazine-binding sites on calmodulin does not significantly decrease the affinity of calmodulin for its target proteins; however, it does affect the ability of calmodulin to activate some enzymes. Previously we demonstrated that a covalent adduct of calmodulin with one molecule of phenothiazine (CAPP 1 -calmodulin) is an antagonist for the calmodulin-dependent enzymes, cAMP phosphodiesterase and myosin kinase, and a partial agonist for calcineurin. We now show that CAPP 1 -calmodulin is a full agonist for glycogen synthase kinase and phosphorylase kinase. Unlike phenothiazines, CAPP 1 -calmodulin is specific for calmodulin-regulated proteins; it has no effect on protein kinase C. With the exception of phosphorylase kinase, occupancy of two phenothiazine-binding sites completely eliminates the ability of calmodulin to activate these proteins. Thus, the study of the interaction of CAPP 1 -calmodulin with calmodulin target proteins demonstrates that calmodulin interacts differently with different proteins. This is confirmed by studies of the effect of calmodulin fragments, 1–77 and 78–148, on calmodulin-regulated enzymes.

The inhibitory protein that binds calmodulin and thus prevents activation of several Ca2+-dependent enzymes by calmodulin is shown to also bind four Ca2+ per mol of protein with high affinity (Kd less than or equal to 10(-6) M). On the basis of its Ca2+- binding properties and its localization to nervous tissue, the inhibitory protein is now called "calcineurin." Calcineurin is composed of two subunits: calcineurin A (61,000 Mr) which interacts with calmodulin in a Ca2+-dependent fashion, and calcineurin B (15,000 Mr) which binds Ca2+. The interaction of calcineurin A with calcineurin B is independent of Ca2+ or Mg2+. The dual interaction of calcineurin A with two different Ca2+-binding components and the high affinity of calcineurin for Ca2+ suggest a possible role for calcineurin in the regulation of free Ca2+ concentrations in the nervous system. Calcineurin may thereby modulate the release and action of neurotransmitters.

The acid stability of calmodulin has been used to devise a rapid and efficient method of decalcification based on trichloroacetic acid precipitation. Study of the competitive binding of K+, Mg2+, and Ca2+ to the Ca2+-binding sites of calmodulin has allowed determination of the intrinsic binding constants of each of the three cations for the four Ca2+-binding sites. The data are compatible with an ordered binding of Ca2+. If the Ca2+ sites are labeled A, B, C, and D starting at the NH2 terminus, the order of binding is postulated to be B, A, C, and D. The ordered binding properties support the suggestion that calmodulin translates quantitative Ca2+ signals into qualitatively different cellular responses.

Subcellular fractions of neuroblastoma x glioma (NG108-15) hybrid cells were used to study the mechanism of inositol 1,4,5-trisphosphate-induced calcium release. A microsomal fraction, enriched in endoplasmic reticulum and plasma membranes and almost devoid of mitochondria, was the most active in inositol trisphosphate- or GTP-dependent release of calcium. Neither GTP nor inositol 1,4,5-trisphosphate affected the calcium efflux mediated by the other reagent, suggesting that inositol trisphosphate and GTP act on different calcium-sequestrating vesicles. The stimulation of calcium release by GTP was relatively slow (t1/2 = 90 s), dependent on polyethyleneglycol, and greater at 2 X 10(-5) M calcium (5 nmol X min-1 X mg-1) than at 10(-6) M calcium (0.8 nmol X min-1 X mg-1). The inositol trisphosphate-induced calcium efflux was not mimicked by inositol monophosphate; it was fast (t1/2 less than 10 s) and unaffected by 3% polyethyleneglycol. The amount of calcium released by inositol trisphosphate was greatest at 10(-6) M external calcium (1 nmol X min-1 X mg-1) and it was undetectable at 2 X 10(-5) M calcium. A feedback inhibition of the inositol trisphosphate-induced calcium release by cytoplasmic calcium provides a safety mechanism preventing deleterious effects of abnormally high calcium levels.

The interaction of calmodulin with calcineurin, a calcium- and calmodulin-stimulated protein phosphatase, was investigated using a solid-phase assay. Binding of 125I-calmodulin by calcineurin immobilized on nitrocellulose membrane filters was of high affinity, reversible, and calcium-dependent. Complex binding kinetics reflected a time- and calcium/calmodulin-dependent conformational change of calcineurin which was shown to be ligand-induced renaturation. After renaturation and removal of calmodulin, immobilized calcineurin exhibited simple 125I-calmodulin binding kinetics with a single class of independent sites. The maximum stoichiometry of 125I-calmodulin binding to immobilized calcineurin was 0.1 mol/mol. The association rate (K1 = 8.9 x 10(3) M-1 S-1) and the dissociation rate (K-1 = 8.5 x 10(-5) s-1) yielded a dissociation constant of Kd = 10 nM. Equilibrium binding analyses gave a Kd value of 16 nM. The affinity of 125I-calmodulin for immobilized calcineurin was half that of unmodified calmodulin. Using equilibrium competition experiments, we determined, for the first time, the dissociation constant for the binding of native calmodulin by calcineurin in solution, Kd less than or equal to 0.1 nM (Kd for 125I-calmodulin = 0.23 +/- 0.09 nM). The effects of ionic strength and pH on 125I-calmodulin binding to immobilized calcineurin were characterized. The dissociation rate was dependent on free calcium concentration, with half-maximal rate at 700 nM calcium. 125I-Calmodulin equilibrium binding by the immobilized A subunit of calcineurin exhibited half the affinity of the holoenzyme, Kd = 30 nM. The described phenomenon, of reversible denaturation associated with immobilization of a protein on nitrocellulose, may be a general one open to exploitation in other systems.

The complete primary structure of the B subunit of calcineurin (protein phosphatase 2B) has been determined by automated sequence analysis. The protein consists of a single polypeptide chain of 168 residues, relative molecular mass 19200. The structure shows 35% identity with the sequence of calmodulin and 29% with troponin C. Homology is mainly confined to the regions of the four putative Ca2+-binding loops. The results demonstrate that the B subunit is a new member of this family of Ca2+-binding proteins. The N-terminal glycine residue is blocked with the C14-saturated fatty acid myristic acid and the first four residues are very similar to those of the catalytic subunit of cyclic-AMP-dependent protein kinase which also contains a myristoyl blocking group.

The alpha,beta eliminations of NH3 from L-histidine and 4-fluoro-L-histidine by histidine ammonia-lyase appear to occur by similar mechanisms, although a large difference in Vmax for the two reactions was observed. Both reactions were shown to be reversible with an equilibrium constant of 4 to 5. The presteady state kinetics of the deamination of 4-fluoro-L-histidine indicates that the rate-determining step precedes the dissociation of ammonia from the enzyme. The isotope effect of 1.4 to 2.0 observed with 4-fluoro-DL-[beta-2-H2]histidine or DL-[beta-2-H2]histidine or DL-[beta-2-H2]histidine indicates that the C-H bond breakage is at least partially rate-determining for the deamination of both substrates.

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.

Myosin light chain kinase and a fraction of type II cAMP-dependent protein kinase have been partially purified from bovine brain by affinity chromatography on calmodulin-Sepharose. The myosin kinase was purified approximately 3700-fold and has an estimated molecular weight of 130,000 +/- 10,000 by sodium dodecyl sulfate gel electrophoresis. A fraction of soluble cAMP-dependent protein kinase also bound to calmodulin-Sepharose and was purified 2300-fold. A fraction of this cAMP-dependent protein kinase after purification by glycerol gradient centrifugation was shown to contain the two subunits of calcineurin, a major calmodulin-binding protein in brain, and the two subunits of type II cAMP-dependent protein kinase in a ratio of 1:1:2:2. Its sedimentation coefficient was 8.1 S and 9.0 S when centrifuged in the absence or presence of calmodulin, suggesting the formation of a complex between calmodulin and protein kinase. Our results suggest the possibility that calcineurin may be involved in the interaction between the protein kinase and calmodulin. Furthermore, our studies imply that the regulatory subunit of the cAMP-dependent protein kinase, but not the catalytic subunit, is the site of interaction with calmodulin since the catalytic subunit of protein kinase was partially resolved from the complex by cAMP.

Using an antibody prepared against performic acid-treated calmodulin, we have localized calmodulin in cultured fibroblastic cells by immunofluorescence and immunoelectron microscopy. In interphase cells, calmodulin was found to be diffusely distributed throughout the cytosol. An increased amount of calmodulin was found in the pericentriolar region of interphase cells. No significant aggregation of calmodulin was found in association with microfilaments, peripheral cytoplasmic microtubules or clathrin-coated structures. Calmodulin was present in moderate amounts in microvilli, ruffles, and zeiotic blebs of the cell surface. In motitic cells, calmodulin was found concentrated in the pericentriolar region, and appeared to concentrate along radiating spindle microtubules proximal to the centrioles. Redistribution of calmodulin was seen between early and late telophase, in which the pericentriolar pattern of calmodulin in early telophase shifted to an aggregation on the intercellular bridge, with a large part of the midbody portion of the bridge being devoid of calmodulin. These results show that calmodulin is distributed throughout the cytosol, but is markedly concentrated in the region of the microtubule organizing center in interphase cells, as well as in elements of the mitotic spindle apparatus. This distribution suggests that calmodulin has a regulatory role in the organization and function of microtubules during interphase, as well as during mitosis.

Monoclonal antibodies have been raised against native calcineurin using conventional in vivo immunization and hybridoma procedures. The relatively high affinity of nonimmune IgG for the two subunits of calcineurin resulted in large nonspecific binding values for immunoassays of native, dissociated and denatured calcineurin, which complicated the antibody screening. Monoclonal aCn5, a high-affinity IgG1 that exhibits specific binding, was characterized. Other calmodulin-binding proteins tested were not recognized by aCn5. Simple binding properties were exhibited in solid-phase experiments, Kd = 26 (+/- 4) pM, but the stoichiometry was low. The loss of immunoreactivity after denaturation of calcineurin indicated that the aCn5 epitope is of the assembled topographic, not segmental, type. The epitope was located to the A subunit and affinity was unaffected by the presence of calcineurin B. The epitope remained intact after proteolytic removal of the amino-terminal 20 residues of calcineurin A essential for phosphatase activity, and the carboxyl-terminal inhibitory and calmodulin-binding domains. The calmodulin-binding peptide derived from calcineurin, cA8, was not recognized by aCn5. Addition of Ca2+, Mn2+, Ni2+, chelators or dithiothreitol did not influence the affinity of aCn5 for the holoenzyme. Phosphatase activity of calcineurin, in the presence and absence of calmodulin and after removal of the inhibitory domain, was little affected by aCn5. Thus, the aCn5 epitope defines a previously unidentified structural domain of calcineurin A located in a region of the proteolytically resistant core that is topologically distinct from the catalytic, inhibitory, calmodulin-binding and calcineurin-B-binding domains, and not functionally connected with calcineurin B or the putative metal-binding domain(s).

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.

We have identified and cloned human cDNA for the Ca2+-binding subunit of calcineurin, the brain isozyme of the Ca2+/calmodulin-stimulated protein phosphatase. The 2.5-kb cDNA has an open reading frame of 510 bp, a leader sequence of at least 500 bp, and a 1,277-bp 3'-noncoding sequence. The deduced sequence of the human protein differs from bovine brain calcineurin B by an additional valine at the carboxyl terminus and substitution of Met-11 and Ser-153 by cysteine. A partial clone of the mouse protein corresponding to amino acids 75-150 was also isolated. This portion of the human and mouse protein sequence is identical, with the DNA sequences showing 94% identity. The respective mRNAs in human and mouse are also of similar size. As was observed with protein levels, mRNA abundance in brain is 20-60 times that found in other tissues with the exception of HeLa cells which, like brain, contain abundant calcineurin B mRNA.

Two types (I and II) of cDNAs encoding the large (A) subunit of calcineurin, a calmodulin-regulated protein phosphatase, were isolated from human basal ganglia and brainstem mRNA. The complete sequences of the two calcineurin clones are identical except for a 54-base-pair insert in the type I clone and different 3' ends including part of the coding sequence for the C termini of the two proteins. These findings suggest that calcineurin A consists of at least two isozymes that may result from alternative splicing events. The two forms of the enzyme differ in the C terminus, which contains an inhibitory domain rapidly severed by limited proteolysis. With the exception of an 18-amino acid insert, the central parts of the molecules, which harbor the catalytic domains, are identical and show extended similarities with the entire catalytic subunits of protein phosphatases 1 and 2A, defining a distinct family of protein phosphatases. The 40-residue N-terminal fragment, specific for calcineurin, contains a sequence of 11 successive prolines that is also found in bovine brain calcineurin by peptide sequencing. A role in the calmodulin activation of calcineurin is proposed for this novel structural element.

Lactose synthetase is made up of a galactosyl transferase (the A protein) and α-lactalbumin which controls the specificity of the reaction by regulating the affinity of the system for acceptor sugars. We have purified the A protein from bovine skim milk so that it is free of contaminating proteins. This preparation is a single chain glycoprotein of molecular weight 44,000 with a sedimentation coefficient of 3.2 S. The enzyme behaves on disc gel electrophoresis as a family of closely related components all of which are enzymically active and contain carbohydrate. A rapid and sensitive method of studying macromolecular interactions is described. In this procedure the band forming cell of Vinograd is used and the migration of a band of A protein through solutions of α-lactalbumin is followed in the analytical ultracentrifuge equipped with a photoelectric scanner. These experiments show that a complex is formed between A protein and α-lactalbumin which contains one molecule of each protein component. The complex is stable enough to be observed only in the presence of one of the substrates of the enzyme such as UDP-galactose or N-acetylglucosamine. Binding of tetranitromethane-treated α-lactalbumin to A protein in the presence of UDP-galactose could be demonstrated by conventional sedimentation velocity techniques using absorption optics at 430 nm.

Mature and immature rat brain mitochondria exhibit marked differences in the rates of oxygen consumption and phosphorylation with dl-β-hydroxybutyrate as substrate. Directly assayed dl-β-hydroxybutyric dehydrogenase activity is 3 to 4 times greater at 15 days than at 180 days of age. This difference is observed also in mitochondrial preparations that have undergone sonic oscillation and is maintained at saturating levels of NAD+. Mixtures of sonically disrupted mitochondria from mature and immature brain exhibit completely additive enzyme activities. The high activity in the immature brain mitochondria has been found to be specific for d(-)-β-hydroxybutyrate. Brain β-hydroxybutyric dehydrogenase activity is low in the immediate postnatal period, rises together with cytochrome oxidase during the first 30 days of life, and then, in contrast to cytochrome oxidase, progressively falls again to the low level of adult brain. The enzyme activity is, therefore, maximal during the period of most rapid growth and maturation of the brain.

Histidine ammonia lyase, obtained by a modification of the purification procedures previously described, is shown to be a mixture of enzymatically active polymeric species which undergo depolymerization on treatment with thiols. The monomeric unit of molecular weight 213,000 is itself composed of four subunits of molecular weight 55,000. The sulfhydryl groups on the enzyme play a role in catalysis as well as in polymerization. Spectrophotometric and circular dichroism measurements have revealed the presence of a hitherto undetected chromophore absorbing near 315 mµ. Inactivation of the enzyme with sodium borohydride or l-cysteine markedly changes the absorption of the protein in this spectral region.

The experiments reported here indicate that polynucleotide phosphorylase from Micrococcus lysodeikticus degrades a given polyribonucleotide chain to completion before releasing a small, resistant oligonucleotide and initiating hydrolysis of another chain. This mode of attack has been termed processive degradation. Two types of experiments support this hypothesis. First, during the phosphorolysis of polynucleotides, the only materials present at all stages of degradation are intact polynucleotide, nucleoside diphosphate, and very short oligonucleotides. Second, in the course of the phosphorolysis of polynucleotides that are radioactively labeled at one or the other end of the chain, the percentage of radioactive product released is equivalent to the over-all percentage of hydrolysis at all stages of the reaction. A procedure which leads to the preparation of polynucleotide phosphorylase (M. lysodeikticus) of a high degree of purity is also described.

Histidine ammonia-lyase from Pseudomonas is a tetrameric protein which contains 4 reactive cysteinyl residues per mole of tetramer; these must be in the reduced form for optimal activity. The reduced enzyme can be selectively alkylated with iodo[14C2]acetate. A single radioactive peptide is found in the tryptic digest of this material, with the following amino acid composition: Cys(Cm), 1; Asp, 2; Glu, 1; Gly, 2; Ala, 1; Leu, 2; Ile, 1; Ser, 2; Thr, 1; Pro, 1; His, 2; and Arg, 1. Four moles of the peptide are recovered per mole of tetrameric enzyme (molecular weight 215,000). This result suggests that the tetrameric enzyme is composed of four very similar or identical polypeptide chains.

Polynucleotide phosphorylase of Micrococcus luteus (formerly Micrococcus lysodeikticus) is converted to a primer-dependent form by limited tryptic hydrolysis. Evidence is presented which indicates that the loss of a portion of the polypeptide is not itself responsible for the loss of ability to initiate polymerization. Thus, the primer-dependent enzyme obtained after tryptic hydrolysis is converted to primer-independent enzyme by reduction with sulfhydryl reagents. It can then be reconverted to primer dependence by reaction with sulfhydryl inhibitors. Under suitable conditions these treatments have a selective effect on the ability of the enzyme to polymerize ADP in the absence of primer. Neither the ability to phosphorolyze polyribonucleotide nor the ability to polymerize in the presence of primer is markedly affected. Therefore it appears that the tryptic hydrolysis is accompanied by the modification of one or more sulfurcontaining amino acids. This modification is incompatible with polymerization unless a primer is present. Preliminary experiments on the nature of these residues in the original enzyme are also presented.

Polynucleotide phosphorylase from Micrococcus luteus (formerly Micrococcus lysodeikticus) can be converted from a primer-independent form (Form-I) to a primer-dependent form (Form-T) by limited proteolysis with trypsin. The conversion of Form-I to Form-T is accompanied by the breaking of between five and six peptide bonds per molecule of enzyme, and results in the removal of a small portion of the peptide chain of each subunit. The two enzymes have molecular weights of 2.6 × 105 and 2.2 × 105, respectively, and both are composed of four subunits. Restoration of primer independence to Form-T is obtained by treatment of Form-T with sulfhydryl reagents. After thiol treatment, one or two enzyme sulfhydryl groups become titratable.

Histidine ammonia-lyase was purified free of metal ions and was shown to be dependent on the addition of a divalent cation (Mn2+, Fe2+, Zn2+, or Cd2+) for optimal activity. The data presented suggest that the increased activity of the enzyme after reduction by thiols is due to an increased affinity for metal ions. Two of the metals, Mn2+ and Cd2+, which were studied in most detail, differ markedly in their binding to the enzyme. In the absence of substrate, Cd2+ ions protect the four reactive —SH groups of the reduced enzyme against 5,5'-dithiobis (2-nitrobenzoic acid) titration, and 2 g atoms of Cd2+ were shown to be bound tightly per mole of enzyme. Mn2+ did not bind to the enzyme in the absence of l-histidine, but in the presence of the substrate a partial protection of the sulfhydryl groups was observed.

A covalent adduct of norchlorpromazine (CAPP) and calmodulin is a very potent antagonist of calmodulin activation of several enzymes. The phenothiazine-calmodulin complex (CAPP-calmodulin) acts as a pure antagonist with phosphodiesterase and myosin kinase or a partial agonist with the phosphoprotein phosphatase, calcineurin. Because of its potency and the selectivity inherent to its calmodulin moiety, CAPP-calmodulin should be a uniquely useful probe of calmodulin actions.

Spectroscopic methods have shown that Ca2+ chelators interact with Ca2(+)-binding proteins. These spectral alterations have been interpreted as evidence for the binding of chelator by the proteins. We show by direct examination of EDTA interaction with calmodulin and alpha-lactalbumin that these proteins repel EDTA rather than bind it. The repulsion is reduced by increased salt concentration but is unaffected by Ca2+ binding to the proteins. The acidic protein, alpha-lactalbumin, repells the negatively charged EDTA and inorganic phosphate whereas the basic protein, lysozyme, repells the positively charged spermine. Thus, spectroscopic changes induced by negatively charged Ca2+ chelators on negatively charged Ca2(+)-binding proteins are due to electrostatic repulsion, and not to binding. These observations underscore the possible pitfalls of using spectroscopic methods alone to analyze protein-ligand interactions.

Calmodulin forms a covalent, one to one, complex with 3H-labeled norchlorpromazine isothiocyanate. Complex formation was monitored by high-performance liquid chromatography using a CN reverse-phase column which resolves calmodulin, the calmodulin-norchlorpromazine adduct, and norchlorpromazine isothiocyanate. Formation of the adduct requires Ca2+ and is not observed with norchlorpromazine. The one to one calmodulin-norchlorpromazine complex does not activate phosphodiesterase but can interact with the enzyme and competitively inhibit its stimulation by calmodulin. High concentrations of trifluoperazine inhibit whereas low concentrations stimulate complex formation. This apparent potentiation of the interaction of calmodulin with norchlorpromazine by another phenothiazine suggests that calmodulin contains at least two phenothiazine binding sites and that the binding of phenothiazine to calmodulin is cooperative.

Publisher Summary Because of the presence of many calmodulin-binding proteins in brain, affinity chromatography based on binding to calmodulin lacks specificity. Additional conventional purification steps are needed, prolonging the length of the purification procedure, causing a reduction in yield and increasing the probability of proteolysis and subsequent decrease of calmodulin stimulation. The lack of specificity of the calmodulin affinity chromatography step can be overcome by the use of calmodulin fragments. The N-terminal half-fragment of calmodulin, fragment 1-77, binds cyclic nucleotide phosphodiesterase but does not interact with most of the other calmodulin binding proteins present in brain extracts. Thus, affinity chromatogramaphy on fragment 1-77 coupled to Sepharose can be used to selectively purify the phosphodiesterase. A three-step method, based on the specific interaction of the enzyme with calmodulin 1-77, allows the rapid (2–3days) purification of cyclic nucleotide phosphodiesterase to a high specific activity and with retention of a large stimulation by calmodulin.

The effect of GTP on Ca2+ uptake and release was studied in a microsomal fraction isolated from neuroblastoma x glioma hybrid NG108-15 cells. GTP did not alter the ATP-dependent initial uptake of Ca2+ but markedly enhanced the efflux of Ca2+ from microsomes. GTP-dependent Ca2+ release requires the presence of millimolar concentration of Mg2+. The effect of GTP was not mimicked by other nucleotides and was competitively blocked by the thiophosphate analogue of GTP, GTP gamma S but not by the non-hydrolyzable nucleotide GMP-PNP. Addition of an inhibiting concentration of GTP gamma S after completion of GTP-induced calcium release did not result in a re-uptake of Ca2+, showing the irreversibility of the releasing effect of GTP. Our data are consistent with the hypothesis of Ca2+-dependent GTP-induced opening of a channel responsible for vectorial transport of Ca2+ ions from one intracellular compartment to another. A model is proposed suggesting that the GTP-binding protein is a GTP-specific diacylglycerol kinase.

Summary Oncomodulin was purified from Morris rat hepatoma according to the procedure of Durkin, J.P., Brewer, L.M. and MacManus, J.P. (1983) Cancer Res. 43 , 5390–5394. The preparation, in general, had the properties and amino acid composition of the material which they described. However, we were unable to confirm the reported stimulation of cyclic nucleotide phosphodiesterase under conditions where calmodulin gave the usual stimulation.

A family of mutant proteins related to calmodulin (CaM) has been produced using cDNA constructs in bacterial expression vectors. The new proteins contain amino acid substitutions in Ca2+-binding domains I, II, both I and II, or both II and IV. The calmodulin-like proteins have been characterized with respect to mobility on SDS-polyacrylamide gels, Ca2+-dependent enhancement of tyrosine fluorescence, and abilities to activate the CaM-dependent phosphatase calcineurin. These studies suggest that an intact Ca2+-binding domain II is minimally required for full activation of calcineurin.

Methods were developed for the isolation and peptide mapping of calmodulin and its chemically modified derivatives by reversed-phase high-performance liquid chromatography (HPLC). Calmodulin and its guanidinated, iodinated, and performic acid-oxidized derivatives can be isolatedon alkylphenyl columns by using gradients of acetonitrile in 10 mM potassium phosphate, pH 6.0, 2mM EGTA. Peptide mapping by HPLC, following complete digestion of the proteins with clostripain, allows identification of the modified amino acids residues. Clostripain peptides are eluted in the order 87–90, 75–86, 91–106, 107–126, 127–148, 107–148, 1–37, and 38–74. Performic acid oxidation of methionines decreases the retention times of the modified peptides, whereas iodination of tyrosines or guanidination of lysines increases retention times of modified peptides. These HPLC methods are applicable to the identification of specific modifications of calmodulin, allowing the assessment of the role of individual amino acid residues in determining the unique physical, chemical, and spectroscopic properties of this ubiquitous intracellular calcium-binding protein.

The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32 P from [γ- 32 P]ATP into calmodulin fragment 1–106. Incorporation was exclusively into serine 101. With fragment 78–148, the extent of phosphorylation was somewhat less and 32 P appeared mainly in threonine residues. Fragment 1–90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca 2+ -binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg 2+ (60–70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca 2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble calmodulin.

Although it has been believed for several years that calcium ions are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past three years that this concept has started to be placed on a firm molecular basis. It appears that the regulation of phosphorylase kinase in vivo is achieved through the interaction of the enzyme with the two calcium binding proteins, calmodulin and troponin-C, and that the relative importance of these proteins depends on the degree of phosphorylation of the enzyme (figure 3). In the dephosphorylated form of the enzyme, troponin-C rather than calmodulin is the dominant calcium dependent regulator providing an attractive mechanism of coupling glycogenolysis and muscle contraction, since the same calcium binding protein activates both processes. On the other hand, the phosphorylated form of the enzyme can hardly be activated at all by troponin-C, although it is still completely dependent on calcium ions. Calmodulin (the delta-subunit) is therefore the dominant calcium dependent regulator of phosphorylase kinase in its hormonally activated state. Recent work has demonstrated that phosphorylase kinase not only activates phosphorylase, but also phosphorylates glycogen synthase thereby decreasing its activity (45-49). The regulation of phosphorylase kinase by calcium ions may therefore also provide a mechanism for co-ordinating the rates of glycogenolysis and glycogen synthesis during muscle contraction.

The K m for the interaction of 4-nitro-L-histidine with histidine ammonia-lyase (reduced enzyme, pH 8.0) is comparable to that for L-histidine, while V max is 1 8 that for the natural substrate. With the analog, addition of Cd +2 effects a small decrease in K m but fails to alter V max ; the normal deuterium isotope effect for removal of the β-hydrogen (1.5–2.0) is eliminated; and enzyme-catalyzed incorporation of solvent tritium into substrate occurs to a much greater extent than into histidine. Thus, the nitro group increases the acidity of the β-hydrogen and the stability of the conjugate carbanion to such a degree that CH bond cleavage now precedes rate-limiting CN bond cleavage.

Calcineurin, a heterodimer of calcineurin B, a 19,000 Mr Ca2+-binding subunit, and calcineurin A, a 61,000 Mr calmodulin-binding subunit, was previously proposed to be a calmodulin- and Ca2+-regulated protein phosphatase. Like other calmodulin-stimulated enzymes, calcineurin can be activated and rendered calmodulin- and Ca2+-independent by limited proteolysis. By glycerol gradient centrifugation, the native enzyme has a s20,w of 4.5 S in EGTA and 5 S in the presence of Ca2+-calmodulin. Under the same conditions, the s20,w of the trypsin-activated enzyme (4.3 S) is not affected by Ca2+ and calmodulin. The trypsin-treated enzyme is a heterodimer of calcineurin B and a 45,000 Mr fragment of calcineurin A that has lost its ability to interact with calmodulin. Phosphatase activity sediments with calcineurin or its proteolytic fragments, providing further evidence that calcineurin is indeed a protein phosphatase. Calmodulin protects calcineurin against tryptic digestion; proteolysis occurs more slowly, yielding fragments with Mr 57,000, 55,000, and 54,000 that have preserved their ability to interact with calmodulin. After trypsin treatment in the presence of calmodulin, the protein phosphatase activity of calcineurin is still regulated by calmodulin. Prolonged trypsin treatment in the presence of calmodulin produces a 46,000 Mr fragment. Unlike the fragments generated in the absence of calmodulin, this 46,000 Mr fragment still interacts weakly with calmodulin. Thus, calcineurin, like other calmodulin-regulated enzymes, consists of a catalytic domain resistant to proteolysis and a calmodulin-binding regulatory domain susceptible to protease action in the absence of calmodulin but not in its presence. In the absence of calmodulin, the regulatory domain exerts an inhibitory effect on the catalytic domain; the inhibition is relieved upon calmodulin binding to or tryptic degradation of the regulatory domain.

Publisher Summary This chapter discusses the domain structure of calcineurin. A calmodulin-stimulated protein phosphatase was first isolated from skeletal muscle and was also shown to be associated with calcineurin, the major calmodulin and Ca 2+ -binding protein of brain extracts. Calmodulin-stimulated phosphatases have been detected in many mammalian tissues. Most, like calcineurin, appear to be composed of two subunits, calcineurin A and calcineurin B, and cross react with polyclonal antibodies to bovine brain calcineurin. The antibodies also recognize calcineurin B-like proteins in several lower eukaryotes such as Paramecium tetraurelia, Drosophila melanogaster, and sea urchins but cross react more poorly with the large subunit in invertebrates and vertebrate tissues other than brain. Calmodulin-stimulated protein phosphatase has a wide tissue and species distribution, but its large subunit may exist as different isozymes. Brain, which contains 10–20 fold more of this enzyme than other tissues, is the richest source of the protein. The affinity of calcineurin for calmodulin is one of the highest reported for calmodulin stimulated enzymes. It is found that calmodulin does not compete with calcineurin B for the calcineurin B binding site on calcineurin A in reconstitution experiments.

Calcineurin, a major calmodulin-binding protein of brain [15], was initially identified as a heat-labile inhibitor of calmodulin-stimulated cyclic nucleotide phosphodiesterase [17,36]. Although the function of this protein was not known, because of its Ca2+ -binding properties [15] and its apparent neural localization [34], it was named calcineurin [15]. The recent discovery of a calmodulin-stimulated protein phosphatase with similar subunit structure [29] led to the identification of calcineurin as a phosphoprotein phosphatase [30]. Calcineurin subunit interactions can now be correlated with observed regulatory properties of the en¬zyme, providing the opportunity to explore the relationship between subunit structure and the mechanisms of phosphatase regulation.

Phosphorylase kinase has the structure (alpha beta gamma delta)4 where the delta-subunit is identical to the calcium-binding protein termed calmodulin [Shenolikar et al. (1979) Eur. J. Biochem. 100, 329--337]. The delta-subunit was tightly bound to phosphorylase kinase in the absence of calcium ions, and its rate of exchange with [14C]calmodulin was only 15% per week. The delta-subunit remained associated with phophorylase kinase in the presence of 8 M urea provided that calcium ions were present and this property enabled electrophoretic techniques to be used which demonstrated that the delta-subunit was associated with the gamma-subunit. This finding was confirmed by cross-linking experiments with dimethylsuberimidate which resulted in the formation of a gamma delta complex. Phosphorylase kinase was shown to bind one additional molecule of calmodulin per alpha beta gamma delta unit, termed the delta'-subunit. Glycerol gradient centrifugation in the presence of [14C]calmodulin indicated that the interaction of the delta'-subunit with phosphorylase kinase only occurred in the presence of calcium ions, and that the Kd value was near 0.01 microM. This was similar to the concentration of delta'-subunit which produced half-maximal activation. The delta'-subunit did not remain associated with phosphorylase kinase in the presence of 8 M urea, either in the presence or absence of calcium ions. The very slow exchange between the delta-subunit and [14C]calmodulin, and the calcium-dependent binding of the delta'-subunit allowed cross-linking experiments to be used which demonstrated that the delta'-subunit was bound to both the alpha and beta subunits. This result was supported by the finding that selective proteolysis of either the alpha-subunit, or the alpha and beta subunits, decreased or abolished the ability of phosphorylase kinase to bind to calmodulin-Sepharose. The roles of the different subunits in the regulation of phosphorylase kinase activity are discussed.

The adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of crude Chinese hamster ovary cell membranes was inhibited 30-40% by low concentrations (6-600 ng/ml) of calcium-dependent regulator (CDR). This inhibitory effect was lost at concentrations of CDR above 600 ng/ml. The adenylate cyclase activity of membranes prepared from low population density Chinese hamster ovary cells was not appreciably altered by CDR. However, with increasing cell population density there was a significant increase in the ability of CDR to inhibit cyclic AMP formation. Further, the intracellular levels of CDR determined in the 12,000 × g supernatant and particulate fractions varied inversely with increasing cell population density. As cell number increased from 2 × 10 6 to 10 × 10 6 cells per dish the CDR concentration present in the supernatant fraction increased from 0.4 to 0.8 μg of CDR per mg of protein, while the amount of endogenous CDR associated with the particulate fraction decreased from 0.6 to 0.4 μg of CDR per mg of protein. This suggests that possible changes in the distribution of CDR between the supernatant and membrane fractions might serve as a regulatory mechanism for activities under CDR control.