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PubMed1990

A calcium-sensitive fluorescent analog of calmodulin based on a novel calmodulin-binding fluorophore.

Structure-activity studies of tetramethinemerocyanine fluorophores enabled the synthesis of novel dyes which showed spectral changes during reversible, calcium-dependent association with calmodulin. These spectral changes were greatly enhanced in dyes with a quaternary nitrogen and specifically placed hydrophobic chains. One such dye was covalently attached to calmodulin, producing a calmodulin analog with calcium-sensitive fluorescence. The analog, MeroCaM, showed a calcium-induced 3.4-fold increase in excitation ratio (608/532 nm excitation, 623 nm emission), which was fully reversed by lowering free calcium levels. MeroCaM's excitation ratio showed a half-maximal change at 300-400 nM calcium, below calcium concentrations reported to produce half-maximal saturation of calcium-calmodulin binding. However, the calcium dependence of MeroCaM's phosphodiesterase activation paralleled that of calmodulin. MeroCaM's fluorescence changes therefore appear to reflect primarily calcium binding to high affinity sites. MeroCaM's maximal phosphodiesterase activation was 30-40% that of calmodulin. In myosin light chain kinase activation, MeroCaM and calmodulin displayed indistinguishable maximal activation levels and concentration dependence of activation. Changes in MeroCaM's calcium affinity induced by magnesium, phosphodiesterase, and melittin were similar to those reported for calmodulin. Experiments with melittin revealed that target protein interaction could alter the fluorescence changes produced by calcium binding. MeroCaM showed promising brightness and photostability when imaged in individual living fibroblasts. The long excitation and emission wavelengths of MeroCaM, and the strong dependence of its excitation ratio on calcium concentrations, suit it well for use as a probe of calmodulin-dependent calcium signaling in living cells, as well as for experiments in vitro.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Toward a model of the calmodulin-myosin light-chain kinase complex: implications for calmodulin function.

We have developed a model for the interaction of calmodulin and the presumptive calmodulin binding domain of rabbit skeletal muscle myosin light-chain kinase. In our model there is a bend in the central helix of calmodulin such that hydrophobic patches associated with the pairs of Ca2+ binding sites: I, II and III, IV; face one another. This was accomplished by altering the psi dihedral angle at one residue: Ser-81. We have made the presumptive calmodulin binding peptide alpha-helical over its entire length. In the model, this basic amphiphilic helix fits into a cavity formed by apposition of the two hydrophobic regions of calmodulin. We suggest that this general type of model may help explain calmodulin's ability to regulate the activities of its many different targets. Small changes in the conformation of a nonhelical bend within the central helix would have large effects on the relative positions of the two halves of the molecule. In this way, calmodulin might adapt itself to a wide range of possible calmodulin binding domains. The literature pertaining to the model is discussed. We also discuss the results of our own recent investigations of calmodulin species that have been altered by site-directed mutagenesis.

Animals↗
PubMed2000

Alternative splice variant of gamma-calmodulin-dependent protein kinase II alters activation by calmodulin.

Calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous, multifunctional enzyme family involved in the regulation of a variety of Ca(2+)-signaling pathways. These family members are expressed from four highly homologous genes (alpha, beta, gamma, and delta) with similar catalytic properties. Additional isoforms of each gene, created by alternative splicing of variable regions I-XI, are differentially expressed in various cell types. gammaB, gammaC, gammaD, gammaE, gammaF, gammaGs, and gammaH CaMKII isoforms are expressed in the biliary epithelium; however, little is known about their roles in these cells. We began our studies into the function of these variable regions by examining the effects of variable region I on kinase activation and calmodulin binding. Activities and calmodulin binding properties of gammaB and gammaGs, which differ only by the exclusion or inclusion of this region, were compared. The K(0.5) for calmodulin was 2.5-fold lower for gammaGs than gammaB. In contrast, gammaB bound calmodulin more tightly in a calmodulin overlay assay. Mutation of variable regions I's charged residue, gammaGs-R318E, resulted in an enzyme with intermediate activation properties but a calmodulin affinity similar to gammaB. Thus, variable region I appears to modulate calmodulin sensitivity, in part, through charge-charge interactions. This altered threshold of activation may modulate cellular responses to gradients of Ca(2+)/calmodulin in the biliary tract.

Alternative Splicing↗
PubMed1992

Synthesis and Accumulation of Calmodulin in Suspension Cultures of Carrot (Daucus carota L.) : Evidence for Posttranslational Control of Calmodulin Expression.

The expression of calmodulin mRNA and protein were measured during a growth cycle of carrot (Daucus carota L.) cells grown in suspension culture. A full-length carrot calmodulin cDNA clone isolated from a lambdagt10 library was used to measure steady-state calmodulin mRNA levels. During the exponential phase of culture growth when mitotic activity and oxidative respiration rates were maximal, calmodulin mRNA levels were 4- to 5-fold higher than they were during the later stages of culture growth, when respiration rates were lower and growth was primarily by cell expansion. Net calmodulin polypeptide synthesis, as measured by pulse-labeling in vivo with [(35)S]methionine, paralleled the changes in calmodulin steady-state mRNA level during culture growth. As a consequence, net calmodulin polypeptide synthesis declined 5- to 10-fold during the later stages of culture growth. The qualitative spectrum of polypeptides synthesized and accumulated by the carrot cells during the course of a culture cycle, however, remained largely unchanged. Calmodulin polypeptide levels, in contrast to its net synthesis, remained relatively constant during the exponential phases of the culture growth cycle and increased during the later stages of culture growth. Our data are consistent with increased calmodulin polypeptide turnover associated with periods of rapid cell proliferation and high levels of respiration.

Journal Article↗
PubMed2002

Calmodulin regulates the transcriptional activity of estrogen receptors. Selective inhibition of calmodulin function in subcellular compartments.

The steroid hormone estrogen elicits biological effects in cells by binding to and activating the estrogen receptor (ER). Estrogen binding induces a conformational change in the receptor, inducing nuclear translocation and transcriptional activation of ER. The ubiquitous Ca(2+)-binding protein calmodulin has been shown to interact directly with ER and enhance its stability. To further elucidate the functional sequelae of the association between calmodulin and ER, we examined the effect on ER transcriptional activation of specifically inhibiting calmodulin. The cell-permeable calmodulin antagonist CGS9343B prevented estrogen-induced transcriptional activation by ER, without altering basal transcription. The inhibition was dose-dependent and independent of the time of estrogen stimulation. To validate these findings, calmodulin function was also neutralized by targeted expression of a specific inhibitor peptide. By inserting localization signals, the inhibitor peptide was selectively targeted to different subcellular domains. Inactivation of calmodulin function in the nucleus virtually eliminated estrogen-stimulated ER transcriptional activation. By contrast, when membrane calmodulin was specifically neutralized, estrogen-stimulated transcriptional activation by ER was only slightly attenuated. Importantly, the inhibitor peptides did not significantly reduce the amount of ER in the cells. Together, these data demonstrate that calmodulin is a fundamental component of ER transcriptional activation.

Animals↗
PubMed1992

Protein A-calmodulin fusions: a novel approach for investigating calmodulin function in yeast.

A novel gene fusion approach which may be of more general use has been developed for investigating the function of calmodulin in the budding yeast Saccharomyces cerevisiae. By fusing a portion of the Staphylococcus aureus spa gene (encoding protein A) to CMD1, the S. cerevisiae gene encoding calmodulin, we have generated a yeast calmodulin with an affinity tag able to bind immunoglobulins. The chimaeric protein A-calmodulin (ProtA-CaM) polypeptide functions in vivo and shows Ca(2+)-dependent binding to calmodulin target proteins. The spa-CMD1 fusion has been used (i) to prepare (by affinity chromatography) a fraction of yeast proteins which interact with calmodulin, (ii) to isolate genes encoding calmodulin target proteins by direct screening of an expression library, and (iii) to visualize calmodulin-binding proteins in crude extracts by Western blot analysis.

Amino Acid Sequence↗
PubMed1987

Affinity-purified melittin antibody recognizes the calmodulin-binding domain on calmodulin target proteins.

Melittin is a 26-amino acid amphipathic peptide which binds to calmodulin in a calcium-dependent manner. The utility of melittin as a peptide replica of the calmodulin-binding region of calmodulin acceptor proteins (CaMBPs) was investigated. Antibody against melittin was raised and purified by antigen affinity chromatography. Interaction of the antibody with CaMBPs was initially suggested by the ability of anti-melittin-Sepharose, but not nonimmune IgG-Sepharose, to bind calmodulin-dependent cyclic AMP phosphodiesterase. Direct interaction of melittin antibody with the calmodulin-binding domain of acceptor proteins was demonstrated by quantitative inhibition of calmodulin binding to the purified CaMBPs, myosin light chain kinase, and eel electric organ CaMBP55. These results indicate that melittin antibody identifies regions of structural similarity between calmodulin acceptor proteins, and this region includes a common calmodulin-binding domain.

Antibodies↗
PubMed1994

Phosphorylation of the calmodulin binding domain of the plasma membrane Ca2+ pump by protein kinase C reduces its interaction with calmodulin and with its pump receptor site.

Two versions of the calmodulin binding domain of the plasma membrane Ca2+ ATPase, a 24-amino acid peptide, C24W (Q-I-L-W-F-R-G-L-N-R-I-Q-T-Q-I-R-V-V-N-A-F-R-S-S-NH2), and the corresponding phosphothreonine containing peptide, C24W-P (Q-I-L-W-F-R-G-L-N-R-I-Q-T(phospho)-Q-I-R-V-V-N-A-F-R-S-S-NH2), were synthesized. They were used to investigate the effect of threonine phosphorylation by protein kinase C on the binding of calmodulin by the calmodulin binding domain and on the inhibitory role of the domain on the activity of the Ca2+ pump. The phosphopeptide C24W-P was obtained after global phosphorylation of the free Thr side chain on the protected resin bound peptide. The phosphorylated calmodulin binding domain failed to bind calmodulin; this was shown by gel shift experiments, by fluorescence energy transfer studies and by competition experiments against calmodulin stimulation of the pump. The inhibition of the Ca2+ pump activity by the calmodulin binding domain in the absence of calmodulin was also affected by the phosphorylation of the threonine; the inhibition of the fully active calpain-truncated pump by the phosphothreonine containing peptide was lower than that by the unphosphorylated synthetic domain.

Adenosine Triphosphatases↗
PubMed2000

Calmodulin-binding peptide PEP-19 modulates activation of calmodulin kinase II In situ.

PEP-19 is a 6 kDa polypeptide that is highly expressed in select populations of neurons that sometimes demonstrate resistance to degeneration. These include the granule cells of the hippocampus and the Purkinje cells of the cerebellum. Its only identified activity to date is that of binding apo-calmodulin. As a consequence, it has been demonstrated to act as an inhibitor of calmodulin-dependent neuronal nitric oxide synthase in vitro, although PEP-19 regulation of calmodulin-dependent enzymes has never been characterized in intact cells. The activation of the calmodulin-dependent enzyme calmodulin kinase II (CaM kinase II) was studied in PC12 cells that had been transfected so as to express physiological levels of PEP-19. The expression of PEP-19 yielded a stable phenotype that failed to activate CaM kinase II upon depolarization in high K(+). However, CaM kinase II could be fully activated when calcium influx was achieved with ATP. The effect of PEP-19 on CaM kinase II activation was not attributable to changes in the cellular expression of calmodulin. The cellular permeability of the transfected cells to calcium ions also appeared essentially unchanged. The results of this study demonstrated that PEP-19 can regulate CaM kinase II in situ in a manner that was dependent on the stimulus used to mobilize calcium. The selective nature of the regulation by PEP-19 suggests that its function is not to globally suppress calmodulin activity but rather change the manner in which different stimuli can access this activity.

Adenosine Triphosphate↗
PubMed1991

The increase of calmodulin in PC12 cells induced by NGF is caused by differential expression of multiple mRNAs for calmodulin.

A rat pheochromocytoma cell line (PC12 cells) was used as a model to investigate the role of calmodulin and its multiple mRNAs in NGF-induced neuronal differentiation. The effect of NGF on the degree of differentiation was assayed using a simple differentiation scoring system. Significant increases in the differentiation score were seen by one day, and the scores increased about 10-fold by 8 days of treatment. NGF also increased calmodulin in the PC12 cells; significant increases were seen by 2 days of treatment, and a maximum increase of 3-fold was seen by 4 days. Northern blot analysis using a calmodulin riboprobe revealed that all five calmodulin mRNAs found in rat tissue were present in PC12 cells. The relative abundance of the calmodulin mRNAs was 1.7 greater than 1.4 greater than 2.3 greater than 4.1 greater than 0.9 kb. NGF treatment caused a differential increase in these mRNAs. The 1.4 kb transcript (from Gene II) was increased earlier (at 1 day) and to a greater extent (3-fold) than any of the other mRNAs. Studies of the half-lives (t1/2) of these mRNAs suggested that the t1/2 varied with the mRNA; the smaller the mRNA, the shorter the t1/2. However, there were no significant effects of NGF on the t1/2 of any of the mRNAs. These studies indicate that NGF elevates calmodulin in PC12 cells by causing a differential increase in the multiple mRNAs for calmodulin and that the increase in calmodulin may play some part in NGF-induced neuronal differentiation in PC12 cells.

Animals↗
PubMed1997

Calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine eye: high calmodulin affinity isozyme immunologically related to the brain 60-kDa isozyme.

Calmodulin-dependent cyclic nucleotide phosphodiesterase was identified in and purified to apparent homogeneity from the total calmodulin-binding protein fraction of bovine eye in a single step by immunoaffinity chromatography. The bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase is immunologically similar to the bovine brain 60-kDa isozyme. The purified enzyme had higher affinity for calmodulin than the 60-kDa phosphodiesterase isozyme from bovine brain, but similar affinity to that of the heart isozyme. When the Ca(2+)-dependence of the eye enzyme was compared to cardiac calmodulin-dependent cyclic nucleotide phosphodiesterase at an identical concentration of calmodulin, the bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase was activated at the same Ca2+ concentration as the bovine heart calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme.

Animals↗
PubMed1995

Gain-of-function mutations in a human calmodulin-like protein identify residues critical for calmodulin action in yeast.

A human epithelial cell-specific transcript (NB-1) encodes a calmodulin-like protein (hCLP), which is identical in length and 85% identical in amino acid sequence to authentic human calmodulin (hCaM). Although hCaM shares only 60% amino acid sequence identity with yeast calmodulin (CMD1 gene product), hCaM was able to substitute functionally for Cmd1 in yeast cells. In contrast, hCLP was unable to support either spore germination or vegetative growth in Cmd1-deficient yeast cells, even when stably expressed at a level at least an order of magnitude above that of hCaM. Thus, hCLP provides an indicator protein for discerning those residues that are critical for calmodulin function in vivo. In addition to 20 conservative amino acid replacements, hCLP differs from hCaM (and other vertebrate calmodulins that are able to complement a cmd1 null mutation) by only three nonconservative substitutions. Site-directed mutagenesis was used to convert these three positions back to residues more typical of those found in authentic calmodulins and to prepare all possible combinations of these three mutations, specifically: three single mutants (R58V, R112N, and A128E), three double mutants (R58V A128E, R112N A128E, and R58V R112N), and the triple mutant (R58V R112N A128E). The triple mutant and one of the double mutants (R58V A128E) were able to restore an apparently normal growth rate to a cmd1 delta strain, indicating that the altered hCLPs have acquired the ability to behave as functional calmodulins in yeast. The other two double mutants were able to support growth of Cmd1-deficient cells only weakly, but cells expressing the R112N A128E mutant grew noticeably better than those expressing the R58V R112N mutant. Remarkably, one single mutant (A128E), but not the other two single mutants, was also reproducibly able to support weak growth of a cmd1 delta strain. The properties of these gain-of-function, or neomorphic, mutations implicate E128, and to a lesser extent V58, as residues critical for calmodulin action in vivo. Molecular modeling of these positions within the structure of a Ca(2+)-calmodulin.peptide complex indicates that E128 projects directly into the central cavity occupied by the bound peptide. Thus, E128 may contribute a contact that is vital for the interaction of Cmd1 with one or more of the targets that are essential for yeast cell growth.

Amino Acid Sequence↗
PubMed2000

Use of fluorescently labelled calmodulins as tools to measure subcellular calmodulin activation in living dorsal root ganglion cells.

We have used fluorescently labelled calmodulins to probe the activity of calmodulin in living dorsal root ganglion cells. Calmodulin labelled with the fluorophore 5-([4,6 dichlorotriazin-2yl]amino)-fluorescein (FL-CaM) does not change its fluorescence when it binds calcium, while calmodulin labelled at lysine 75 with 2-chloro-(6-(4-N,N-diethylamino-phenyl)-1,4,5-triazin-4-yl (TA-CaM), an environment-sensitive probe, increases its fluorescence when it binds calcium. We micro-injected FL-CaM or TA-CaM into rat dorsal root ganglion cells and found that both probes localise to the cell nucleus. In contrast, endogenous cellular calmodulin, in dorsal root ganglion cells as in hippocampal neurones, is predominantly cytosolic unless the neurones are depolarised, then it moves to the nucleus. FL-CaM and TA-CaM, introduced into dorsal root ganglion cells via a patch pipette, also immediately move to the nucleus, indicating that the nuclear localisation is a property of the labelled calmodulins. Although the subcellular distribution of FL-CaM and TA-CaM does not necessarily match that of endogenous calmodulin, we show that FL-CaM can be used as a control for TA-CaM when studying calmodulin activation in different cellular compartments.

Animals↗
PubMed1990

Formation of cyclic imide-like structures upon the treatment of calmodulin and a calmodulin peptide with heat.

Protein cyclic imide is the putative intermediate in the formation of sites of carboxyl-methylation in eukaryotic proteins. Conditions known to induce the formation of a cyclic imide in model peptides have been applied to a protein, calmodulin. Heating of calmodulin in the dry state at 100 degrees C for 24 h after lyophilization from a pH 2.0 or pH 6.0 solution produces derivatives with altered chromatographic properties in anion-exchange HPLC. At pH 6.0, complete activity of calmodulin was retained. Analysis with Fourier transform infrared (FTIR)-photoacoustic spectroscopy demonstrated the presence of a new structure in the calmodulin molecule consistent with modification of carboxylic acid groups. The conversion of calmodulin is dependent upon the absence of Ca2+ (the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid). A peptide analogous to the calcium binding regions of calmodulin, Asp-Lys-Asp-Gly-Asn-Gly-Thr-Ile-Thr-Thr-Lys-Glu, is also converted, upon heating, to chromatographically different forms in reversed-phase chromatography. This process is also dependent upon the absence of calcium. Sequence analysis of the peptide derivatives reveals a second amino terminus, implicating peptide bond hydrolysis in the product. A dipeptide, Asp-Gly, known to form a cyclic imide structure under similar conditions is also hydrolyzed during sequence analysis consistent with cleavage occurring at the position of the cyclic imide structure. Asp3 is suggested to be the site of cyclic imide formation in the calmodulin peptide. The presence of a cyclic imide structure is also confirmed by the application of FTIR-photoacoustic spectroscopy. These data suggest that cyclic imide formation in calmodulin has been induced, possibly at one, or more, of the calcium binding loops of the protein. These modification reactions may provide a basis for future investigations of cyclic imide formation in proteins.

Amino Acid Sequence↗
PubMed1981

Calmodulin ligands. The interaction of muscle phosphorylase kinase with phosphodiesterase. Comparison of calmodulin ligands in muscle extracts from normal and phosphorylase kinase-deficient mice.

Interactions between phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38) and calmodulin were studied with pure preparations of muscle phosphorylase kinase, and with crude extracts from muscles of control (C57 Black) and deficient (ICR/IAn) mice, which lack muscle phosphorylase kinase activity. Calmodulin was determined by its ability to stimulate a calmodulin-dependent phosphodiesterase. The amount of calmodulin bound to phosphorylase kinase in muscle extract was estimated to a maximum of 30% of the total amount of calmodulin. In the muscle of the deficient strain a decrease of 35% in the total amount of calmodulin was observed. This correlates with the absence of the calmodulin fraction specifically bound to phosphorylase kinase. From sucrose gradient studies we demonstrated that in the presence of Ca2+ the amount of calmodulin bound to phosphorylase kinase was enhanced, compared to the control in the presence of EGTA. This observation was made both in crude extracts and in pure phosphorylase kinase preparations. Sucrose gradient also showed that muscle phosphorylase kinase can be dissociated to low molecular species when extracts are made in the presence of Ca+; this dissociation was found to be related to a Ca2+-dependent proteolytic effect.

Animals↗
PubMed1991

Quantitative evaluation of indirect ELISA. Effect of calmodulin antagonists on antibody binding to calmodulin.

A simple linearization procedure has been developed to determine the apparent dissociation constant of the interaction between antigen and antibody from the data of indirect, non-competitive enzyme-linked immunosorbent assays (ELISA). Applying this dissociation constant the binding constant of ligands to antigen can be determined and the quantitative evaluation of the competitive ELISA experiments makes it possible to analyse the affinity of antibody to antigen on the surface and in solution. The binding of the monospecific anti-calmodulin antibody to calmodulin and to solid-phase bound calmodulin has been tested by non-competitive and competitive assays. We have developed an experimental system where binding of the antibody to the solid-phase bound calmodulin has been studied under equilibrium conditions. Competitive ELISA experiments showed that the affinity of antibody to calmodulin on the surface and in solution was almost the same. The binding constant of a hypothalamic neuropeptide to calmodulin was determined using the quantitative ELISA approach. The neuropeptide was found to be of very high inhibitory potency (Kd = 2 nM) and competed with the antibody for calmodulin binding. This simple and sensitive procedure is suitable for screening molecules with anti-calmodulin activity and comparing their efficacy.

Antigen-Antibody Reactions↗
PubMed1983

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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Direct interaction between the catalytic subunit of the calmodulin-sensitive adenylate cyclase from bovine brain with 125I-labeled wheat germ agglutinin and 125I-labeled calmodulin.

A calmodulin-sensitive adenylate cyclase has been purified to apparent homogeneity from bovine cerebral cortex using calmodulin-Sepharose followed by forskolin-Sepharose and wheat germ agglutinin-Sepharose. The final product appeared as one major polypeptide of approximately 135,000 daltons on sodium dodecyl sulfate-polyacrylamide gels. This polypeptide was a major component of the protein purified through calmodulin-Sepharose. The catalytic subunit was stimulated 3-4-fold by calmodulin (CaM) with a turnover number greater than 1000 min-1 and was directly inhibited by adenosine. The catalytic subunit of the enzyme interacted directly with 125I-CaM on a sodium dodecyl sulfate-polyacrylamide gel overlay system, and this interaction was Ca2+ concentration dependent. In addition, the catalytic subunit was shown to directly bind 125I-labeled wheat germ agglutinin using a sodium dodecyl sulfate-polyacrylamide gel overlay technique, and N-acetylglucosamine inhibited binding of the lectin to the catalytic subunit. Calmodulin did not inhibit binding of wheat germ agglutinin to the catalytic subunit, and the binding of calmodulin was unaffected by wheat germ agglutinin. These data illustrate that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is a glycoprotein which interacts directly with calmodulin and that adenosine can inhibit the enzyme without intervening receptors or G coupling proteins. It is concluded that the catalytic subunit of adenylate cyclase is a transmembrane protein with a domain accessible from the outer surface of the cell.

Animals↗