Ubiquitination of endogenous calmodulin in rabbit tissue extracts.

Previously we were able to show that purified calmodulins from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner. It was therefore pertinent to answer the question if a tissue extract contains all the components necessary for the endogenous synthesis of ubiquityl calmodulin (uCaM). Therefore [125I]ubiquitin, ATP/Mg2+ and Ca2+ were added to tissue extracts enriched by a single ion exchange step. In such extracts of red blood cells, skeletal muscle and testis a novel ubiquitin conjugate of 27-29 kDa is formed. This novel band could be identified as ubiquityl-calmodulin by the following methods: (i) identical Rf-value of novel conjugate and standard uCaM in SDS-PAGE; (ii) Ca(2+)-dependent conjugate formation; (iii) Ca(2+)-dependent adsorption to fluphenazine-Sepharose; (iv) Ca(2+)-dependent mobility change of the novel conjugate during SDS-PAGE; and (v) inhibition of conjugate band formation by phosphorylase kinase. These experiments clearly demonstrate that ubiquityl calmodulin can be endogenously generated in enriched cellular extracts and strongly indicate that this reaction is of importance in vivo.     

Plant and fungus calmodulins are polyubiquitinated at a single site in a Ca2(+)-dependent manner

In plants Ca2+ plays a crucial role as second messenger. Thus calmodulin is one of the most important signal transducing molecules for metabolic regulation in plants. Previously we showed that bovine testis calmodulin can be covalently coupled at one site to ubiquitin in a Ca2(+)-dependent manner in the presence of ATP/Mg2+ by ubiquityl-calmodulin synthetase. Since calmodulin from spinach has 13 amino acid sequence differences to bovine calmodulin - two of them in Ca2(+)-binding loops - it was unclear, whether a conjugation of ubiquitin to this molecule would be possible. In this paper it is shown that calmodulin from spinach and a similar calmodulin from the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca2(+)-dependent manner. It is shown that higher molecular mass conjugates containing up to three ubiquitin molecules per calmodulin are obtained. Experiments with methylated ubiquitin demonstrate that, as with vertebrate calmodulins, only one lysine residue is linked to ubiquitin and that the incorporation of additional ubiquitin molecules leads to a polyubiquitin chain.     

Covalent conjugation of mammalian calmodulin with ubiquitin

In this paper it is shown that mammalian calmodulin from bovine testis is a substrate for reticulocyte ubiquitin conjugating activity (UCA) forming a 1:1 covalent conjugate between bovine calmodulin and ubiquitin (uCaM). There is an absolute requirement for Ca2+ in the range of approximately 10 microM for ubiquitination of calmodulin to occur. This novel conjugate (uCaM) shows a Ca2+-dependent mobility change in polyacrylamide gel electrophoresis in the presence of SDS, indicating that the calmodulin-ubiquitin conjugate still retains the mobility change of native calmodulin. This conjugation reaction could be of prime importance for the intracellular turnover of calmodulin in the mammalian cell, although it cannot be excluded that the ubiquitin-calmodulin conjugate might in itself be of biological relevance.     

Bacterially synthesized vertebrate calmodulin is a specific substrate for ubiquitination

Calmodulin purified from bacteria which express a cloned chicken calmodulin gene can be selectively conjugated with ubiquitin, using enzymes present in reticulocyte extracts. Analyses of peptide products generated from limited proteolytic digestion of the calmodulin conjugate containing a single ubiquitin indicate that lysine 115 on calmodulin is the site of linkage. This linkage site is identical to that previously reported for calmodulin purified from Dictyostelium discoideum. Substrate-dependent ATP hydrolysis by a partially purified ubiquitin conjugation enzyme system from reticulocyte extracts was used to determine the enzyme affinity to calmodulin. Km values of 7 and 9 microM were determined for dictyostelium and the bacterially expressed calmodulin, respectively. The bacterially expressed calmodulin, unlike the Dictyostelium protein, can also form conjugates containing a 2-5 molar ratio of ubiquitin but at a slower rate than that observed for conjugation at lysine 115. Results from these studies further support our hypothesis that the post-translational methylation of lysine 115 found in most forms of calmodulin serves the important function of protecting calmodulin from ubiquitination and from degradation by the cytoplasmic ubiquitin-dependent proteolytic pathway. The capability of the bacterially expressed calmodulin to form conjugates with a high molar ratio of ubiquitin suggests that the post-translational acetylation of the N terminus of calmodulin may serve a similar function.     

Multiple ubiquitination of vertebrate calmodulin by reticulocyte lysate and inhibition of calmodulin conjugation by phosphorylase kinase

Mammalian calmodulin containing trimethyllysine 115 can be covalently coupled to ubiquitin in a Ca2+-dependent manner in the presence of ATP/Mg2+ by reticulocyte lysate. This conjugation reaction can be quantitated in a novel test employing fluphenazine-Sepharose. It is shown that at least 3 ubiquitin molecules can be coupled to calmodulin indicating that more than one lysine residue is involved in the ubiquitination reaction. In addition only the free form of calmodulin can be ubiquitinated. Neither calmodulin bound to phosphorylase kinase as an integral subunit (delta-subunit) nor that bound as a peripheral subunit (delta'-subunit) is ubiquitinated. A total binding of equimolar calmodulin to phosphorylase kinase occurs since the affinity of binding of calmodulin to phosphorylase kinase as integral (KCaMm unknown) or peripheral subunit (KCaMm ca. 30-50nM) is several order of magnitude higher than the corresponding affinity of calmodulin for the ubiquitin-conjugating enzyme (KCaMm ca. 8 microM). We conclude that the "protective" effect of phosphorylase kinase towards calmodulin conjugation is due to a changed conformation of bound calmodulin and/or inacessibility of the ubiquitination sites (e.g. at subunit-subunit interface). Thus Ca2+-dependent ubiquitination only of free calmodulin may provide an efficient scavanging mechanism (with subsequent breakdown) for all free calmodulin in excess of that amount which can be bound by the calmodulin-binding proteins in the cell.     

Specific recognition of calmodulin from Dictyostelium discoideum by the ATP, ubiquitin-dependent degradative pathway

Calmodulin purified from Dictyostelium discoideum is selectively degraded by rabbit reticulocyte extracts in the presence of ubiquitin and ATP. This protein forms a 1:1 covalent conjugate with ubiquitin. Analyses of the cyanogen bromide fragments of the protein conjugate indicate that lysine 115 on calmodulin is the ubiquitin conjugation site. Bovine brain calmodulin which contains a trimethyllysine residue at this position is not a substrate for conjugation with ubiquitin, and its degradation rate is not affected by ATP and ubiquitin. These results suggest that the trimethyllysine residue in mammalian calmodulin may function in protecting the protein from degradation by the ATP, ubiquitin-dependent pathway. Since there are eight lysine residues in Dictyostelium calmodulin, the specific conjugation of ubiquitin to lysine 115 may provide a good model system to delineate the structural features required for the conjugation and to follow the degradative steps in the pathway.     

Synthesis and decay of calmodulin-ubiquitin conjugates in cell-free extracts of various rabbit tissues

Calmodulin is the natural substrate for ubiquitin-ligation by the enzyme ubiquitin-calmodulin ligase (uCaM-synthetase; EC 6.3.2.21). The activity of this ligase is regulated by the binding of the second messenger Ca²⁺ to the substrate calmodulin, which increases the activity ca. 10-fold. Up till now, two components of the ligase could be identified: uCaM Syn-F1 and uCaM Syn-F2, the first of which binds to ubiquitin and the second which binds to calmodulin. Since the physiological role of this enzyme is still unclear, this study was designed to examine whether the activity of uCaM-Synthetase in 40 000×g tissue supernatants correlates with the calmodulin content in the various tissues. In reticulocytes, spleen, erythrocytes, testis and brain, which are rich in uCaM synthetase, the tissue contents calculated on the basis of activity measurements were between 4–80-fold higher than in red and white skeletal muscle. These activities did not correlate with the respective calmodulin contents of the tissues indicating that other factors were determining these enzyme levels. A second aim was to gain information on the role of the ATP-ubiquitin-dependent proteolytic pathway in those tissues displaying uCaM synthetase activity. In the reticulocyte system which contains the classical ATP-ubiquitin-dependent proteolytic pathway as measured with ¹²⁵I-BSA, no ubiquitin-dependent degradation of calmodulin could be detected. We therefore examined the other tissues of the rabbit with the substrate ¹²⁵I-BSA and succeeded in finding a ubiquitin-independent ATP-dependent proteolytic activity in every case but no ubiquitin-dependent activity. The ubiquitin-independent activity was highest in smooth muscle and red skeletal muscle being ca. 3–4-fold higher than in lung and testis. In 50% of the tissue crude extracts the time curve of calmodulin ubiquitylation progressed through a maximum indicating a dynamic steady state based on conjugate synthesis and decay. If a ubiquitylation pulse of 30 min was followed in liver crude extracts by the addition of EGTA, which specifically inhibits ubiquityl-calmodulin synthesis, a half-life of calmodulin-conjugate decay of 15–20 min is observed. A similar conjugate half-life of ca. 30 min was observed after addition of EDTA excluding that conjugate decay is due to an ATP-dependent proteolytic process. Studying the decay of purified ubiquitin-¹²⁵I-BH-calmodulin conjugates in cell-free reticulocyte extracts led to the discovery of an ATP-independent isopeptidase activity which splits ubiquitin-calmodulin conjugates without leading to detectable calmodulin fragments. The rapid decay of ubiquitin-calmodulin conjugates in tissue extracts can therefore be plausibly explained by a ubiquityl-calmodulin splitting isopeptidase activity.     

Calcium negatively modulates calmodulin interaction with IQGAP1

IQGAP1 regulates cytoskeletal dynamics through interactions with the Rho family GTPases Rac1 and Cdc42, F-actin, and beta-catenin. Calmodulin interaction with IQ motifs of IQGAP1 negatively influences these IQGAP1 interactions. Although, calmodulin interacts with IQGAP1 in the absence of Ca(2+) and was suggested to exhibit reduced binding when Ca(2+) bound, recent reports show substantially greater binding when Ca(2+) is present. Binding evaluations have primarily relied on IQGAP1 interaction with calmodulin conjugated to Sepharose 4B. In this study we evaluated the Ca(2+)-dependence of calmodulin interaction with native IQGAP1 using a series of independent biochemical approaches. We found the apparent binding of calmodulin to IQGAP1 was Ca(2+)-independent, being between 5- and 20-fold greater in the absence than in the presence of Ca(2+). In addition, calmodulin interaction with IQGAP1 was negatively regulated by buffer [Ca(2+)] (IC(50)=3.4x10(-7)M). Regulation was specific to Ca(2+), as Ba(2+) was approximately 400-fold less effective than Ca(2+) at modulating the interaction. Moreover, testing of calmodulin mutants demonstrated that apocalmodulin tightly binds IQGAP1 and that the N- and C-terminal pair of EF hands are important for Ca(2+) sensitivity. These data indicate that calmodulin may disassemble from IQGAP1 to facilitate IQGAP1 interaction with effectors of cytoskeletal reorganization during conditions of cell activation that promote increased cytosolic [Ca(2+)].     

Ubiquitin-mediated degradation of histone H3 does not require the substrate-binding ubiquitin protein ligase, E3, or attachment of polyubiquitin chains

Radioiodinated histone H3 was incubated with ubiquitin, the ubiquitin-activating enzyme E1, and one of three ubiquitin carrier proteins, reticulocyte E2(20K) or E2(32K) or the yeast RAD6 product. Although the resulting ubiquitin-histone conjugates were synthesized in the absence of the substrate-binding protein E3, they were nevertheless degraded by purified rabbit reticulocyte 26 S protease. In contrast, unmodified histone H3 remained intact upon challenge with the 26 S ubiquitin/ATP-dependent enzyme. Conjugates produced by the RAD6 protein were better proteolytic substrates than those formed by reticulocyte E2 unless ubiquitin molecules with altered lysines were used for conjugate synthesis. Substitution of methylated ubiquitin or ubiquitin molecules in which lysine 48 was converted to arginine by site-directed mutation produced histone conjugates that were degraded at slow but measurable rates. Since methylated ubiquitin molecules are incapable of forming branched polyubiquitin chains, these results demonstrate that neither ubiquitin "trees" nor the substrate binding factor E3 is absolutely required for ubiquitin-dependent degradation of histone H3 in vitro.     

A thapsigargin-sensitive Ca(2+) pump is present in the pea Golgi apparatus membrane

The Golgi apparatus behaves as a bona fide Ca(2+) store in animal cells and yeast (Saccharomyces cerevisiae); however, it is not known whether this organelle plays a similar role in plant cells. In this work, we investigated the presence of an active Ca(2+) accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca(2+) uptake in subcellular fractions isolated from the elongating zone of etiolated pea (Pisum sativum) epicotyls. Separation of organelles using sucrose gradients showed a strong correlation between the distribution of an ATP-dependent Ca(2+) uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca(2+) uptake of 2.5 nmol mg min(-1) and an apparent K(m) for Ca(2+) of 209 nM. The ATP-dependent Ca(2+) uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I(50)] = 126 microM) and cyclopiazonic acid (I(50) = 0.36 nmol mg protein(-1)) and was not stimulated by calmodulin (1 microM). Addition of Cd(2+) and Cu(2+) at nanomolar concentration inhibited the Ca(2+) uptake, whereas Mn(2+), Fe(2+), and Co(2+) had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I(50) = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca(2+) ATPase from mammalian cells. A thapsigargin-sensitive Ca(2+) uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca(2+) pumps. To our knowledge, this is the first report of a plant Ca(2+) pump activity that shows sensitivity to low concentrations of thapsigargin.     

Calcium and calmodulin in membrane fusion

Regulated exocytosis was the first intracellular membrane fusion step that was suggested to involve both Ca(2+) and calmodulin. In recent years, it has become clear that calmodulin is not an essential Ca(2+) sensor for exocytosis but that it is likely to have a more regulatory role. A requirement for cytosolic Ca(2+) in other vesicle fusion events within cells has become apparent and in certain cases, such as homotypic fusion of early endosomes and yeast vacuoles, calmodulin may be the primary Ca(2+) sensor. A number of distinct targets for calmodulin have been identified including SNARE proteins and subunits of the vacuolar ATPase. The extent to which calmodulin regulates different intracellular fusion events through conserved SNARE-dependent or other mechanisms remains to be resolved.     

Maize cap1 encodes a novel SERCA-type calcium-ATPase with a calmodulin-binding domain

A cDNA (CAP1) isolated from maize roots shares sequence identity with genes encoding P-type Ca(2+)-ATPases and restores the growth phenotype of yeast mutants defective in Ca(2+)-pumps. CAP1 was transcribed and translated in the yeast mutant. Furthermore, the membrane-integrated product formed a Ca(2+)-dependent phosphorylated intermediate and supported Ca(2+) transport. Although CAP1 shares greater sequence identity with mammalian "endoplasmic reticulum-type" Ca(2+)-pumps, it differs from these genes by having features of calmodulin (CaM)-regulated Ca(2+)-pumps. CAP1 from yeast microsomes bound CaM, and the CAP1-dependent Ca(2+) transport in yeast was stimulated by CaM. Peptides from the C terminus of CAP1 bound CaM. Anti-CAP1 antibodies specifically recognized a maize microsomal polypeptide that also bound CaM. A similar polypeptide also formed a Ca(2+)-dependent phosphoenzyme. Our results suggest that cap1 encodes a novel form of CaM-regulated Ca(2+)-ATPase in maize. CAP1 appears to be encoded by one or two genes in maize. CAP1 RNA is induced only during early anoxia, indicating that the Ca(2+)-pump may play an important role in O(2)-deprived maize cells.     

Calmodulin X (Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM X (Ca2+)4 is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E X CaM X (Ca2+)4 complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01-1 microM) was 0.9 nM; the respective apparent dissociation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1-50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by 125I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM X (Ca2+)4. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E X CaM) is at least 100-fold greater than the apparent dissociation constant of the E X CaM X (Ca2+)4 complex, as judged from non-saturation 125I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.     

Truncation of a mammalian myosin I results in loss of Ca2+-sensitive motility

MYR-1, a mammalian class I myosin, consisting of a heavy chain and 4-6 associated calmodulins, is represented by the 130-kDa myosin I (or MI(130)) from rat liver. MI(130) translocates actin filaments in vitro in a Ca(2+)-regulated manner. A decrease in motility observed at higher Ca(2+) concentrations has been attributed to calmodulin dissociation. To investigate mammalian myosin I regulation, we have coexpressed in baculovirus calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic "motor" domain and the first calmodulin-binding IQ domain of rat myr-1; we refer to this truncated molecule here as MI(1IQ). Association of calmodulin to MI(1IQ) is Ca(2+)-insensitive. MI(1IQ) translocates actin filaments in vitro at a rate resembling MI(130), but unlike MI(130), does not exhibit sensitivity to 0.1-100 micrometer Ca(2+). In addition to demonstrating successful expression of a functional truncated mammalian myosin I in vitro, these results indicate that: 1) Ca(2+)-induced calmodulin dissociation from MI(130) in the presence of actin is not from the first IQ domain, 2) velocity is not affected by the length of the IQ region, and 3) the Ca(2+) sensitivity of actin translocation exhibited by MI(130) involves 1 or more of the other 5 IQ domains and/or the carboxyl tail.     

Ca2+-free calmodulin and calmodulin damaged by in vitro aging are selectively degraded by 26 S proteasomes without ubiquitination

The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca(2+)-binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca(2+)). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca(2+) stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca(2+)-free (apo) CaM without ubiquitination. Addition of Ca(2+) dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca(2+) binding, native CaM assumes a non-degradable conformation, which most of the age-damaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.     

Molecular regulation of cardiac ryanodine receptor ion channel

The release of Ca(2+) ions from intracellular stores is a key step in a wide variety of cellular functions. In striated muscle, the release of Ca(2+) from the sarcoplasmic reticulum (SR) leads to muscle contraction. Ca(2+) release occurs through large, high-conductance Ca(2+) release channels, also known as ryanodine receptors (RyRs) because they bind the plant alkaloid ryanodine with high affinity and specificity. The RyRs are isolated as 30S protein complexes comprised of four 560 kDa RyR2 subunits and four 12 kDa FK506 binding protein (FKBP12) subunits. Multiple endogenous effector molecules and posttranslational modifications regulate the RyRs. This review focuses on current research toward understanding the control of the isolated cardiac Ca(2+) release channel/ryanodine receptor (RyR2) by Ca(2+), calmodulin, thiol oxidation/reduction and nitrosylation, and protein phosphorylation.     

Can calmodulin function without binding calcium?

Calmodulin is a small Ca(2+)-binding protein proposed to act as the intracellular Ca2+ receptor that translates Ca2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca(2+)-binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca2+; one does not bind detectable levels of 45Ca2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca2+.     

Protein kinase C phosphorylates the carboxyl terminus of the plasma membrane Ca(2+)-ATPase from human erythrocytes.

Purified Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase) from human erythrocytes was phosphorylated with a stoichiometry of about 1 mol of phosphate/mol of ATPase at both threonine and serine residues by purified rat brain type III protein kinase C. In the presence of calmodulin, the phosphorylation was markedly reduced. Labeled phosphate from [gamma-32P]ATP was retained on an 86-kDa calmodulin-binding tryptic fragment of Ca(2+)-ATPase but not on 82- and 77-kDa non-calmodulin-binding fragments. Similarly, fragmentation of the phosphorylated Ca(2+)-ATPase by calpain I revealed that calmodulin-binding fragments (127 and 125 kDa) retained phosphate label whereas a non-calmodulin-binding fragment (124 kDa) did not. The calmodulin-binding domain, located about 12 kDa from the carboxyl terminus of the Ca(2+)-ATPase, was thus located as a site of protein kinase C phosphorylation. A synthetic peptide corresponding to a segment of the calmodulin-binding domain (H2 N-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-COOH) was indeed phosphorylated at the single threonine residue within this sequence. The additional serine phosphorylation site was carboxyl terminal to the calmodulin domain. Phosphorylation by purified type III protein kinase C (canine heart) antagonized the calmodulin activation of the Ca(2+)-ATPase, particularly at lower Ca2+ concentrations (0.2-1.0 microM). By contrast, a purified but unresolved protein kinase C isoenzyme mixture from rat brain stimulated the activity of Ca(2+)-ATPase prepared in asolectin, but not glycerol, by more than 2-fold in the presence of the ionophore A23187, without increasing its Ca2+ sensitivity. The results clearly indicate that human erythrocyte Ca(2+)-ATPase is a substrate of protein kinase C, but the effect of phosphorylation on the activity of the enzyme depends on the isoenzyme form of protein kinase C used and on the lipid associated with the Ca(2+)-ATPase.     

Ubiquitin-calmodulin conjugating activity from cardiac muscle

Enzyme activity capable of covalently linking ubiquitin to bovine calmodulin in an ATP-dependent manner has been detected in rabbit cardiac muscle demonstrating that this enzyme occurs not only in reticulocytes but also in other tissues and possibly all tissues and cells which contain calmodulin as intracellular Ca2+-acceptor protein. This is of special interest since a ubiquitin-dependent proteolytic activity could previously not be detected in cardiac muscle. The name ubiquityl-calmodulin synthetase [uCaM-synthetase, ubiquityl:calmodulin ligase (EC 6.3.?.?)] is therefore suggested for this enzyme. In crude cardiac muscle extracts uCaM-Synthetase displays a specific activity of 93 nUnits/mg in comparison to reticulocyte lysate with 270 nUnits/mg as measured by the fluphenazine-Sepharose affinity adsorbent test (FP-test). Analysis of the ubiquitination product (125I-uCaM) by polyacrylamide electrophoresis in the presence of SDS followed by autoradiography reveals a major double band with molecular masses of 27 and 29 kDa (mono-ubiquitination products) respectively. In addition two novel minor bands (17 and 20 kDa) of smaller molecular mass than the monoubiquitination products were detected. These are probably proteolytic breakdown products of uCaM. A model is suggested for a specific function of this synthetase in the Ca2+-dependent breakdown of calmodulin in vertebrate (eukaryotic) cells.     

ATP-dependent proteolysis and the role of ubiquitin in rabbit cardiac muscle

A soluble ATP/Mg2-dependent proteolytic system from rabbit cardiac muscle has been identified (m ca. 310 kDa) and purified ca. 9-fold. This enzyme which splits the substrate [3H]globin and 125I-bovine serum albumin (125I-BSA) has many similarities to the ATP-dependent proteolytic enzyme system from reticulocytes which utilizes ubiquitin: 1) The specific activities in reticulocyte lysates and cardiac muscle extracts are of the same magnitude (0.5-1 arb. unit/mg). 2) The binding and elution behavior on DEAE-cellulose is similar. 3) In both cases the pH optimum (substrate 125I-BSA) is pH 7.6. 4) Both enzymes are inhibited by hemin, NEM and iodoacetate but not e.g. by leupeptin, or inhibitors of serine proteases. 5) Neither enzyme system can utilize ATP-analogs such as AMP-CPP, AMP-PCP, AMP-PNP or ATP-gamma-S. There are however also significant differences: 1) The enzyme system from cardiac muscle is fully active in the absence of ubiquitin and cannot be activated by this peptide. 2) The enzyme from cardiac muscle can degrade methylated BSA. 3) The cardiac muscle enzyme can be further purified on Sepharose 4B; the enzyme from reticulocytes is inactivated by this procedure. 4) The cardiac enzyme cannot be inactivated by ribonuclease as the reticulocyte counterpart. Although ubiquitin does not appear to play a role in the isolated ATP/Mg2-dependent proteolytic system from cardiac muscle, it is demonstrated for the first time that 125I-ubiquitin can be conjugated to a wide variety of cardiac muscle proteins in vitro in an ATP-dependent manner. Apparent molecular masses of major conjugates were: 185 kDa, 140 kDa, 85 kDa, 65 kDa, 46 kDa, 38 kDa and 36 kDa as estimated by discontinuous SDS gel electrophoresis. Addition of purified phosphorylase kinase to cardiac muscle extract changed the ubiquitination pattern by the appearance of two novel protein bands. It is concluded that the ATP/Mg2-dependent proteolytic system of cardiac muscle must be differentiated from the proteolytic system of reticulocytes mainly because of its ubiquitin-independence. Nevertheless the conjugation of 125I-ubiquitin to many muscle proteins is a strong indication for a crucial role of this interesting peptide in striated muscle.