Solubilization of guanylyl cyclase from bovine rod outer segments and effects of lowering Ca2+ and nitro compounds.

Guanylyl cyclase from bovine rod outer segments was solubilized using Triton X-100 and a high concentration of KCl, and its regulation was studied. The efficiency of solubilization was about 50-90% of total activity. When the Ca2+ content was lowered (less than 80 nM), guanylyl cyclase was activated about 2-fold. In the presence of higher concentrations of Ca2+ (greater than 140 nM), the activity was decreased. The regulation by Ca2+ was also demonstrated with solubilized preparations. In the presence of 186 nM Ca2+ which inhibited guanylyl cyclase, La3+ activated the enzyme about 2-fold, suggesting that the Ca2(+)-binding protein similar to other Ca2(+)-binding proteins associates with guanylyl cyclase regulation. Sodium nitroprusside and nitric oxide which are activators of soluble guanylyl cyclase in other tissues also activated the retinal guanylyl cyclase. Maximum activation by sodium nitroprusside was 20-fold using Mg2+ as a cofactor. Activation by nitric oxide and related compounds suggests that retinal guanylyl cyclase contains a heme prosthetic group that may participate in a novel regulatory mechanism for this enzyme.     

Quantitative analysis of the free energy coupling in the system calmodulin, calcium, smooth muscle myosin light chain kinase

Interactions between Ca2+, calmodulin and turkey gizzard myosin light chain kinase have been studied by equilibrium gel filtration and analyzed in terms of the theory of free energy coupling as formulated by Huang and King for calmodulin-regulated systems (Current Topics in Cellular Regulation 27, 1966-1971, 1985). Direct binding studies revealed that upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding. The determination of the Ca2+-binding constants is inherently approximative due to the apparent homotropic cooperativity; therefore a statistical chi 2 analysis was carried out to delimit the formation-, and subsequently the stoichiometric Ca2+-binding constants. Whereas the first two stoichiometric Ca2+-binding constants of enzyme-bound CaM do not differ or are at the upmost 10-fold higher than those in free calmodulin, the third Ca2+ ion binds with an at least 70-fold and more likely 3000-fold higher affinity constant. The binding constant for the fourth Ca2+ is only 5-fold higher than the corresponding one in free calmodulin, thus creating a plateau at 3 bound Ca2+ in the isotherm. Direct binding of Ca2+-free calmodulin to myosin light chain kinase at 10(-7) M free Ca2+ yielded a l/l stoichiometry and an affinity constant of 2.2 x 10(5) M-1. It is thus anticipated that in resting smooth muscle ([Ca2+] less than or equal to 10(-7) M) more than half of the enzyme is bound to metal-free calmodulin. Analysis of the enzymatic activation of myosin light chain kinase at different concentrations of calmodulin and Ca2+ revealed that this Ca2+-free complex is inactive and that activation is concomitant with the formation of the enzyme.calmodulin.Ca3 complex.     

Functional consequences of alterations to polar amino acids located in the transmembrane domain of the Ca2(+)-ATPase of sarcoplasmic reticulum

Glu309, Glu771, Asn796, Thr799, Asp800, and Glu908 (ligands 1 to 6, respectively) appear to form the high affinity Ca2(+)-binding sites of the Ca2(+)-ATPase. The plasticity of the Ca2(+)-binding sites was tested by separate replacement of each of the ligands with a structurally similar oxygen-containing residue using site-specific mutagenesis. Mutant cDNAs were transfected into COS-1 cells, and ATP-dependent Ca2+ transport or partial reactions were studied in microsomes containing the expressed Ca2(+)-ATPases. In most cases where amino acid substitutions were carried out, the expressed enzymes lacked Ca2+ transport function and Ca2(+)-dependent phosphorylation by ATP. Furthermore, the mutant enzymes were phosphorylated by inorganic phosphate, even in the presence of Ca2+, which inhibits phosphorylation of the wild-type enzyme possessing intact Ca2(+)-binding sites. On mutant, however, containing an isosteric replacement of Glu by Gln at ligand 6, exhibited wild-type levels of Ca2+ transport activity and Ca2+ affinity. Two mutants exhibited properties consistent with a reduction in Ca2+ affinity. In the mutant in which Thr was replaced by Ser at ligand 4, Ca2+ transport activity was 70% of wild-type, while half-maximal activation by Ca2+ occurred at 0.8 microM as compared to 0.3 microM for the wild-type enzyme. In the mutant Glu309----Asp at ligand 1, Ca2+ transport activity was lost, but Ca2(+)-activated phosphorylation by ATP was retained. The concentration of Ca2+ required to activate phosphorylation was increased about 10-fold, however, compared to wild type. These results support our hypothesis that ligands 1 to 6, believed to reside within the transmembrane domain, interact with Ca2+ ions during the transport process. The roles of 12 other oxygen-containing residues and of His278 located in the transmembrane domain were also examined by mutation. Although the oxygen-containing side chains of these residues are potential Ca2+ ligands, their replacement with nonpolar amino acids did not abolish Ca2+ transport function, leading to the conclusion that they are not essential ligands for high affinity Ca2+ binding by the Ca2+ pump.     

Modification of ATP regulatory function in sarcoplasmic reticulum Ca2(+)-ATPase by hydrophobic molecules

The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3-nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 microM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2+ uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low-affinity Ca2(+)-binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3-nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1-1 mM ATP). A striking difference was observed when both the high- and low-affinity Ca2(+)-binding sites were saturated. In this condition, triphenylphosphine and 3-nitrophenol promoted a 3-4-fold increase in the apparent affinity for ATP at its regulatory site.     

The N-terminal portion of the main cytosolic loop mediates K+ sensitivity in the retinal rod Na+/Ca2+-K+-exchanger.

Two types of Na+/Ca2+-exchangers have been characterized in the literature: The first is the cardiac, skeletal muscle and brain type, which exchanges 1 Ca2+ for 3 Na+, the second, found in retinal photosensor cells, transports 1 Ca2+ and 1 K+ in exchange for 4 Na+. The present work describes the properties of chimeric constructs of the two exchanger types. Ca2+ gel overlay experiments have identified a high affinity (Kd in the 1 microM range) Ca2+-binding domain between Glu601 and Asp733 in the main cytosolic loop of the retinal protein, just after transmembrane domain 5. Insertion of the retinal Ca2+-binding domain in the cytosolic loop of the cardiac exchanger conferred K+-dependence to the Ca2+ uptake activity of the chimeric constructs expressed in HeLa cells. The apparent Km of the K+ effect was about 1 mM. Experiments with C-terminally truncated versions of the retinal insert indicated that the sequence between Leu643 and Asp733 was critical in mediating K+ sensitivity of the recombinant chimeras. Thus, the high affinity Ca2+-binding domain in the main cytosolic loop of the retinal exchanger may regulate the activity of the retinal protein by binding Ca2+, and by conferring to it K+ sensitivity.     

Evidence of intramolecular regulation of the Dictyostelium discoideum 34 000 Da F-actin-bundling protein

Intramolecular interaction within the Ca(2+)-regulated 34 kDa actin-bundling protein from Dictyostelium discoideum was found to contribute to the regulation of its actin-binding activity. Recombinant N-terminally truncated proteins aa77-295, 124-295, and 139-295 bound actin at > or = 2:1 stoichiometry, which is 5-fold greater than the intact protein aa1-295 as assessed by cosedimentation with F-actin. These proteins also have enhanced cross-linking activity as assessed by viscometry and electron microscopy. All truncated 34 kDa proteins failed to bind (45)Ca(2+) on blots and displayed Ca(2+)-insensitive binding with actin, although most proteins possessed intact putative EF-hand Ca(2+)-binding motifs. An intramolecular interaction within the 34 kDa protein was inferred from direct demonstrations of domain-domain interaction among the truncated 34 kDa proteins both in the presence and absence of actin. The intramolecular interaction between interaction zone 1 (aa71-123) and interaction zone 2 (aa193-254) is proposed to maintain the N-terminal inhibitory region (aa1-76) in close proximity with the strong actin-binding site (aa193-254) in order to modulate the interaction of the intact protein with actin filaments.     

The calcium requirement for stability and enzymatic activity of two isoforms of barley aleurone alpha-amylase

alpha-Amylases (EC 3.2.1.1) secreted by the aleurone layer of barley grains are Ca2+-containing metalloenzymes. We studied the effect of Ca2+ on the activity and structure of the two major groups of aleurone alpha-amylase by incubating affinity purified enzyme in solutions containing Ca2+ from pCa 4 to 7. Both groups of isoforms required one atom of Ca2+/molecule of enzyme as determined by isotope exchange, but the two groups differed by more than 10-fold in their affinity for Ca2+. Both groups of alpha-amylase were irreversibly inactivated by incubation in low Ca2+ (pCa 7). This inactivation was not due to changes in primary structure, as measured by molecular weight, but appeared to be the result of changes in secondary and tertiary structure as indicated by circular dichroism spectra, serology, lability in the presence of protease, and fluorescence spectra. Analysis of the predicted secondary structure of barley aleurone alpha-amylase indicates that the Ca2+-binding region of barley amylases is structurally similar to that of mammalian alpha-amylases. Our data indicate that micromolar levels of Ca2+ are required to stabilize the structure of barley alpha-amylases in the endoplasmic reticulum of the aleurone layer where these enzymes are synthesized.     

Characterization of a calmodulin-stimulated adenylate cyclase from abalone spermatozoa

Abalone sperm adenylate cyclase activity is particulate in nature and displays a high Mg2+-supported activity (Mg2+/Mn2+ = 0.8) as compared to other sperm adenylate cyclases. Approximately 90% of the enzyme activity in crude homogenates is inhibited by EGTA in a concentration-dependent manner which is overcome by added micromolar free Ca2+. The EGTA-inhibited Ca2+-stimulated enzyme activity is also inhibited by phenothiazines. Added calmodulin, however, has no effect on enzyme activity prepared from crude homogenates. Preparation of a twice EGTA-extracted 48,000 X g pellet fraction yields a particulate enzyme activity that can be stimulated 10-65% by added calmodulin in the presence of micromolar free Ca2+. Detergent extraction (1% Lubrol PX) of the EGTA-washed 48,000 X g pellet solubilizes 2-5% of the total particulate adenylate cyclase activity, and this solubilized enzyme is activated up to 125% by calmodulin. The ability of the different enzyme preparations to be stimulated by calmodulin is inversely proportional to the endogenous calmodulin concentration. Calmodulin stimulation of the Lubrol PX-solubilized enzyme is specific to this Ca2+-binding protein and is mediated as an effect on the velocity of the enzyme. This stimulation is completely Ca2+ dependent and is fully reversible. These data suggest that the control of sperm cAMP synthesis by changes in Ca2+ conductance may be mediated via this Ca2+-binding protein.     

Isolation and characterization of myosin from amoebae of Physarum polycephalum

Myosin was isolated from amoebae of Physarum polycephalum and compared with myosin from plasmodia, another motile stage in the Physarum life cycle. Amoebal myosin contained heavy chains (Mr approximately 220,000), phosphorylatable light chains (Mr 18,000), and Ca2+-binding light chains (Mr 14,000) and possessed a two-headed long-tailed shape in electron micrographs after rotary shadow casting. In the presence of high salt concentrations, myosin ATPase activity increased in the following order: Mg-ATPase activity less than K-EDTA-ATPase activity less than Ca-ATPase activity. In the presence of low salt concentrations, Mg-ATPase activity was activated approximately 9-fold by skeletal muscle actin. This actin-activated ATPase activity was inhibited by micromolar levels of Ca2+. Amoebal myosin was indistinguishable from plasmodial myosin in ATPase activities and molecular shape. However, the heavy chain and phosphorylatable light chains of amoebal myosin could be distinguished from those of plasmodial myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunological studies, suggesting that these are different gene products. Ca2+-binding light chains of amoebal and plasmodial myosins were found to be identical using similar criteria, supporting our hypothesis that the Ca2+-binding light chain plays a key role in the inhibition of actin-activated ATPase activity in Physarum myosins by micromolar levels of Ca2+.     

The regulatory role of EF-hand motifs of pig 80K diacylglycerol kinase as assessed using truncation and deletion mutants.

To elucidate the regulatory function of EF-hand motifs of pig 80K diacylglycerol (DG) kinase, we constructed and expressed several truncation and deletion mutants of the enzyme in E. coli or COS-7 cells. The bacterially expressed EF-hand region could bind Ca2+ and was suggested to undergo conformational change like calmodulin. A mutant enzyme lacking EF-hands lost Ca(2+)-binding activity, but could be fully activated by phosphatidylserine (PS) or deoxycholate in the absence of Ca2+. The full activation of the wild-type enzyme by PS, on the other hand, was totally dependent on Ca2+. Further, the wild-type enzyme expressed in COS-7 cells was exclusively soluble, whereas the EF-hand-deleted mutant was considerably associated with the membranes. The results suggest that under Ca(2+)-free condition, the EF-hand masks the PS-binding site of the DG kinase, and that the Ca(2+)-binding results in the exposure of the PS-binding site through the conformational change of the EF-hand region.     

Regulation of the erythrocyte Ca(2+)-ATPase by mutant calmodulins with Glu----Ala substitutions in the Ca(2+)-binding domains.

We have used four mutant calmodulins to study the regulation of human erythrocyte Ca(2+)-ATPase by the calmodulin-dependent pathway; the conserved Glu at position 12 in each of the four Ca(2+)-binding domains of calmodulin (Glu31, Glu67, Glu104, or Glu140) was replaced by Ala. At pCa 7, where unmodified calmodulin maximally activates the erythrocyte Ca(2+)-ATPase, all four mutants stimulated Ca(2+)-ATPase activity to the same maximal velocity. However, the concentrations of mutant calmodulins required for half-maximal activation (KCaM) were significantly higher than that for unmodified calmodulin and were strongly dependent on the domain in which the mutated Glu was located; substitution in either the first or second Ca(2+)-binding domain had little effect (2-3-fold increase in KCaM), whereas substitution in either the third or fourth domain resulted in a dramatic, 25-71-fold increase in KCaM. The same order of sensitivity was observed when the Ca2+ dependence of enzyme activation was measured at a constant 100 nM concentration of mutant calmodulin. These data point to dramatic differences in the functional significance of the replacement of the Glu at position 12 in each of the four Ca(2+)-binding domains for activation of the Ca(2+)-ATPase. The 2 Glu residues located in the carboxyl-terminal half of calmodulin (particularly Glu140) are crucial for activation of the Ca(2+)-ATPase at physiologically significant Ca2+ concentrations.     

Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum

The complete nucleotide sequence of the celH gene of Clostridium thermocellum was determined. The open reading frame extended over 2.7-kb DNA fragment and encoded a 900-amino acid (aa) protein (Mr 102,301) which hydrolyzes carboxymethylcellulose, p-nitrophenyl-beta-D-cellobioside, methylumbelliferyl- beta-D-cellobioside, barley beta-glucan, and larchwood xylan. The N terminus showed a typical signal peptide, and a cleavage site after Ser44 was predicted. Two Pro-Thr-Ser-rich regions divided the protein into three approximately equal domains. The central 328-aa region was similar to the N-terminal part, carrying the active site, of C. thermocellum endoglucanase E (EGE; 30.2%). The C-terminal region ended with two conserved 24-aa stretches showing close similarity with those previously described in EGA, EGB, EGD, EGE, EGX, and xylanase from C. thermocellum. Deletions of celH removing up to 327 codons from the 5' end and up to 245 codons from the 3' end of the coding sequence did not affect enzyme activity, confirming that the central domain was indeed responsible for catalytic activity. Production of truncated EGH in Escherichia coli was increased up to 120-fold by fusing fragments containing the 3' portion of the gene with the start of lacZ' present in pTZ19R.     

Crayfish abdominal muscle adenylate cyclase. Studies on the stimulation by a Ca2+-binding protein.

A plasma-membrane preparation of crayfish muscle showed an adenylate cyclase activity which is inhibited to about 80% of its original activity by 100 microM-EGTA. Measurements of the enzyme activity in the presence of 100 microM-EGTA and various concentrations of Ca2+ revealed an increase in enzyme activity of about 400%, indicating an adenylate cyclase which is dependent on Ca2+ for activity. Fluphenazine (1 mM), a blocker of the Ca2+-binding protein calmodulin, decreased enzyme activity to zero. The enzyme can be re-activated by the addition of certain concentrations of calmodulin to the assay medium. This suggests that crayfish muscle adenylate cyclase is dependent on Ca2+ and calmodulin for activity.     

Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca(2+)-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca(2+)-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca(2+)-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.     

Terbium as a luminescent probe of metal-binding sites in protein kinase C

In the present report, we demonstrate that Tb3+ binds to protein kinase C and serves as a luminescent reporter of certain cationic metal-binding sites. Tb3+ titration of 50 nM protein kinase C results in a 20-fold enhancement of Tb3+ luminescence which is half-maximal at 12 microM Tb3+. A Kd of approximately 145 nM was determined for Tb3+ binding to the enzyme. The excitation spectrum of bound Tb3+ exhibits a peak at 280 nm characteristic of energy transfer from protein tryptophan or tyrosine residues. The luminescence of this complex can be markedly decreased by other metals, including Pb2+ (IC50 = 25 microM), La3+ (IC50 = 50 microM), Hg2+ (IC50 = 300 microM), Ca2+ (IC50 = 6 mM), and Zn2+ (IC50 greater than 10 mM), and chelation of Tb3+ by 2 mM EGTA. Tb3+ binding to protein kinase C is correlated with its inhibition of protein kinase activity (IC50 = 8 microM), r = 0.99) and phorbol ester binding (IC50 = 15 microM, r = 0.98). Tb3+ inhibition of protein kinase C activity cannot be overcome by excess Ca2+, but can be partially overcome with excess phosphatidylserine or by chelation of Tb3+ with EGTA. Tb3+ noncompetitively inhibits phorbol ester binding by decreasing the maximal extent of binding without significantly altering binding affinity. The results suggest that the Tb3(+)-binding site is at or allosterically related to the enzyme's phosphatidylserine-binding site, but is distinct from the phorbol ester-binding domain and the Ca2(+)-binding site that regulates enzyme activity.     

Interactions of calcineurin A, calcineurin B, and Ca2+.

Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.     

Characterization of the cation-binding properties of porcine neurofilaments

In the presence of physiological levels of Na+ (10 mM), K+ (150 mM), and Mg2+ (2 mM), dephosphorylated neurofilaments contained two Ca2+ specific binding sites with Kd = 11 microM per unit consisting of eight low, three middle, and three high molecular subunits, as well as 46 sites with Kd = 620 microM. Only one class of 126 sites with Kd = 740 microM was detected per unit of untreated neurofilaments. A chymotryptic fraction enriched in the alpha-helical domains of neurofilament subunits contained one high-affinity Ca2+-binding site (Kd = 3.6 microM) per domain fragment of approximately 32 kDa. This site may correspond to a region in coil 2b of the alpha-helical domain, which resembles the I-II Ca2+-binding site in intestinal Ca2+-binding protein. Homopolymeric filaments composed of the low or middle molecular weight subunits contained low-affinity Ca2+-binding sites with Kd = 37 microM and 24 microM, respectively, while the Kd values for the low-affinity sites in heteropolymeric filaments were 8-10-fold higher. Competitive binding studies, using the chymotryptic fraction to assay the high-affinity Ca2+-binding sites and 22Na+ to monitor binding to the phosphate-containing low-affinity sites, yielded Kd values for Al3+ of 0.01 microM and 4 microM, respectively. This suggests that the accumulation of Al3+ in neurons may be due in part to its binding to neurofilaments.