Purification and Characterization of α-Amylase from Bacillus licheniformis CUMC305

α-Amylase produced by Bacillus licheniformis CUMC305 was purified 212-fold with a 42% yield through a series of four steps. The purified enzyme was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme showed maximal activity at 90°C and pH 9.0, and 91% of this activity remained at 100°C. The enzyme retained 91, 79, and 71% maximal activity after 3 h of treatment at 60°C, 3 h at 70°C, and 90 min at 80°C, respectively, in the absence of substrate. On the contrary, in the presence of substrate (soluble starch), the α-amylase enzyme was fully stable after a 4-h incubation at 100°C. The enzyme showed 100% stability in the pH range 7 to 9; 95% stability at pH 10; and 84, 74, 68, and 50% stability at pH values of 6, 5, 4, and 3, respectively, after 18 h of treatment. The activation energy for this enzyme was calculated as 5.1 × 10⁵ J/mol. The molecular weight was estimated to be 28,000 by sodium dodecyl sulfate-gel electrophoresis. The relative rates of hydrolysis of soluble starch, amylose, amylopectin, and glycogen were 1.27, 1.8, 1.94, and 2.28 mg/ml, respectively. V_max values for hydrolysis of these substrates were calculated as 0.738, 1.08, 0.8, and 0.5 mg of maltose/ml per min, respectively. Of the cations, Na⁺, Ca²⁺, and Mg²⁺, showed stimulatory effect, whereas Hg²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Ag⁺, Fe²⁺, Co²⁺, Cd²⁺, Al³⁺, and Mn²⁺ were inhibitory. Of the anions, azide, F⁻, SO₃²⁻, SO₄³⁻, S₂O₃²⁻, MoO₄²⁻, and Wo₄²⁻ showed an excitant effect. p-Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, β-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. α-Amylase was fairly resistant to EDTA treatment at 30°C, but heating at 90°C in presence of EDTA resulted in the complete loss of enzyme activity, which could be recovered partially by the addition of Cu²⁺ and Fe²⁺ but not by the addition of Ca²⁺ or any other divalent ions.     

Purification and Properties of a Thermoactive Glucoamylase from Clostridium thermosaccharolyticum

A bacterial glucoamylase was purified from the anaerobic thermophilic bacterium Clostridium thermosaccharolyticum and characterized. The enzyme, which was purified 63-fold, with a yield of 36%, consisted of a single subunit with an apparent molecular mass of 75 kDa. The purified enzyme was able to attack α-1,4- and α-1,6-glycosidic linkages in various α-glucans, liberating glucose with a β-anomeric configuration. The purified glucoamylase, which was optimally active at 70°C and pH 5.0, attacked preferentially polysaccharides such as starch, glycogen, amylopectin, and maltodextrin. The velocity of oligosaccharide hydrolysis decreased with a decrease in the size of the substrate. The K_m values for starch and maltose were 18 mg/ml and 20 mM, respectively. Enzyme activity was not significantly influenced by Ca²⁺, EDTA, or α- or β-cyclodextrins.     

Studies on Cellulose Hydrolysis by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus extracellular cellulase extensively hydrolyzed crystalline celluloses such as Avicel (FMC Corp., Food and Pharmaceutical Products Div., Philadelphia, Pa.) but only if it was desalted and supplemented with Ca²⁺. The Ca²⁺ effect was one of increased enzyme stability in the presence of the ion. Although preincubation of the cellulase complex at 40°C for 5 h without added Ca²⁺ had a negligible effect on endoglucanase activity or on the subseqent hydrolysis of amorphous cellulose, the capacity of the enzyme to hydrolyze crystalline cellulose was almost completely lost. Adsorption studies showed that 90% of the Avicel-solubilizing component of the total enzyme preparation bound to 2% Avicel at 40°C. Under these conditions, only 15% of the endoglucanase and 25% of the protein present in the enzyme preparation adsorbed to the substrate. The protein profile of the bound enzyme, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was complex and distinctly different from the profile observed for total cellulase preparations. The specific activity of A. cellulolyticus cellulase with respect to Avicel hydrolysis was compared with that of commercially available Trichoderma reesei cellulase.     

Distinctive Features of Catalytic and Transport Mechanisms in Mammalian Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases

Ca²⁺ (sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA)) and Cu⁺ (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca²⁺, demonstrated by the addition of ATP and Ca²⁺ to SERCA deprived of Ca²⁺ (E2) as compared with ATP to Ca²⁺-activated enzyme (E1·2Ca²⁺). Activation by Ca²⁺ is slower at low pH (2H⁺·E2 to E1·2Ca²⁺) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca²⁺ translocation. A “H⁺-gated pathway,” demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca²⁺ release by H⁺/Ca²⁺ exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu⁺/H⁺ exchange. As opposed to SERCA after Ca²⁺ chelation, ATP7A/B does not undergo reverse phosphorylation with P_i after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.     

Calcium binding by human erythrocyte membranes

1. The characteristics of Ca²⁺ binding to haemoglobin-free human erythrocyte membranes were investigated by using ⁴⁵Ca and centrifugation partition of `ghosts' from their external incubation medium. Equilibrium of `ghosts' with external Ca²⁺ required less than 15min. 2. The binding did not vary with temperature in the range 0–37°C. 3. At pH7.4 `ghosts' bound a maximum of 283μmol of Ca<sup>2+</sup>/g of `ghost' protein, equivalent to 6.85×10⁷ Ca²⁺ ions per cell. 4. Increasing the ionic strength from 0.01 to 0.46 diminished Ca²⁺ binding, as did ATP in concentrations ranging from 0 to 15mm in the incubation medium. 5. An increase of the pH from 3.0 to 9.3 caused a marked increase in the amount of Ca²⁺ bound. 6. Extraction of ⁴⁵Ca-labelled `ghosts' with chloroform–methanol showed that the distribution of Ca²⁺ was: 79% protein-bound, 16% lipid-bound, 5% in the aqueous phase, presumably non-bound. Most of the lipid-bound Ca²⁺ (about 80%) was associated with a phospholipid fraction containing phosphatidylserine, phosphoinositides and phosphatidylethanolamine, giving a molar Ca²⁺: phosphorus ratio of about 1:2.     

Modulation of the organization of erythrocyte membrane phospholipids by cytoplasmic ATP. The susceptibility of isoionic human erythrocyte ghosts to attack by detergents and phospholipase C

Human erythrocyte ghosts were prepared in media of physiological ionic composition, and these “isoionic” ghosts were then lysed and resealed in media of varying Ca²⁺, Mg²⁺ and ATP concentrations. The susceptibilities of these ghosts to limited attack by various detergents and by phospholipases C were then compared with the susceptibilities of intact cells to similar attack: attack was assessed by measurements of lysis and of phospholipid hydrolysis. Ghosts were more readily attacked than cells by anionic detergents (cholate, glycocholate, dodecyl sulphate) and by phospholipases C, but Triton X-100 and cetyltrimethylammonium attacked cells and ghosts to the same extent. Mg · ATP^2? partially protected ghosts from attack by the anionic detergents and by the phospholipases C of Bacillus cereus and of Clostridium perfringens. Protection by Mg · ATP^2? occurred only if Mg · ATP^2? had access to the cytoplasmic surface of the membrane. Adenylyl(β-γ-methylene)diphosphonate, a non-hydrolysable ATP analogue, protected as effectively as did Mg · ATP^2?. Internal Mg · ATP^2? caused a marked reduction in the hydrolysis by phospholipases of phosphatidylethanolamine and sphingomyelin, but had no appreciable effect upon the simultaneous hydrolysis of phosphatidylcholine. It therefore seems that interaction of ATP with sites on the cytoplasmic surface of the erythrocyte membrane can, without ATP hydrolysis, cause changes in the organization of the outer surface of the membrane that specifically render phosphatidylethanolamine and sphingomyelin less accessible to attack by extracellular phospholipases.     

Halophilic Nuclease of a Moderately Halophilic Bacillus sp.: Production, Purification, and Characterization

A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular weight was estimated to be 138,000 by Sephadex G-200 gel filtration. The enzyme had marked halophilic properties, showing maximal activities in the presence of 1.4 to 3.2 M NaCl or 2.3 to 3.2 M KCl. The enzyme hydrolyzed thymidine-5′-monophosphate-p-nitrophenyl ester at a rate that increased with NaCl concentration up to 4.8 M. In the presence of both Mg²⁺ and Ca²⁺, activity was greatly enhanced. The activity was lost by dialysis against water and low-salt buffer, but it was protected when 10 mM Ca²⁺ was added to the dialysis buffer. When the inactivated enzyme was dialyzed against 3.5 M NaCl buffer as much as 68% of the initial activity could be restored. The enzyme exhibited maximal activity at pH 8.5 and at 50°C on DNA and at 60°C on RNA and attacked RNA and DNA exonucleolytically and successively, producing 5′-mononucleotides.     

The binding of calcium and yttrium ions to a glycoprotein from bovine cortical bone

The binding of Ca²⁺ and Y³⁺ to an acidic glycoprotein from bovine cortical bone, bone sialoprotein, was determined from the titration curves at I 0·2 in the presence and absence of the cations. The binding of Y³⁺ was greater than that of Ca²⁺. The value for the association constant, k, for the interaction with Y³⁺ increased with pH, from log k 2·93 at pH3·4 to log k 3·50 at pH4·4, and the number of binding sites/mol. increased from 4·6 at pH3·4 to 9·1 at pH4·4. It is proposed that the binding site consists of three carboxyl groups, but it is likely that the binding is a strong electrostatic interaction rather than a co-ordination linkage. A chondroitin sulphate–protein complex also extracted from bovine cortical bone interacted with Y³⁺ and Ca²⁺ to a similar extent as did bone sialoprotein. It is suggested that these materials are present in bone at the resting and resorbing surfaces and that they contribute to the deposition of yttrium, americium and plutonium at these sites.     

Partial Purification and Characterization of a Thermostable Actinomycete β-Amylase

A thermostable amylase, possibly a β-amylase from Thermoactinomyces sp. no. 2 isolated from soil, is reported. The enzyme was purified 36-fold by acetone precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration, and the molecular weight was estimated at 31,600. The enzyme was characterized by demonstration of optimum activity at 60°C and pH 7 and by retention of 70% activity at 70°C (30 min). It was stimulated by Mn²⁺ and Fe²⁺ but strongly inhibited by Hg²⁺. Maltose was the only detectable product of hydrolysis of starches and was quantitatively highest in plantain starch hydrolysate.     

Purification and Properties of a Xylan-Binding Endoxylanase from Alkaliphilic Bacillus sp. Strain K-1

An alkaliphilic bacterium, Bacillus sp. strain K-1, produces extracellular xylanolytic enzymes such as xylanases, β-xylosidase, arabinofuranosidase, and acetyl esterase when grown in xylan medium. One of the extracellular xylanases that is stable in an alkaline state was purified to homogeneity by affinity adsorption-desorption on insoluble xylan. The enzyme bound to insoluble xylan but not to crystalline cellulose. The molecular mass of the purified xylan-binding xylanase was estimated to be approximately 23 kDa. The enzyme was stable at alkaline pHs up to 12. The optimum temperature and optimum pH of the enzyme activity were 60°C and 5.5, respectively. Metal ions such as Fe²⁺, Ca²⁺, and Mg²⁺ greatly increased the xylanase activity, whereas Mn²⁺ strongly inhibited it. We also demonstrated that the enzyme could hydrolyze the raw lignocellulosic substances effectively. The enzymatic products of xylan hydrolysis were a series of short-chain xylooligosaccharides, indicating that the enzyme was an endoxylanase.     

Brain arylamidase. Purification and characterization of the soluble bovine enzyme

1. An enzyme acting on aminoacyl-β-naphthylamides has been isolated from the soluble fraction of bovine brain and purified 205-fold by means of ammonium sulphate fractionation, hydroxyapatite adsorption and DEAE-Sephadex column chromatography. 2. Arylamidase requires thiol groups for retention of its activity, is heat-labile and is susceptible to freezing. p-Chloromercuribenzoate and N-ethylmaleimide inactivate the enzyme rapidly. 3. Metal ions are not required for its activity, but stimulation by Mn²⁺ and Mg²⁺ and inactivation by Co²⁺ and Zn²⁺ are observed. 4. Optimum pH7·5 in phosphate buffer was exhibited for all substrates tested except l-leucyl-β-naphthylamide, for which optimum pH is 6·5. 5. K_m values for a number of substrates have been obtained and substrate inhibition at high concentrations was demonstrated. 6. The molecular weight is approx. 70000 as determined by Sephadex-gel filtration.     

Direct fluorimetric measurement on the hydrolysis of β-naphthyl triphosphate, a fluorescent ATP analog, by heavy meromyosin

A fluorescent ATP analog, β-naphthyl triphosphate, was hydrolyzed to β-naphthyl diphosphate and orthophosphate by heavy meromyosin ATPase. In the process of hydrolysis the fluorescence intensity of β-naphthyl triphosphate changed remarkably. Thus, the rate of β-naphthyl triphosphate hydrolysis is evaluated directly and continuously by measuring the time course of fluorescence intensity.In the presence of Ca²⁺, the Michaelis constant (K_m) of β-naphthyl triphosphate hydrolysis by heavy meromyosin was similar to that of ATP hydrolysis. While, in the presence of Mg²⁺ the K_m of β-napthyl triphosphate hydrolysis was 9.0·10⁻⁶ M, much larger than the value of ATP hydrolysis, indicating that the apparent affinity of the enzyme for β-naphthyl triphosphate is less than that for ATP.The pH dependence of β-naphthyl triphosphatase activity resembled that of ATPase activity, suggesting a similarity in the mechanism of hydrolysis of the two substrates.     

Effects of low temperature and respiratory inhibitors on calcium flux in plant mitochondria

The effects of low temperature on uptake and release of ⁴⁵Ca²⁺ were studied with sound, well-coupled mitochondria extracted at room temperature from avocado (Persea americana Mill, cv Fuerte) fruits. Low Ca²⁺ concentrations (10 micromolar) were employed to simulate physiological conditions. At 25°C, the rate of Ca²⁺ uptake decreased with time, whereas at 5°C the initial rate, though lower, remained linear. As a consequence total uptake at 5°C was substantially greater than at 25°C for periods greater than 5 min. Preincubation of mitochondria at 5°C enhanced subsequent Ca²⁺ uptake at 25°C. Ca²⁺ uptake was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and by ruthenium red, but neither KCN nor salicylhydroxamic acid separately or together had any major inhibitory effect. Preloaded mitochondria held for 60 min in a Ca-free medium lost little Ca²⁺ at 25°C and none at 5°C, except in the presence of ruthenium red or CCCP.     

L-Type Ca2+ Channel Sparklets Revealed by TIRF Microscopy in Mouse Urinary Bladder Smooth Muscle

Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca²⁺, referred to as Ca²⁺ sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca²⁺ imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca²⁺ (0.035±0.01 vs. 0.23±0.07 Hz/mm²; P<0.05). Co-loading of smooth muscle strips with the slow Ca²⁺ chelator EGTA-AM (10 mM) confirmed that Ca²⁺ sparklets are restricted to the cell membrane. Ca²⁺ sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 μM) (0.034±0.02 vs. 0.21±0.08 Hz/mm²; P<0.05), and diltiazem (10 μM) (0.097±0.04 vs. 0.16±0.06 Hz/mm²; P<0.05). Ca²⁺ sparklets were unaffected by inhibition of P2X₁ receptors α,β-meATP (10 μM) whilst sparklet frequencies were significantly reduced by atropine (1 μM). Ca²⁺ sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) (0.030±0.01 vs. 0.30±0.1 Hz/mm²; P<0.05), demonstrating that Ca²⁺ sparklets are PKC dependant. In the presence of CPA (10 μM), there was no apparent change in the overall frequency of Ca²⁺ sparklets, although the sparklet frequencies of each UBSM became statistically independent of each other (Spearman''s rank correlation 0.2, P>0.05), implying that Ca²⁺ store mediated signals regulate Ca²⁺ sparklets. Under control conditions, inhibition of store operated Ca²⁺ entry using ML-9 (100 μM) had no significant effect. Amplitudes of Ca²⁺ sparklets were unaffected by any agonists or antagonists, suggesting that these signals are quantal events arising from activation of a single channel, or complex of channels. The effects of CPA and ML-9 suggest that Ca²⁺ sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca²⁺ concentrations.     

The Effect of SERCA1b Silencing on the Differentiation and Calcium Homeostasis of C2C12 Skeletal Muscle Cells

The sarcoplasmic/endoplasmic reticulum Ca²⁺ATPases (SERCAs) are the main Ca²⁺ pumps which decrease the intracellular Ca²⁺ level by reaccumulating Ca²⁺ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca²⁺ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca²⁺-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca²⁺]i measured at the 20^th and 40^th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca²⁺-uptake capability which was quantified by extracting the maximal pump rate (454±41 μM/s vs. 144±24 μM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca²⁺]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.     

Nucleotide Activation of the Ca-ATPase

We have used fluorescence spectroscopy, molecular modeling, and limited proteolysis to examine structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA). The Ca-ATPase in sarcoplasmic reticulum vesicles from fast twitch muscle (SERCA1a isoform) was selectively labeled with fluorescein isothiocyanate (FITC), a probe that specifically reacts with Lys-515 in the nucleotide-binding site. Conformation-specific proteolysis demonstrated that FITC labeling does not induce closure of the cytoplasmic headpiece, thereby assigning FITC-SERCA as a nucleotide-free enzyme. We used enzyme reverse mode to synthesize FITC monophosphate (FMP) on SERCA, producing a phosphorylated pseudosubstrate tethered to the nucleotide-binding site of a Ca²⁺-free enzyme (E2 state to prevent FMP hydrolysis). Conformation-specific proteolysis demonstrated that FMP formation induces SERCA headpiece closure similar to ATP binding, presumably due to the high energy phosphoryl group on the fluorescent probe (ATP·E2 analog). Subnanosecond-resolved detection of fluorescence lifetime, anisotropy, and quenching was used to characterize FMP-SERCA (ATP·E2 state) versus FITC-SERCA in Ca²⁺-free, Ca²⁺-bound, and actively cycling phosphoenzyme states (E2, E1, and EP). Time-resolved spectroscopy revealed that FMP-SERCA exhibits increased probe dynamics but decreased probe accessibility compared with FITC-SERCA, indicating that ATP exhibits enhanced dynamics within a closed cytoplasmic headpiece. Molecular modeling was used to calculate the solvent-accessible surface area of FITC and FMP bound to SERCA crystal structures, revealing a positive correlation of solvent-accessible surface area with quenching but not anisotropy. Thus, headpiece closure is coupled to substrate binding but not active site dynamics. We propose that dynamics in the nucleotide-binding site of SERCA is important for Ca²⁺ binding (distal allostery) and phosphoenzyme formation (direct activation).     

Purification and properties of diaminopimelate decarboxylase from Escherichia coli

1. Diaminopimelate decarboxylase from a soluble extract of Escherichia coli A.T.C.C. 9637 was purified 200-fold by precipitation of nucleic acids, fractionation with acetone and then with ammonium sulphate, adsorption on calcium phosphate gel and chromatography on DEAE-cellulose or DEAE-Sephadex. 2. The purified enzyme showed only one component in the ultracentrifuge, with a sedimentation coefficient of 5·4s. One major peak and three much smaller peaks were observed on electrophoresis of the enzyme at pH8·9. 3. The mol.wt. of the enzyme was approx. 200000. The catalytic constant was 2000mol. of meso-diaminopimelic acid decomposed/min./mol. of enzyme, at 37°. The relative rates of decarboxylation at 25°, 37° and 45° were 0·17:1·0:1·6. At 37° the Michaelis constant was 1·7mm and the optimum pH was 6·7–6·8. 4. There was an excess of acidic amino acids over basic amino acids in the enzyme, which was bound only on basic cellulose derivatives at pH6·8. 5. The enzyme had an absolute requirement for pyridoxal phosphate as a cofactor; no other derivative of pyridoxine had activity. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was also needed as an activator. 6. In the presence of 2,3-dimercaptopropan-1-ol (1mm), heavy-metal ions (Cu²⁺, Hg²⁺) did not inhibit the enzyme, but there was inhibition by several amino acids with analogous structures to diaminopimelate, generally at high concentrations relative to the substrate. Penicillamine was inhibitory at relatively low concentrations; its action was prevented by pyridoxal phosphate.     

The Small G Protein Rac1 Activates Phospholipase Cδ1 through Phospholipase Cβ2

Rac1, which is associated with cytoskeletal pathways, can activate phospholipase Cβ2 (PLCβ2) to increase intracellular Ca²⁺ levels. This increased Ca²⁺ can in turn activate the very robust PLCδ1 to synergize Ca²⁺ signals. We have previously found that PLCβ2 will bind to and inhibit PLCδ1 in solution by an unknown mechanism and that PLCβ2·PLCδ1 complexes can be disrupted by Gβγ subunits. However, because the major populations of PLCβ2 and PLCδ1 are cytosolic, their regulation by Gβγ subunits is not clear. Here, we have found that the pleckstrin homology (PH) domains of PLCβ2 and PLCβ3 are the regions that result in PLCδ1 binding and inhibition. In cells, PLCβ2·PLCδ1 form complexes as seen by Förster resonance energy transfer and co-immunoprecipitation, and microinjection of PHβ2 dissociates the complex. Using PHβ2 as a tool to assess the contribution of PLCβ inhibition of PLCδ1 to Ca²⁺ release, we found that, although PHβ2 only results in a 25% inhibition of PLCδ1 in solution, in cells the presence of PHβ2 appears to eliminates Ca²⁺ release suggesting a large threshold effect. We found that the small plasma membrane population of PLCβ2·PLCδ1 is disrupted by activation of heterotrimeric G proteins, and that the major cytosolic population of the complexes are disrupted by Rac1 activation. Thus, the activity of PLCδ1 is controlled by the amount of bound PLCβ2 that changes with displacement of the enzyme by heterotrimeric or small G proteins. Through PLCβ2, PLCδ1 activation is linked to surface receptors as well as signals that mediate cytoskeletal pathways.     

Excitation-Contraction Coupling in Zebrafish Ventricular Myocardium Is Regulated by Trans-Sarcolemmal Ca2+ Influx and Sarcoplasmic Reticulum Ca2+ Release

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca²⁺-flux and sarcoplasmic reticulum Ca²⁺-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca²⁺-concentration, trans-sarcolemmal Ca²⁺-flux via L-type Ca²⁺-channels and Na⁺-Ca²⁺-exchanger, and Ca²⁺-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca²⁺-channels by verapamil (1 μM) decreased force of contraction to 22±7% compared to baseline (n=4, p<0.05). Ni²⁺ was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73±3 ms vs. 101±8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca²⁺-release decreased force development to 54±3% in ventricular (n=13, p<0.05) and to 52±8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca²⁺-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169±7% force compared to baseline, n=8, p<0.05) and 10 s (198±9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca²⁺-flux, but also intact sarcoplasmic reticulum Ca²⁺-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.     

Unitary Ca2+ Current through Cardiac Ryanodine Receptor Channels under Quasi-Physiological Ionic Conditions

Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1–5 kHz and filtered at 0.2–1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca²⁺ currents were monitored when 2–30 mM Ca²⁺ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca²⁺ current (at 0 mV holding potential) and lumenal [Ca²⁺] was hyperbolic and saturated at ∼4 pA. This relationship was then defined in the presence of different symmetrical CsCH₃SO₃ concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca²⁺; and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca²⁺ (n > 6). Unitary Ca²⁺ current was also defined in the presence of symmetrical [Mg²⁺] (1 mM) and low [Cs⁺] (5 mM). Under these conditions, unitary Ca²⁺ current in 2 and 10 mM lumenal Ca²⁺ was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs⁺] (50 mM), Mg²⁺ (1 mM), and lumenal [Ca²⁺] (10 mM), unitary Ca²⁺ current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs⁺ and Mg²⁺ on unitary Ca²⁺ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg²⁺ effectively compete with Ca²⁺ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca²⁺ is 2 mM, then our results indicate that unitary Ca²⁺ current under physiological conditions should be <0.6 pA.