Ca2+ and endoplasmic reticulum Ca2+-ATPase regulate the formation of silk fibers with favorable mechanical properties

Calcium ions (Ca²⁺) are crucial for the conformational transition of silk fibroin in vitro, and silk fibroin conformations correlate with the mechanical properties of silk fibers. To investigate the relationship between Ca²⁺ and mechanical properties of silk fibers, CaCl₂ was injected into silkworms (Bombyx mori). Fourier-transform infrared spectroscopy (FTIR) analysis and mechanical testing revealed that injection of CaCl₂ solution (7.5 mg/g body weight) significantly increased the levels of α-helix and random coil structures of silk proteins. In addition, extension of silk fibers increased after CaCl₂ injection. In mammals, sarcoplasmic reticulum Ca²⁺-ATPase in muscle and endoplasmic reticulum Ca²⁺-ATPase in other tissues (together denoted by SERCA) are responsible for calcium balance. Therefore, we analyzed the expression pattern of silkworm SERCA (BmSERCA) in silk glands and found that BmSERCA was abundant in the anterior silk gland (ASG). After injection of thapsigargin (TG) to block SERCA activity, silkworms showed a silk-spinning deficiency and their cocoons had higher calcium content compared to that of controls. Moreover, FTIR analysis revealed that the levels of α-helix and β-sheet structures increased in silk fibers from TG-injected silkworms compared to controls. The results provide evidence that BmSERCA has a key function in calcium transportation in ASG that is related to maintaining a suitable ionic environment. This ionic environment with a proper Ca²⁺ concentration is crucial for the formation of silk fibers with favorable mechanical performances.     

Effect of Ca2+ ions on hydrodynamic properties of pentamer and decamer of C-reactive protein in solution

The molecular mass and sedimentation coefficient of native C-reactive protein in solution were determined by analytical ultracentrifugation in the presence and absence of calcium ions. Pentameric C-reactive protein was shown to be the major macroscopic form of this protein in solution. The removal of calcium ions from solution caused decompaction of the protein accompanied by changes in its hydrodynamic parameters. The sedimentation coefficient s20(0), w of pentameric C-reactive protein in solution containing 2 mM--Ca2+ (6.6S) exceeded that for C-reactive protein in solution containing 2 mM EDTA (6.4S). Analysis of average molecular masses Mw and Mz obtained from sedimentation data demonstrated that the solution of highly purified protein was not homogeneous. As shown by intermolecular crosslinking, the solution also contained the 241-kDa decamer of C-reactive protein (9.5S) as a separate macroscopic form, whose share hardly reached 10% in the presence of 2 mM Ca2+ and increased after removal of calcium ions. The decamers were shown to result from intermolecular association of the pentamers.     

Formation of polysaccharide gel layers in the presence of Ca2+ and K+ ions: measurements and mechanisms

Understanding the adsorption properties of polysaccharides in terms of substrate affinity, kinetics, and layer structure is of paramount importance in numerous industrial and natural systems. The structural growth of the layers of two model polysaccharides--sodium alginate and polygalacturonic acid (PGA)--was characterized by quartz crystal microbalance with dissipation and atomic force microscopy. Monitoring the variations in frequency and dissipation energy provides insights into both the average adsorbed mass and the viscoelastic properties of the adsorbed layer of polyelectrolytes along with the associated ions and water molecules. Both polysaccharides had similar adsorption patterns with increasing ionic strengths and showed significant complexation of calcium ions. In the presence of calcium, the alginate gel layer exhibited significant swelling with an increasing concentration of monovalent salt that the PGA gel layer did not manifest. Basing our discussion on the "egg-box model", we interpreted these different swelling behaviors as resulting from differences in the complexation modes of the two polysaccharides. The dimerization of the polymers by cross-linking and the weaker dimer-dimer associations play major roles in the sensitivity of the polysaccharide gel matrix to high salt concentration environments.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+-transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca²⁺ + Mg²⁺)-ATPase activity and in Ca²⁺ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K⁺ and Na⁺ were placed. Under the conditions used, (Ca²⁺ + Mg²⁺)-ATPase activity and Ca²⁺ efflux was highest when K⁺ ($\text{35 ± 0.5}\text{mM (± S.E.)}$, mean of four experiments) was at the inside and Na⁺ (130 mM) at the outside of the ghost membrane.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+ -transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca2+ + Mg2+)-ATPase activity and in Ca2+ efflux at 37 degrees C, depending on the side of the membrane at which the monovalent cations K+ and Na+ were placed. Under the conditions used, (Ca2+ + Mg2+)-ATPase activity and Ca2+ efflux was highest when K+ (35 +/- 0.5 mM (+/- S.E.), mean of four experiments) was at the inside and Na+ (130 mM) at the outside of the ghost membrane.     

Conformational transitions in the Ca2+ + Mg2+-activated ATPase and the binding of Ca2+ ions.

We have studied the fluorescence of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate. The change in intensity of fluorescein fluorescence caused by addition of Ca2+ to the labelled ATPase can be interpreted in terms of a two-conformation model for the ATPase, one conformation (E1) having a high affinity for Ca2+, the other (E2) a low affinity. Effects of Ca2+ as a function of pH allow an estimate of the effect of pH on the E1/E2 ratio, consistent with kinetic studies. A model is presented for binding of Ca2+ to the ATPase as a function of pH that is consistent both with the data on the E1/E2 equilibrium and with literature data on Ca2+ binding.     

Effect of Ca2+ ions on plasmid transformation of Streptococcus lactis protoplasts

The effects of Mg2+ and Ca2+ ions on the efficiency of the plasmid transformation of lysozyme-treated Streptococcus lactis protoplasts were compared. A 33-megadalton plasmid, pLP712, coding for lactose fermentation and a 6.5-megadalton plasmid, pGB301, coding for erythromycin and chloramphenicol resistance were used as model plasmids, and S. lactis MG1614 was the recipient. Replacing Mg2+ with Ca2+ in the transformation buffer was found to increase transformant frequency more than 10-fold with both plasmids.     

Ba2+ ions inhibit the release of Ca2+ ions from rat liver mitochondria

The release of Ca2+ from respiring rat liver mitochondria following the addition of either ruthenium red or an uncoupler was measured by a Ca2+-selective electrode or by 45Ca2+ technique. Ba2+ ions are asymmetric inhibitors of both Ca2+ release processes. Ba2+ ions in a concentration of 75 microM inhibited the ruthenium red and the uncoupler induced Ca2+ release by 80% and 50%, respectively. For the inhibition, it was necessary that Ba2+ ions entered the matrix space: Ba2+ ions did not cause any inhibition of Ca2+ release if addition of either ruthenium red or the uncoupler preceded that of Ba2+. The time required for the development of the inhibition of the Ca2+ release and the time course of 140Ba2+ uptake ran in parallel. Ba2+ accumulation is mediated through the Ca2+ uniporter as 140Ba2+ uptake was competitively inhibited by extramitochondrial Ca2+ and prevented by ruthenium red. Due to the inhibition of the ruthenium red insensitive Ca2+ release, Ba2+ shifted the steady-state extramitochondrial Ca2+ concentration to a lower value. Ba2+ is potentially a useful tool to study mitochondrial Ca2+ transport.     

Silver ions induce Ca2+ release from the SR in vitro by acting on the Ca2+ release channel and the Ca2+ pump.

Silver nitrate (AgNO3) is a sulfhydryl oxidizing agent that induces a biphasic Ca2+ release from isolated sarcoplasmic reticulum (SR) vesicles by presumably oxidizing critical sulfhydryl groups in the Ca2+ release channel (CRC), causing the channel to open. To further examine the effects of AgNO3 on the CRC and the Ca2+-ATPase, Ca2+ release was measured in muscle homogenates prepared from rat hindlimb muscle using indo 1. Cyclopiazonic acid (CPA) and ruthenium red (RR) were used to inhibit the Ca2+-ATPase and block the CRC, respectively, before inducing Ca2+ release with both AgNO3 and 4-chloro-m-cresol (4-CMC), a releasing agent specific for the CRC. With AgNO3 and CPA, the early rapid rate of release (phase 1) was increased (P < 0.05) by 42% (314 +/- 5 vs. 446 +/- 39 micromol x g protein(-1) x min(-1)), whereas the slower, more prolonged rate of release (phase 2) was decreased (P < 0.05) by 72% (267 +/- 39 vs. 74 +/- 7.7 micromol x g protein(-1) x min(-1)). RR, in combination with AgNO3, had no effect on phase 1 (P > 0.05) (314 +/- 51 vs. 334 +/- 43 micromol x g protein(-1) x min(-1)) and decreased phase 2 (P < 0.05) by 65% (245 +/- 34 vs. 105 +/- 8.2 micromol x g protein(-1) x min(-1)). With 4-CMC, CPA had no effect (P > 0.05) on either phase 1 or 2. With addition of RR, phase 1 was reduced (P < 0.05) by 59% (2,468 +/- 279 vs. 1,004 +/- 87 micromol x g protein(-1) x min(-1)), and RR completely blocked phase 2. Both AgNO3 and 4-CMC fully inhibited Ca2+-ATPase activity measured in homogenates. These findings indicate that AgNO3, but not 4-CMC, induces Ca2+ release by acting on both the CRC and the Ca2+-ATPase.     

Release of Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle induced by heparin. Relation between the Ca2+ release caused by Ca2+ ions and caffeine

Using a Ca2+-selective electrode and Quin 2 and chlortetracycline fluorescence, a Ca2+ release from terminal cysterns of skeletal muscle sarcoplasmic reticulum under effects of heparin, caffeine and Ca2+ has been studied. It was shown that Ca2+ release induced by heparin is insensitive to the blockers of Mg2+-dependent system of Ca2+-induced Ca2+ release, i.e., Mg2+, tetracaine and dimethylsulfoxide. Preliminary release of Ca2+ in the presence of caffeine, which activates Mg2+-dependent Ca2+ release, does not prevent the heparin-induced Ca2+ release. At the same time, after Ca2+ release caused by Ca2+ in a Mg2+-independent system, heparin cannot cause additional efflux of Ca2+. It has been shown that the heparin-induced release of Ca2+ diminishes with a decrease in a decrease in Ca2+ concentration. This effect is less pronounced in the presence of Na+ than with K+. The data obtained suggest that sarcoplasmic reticulum terminal cysterns contain two systems of Ca2+-induced release of Ca2+, i.e., a Mg2+-dependent, caffeine-sensitive and a Mg2+-independent heparin-sensitive ones. The mechanism of activation of both systems by caffeine and heparin consists, in all probability, in their increased affinity for Ca2+.     

Effect of Ca2+ ions on plasmid transformation of Streptococcus lactis protoplasts.

The effects of Mg2+ and Ca2+ ions on the efficiency of the plasmid transformation of lysozyme-treated Streptococcus lactis protoplasts were compared. A 33-megadalton plasmid, pLP712, coding for lactose fermentation and a 6.5-megadalton plasmid, pGB301, coding for erythromycin and chloramphenicol resistance were used as model plasmids, and S. lactis MG1614 was the recipient. Replacing Mg2+ with Ca2+ in the transformation buffer was found to increase transformant frequency more than 10-fold with both plasmids.     

Ca2+-dependent neutral proteinase from human erythrocytes: activation by Ca2+ ions and substrate and regulation by the endogenous inhibitor

Ca2+-dependent neutral proteinase purifies from human erythrocytes as an inactive proenzyme, that can be converted in an active low Ca2+ requiring form either by high concentrations of Ca2+ (0.1-1 mM) in the absence of the substrate, or by low concentrations of Ca2+ (1-5 microM) in the presence of digestible substrates. Activation requires dissociation to constituent inactive proenzyme subunits which are then converted to the active proteinase species still retaining their monomeric structure. The activation process produced by high Ca2+ concentrations is controlled by the endogenous inhibitor which also dissociates into constituent subunits in order to exert its inhibitory effect. An additional regulation of the activated proteinase involves an autoproteolytic process, Ca2+ and substrate dependent, producing enzyme inactivation.     

The Staphylococcus aureus alpha-toxin channel complex and the effect of Ca2+ ions on its interaction with lipid layers.

Using the techniques of two-dimensional crystallization on supported lipid bilayers together with computer image processing, two distinct two-dimensional crystal types of staphylococcal alpha-toxin complex are formed depending on the presence or absence of Ca2+ ions. Without Ca2+, these are hexagonally packed (in A, a = b = 89.5 +/- 2.5 A; theta = 119.7 degrees) With Ca2+ present, rectangular crystal packing is seen (in A, a = 114.8 +/- 1.6 A, b = 140.2 +/- 0.7 A; theta = 89.1 degrees). A third, banded crystal type is also seen which is interpreted as a side-to-side packing of regular tubules. We use these tubular crystals for cross-correlation searches with top and side-on views of the complex from single particle reconstructions, and with the repeating units from the two-dimensional crystal types. The results lead us to propose a model in which the different two-dimensional crystal types are formed as a result of alpha-toxin hexamers packing in different orientations. In the hexagonal crystals the hexamers lie end-on with a 6-fold axis in projection. On the addition of Ca2+, the hexamers reorient to lie tilted with respect to the support, thus giving rise to a rectangular projection.     

The reversible activation by Mn2+ ions of the Ca2+-requiring neutral proteinase of human erythrocytes

Mn2+ (50 microM) satisfies the requirement for activity of the purified Ca2+-dependent neutral proteinase from human erythrocytes. Unlike the activation by Ca2+ [E. Melloni et al. (1984) Biochem. Int. 8, 477-489], the effect of Mn2+ is fully reversible and does not involve autodigestion of the native 80-kDa catalytic subunit. However, the native dimeric proenzyme (procalpain), which contains both the 80-kDa subunit and a smaller 30-kDa subunit, is not activated by Mn2+ alone but also requires the presence of micromolar concentrations of Ca2+. Under these conditions, 40% of the maximum activity is expressed without dissociation of the 80- and 30-kDa subunits. Mn2+, but not micromolar Ca2+, can also partially satisfy the metal requirement of the native 80-kDa subunit isolated after dissociation of the heterodimer. This activity is further enhanced by the addition of 5 microM Ca2+, which is ineffective in the absence of Mn2+. After procalpain is converted to active calpain by incubation with Ca2+ and substrate [S. Pontremoli et al. (1984) Biochem. Biophys. Res. Commun. 123, 331-337] full activity is observed with 5 microM Mn2+, which now substitutes completely for Ca2+. Activation of procalpain by Mn2+ represents a new mechanism for modulation of the Ca2+-dependent proteinase activity.     

Solubilization and properties of Ca2+-dependent human platelet phospholipase A2

Using a sonicated dispersion of radiolabeled 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate, we found that phospholipase A2 activity of human platelets was enhanced 2.4-fold by albumin (1 mg/ml). The enzyme was recovered predominantly in the cytosolic fraction of platelets with less than a third of its activity being associated with the membrane fraction. In the presence of 24 mM n-octyl-beta-D-glucopyranoside (octylglucoside) phospholipase A2 was effectively (more than 90%) extracted from platelet lysates without solubilization of platelet membranes. Ion exchange chromatography of the soluble enzyme yielded a phospholipase A2 of unchanged total activity and great stability. This phospholipase A2 was active only in the presence of divalent cations (Ca2+ greater than Sr2+ greater than Mg2+ = 0), required albumin for optimal activity and exhibited exclusive positional specificity for the acyl ester bond at the 2-position of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. Indomethacin (500 microM), mepacrine (500 microM) and N-ethylmaleimide (4 mM) inhibited the phospholipase A2 by 69, 62 and 19%, respectively. The results are discussed in the light of previous findings on human platelet phospholipase A2.     

Effect of Ca2+ on thermotropic properties of saturated phosphatidylcholine liposomes

The effect of various concentrations of calcium ion (Ca²⁺) on dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and mixed DPPC/DSPC (1:1) liposomes was studied by differential scanning calorimetry. Ca²⁺ concentrations of 10 mM and above split the main transition peak of DPPC into two distinguishable components, and, at 30 mM and above, also resulted in the disappearance of a pre-transition peak. The effect of Ca²⁺ on DSPC liposomes was even more dramatic in that it induced a more definitive split in the main transition peak and caused the loss of the pretransition peak at only 10 mM concentration. The thermograms of the DPPC/DSPC mixed liposomes were unaltered in the presence of Ca²⁺, even at a concentration of 50 mM. Whether or not Ca²⁺ is able to alter thermograms of phosphatidylcholine liposomes appears to be dependent on the degree of molecular order of the bilayer prior to interaction with Ca²⁺.     

Effect of ethylmaleimide on the transport of Ca+ and K+ ions across mitochondrial membranes

As already reported, it has been found that the gradient of protons, set up across the inner membrane during the Ca2+ uptake by rat liver mitochondria, can be completely reversed by the addition of NEM. Identical results have been obtained by following the energy dependent K+ uptake. In these last conditions, the rate of H+ efflux supported by succinate oxidation is greatly enhanced only when NEM is added after rotenone. It is proposed that the increased rate other than to the inhibition of Pi uptake, as suggested by Reynafarje and Lehninger, could also be ascribed to a further decrease in the energetic level of the membrane as well as to an increased rate of succinate-Pi exchange diffusion reaction induced by NEM. A possible direct effect of NEM on succinate oxidation has been also considered to account for the inhibition observed when it is added before rotenone.     

Mechanisms of the effects of Ca2+ ions on lipid peroxidation

Mechanisms underlying Ca2+ effects on lipid peroxidation (LPO) induced in liposomes (from egg yolk lecithin) and UFsomes (from linolenic acid, methyl linolenate) with the aid of O2- -system (Fe2+ + ascorbate) were studied. It was shown that stimulation of lipid peroxidation by low Ca2+ concentrations (10(-6)-10(-5) M) was due to its ability to release Fe2+-ions bound to negatively charged (phosphate, carboxylic) lipid groups (of licethin, linolenic acid), thus increasing the concentration of catalytically active Fe2+. The inhibitory effect of high Ca2+ concentrations was caused by its interaction with superoxide anion-radicals and was not observed in LPO-systems, independent of O2- generation (e. g. Fe2+ + cumol hydroperoxide).     

Sorption of alkaline earth metal ions Ca2+ and Mg2+ on lyocell fibres

Ca²⁺ and Mg²⁺ content of cellulose fibres is of relevance for a wide range of applications e.g. textile processing, pulp/paper, food. Sorption of Ca²⁺ and Mg²⁺ ions were found on lyocell type regenerated cellulose fibres. Higher affinity was found for Ca²⁺ ions compared to Mg²⁺ ions. At pH 9, fibre saturation was observed at a calcium binding capacity of 18–20 mmol/kg. A carboxylic group content of 18 mmol COOH per kg fibre material was determined based on the Methylene Blue absorption. This indicates a 1:1 molar stoichiometry between the carboxylic groups present in the fibres and the bound Ca²⁺ ions. Thus it is proposed that the salt in fibre shows the general composition (Cell-O^? Ca²⁺ X^?), X^? being an anion bound in the salt to achieve charge neutrality.The sorption of Ca²⁺ also can be demonstrated by complex formation with 1,2-dihydroxy-9,10-anthraquinone (alizarin) which forms a red-violet Ca²⁺-complex. Colour fixation thus can be used as an indicator for the Ca²⁺-ions bound in the fibre.