Calcium-induced release of cytochrome c from cardiolipin nanodisks: Implications for apoptosis

Miniature bilayer membranes comprised of phospholipid and an apolipoprotein scaffold, termed nanodisks (ND), have been used in binding studies. When ND formulated with cardiolipin (CL), but not phosphatidylcholine, were incubated with cytochrome c, FPLC gel filtration chromatography provided evidence of a stable binding interaction. Incubation of CL ND with CaCl₂ resulted in a concentration-dependent increase in sample turbidity caused by ND particle disruption. Prior incubation of CL ND with cytochrome c increased CL ND sensitivity to CaCl₂-induced effects. Centrifugation of CaCl₂-treated CL ND samples yielded pellet and supernatant fractions. Whereas the ND scaffold protein, apolipophorin III, was recovered in the pellet fraction along with CL, the majority of the cytochrome c pool was in the supernatant fraction. Moreover, when cytochrome c CL ND were incubated with CaCl₂ at concentrations below the threshold to induce ND particle disruption, FPLC analysis showed that cytochrome c was released. Pre-incubation of CL ND with CaCl₂ under conditions that do not disrupt ND particle integrity prevented cytochrome c binding to CL ND. Thus, competition between Ca²⁺ and cytochrome c for a common binding site on CL modulates cytochrome c binding and likely plays a role in its dissociation from CL-rich cristae membranes in response to apoptotic stimuli.     

Evidence that a light-accelerated abrupt change in membrane characteristics of bovine rod outer segment fragments takes place at acidic pH

Changes in the turbidity of suspensions of bovine rod outer segment fragments induced by rhodopsin bleaching were measured in the presence of various concentrations of divalent cations at acidic pH (4.7–5.4). Unlike the situation at neutral pH, the turbidity of the suspensions increased drastically by bleaching at acidic pH. It was found that the extent of turbidity change became maximum at a particular concentration of divalent cations (i.e., 5 mM CaCl₂, 5 mM MgCl₂, or 5 mM mixed divalent cations). However, the turbidity increment in the presence of 5 mM MgCl₂ was greatly enhanced by the addition of a minute amount of CaCl₂. These results evidently show that the membrane characteristic is abruptly changed by bleaching at acidic pH in particular. It is also suggested that there are two kinds of binding sites for Ca ions: one is a Ca²⁺ specific site, and the other is a nonspecific site to which Mg²⁺ can also bind.     

Isolation of calcium-transporting lipid from the mitochondrial glycolipoprotein

Summary From the mitochondrial Ca²⁺-transporting glycolipoprotein (GLP) the lipid was isolated which induced Ca²⁺-translocation through bilayer lipid membranes. Electroconductivity of modified phospholipid membranes in the presence of CaCl₂ is increased 150-200 times. At 10-fold CaCl₂ gradient a generation of membrane potential is observed close to its theoretical value. It is shown that the lipid forms separate conductivity channels of 10 and 20 pS in the bilayer. The mode of action of GLP in the membrane is proposed It is assumed that the carbohydrate part of GLP is a selective receptor-accumulator for Ca²⁻, whereas the function of the lipid component consists in forming channels in the bilayer.     

Divalent cations stabilize GroEL under conditions of oxidative stress

The divalent cations Mg²⁺, Mn²⁺, Zn²⁺, Ca²⁺, and Ni²⁺ were found to protect against proteolysis a form of GroEL (ox-GroEL) prepared by exposing GroEL for 16 h to 6 mM hydrogen peroxide (H₂O₂). K⁺ and other monovalent cations did not have any effect. Divalent cations also induced a conformational change of ox-GroEL that led to the decrease of its large exposed hydrophobic surfaces (exposed with H₂O₂). Ox-GroEL incubated with a divalent cation behaved like N-GroEL in that it could transiently interact with H₂O₂-inactivated rhodanese (ox-rhodanese), whereas ox-GroEL alone could strongly interact with ox-rhodanese. Although, ox-GroEL incubated with a divalent cation could not recover the ATPase activity (66%) lost with H₂O₂, it could facilitate the reactivation of ox-rhodanese (>86% of active rhodanese recovered), without requiring ATP or the co-chaperonin, GroES. This is the first report to demonstrate a role for the divalent cations on the structure and function of ox-GroEL.     

The interaction of lanthanide and calcium salts with phospholipid bilayer vesicles: The validity of the nuclear magnetic resonance method for determination of vesicle bilayer phospholipid surface ratios

Contrary to a recent report (B. Sears et al., Biochemistry 15 (1976) 1635), it has been determined that the ratio of the number of phospholipids on the inner and outer surfaces of phospholipid bilayer vesicles can be accurately determined by NMR paramagnetic ion shift reagent studies of vesicles. It is concluded that the metal interacts with all of the phospholipid on the exposed bilayer surface. A ratio of outer phospholipid to inner surface phospholipid of 2.1 ± 0.1 is obtained regardless of the nucleus studies, position of the nucleus relative to the metal ion binding site, molar ratio of metal to phospholipid over three orders of magnitude, or location of the metal ion on the inside or outside of the vesicle. Additionally, P-31 NMR studies using LaCl₃ and CaCl₂ indicate that Ca²⁺ weakly interacts with egg PC vesicles and than the lanthanides are adequate substitutes for Ca²⁺ since neither metal is found to perturb measurably the average polar head group conformation.     

Differences in the interaction of inorganic and organic (hydrophobic) cations with phosphatidylserine membranes

The interaction of phosphatidylserine dispersions with “hydrophobic”, organic cations (acetylcholine, tetraethylammonium ion) is compared with that of simple inorganic cations (Na⁺, Ca²⁺); differences in the hydration properties of the two classes of ions exist in the bulk phase as evident from spin-lattice relaxation time T₁, measurements. It is shown that the reaction products (cation-phospholipid) differ markedly in their physicochemical behaviour. With increasing concentration both classes of ions reduce the ζ-potential of phosphatidylserine surfaces, the monovalent inorganic cations being only slightly more effective than the hydrophobic cations. Inorganic cations cause precipitation of the lipid once the surface charge of the bilayer is reduced to a certain threshold value. This is not the case with the organic cations. The difference is probably associated with the different hydration properties of the resulting complexes. Thus binding of Ca²⁺ causes displacement of water of hydration and formation of an anhydrous, hydrophobic calcium-phosphatidylserine complex which is insoluble in water, whereas the product of binding of the organic cations is hydrated, hydrophilic and water soluble. The above findings are consistent with NMR results which show that the phosphodiester group is involved in the binding of both classes of cations as well as being the site of the primary hydration shell. Besides affecting interbilayer membrane interactions such as those involved in cell adhesion and membrane fusion, the binding of both classes of cation can affect the molecular packing within a bilayer.     

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA₃) with or without 5 millimolar CaCl₂ shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca²⁺. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca²⁺. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA₃ with 5 millimolar CaCl₂ and layers incubated in GA₃ alone. RNA isolated from layers incubated for 12 hours in GA₃ with and without Ca²⁺ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca²⁺ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca²⁺-deprived aleurone layers was translatable. Ca²⁺ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.     

Inhibition by ethylenediamine tetraacetate and restoration by Mn2+ and Ca2+ of oxygen-evolving activity in Photosystem II preparation from the thermophilic cyanobacterium,Synechococcus sp

The Photosystem II particles capable of evolving oxygen with ferricyanide as an electron acceptor were isolated from β-octylglucoside-solubilized thylakoid membranes of the thermophilic cyanobacterium Synechococcus sp. High and steady rates of oxygen evolution were observed only in the presence of 0.5% digitonin and 1 M sucrose. The activity was totally lost when the particles had been treated with 1 mM ethylenediamine tetraacetate for 1 min in a hypotonic medium. The treatment removed two out of four Mn atoms present per the Photosystem II reaction center in the particles. A significant rate of oxygen evolution was restored almost immediately after the addition of 5 mM MnCl₂ to the treated particles. The activity was also slowly and partially restored when the treated particles had been incubated with 5 mM CaCl₂. MgCl₂ was much less effective irrespective of the incubation time. The addition of MnCl₂ to the CaCl₂-restored particles increased the activity to a level which is significantly larger than the sum of the MnCl₂- and the CaCl₂-induced increases in the activity. The Mn- and Ca-effects were largely suppressed by the simultaneous addition of CaCl₂ and MnCl₂, respectively. It is concluded that both Mn²⁺ and Ca²⁺ are essential components of the water oxidation of photosynthesis, but the two cations support oxygen evolution through different mechanisms.     

Calcium Transport and Local Pool Regulate Polycystin-2 (TRPP2) Function in Human Syncytiotrophoblast

Polycystin-2 (PC2, TRPP2) is a Ca²⁺-permeable, nonselective cation channel implicated in Ca²⁺ transport and epithelial cell signaling. Although PC2 may contribute to Ca²⁺ transport in human term placenta, the regulatory mechanisms associated with Ca²⁺ handling in this tissue are largely unknown. In this work we assessed the regulation by Ca²⁺ of PC2 channel function from a preparation of apical membranes of human syncytiotrophoblast (PC2_hst) reconstituted in a lipid bilayer system. Addition of either EGTA or BAPTA to the cis hemi-chamber, representing the cytoplasmic domain of the channel, and lowering Ca²⁺ to ∼0.6–0.8 nM, inhibited spontaneous PC2_hst channel activity, with a time response dependent on the chelator tested. EGTA reduced PC2_hst channel currents by 86%, with a t_1/2 = 3.6 min, whereas BAPTA rapidly and completely (100%) eliminated channel activity with a t_1/2 = 0.8 min. Subsequent titration with Ca²⁺ reversed the inhibition, which followed a Hill-type function with apparent dissociation constants of 1–5 nM, and 4 Ca²⁺ binding sites. The degree of inhibition by the cis Ca²⁺ chelator largely depended on increasing trans Ca²⁺. This was consistent with measurable Ca²⁺ transport through the channel, feeding the regulatory sites in the cytoplasmic domain. Interestingly, the reconstituted in vitro translated PC2 (PC2_iv) was completely insensitive to Ca²⁺ regulation, suggesting that the regulatory sites are not intrinsic to the channel protein. Our findings demonstrate the presence of a Ca²⁺ microdomain largely accessible through the channel that controls PC2 function in human syncytiotrophoblast of term placenta.     

CaCO3 and SrCO3 bioprecipitation by fungi isolated from calcareous soil

The urease‐positive fungi Pestalotiopsis sp. and Myrothecium gramineum, isolated from calcareous soil, were examined for their properties of CaCO₃ and SrCO₃ biomineralization. After incubation in media amended with urea and CaCl₂ and/or SrCl₂, calcite (CaCO₃), strontianite (SrCO₃), vaterite in different forms [CaCO₃, (Ca_xSr_1?x)CO₃] and olekminskite [Sr(Sr,Ca)(CO₃)₂] were precipitated, and fungal ‘footprints’ were observed on mineral surfaces. The amorphous precipitate mediated by Pestalotiopsis sp. grown with urea and equivalent concentrations of CaCl₂ and SrCl₂ was identified as hydrated Ca and Sr carbonates by Fourier transform infrared spectroscopy. Liquid media experiments showed M. gramineum possessed the highest Sr²⁺ removal ability, and ～ 49% of supplied Sr²⁺ was removed from solution when grown in media amended with urea and 50 mM SrCl₂. Furthermore, this organism could also precipitate 56% of the available Ca²⁺ and 28% of the Sr²⁺ in the form of CaCO₃, SrCO₃ and (Ca_xSr_1?x)CO₃ when incubated in urea‐amended media and equivalent CaCl₂ and SrCl₂ concentrations. This is the first report of biomineralization of olekminskite and coprecipitation of Sr into vaterite mediated by fungi. These findings suggest that urease‐positive fungi could play an important role in the environmental fate, bioremediation or biorecovery of Sr or other metals and radionuclides that form insoluble carbonates.     

The calcium requirement in GnRH-stimulated LH release is not mediated through a specific action on receptor binding

A radioligand receptor assay was used to determine the effect of Ca⁺² and other cations on the binding of D-Ser-(t-Bu)⁶-desGly¹⁰EA GnRH (Buserelin acetate, Hoechst) to the gonadotropin releasing hormone receptor in rat pituitary membrane. Specific binding was reduced in the presence of CaCl₂, BaCl₂, MnCl₂, Co(CH₃COO)₂, MgCl₂, NaCl, or LaCl₃, although these salts have markedly different effects on receptor mediated gonadotropin release from intact cells. Consistent with this finding, compounds which chelate cations increased specific binding of the ligand. The decreased binding is explained solely by an effect on receptor affinity, which was reduced from 4 × 10⁹ M^?1 in the presence of 10 mM CaCl₂. Total receptor number was unchanged. These data suggest that the requirement for Ca⁺² in GnRH-stimulated LH release is not mediated through a specific action of this ion at the level of receptor binding.     

Effects of calcium on the activities of cytosolic antioxidative enzymes and IAA oxidase during in vitro adventitious rooting of mung bean seedlings

The effects of Ca²⁺ on antioxidative enzymes and indole-3-acetic acid (IAA) oxidase during adventitious rooting were investigated in mung bean (Vigna radiata). CaCl₂ significantly promoted the formation and growth of adventitious roots. EGTA (a Ca²⁺ chelator) or ruthenium red (a Ca²⁺-channel blocker) significantly inhibited root formation and growth, but these inhibitory effects could be partially reversed by CaCl₂. Furthermore, inclusion of 5 mM CaCl₂ significantly increased superoxide dismutase (SOD) activity by 10% at 3 h and catalase (CAT) activity by an average of 29.6% at each time point. CaCl₂ decreased peroxidase (POD) activity by 9.4% and 21% at 12 and 24 h, respectively, and ascorbate peroxidase (APX) activity by an average of 13.9% at each time point. These CaCl₂-induced changes in enzymatic activities were similar to changes caused by indole-3-butyric acid (IBA). Treatment with EGTA or ruthenium red decreased SOD activity by an average of 18.4% and 15.2%, respectively; POD activity by 27.4% and 57.6%, respectively; APX activity by 10.3% and 15.6%, respectively; and CAT activity by 19.3% and 5.2%, respectively, when compared with CaCl₂. In addition, CaCl₂ increased IAA oxidase activity by an average of 5.5% beginning at 6 h, whereas EGTA significantly decreased IAA oxidase activity by 29.2%, 22.9%, and 13.5% at 6, 9, and 12 h, respectively. The inhibitory effects of EGTA could be partially suppressed by addition of CaCl₂. These results imply that the stimulative effect of Ca²⁺ on adventitious rooting is partially related to Ca²⁺-induced changes in the activities of antioxidative enzymes and IAA oxidase.     

Alleviation of the adverse effects of NaCl on germination of maize grains by calcium

The lengths of roots and shoots, fresh and dry matter yield, and the contents of insoluble saccharides and free amino acids were reduced with the rise in NaCl concentration. However, under combination of NaCl with Ca²⁺ ions, these parameters generally raised. Contents of soluble saccharides, proline and quaternary ammonium compounds increased with increasing NaCl concentration, but under addition of CaCl₂ or CaSO₄, contents of these compounds were decreased. Low concentrations of NaCl stimulated soluble proteins, production, but higher concentrations decreased the content of soluble proteins. Addition of Ca²⁺ in the media did not improve the soluble protein production. Insoluble proteins content was increased with the rise of salinity level, but these effects were more pronounced with NaCl and CaCl₂ or CaSO₄ than with NaCl only.     

Effects of trypsin and bivalent cations on P-680+-reduction,fluorescence induction and oxygen evolution in Photosystem II membrane fragments from spinach

Laser-flash-induced absorption changes at 830 nm, fluorescence-induction curves and the average oxygen yield per flash have been measured in spinach Photosystem II membrane fragments as a function of trypsin treatment and its modification by CaCl₂. The following was found. (i) The relative contribution of the nanosecond relaxation to the overall decay kinetics of 830 nm absorption changes reflecting the P-680⁺-reduction decreases as a function of incubation time with trypsin. Simultaneously, mild treatment at pH = 6.0 markedly increases the extent of 200 μs kinetics that highly revert back to nanosecond kinetics by CaCl₂ addition. After harsher trypsin treatment (pH = 7.5) pH-dependent 2–20 μs kinetics appear that cannot be reverted to nanosecond kinetics by CaCl₂. (ii) The CaCl₂-induced restoration of nanosecond kinetics is mainly due to a Ca²⁺-induced effect rather than to a functional role of Cl⁻. Sr²⁺ can substantially substitute for Ca²⁺, whereas Mg²⁺, Mn²⁺ and monovalent ions are almost inefficient. (iii) A quantitative correlation between the extent of the nanosecond kinetics and the average oxygen yield per flash was not observed. (iv) If CaCl₂ is present in the assay medium for trypsin treatment the samples are markedly protected to proteolytic degradation. This effect mainly refers to the reaction pattern of the acceptor side. Other bivalent cations can substitute Ca²⁺ for its protective function. (v) The CaCl₂-induced protection to proteolytic attack is extremely sensitive to a very short trypsin pretreatment that does hardly affect the shape of the fluorescence induction curve. The results are discussed in relation to the functional and structural organization of Photosystem II.     

Polycystin-2 (TRPP2) Regulation by Ca2+ Is Effected and Diversified by Actin-Binding Proteins

Calcium regulation of Ca²⁺-permeable ion channels is an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the transient receptor potential superfamily, is a nonselective cation channel with Ca²⁺ permeability. The molecular mechanisms associated with PC2 regulation by Ca²⁺ remain ill-defined. We recently demonstrated that PC2 from human syncytiotrophoblast (PC2_hst) but not the in vitro translated protein (PC2_iv), functionally responds to changes in intracellular (cis) Ca²⁺. In this study we determined the regulatory effect(s) of Ca²⁺-sensitive and -insensitive actin-binding proteins (ABPs) on PC2_iv channel function in a lipid bilayer system. The actin-bundling protein α-actinin increased PC2_iv channel function in the presence of cis Ca²⁺, although instead was inhibitory in its absence. Conversely, filamin that shares actin-binding domains with α-actinin had a strong inhibitory effect on PC2_iv channel function in the presence, but no effect in the absence of cis Ca²⁺. Gelsolin stimulated PC2_iv channel function in the presence, but not the absence of cis Ca²⁺. In contrast, profilin that shares actin-binding domains with gelsolin, significantly increased PC2_iv channel function both in the presence and absence of Ca²⁺. The distinct effect(s) of the ABPs on PC2_iv channel function demonstrate that Ca²⁺ regulation of PC2 is actually mediated by direct interaction(s) with structural elements of the actin cytoskeleton. These data indicate that specific ABP-PC2 complexes would confer distinct Ca²⁺-sensitive properties to the channel providing functional diversity to the cytoskeletal control of transient receptor potential channel regulation.     

Phase behavior of isolated photoreceptor membrane lipids is modulated by bivalent cations

The phase behavior of isolated photoreceptor membrane lipids is further investigated by ³¹P-NMR, in view of earlier discrepant results [(1979) Biochim. Biophys. Acta 558, 330–337; (1982) FEBS Lett. 124, 93–99]. We present evidence that the discrepancy is due to bivalent cations. When resuspended in aqueous media at neutral pH in the absence of bivalent cations, the isolated photoreceptor membrane lipids largely adopt the bilayer configuration. However, upon addition of such cations (Ca²⁺ Mg²⁺) or when resuspended in their presence, the formation of other phases (hexagonal H_II, lipidic particles) results. The rate of this transition depends on cation concentration and temperature. The transition is not easily reversed by addition of EDTA. Implications with regard to photoreceptor membrane structure and function need further study.     

Structural preferences of phosphatidylinositol and phosphatidylinositol-phosphatidylethanolamine model membranes influence of Ca2+ and Mg2+

The structural preferences of soya phosphatidylinositol in isolation and in mixtures with soya phosphatidylethanolamine, and the influence of Ca²⁺ and Mg²⁺ on these preferences, have been examined employing ³¹P-NMR and freeze-fracture techniques. It is shown that phosphatidylinositol assumes the bilayer organization on hydration both in the presence and absence of Ca²⁺ and Mg²⁺. In mixed systems with H_II phase) phosphatidylethanolamine, phosphatidylinositol induces lipidic particle structure at low (<10 mol%) concentrations and bilayer structure at higher levels. In systems containing 15 or 20 mol% phosphatidylinositol, Ca²⁺ (but not Mg²⁺) can induce H_II phase structure. The results indicate that phosphatidylinositol is a more effective agent than other acidic phospholipids for stabilizing bilayer structure, particularly when high levels of divalent cations are present. These findings are discussed in terms of functional roles of phosphatidylinositol and mechanisms whereby Ca²⁺ induces structural reorganization in mixed systems containing acidic phospholipids and phosphatidylethanolamine.     

Free radical fragmentation of cardiolipin by cytochrome c

The effect of cytochrome c (cyt c) on degradation of cardiolipin in its polar part was investigated in cardiolipin/phosphatidylcholine (CL/PC) liposomes incubated with cyt c/H₂O₂/and (or) ascorbate by high-performance thin layer chromatography and MALDI-TOF mass spectrometry. It has been shown that phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were formed in the system under conditions where hydrogen peroxide favours a release of heme iron from cyt c. The formation of PA and PHA occurs via an OH-induced fragmentation taking place in the polar moiety of cardiolipin. Formation of fragmentation products correlated with the loss of CL in CL/PC liposomes incubated with cyt c/H₂O₂/ascorbate or with Cu²⁺/H₂O₂/ascorbate.     

Role of calcium in the initiation of fast axonal transport of protein: Effects of divalent cations

Fast axonal transport of [³H]protein has been examined in bullfrog primary afferent neurons incubated in media supplemented with divalent cations that can act as agonists or antagonists of calcium ions. Incubation in calcium-free medium (CFM) had no effect on the rate of transport, but reduced the amount of transported [³H]protein by 40–60% relative to transport in the contralateral preparation maintained in normal medium. Preparations incubated in CFM supplemented with 1.8 mM SrCl₂ (equimolar to the CaCl₂ concentration in normal medium) carried out transport at control levels. Incubation conditions in which primary afferent somata were exposed to the Sr²⁺-medium while nerve trunks were maintained in CFM also supported normal transport. By contrast, selective exposure of nerve trunks to Sr²⁺-medium, and somata to CFM resulted in a reduced level of transport similar to that observed when the whole preparation was incubated in CFM. The depression of transport resulting from incubation in CFM was shown to be reversible when preparations were transferred from CFM to either Sr²⁺-supplemented CFM or to normal medium. By contrast to the effects of Sr²⁺, Ba²⁺ (up to 18 mM) did not substitute for Ca²⁺ in the transport process. When normal medium was supplemented with calciumantagonist cations, the amount of transport was depressed (Co²⁺ > Mn²⁺ >> Mg²⁺), with no concomitant effect on the rate of transport. Results of studies with Co²⁺, as well as those with Sr²⁺, suggest that a major locus of action of these cations is within the neuronal soma at a step subsequent to protein synthesis, and prior to the onset of protein translocation via the transport system. Thus, it is inferred that these divalent cations affect a calcium-dependent step that occurs during the initiation phase of fast axonal transport.     

Calcium Ion-Dependent Increase in Thermostability of Dextran Glucosidase from Streptococcus mutans

Dextran glucosidase from Streptococcus mutans (SmDG), which belongs to glycoside hydrolase family 13 (GH13), hydrolyzes the non-reducing terminal glucosidic linkage of isomaltooligosaccharides and dextran. Thermal deactivation of SmDG did not follow the single exponential decay but rather the two-step irreversible deactivation model, which involves an active intermediate having 39% specific activity. The presence of a low concentration of CaCl₂ increased the thermostability of SmDG, mainly due to a marked reduction in the rate constant of deactivation of the intermediate. The addition of MgCl₂ also enhanced thermostability, while KCl and NaCl were not effective. Therefore, divalent cations, particularly Ca²⁺, were considered to stabilize SmDG. On the other hand, CaCl₂ had no significant effect on catalytic reaction. The enhanced stability by Ca²⁺ was probably related to calcium binding in the β→α loop 1 of the (β?α)₈ barrel of SmDG. Because similar structures and sequences are widespread in GH13, these GH13 enzymes might have been stabilized by calcium ions.