Chlorogenic acid attenuates pro-inflammatory response in the blood of streptozotocin-induced diabetic rats.

BACKGROUND: Chlorogenic acid (CGA) has been shown to reduce pro-inflammation by scavenging reactive oxygen species (ROS) and reactive nitrogen species. In this study, the anti-inflammatory effect of CGA was expanded to streptozotocin (STZ)-induced diabetic rats. The inter-relationships among oxidative stress, pro-inflammation, and cytochrome P450 (CYP) 1A enzymes were also investigated in peripheral blood mononuclear cells (PBMC) of STZ-diabetic rats. RESULTS: The levels of pro-inflammatory cytokines, interleukin-6 and tumor necrosis factor-alpha, increased by approximately 3.4- and 2.9-fold, respectively, and the albumin concentration decreased in the serum of STZ-induced diabetic rats compared to normal rats. The C-reactive protein (CRP) values also increased by about 3.8-fold higher, indicating that STZ induced an inflammation in the blood of STZ-diabetic rats. The expression levels and catalytic activities of CYP1A enzymes were elevated by approximately 2.2-2.5- and 4.3-6.7-fold, respectively, in the PBMC of STZ-treated rats. A decrease in the amount of PBMC-bound albumin was also observed. In contrast, the levels of cytokines and CRP in serum and the activities of CYP1A enzymes in PBMC were significantly reduced in CGA-treated diabetic rats in a CGA concentration-dependent manner. In addition, STZ-mediated elevation of ROS in serum and PBMC was decreased by the CGA administration. However, the CGA treatment did not change the enhanced blood glucose level and expression of CYP1A enzymes by STZ. STZ-mediated decrease in the levels of serum and PBMC-bound albumin was not also restored by the CGA administration. CONCLUSIONS: These results suggest that CGA could be used to treat type 1 diabetes-induced inflammation.

Protective effects of Erythronium japonicum and Corylopsis coreana Uyeki extracts against 1,3-dichloro-2-propanol-induced hepatotoxicity in rats.

Erythronium japonicum (E. japonicum) and Corylopsis coreana Uyeki (C. coreana Uyeki, Korean winter hazel) have been shown to significantly decrease 1,3-dichloro-2-propanol (1,3-DCP)-induced generation of reactive oxygen species and CYP2E1 activity in HuH7, human hepatocytes. In this study, we expanded upon the previous study and investigated the effects of E. japonicum and C. coreana Uyeki extracts on 1,3-DCP-induced liver damage in rats. The pre-treatment of rats with these extracts alleviated a decrease in body weight and reduced 1,3-DCP-induced increase in catalytic activities of hepatic enzymes, such as aspartate aminotransferase and alanine aminotransferase, in the serum. Moreover, treatment with the extracts restored the 1,3-DCP-induced decreases in anti-oxidant enzyme activities, such as the activities of superoxide dismutase and catalase, in the rat liver. Histopathological studies also strongly supported the results of enzyme activities. These results suggest a possibility that the extracts of E. japonicum and C. coreana Uyeki can be a remedy for alleviating 1,3-DCP-induced liver damage in animals.

Extracts from Erythronium japonicum and Corylopsis coreana Uyeki reduce 1,3-dichloro-2-propanol-mediated oxidative stress in human hepatic cells.

In this study, it was demonstrated that 1,3-dichloro-2-propanol (1,3-DCP) induced oxidative stress and cell death in HuH7, human hepatocytes. The protective effects of Erythronium japonicum (E. japonicum) and Corylopsis coreana Uyeki (C. coreana Uyeki) extracts against 1,3-DCP-treated cells were also investigated. First, the activities of superoxide dismutase (SOD) and catalase (CAT) were diminished by the treatment of 1,3-DCP. Moreover, 1,3-DCP stimulated the expression and catalytic activity of cytochrome P450 2E1 (CYP2E1), an enzyme that generates reactive oxygen species in the liver. In contrast, co-treatment of 1,3-DCP with the extracts significantly decreased ROS generation and inhibited CYP2E1 activity without affecting its expression. The co-administration of extracts also restored the activities of SOD and CAT reduced by 1,3-DCP and protected against 1,3-DCP-mediated cell death. In conclusion, these results suggest that 1,3-DCP induces oxidative stress through the elevated CYP2E1 level, which is inhibited by the extracts, protecting cells against the effects of 1,3-DCP.

Wicking Property of Graft Material Enhanced Bone Regeneration in the Ovariectomized Rat Model.

BACKGROUND: Recruitment and homing cells into graft materials from host tissue is crucial for bone regeneration. METHODS: Highly porous, multi-level structural, hydroxyapatite bone void filler (HA-BVF) have been investigated to restore critical size bone defects. The aim was to investigate a feasibility of bone regeneration of synthetic HA-BVF compared to commercial xenograft (Bio-Oss). HA-BVF of 0.7 mm in average diameter was prepared via template coating method. Groups of animals (n = 6) were divided into two with normal (Sham) or induced osteoporotic conditions (Ovx). Subsequently, subdivided into three treated with HA-BVF as an experiment or Bio-Oss as a positive control or no treatment as a negative control (defect). The new bone formation was analyzed by micro-CT and histology. RESULTS: At 4 weeks post-surgery, new bone formation was initiated from all groups. At 8 weeks post-surgery, new bone formation in the HA-BVF groups was greater than Bio-Oss groups. Extraordinarily greater bone regeneration within the Ovx-HA group than Sham-Bio-Oss or Ovx-Bio-Oss group (p < 0.05). CONCLUSION: This study suggests that the immediate wicking property of HA-BVF from host tissue activates a natural healing cascade without the addition of exogeneous factors or progenitor cells. HA-BVF may be an effective alternative for repairing bone defects under both normal and osteoporotic bone conditions.

Diacylglycerol in Cationic Nanoparticles Stimulates Oxidative Stress-Mediated Death of Cancer Cells.

Previous reports have suggested that cargo-free cationic nanoparticles (cNP) consisting of cationic monovalent lipids, such as 1,2-oleoyl-3-trimethylammonium-propane (DOTAP), induce reactive oxygen species (ROS) generation and toxicity in cells. In addition, cNP containing six lysine residues (6K) and cargo (6K-cNP) exerted synergistic effects on ROS production and cell death in cancer cells. In this study, we investigated the effect of diacylglycerol (DAG) derived from egg phosphatidylcholine in nanoparticles (NP) on ROS-mediated cellular toxicity. When DAG was incorporated into cNP (D-cNP) or 6K-cNP (6K-D-cNP) up to 7.8 mol% at the expense of DOTAP, and treated with cells, ROS generation in cancer cells increased further in a DAG concentration-dependent manner compared with those of both cNP without DAG. Concomitantly, cancer cell viability was more decreased upon the treatment with DAG-containing cNP. Moreover, D-cNP or 6K-D-cNP exhibited enhanced uptake into cells under endocytosis-inhibited conditions. Taken together, these results suggested that the presence of DAG in NP stimulated the interaction of NP with cancer cells and the resulting ROS-mediated cytotoxicity.

Cargo-Free Nanoparticles Containing Cationic Lipids Induce Reactive Oxygen Species and Cell Death in HepG2 Cells.

Nanoparticles (NPs) containing cationic monovalent lipids such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA), have been widely used for the delivery of nucleic acid such as small-interfering RNA and polypeptide to cells as cancer therapies and vaccine development. Several previous reports have suggested that cationic liposomes induce reactive oxygen species (ROS) and ROS-mediated toxicity in cells. Here, we systematically investigated the effects of DOTAP- or DOTMA-containing NPs without any cargo on the human carcinoma cells, HepG2. Treatment with NPs containing DOTAP or DOTMA increased the production of cellular ROS, such as H2O2 and lipid peroxidation, in HepG2 cells and concomitantly decreased cell viability. These effects were dependent on the lipid concentration, surface density of cationic lipids, and particle size of NPs. However, neutral NPs consisting of 1,2-dioleoyl-3-phosphocholine did not elicit the effective ROS generation or cell death regardless of the lipid concentration and particle size. The present study suggests that DOTAP- and DOTMA-NPs are able to induce cancer cell death through production of ROS in the absence of any therapeutic cancer reagents. These results also provide a rational background for the design of delivery systems using cationic lipid-based NP formulations.

Cloning and characterization of a serotonin N-acetyltransferase from a gymnosperm, loblolly pine (Pinus taeda).

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N-acetylserotonin, an immediate precursor for melatonin biosynthesis by N-acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K(m) (428 µM) and Vmax (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis.

Regioselective hydroxylation of 17ß-estradiol by mutants of CYP102A1 from Bacillus megaterium.

A large set of mutants of CYP102A1 from Bacillus megaterium have human cytochrome P450-like activities and the ability to metabolize a number of marketed drugs and steroids. Here, we tested whether the CYP102A1 mutants could be used to produce hydroxylated human metabolites of 17ß-estradiol (E2). A set of the mutants, which were generated by site-directed and random mutagenesis, was used to produce hydroxylated human metabolites of E2 in this study. Some of the tested mutants could regioselectively generate 2-OH E2 as a major metabolite but not other hydroxylated products. These results suggest that CYP102A1 mutants would be useful for the bioconversion of steroid hormones to hydroxylated products, which can be used for industrial applications.

Decreased level of albumin in peripheral blood mononuclear cells of streptozotocin-induced diabetic rats.

We investigated the phenotypic level of albumin in peripheral blood mononuclear cells (PBMC) of streptozotocin (STZ)-induced diabetic rats. A specific reduction of albumin was identified by 2-dimensional electrophoresis and mass spectrometry. Decreased albumin content was also confirmed by immunoblotting and quantitative real-time PCR. Since albumin is a major and predominant antioxidant in plasma, the PBMC albumin may also contribute to their antioxidant activity. By measuring the amount of H2O2, lipid peroxidation and the redox form of glutathione, it was found that the production of the oxidative stress was elevated in STZ-diabetic rats compared to that of normal control. We suggest, therefore, that decreased albumin content may lead to the decreased antioxidant activity in the PBMC of type 1 diabetic rats.

Mechanical Characterization of High-Performance Steel-Fiber Reinforced Cement Composites with Self-Healing Effect.

The crack self-healing behavior of high-performance steel-fiber reinforced cement composites (HPSFRCs) was investigated. High-strength deformed steel fibers were employed in a high strength mortar with very fine silica sand to decreasing the crack width by generating higher interfacial bond strength. The width of micro-cracks, strongly affected by the type of fiber and sand, clearly produced the effects on the self-healing behavior. The use of fine silica sand in HPSFRCs with high strength deformed steel fibers successfully led to rapid healing owing to very fine cracks with width less than 20 µm. The use of very fine silica sand instead of normal sand produced 17%-19% higher tensile strength and 51%-58% smaller width of micro-cracks.

Increase of human CYP1B1 activities by acidic phospholipids and kinetic deuterium isotope effects on CYP1B1 substrate oxidation.

The effect of phospholipids on the kinetic parameters of three substrates, 7-ethoxy-4 -(trifluoromethyl)coumarin (7-EFC), 7-ethoxycoumarin (7-EC) and 17ß-estradiol (E(2)), of human CYP1B1 was studied. In general, anionic phospholipids, phosphatidic acid and cardiolipin increased catalytic efficiency by increasing k(cat) values or decreasing K(m) values. The advantages of using the 7-EFC as a substrate over 7-EC and E(2) include high k(cat), low K(m) and high catalytic efficiency. Spectral binding titrations indicated that the binding affinity of 7-EFC to CYP1B1 in the presence or absence of phospholipids is higher than that of 7-EC or E(2). Furthermore, phosphatidylcholine increased the binding affinity of the substrates to the CYP1B1. High non-competitive intermolecular kinetic deuterium isotope effects (values 5.4-12) were observed for O-deethylation of 7-EFC and 7-EC with deuterium substitution at the ethoxy group, indicating that the C-H bond-breaking step makes a major contribution to the rate of these CYP1B1-catalyzed reactions. However, the intermolecular kinetic deuterium isotope effect is ~2 for the E(2) 4-hydroxylation reaction, indicating that the C-H bond-breaking step contributes only partially to the rate of this CYP1B1-catalyzed reaction. These results indicate that the reaction mechanism of CYP1B1-catalyzed reactions is distinct for each substrate.

The Flavin-Containing Reductase Domain of Cytochrome P450 BM3 Acts as a Surrogate for Mammalian NADPH-P450 Reductase.

Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase that consists of a heme domain and FAD/FMN-containing reductase domain (BMR). In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by BMR was evaluated as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to BMR substrates, MTT and CTC. MTT and CTC are monotetrazolium salts that form formazans upon reduction. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics (kcat =4120 min(-1), Km =77 µM for MTT and kcat =6580 min(-1), Km =51 µM for CTC). Our continuous assay using MTT and CTC allows the simple, rapid measurement of BMR activity. The BMR was able to metabolize mitomycin C and doxorubicin, which are anticancer drug substrates for CPR, producing the same metabolites as those produced by CPR. Moreover, the BMR was able to interact with CYP1A2 and transfer electrons to promote the oxidation reactions of substrates by CYP1A2 and CYP2E1 in humans. The results of this study suggest the possibility of the utilization of BMR as a surrogate for mammalian CPR.

Doxorubicin- and daunorubicin-induced regulation of Ca2+ and H+ fluxes through human bax inhibitor-1 reconstituted into membranes.

Bax inhibitor-1 (BI-1) is an evolutionarily conserved cell death suppressor in both animals and plants. We examined the effect of doxorubicin (DXR) and daunorubicin (DNR), which are clinically important anthracycline compounds, on the functional regulation of BI-1 reconstituted into membranes. DXR and DNR inhibited the proton-induced efflux of encapsulated Ca(2+) from membranes in a drug concentration-dependent manner. Both compounds also reduced the H(+) influx activity of BI-1. The proteoliposomes containing BI-1 increased the quenching of DXR fluorescence by Cu(2+), and the fluorescence energy transfer between pyrene-labeled BI-1 and DXR was enhanced with increasing DXR concentrations. The dissociation constants and the number of binding sites for both drugs in BI-1 were determined to be in the range of 3.7-4.5 × 10(-6) m and approximately 4-5/BI-1 molecule, respectively, using a proteomicelle system. DXR also induced secondary structural changes in reconstituted BI-1 and abolished the ability of BI-1-overexpressing cells to protect against endoplasmic reticulum stress-induced cell death. However, when mitoxantrone was used instead of DNR and DXR as an anthracycline analog, no significant effects were observed. These results suggest that BI-1 can be considered to be a new cancer therapeutic target by anthracyclines because of its stimulatory effects in cancer/tumor progression.

Enhanced lysosomal activity is involved in Bax inhibitor-1-induced regulation of the endoplasmic reticulum (ER) stress response and cell death against ER stress: involvement of vacuolar H+-ATPase (V-ATPase).

Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H(+)-ATPase(V-ATPase), a proton pump, was activated, suggesting high H(+) uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.

Surface display of heme- and diflavin-containing cytochrome P450 BM3 in Escherichia coli: a whole cell biocatalyst for oxidation.

Cytochrome P450 enzymes (P450s) are involved in the synthesis of a wide variety of valuable products and in the degradation of numerous toxic compounds. The P450 BM3 (CYP102A1) from Bacillus megaterium was the first P450 discovered to be fused to its redox partner, a mammalian-like diflavin reductase. Here, we report the development of a whole cell biocatalyst using ice-nucleation protein (Inp) from Pseudomonas syringae to display a heme- and diflavin-containing oxidoreductase, P450 BM3 (a single, 119-kDa polypeptide with domains of both an oxygenase and a reductase) on the surface of Escherichia coli. Surface localization and functionality of the fusion protein containing P450 BM3 were verified by flow cytometry and measurement of enzymatic activities. The results of this study comprise the first report of microbial cell-surface display of heme- and diflavin-containing enzyme. This system should allow us to select and develop oxidoreductases containing heme and/or flavins, into practically useful whole-cell biocatalysts for extensive biotechnological applications including selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, live vaccine development, and bio-chip development.

Cardiolipin, phosphatidylserine, and BH4 domain of Bcl-2 family regulate Ca2+/H+ antiporter activity of human Bax inhibitor-1.

We investigated the effects of phospholipid composition in membranes and Bcl-2 homology (BH) domains of the Bcl-2 family on Ca2+/H+ antiporter activity of human recombinant Bax inhibitor-1 (BI-1) reconstituted into membranes. Cardiolipin (CL) and phosphatidylserine (PS) stimulated the proton-mediated efflux of Ca2+ ions encapsulated into proteoliposomes when compared to Ca2+ efflux from 100% phosphatidylcholine (PC) membranes in a CL or PS concentration-dependent manner. Concomitantly, the anionic phospholipids also enhanced H+ ion influx into the membranes. Lateral segregations of CL and PS were observed through the fluorescence properties of fluorophore-labeled phospholipids upon BI-1 reconstitution in PC/CL or PC/PS binary systems. However, other anionic phospholipids, such as phosphatidic acid, phosphatidylglycerol, and phosphatidylinositol did not influence the stimulation of BI-1 functions in membranes. The peptide corresponding to the BH4 domain of Bcl-2 and Bcl-xL proteins stimulated the BI-1 activities in 100% PC membranes. The peptide also showed an additive effect with CL or PS. Furthermore, the CL, PS, and BH4 domains specifically increased oligomerization levels such as dimer and tetramer of BI-1 in membranes. Taken together, these results suggest that the CL, PS, and BH4 domains were stimulating factors for the Ca2+/H+ antiporter activities of BI-1 through protein oligomerization.

Bax inhibitor 1 increases cell adhesion through actin polymerization: involvement of calcium and actin binding.

Bax inhibitor 1 (BI-1), a transmembrane protein with Ca2+ channel-like activity, has antiapoptotic and anticancer activities. Cells overexpressing BI-1 demonstrated increased cell adhesion. Using a proteomics tool, we found that BI-1 interacted with gamma-actin via leucines 221 and 225 and could control actin polymerization and cell adhesion. Among BI-1-/- cells and cells transfected with BI-1 small interfering RNA (siRNA), levels of actin polymerization and cell adhesion were lower than those among BI-1+/+ cells and cells transfected with nonspecific siRNA. BI-1 acts as a leaky Ca2+ channel, but mutations of the actin binding sites (L221A, L225A, and L221A/L225A) did not change intra-endoplasmic reticulum Ca2+, although deleting the C-terminal motif (EKDKKKEKK) did. However, store-operated Ca2+ entry (SOCE) is activated in cells expressing BI-1 but not in cells expressing actin binding site mutants, even those with the intact C-terminal motif. Consistently, actin polymerization and cell adhesion were inhibited among all the mutant cells. Compared to BI-1+/+ cells, BI-1-/- cells inhibited SOCE, actin polymerization, and cell adhesion. Endogenous BI-1 knockdown cells showed a similar pattern. The C-terminal peptide of BI-1 (LMMLILAMNRKDKKKEKK) polymerized actin even after the deletion of four or six charged C-terminal residues. This indicates that the actin binding site containing L221 to D231 of BI-1 is responsible for actin interaction and that the C-terminal motif has only a supporting role. The intact C-terminal peptide also bundled actin and increased cell adhesion. The results of experiments with whole recombinant BI-1 reconstituted in membranes also coincide well with the results obtained with peptides. In summary, BI-1 increased actin polymerization and cell adhesion through Ca2+ regulation and actin interaction.

Functional and conformational modulation of human cytochrome P450 1B1 by anionic phospholipids.

We investigated the interaction of human P450 1B1 (CYP1B1) with various phospholipid bilayers using the N-terminally deleted (Delta2-4)CYP1B1 and (Delta2-26)CYP1B1 enzymes. Among anionic phospholipids, phosphatidic acid (PA) and cardiolipin specifically increased the catalytic activities, membrane binding affinities, and thermal stabilities of both CYP1B1 proteins when phosphatidylcholine matrix was gradually replaced with these anionic phospholipids. PA- or cardiolipin-dependent changes of CYP1B1 conformation were revealed by altered Trp fluorescence and CD spectra. However, both PA and cardiolipin exerted more significant effects with the (Delta2-4)CYP1B1 than the (Delta2-26)CYP1B1 implying the functional importance of N-terminal region for the interaction with the phospholipid membranes. In contrast, other anionic phospholipids such as phosphatidylserine and the neutral phospholipid phosphatidylethanolamine had no apparent effects on the catalytic activity or conformation of CYP1B1. These data suggest that the chemical and physical properties of membranes influenced by PA or cardiolipin composition are critical for the functional roles of CYP1B1.

Conformational change of Escherichia coli signal recognition particle Ffh is affected by the functionality of signal peptides of ribose-binding protein.

We examined the effects of synthetic signal peptides, wild-type (WT) and export-defective mutant (MT) of ribose-binding protein, on the conformational changes of signal recognition particle 54 homologue (Ffh) in Escherichia coli. Upon interaction of Ffh with WT peptide, the intrinsic Tyr fluorescence, the transition temperature of thermal unfolding, and the GTPase activity of Ffh decreased in a peptide concentration-dependent manner, while the emission intensity of 8-anilinonaphthalene-1-sulfonic acid increased. In contrast, the secondary structure of the protein was not affected. Additionally, polarization of fluorescein-labeled WT increased upon association with Ffh. These results suggest that WT peptide induces the unfolded states of Ffh. The WT-mediated conformational change of Ffh was also revealed to be important in the interaction between SecA and Ffh. However, MT had marginal effect on these conformational changes suggesting that the in vivo functionality of signal peptide is important in the interaction with Ffh and concomitant structural change of the protein.

Ca(2+)-induced stimulation of the membrane binding of Escherichia coli SecA and its association with signal peptides of secretory proteins.

Previously, it was found that Ca(2+) stimulates the intrinsic Escherichia coli SecA ATPase activity [Kim et al., FEBS Lett. 493 (2001) 12-16]. Now, we suggest that Ca(2+) is required for efficient interaction of SecA with membranes and the signal peptide of ribose-binding protein. When the amount of external Ca(2+) was enhanced, the amounts of membrane-bound SecA and its lipid/ATPase activity increased. In the presence of entrapped Ca(2+) in liposomes, the binding was also stimulated in a Ca(2+) concentration-dependent manner. The effect of Ca(2+) on the functional regulation of SecA was also evident in the presence of the signal peptides of secretory proteins, which the interaction of SecA with the signal peptide increased with increasing Ca(2+) concentration in the presence of membranes. However, other divalent cations including Mg(2+), Mn(2+), and Zn(2+) had inhibitory or no effect, suggesting a specific role of Ca(2+) in SecA interaction with lipid bilayers and signal peptides.