Dexamethasone suppresses osteogenesis of osteoblast via the PI3K/Akt signaling pathway in vitro and in vivo

The hypofunction of osteoblasts induced by glucocorticoids (GCs) has been identified as a major contributing factor for GC-induced osteoporosis (GIO). However, the biological mechanism underlying the effect of GC in osteoblasts are not fully elucidated. Recent studies implicated an important role of phosphoinositide 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway in the regulation of bone growth. We propose that the PI3K/Akt signaling may be implicated in the process of GC-induced osteogenic inhibition in osteoblasts. In this study, primary osteoblasts were used in vitro and in rats in vivo to evaluate the biological significance of the PI3K/Akt pathway in GC-induced bone loss. In vivo, dexamethasone (Dex)-treated rats had low bone mineral density and decreased expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and phosphorylated Akt (p-Akt) in bone tissue. In vitro study shows that Dex over the dose of 10^–8 M remarkably inhibited cellular osteogenesis, as represented by decreased cell viability, lessened ALP activity, and suppressed osteogenic protein expressions including ALP and OCN. Meanwhile, a dramatic downregulation in the PI3K/Akt pathway phosphorylation was also observed in Dex-treated osteoblasts. These changes were marked rescued by treatment with a PI3K agonist 740Y-P. Moreover, downregulation of ALP and OCN expressions by LY294002 can mimic the suppressive effects of Dex. These data together reveal that the suppressed PI3K/Akt pathway is involved in the regulatory action of Dex on osteogenesis.     

Starch and sugar accumulation in Sulla carnosa leaves upon Mg2+ starvation

In the present work, magnesium deficiency effects were studied in Sulla carnosa plants grown in nutrient solution containing 1.50, 0.05, 0.01, and 0.00 mM Mg²⁺. After 5 weeks of treatment, fully expanded leaves were harvested to study their morphological and ultrastructural changes, as well as their carbohydrate, pigment, and Mg²⁺ concentrations. In control plants, leaves were well developed with remarkable green color. Down to 0.05 mM Mg²⁺, no chlorosis symptom was recorded, but below this concentration, mature leaves showed an appearance of interveinal chlorosis that was much more pronounced at 0.00 mM Mg²⁺ with the development of necrotic spots. Optima of chlorophyll a, chlorophyll b, and carotenoid concentrations were observed at 0.05 and 1.50 mM Mg²⁺; leaf magnesium concentration was severely reduced at 0.05 mM Mg²⁺. A significant decrease in pigment concentrations was noticed at 0.01 mM Mg²⁺, but the lowest values were recorded at 0.00 mM Mg²⁺. Enzymatic assays showed an increase in the accumulation of soluble sugars and starch with decreasing Mg²⁺ concentration. These results were in accordance with those of ultrastructural studies that revealed a marked alteration of chloroplasts in leaves of deficient plants. These chloroplasts were round and bigger as a result of a massive accumulation of oversized starch grains with disrupted thylakoids. As a whole, 1.50, 0.05, and 0.01 mM Mg²⁺ were found optimal, suboptimal, and deficient concentrations, respectively, the latter showing no significant difference with absolute Mg²⁺ absence (0.00 mM Mg²⁺).     

镁离子对成骨细胞活力和分化的促进作用及其机制研究

目的:研究不同浓度镁离子对成骨细胞活力和分化的影响,并探讨镁基生物材料促进骨再生的机制。方法:分离培养大鼠乳鼠颅骨成骨细胞,之后将细胞分别在DMEM培养基(含有0.8 m M镁离子;对照组)和含有6 m M、10 m M、18 m M镁离子(实验组)的培养基中进行培养,通过MTT法测定细胞活力,ALP活力、茜素红染色法测定成骨细胞的分化,通过western blot法测定不同浓度镁离子组中PI3K/Akt信号通路的表达情况。结果:6 m M、10 m M镁离子组成骨细胞活力、ALP活力、基质矿化水平较对照组明显增加(P0.05),18 m M镁离子组成骨细胞活力、ALP活力、基质矿化水平对照组明显降低(P0.05)。在10 m M镁离子组加入wortmannin后,上述增强的结果受到抑制。结论:6-10 m M镁离子促进成骨细胞的活力和分化,而过高浓度镁离子(18 m M)对成骨细胞的活力和分化具有抑制作用。10 m M镁离子通过激活PI3K/Akt信号通路促进成骨细胞的活力和分化。这项研究为医用镁基生物材料的进一步研究提供了很好的参考作用。     

Glimepiride Promotes Osteogenic Differentiation in Rat Osteoblasts via the PI3K/Akt/eNOS Pathway in a High Glucose Microenvironment

Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS) plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM) inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a PI3K inhibitor) led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment.     

Improving magnesium uptake,photosynthesis and antioxidant enzyme activities of watermelon by grafting onto pumpkin rootstock under low magnesium

Background and aims

Magnesium (Mg) is an essential macronutrient that plays an important role in numerous physiological and biochemical processes of plant. However, Mg deficiency commonly occurs worldwide. Watermelon is an important crop that often suffers from Mg deficiency. This study aims to test whether watermelon performance can be improved by grafting onto rootstocks under low Mg and to clarify the underlying physiological mechanism.

Methods

Self-grafted, bottle gourd (Jingxinzhen No.1) and pumpkin (Jingxinzhen No.4) rootstock-grafted plants were treated with three Mg concentrations: 2.0 mM (normal condition), 0.4 mM (moderate stress), and 0.04 mM (severe stress) for 16 days under hydroponic conditions. Ungrafted watermelon and pumpkin were treated with 2.0 mM and 0.04 mM for 12 days.

Results

The growth of the plants was not affected by 0.4 mM Mg; however, plant growth decreased under 0.04 mM Mg in all graft combinations compared with control (2.0 mM Mg). Pumpkin rootstock grafting significantly increased watermelon growth under low Mg stress (0.04 mM Mg), compared with self-grafted and bottle gourd-grafted plants. The Mg²⁺ uptake of watermelon plants was increased by grafting onto pumpkin rootstocks, however, root-to-shoot transport capacity of Mg²⁺ was similar compared with self-grafted plants under 0.04 mM Mg. Gene expression analysis showed that magnesium transporter genes MGT1, MGT3, MGT4, and MGT5 may play an important role in higher Mg²⁺ uptake of pumpkin root. The photosynthetic parameters and activities of superoxide dismutase, peroxidase and catalase were significantly higher, but malonaldehyde (MDA) content were lower in the pumpkin rootstock grafted plants compared with other graft combinations under 0.04 mM Mg.

Conclusion

Our results provide strong evidence that pumpkin rootstock ‘Jinxinzhen No. 4’ grafting can improve watermelon performance under low Mg stress. The enhanced plant performance is attributed to higher root Mg²⁺ uptake and the improvement of photosynthesis and antioxidant enzyme activities.

     

Strontium ranelate stimulates the activity of bone-specific alkaline phosphatase: interaction with Zn2+ and Mg2+

Strontium ranelate (SR) is an orally administered and bone-targeting anti-osteoporotic agent that increases osteoblast-mediated bone formation while decreasing osteoclastic bone resorption, and thus reduces the risk of vertebral and femoral bone fractures in postmenopausal women with osteoporosis. Osteoblastic alkaline phosphatase (ALP) is a key enzyme involved in the process of bone formation and osteoid mineralization. In this study we investigated the direct effect of strontium (SR and SrCl₂) on the activity of ALP obtained from UMR106 osteosarcoma cells, as well as its possible interactions with the divalent cations Zn²⁺ and Mg²⁺. In the presence of Mg²⁺, both SR and SrCl₂ (0.05–0.5 mM) significantly increased ALP activity (15–66 % above basal), and this was dose-dependent in the case of SR. The stimulatory effect of strontium disappeared in the absence of Mg²⁺. The cofactor Zn²⁺ also increased ALP activity (an effect that reached a plateau at 2 mM), and co-incubation of 2 mM Zn²⁺ with 0.05–0.5 mM SR showed an additive effect on ALP activity stimulation. SR induced a dose-dependent decrease in the Km of ALP (and thus an increase in affinity for its substrate) with a maximal effect at 0.1 mM. Co-incubation with 2 mM Zn²⁺ further decreased Km in all cases. These direct effects of SR on osteoblastic ALP activity could be indicating an alternative mechanism by which this compound may regulate bone matrix mineralization.     

Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation

We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPβS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca²⁺-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca²⁺ induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca²⁺-containing but not in a Ca²⁺-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y₂ receptors and PI3K/Akt pathway activation involving Ca²⁺, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca²⁺ influx/CaM pathway.     

The role of Ca2+/Calcineurin/NFAT signalling pathway in osteoblastogenesis

The bone remodelling process is closely related to bone health. Osteoblasts and osteoclasts participate in the bone remodelling process under the regulation of various factors inside and outside. Excessive activation of osteoclasts or lack of function of osteoblasts will cause occurrence and development of multiple bone‐related diseases. Ca²⁺/Calcineurin/NFAT signalling pathway regulates the growth and development of many types of cells, such as cardiomyocyte differentiation, angiogenesis, chondrogenesis, myogenesis, bone development and regeneration, etc. Some evidences indicate that this signalling pathway plays an extremely important role in bone formation and bone pathophysiologic changes. This review discusses the role of Ca²⁺/Calcineurin/NFAT signalling pathway in the process of osteogenic differentiation, as well as the influence of regulating each component in this signalling pathway on the differentiation and function of osteoblasts, whereby the relationship between Ca²⁺/Calcineurin/NFAT signalling pathway and osteoblastogenesis could be deeper understood.     

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca²⁺ on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca²⁺ on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca²⁺ (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca²⁺ stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca²⁺ significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.     

Enamel matrix derivative enhances the proliferation and osteogenic differentiation of human periodontal ligament stem cells on the titanium implant surface

Periodontal ligament stem cells (PDLSCs) have mesenchymal-stem-cells-like qualities, and are considered as one of the candidates of future clinical application in periodontal regeneration therapy. Enamel matrix derivative (EMD) is widely used in promoting periodontal regeneration. However, the effects of EMD on the proliferation and osteogenic differentiation of human PDLSCs grown on the Ti implant surface are still no clear. Therefore, this study examined the effects of EMD on human PDLSCs in vitro. Human PDLSCs were isolated from healthy participants, and seeded on the surface of Ti implant disks and stimulated with various concentrations of EMD. Cell proliferation was determined with Cell Counting Kit-8 (CCK-8). The osteogenic differentiation of PDLSCs was evaluated by the measurement of alkaline phosphatase (ALP) activity, Alizarin red staining, and real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. The results indicated that EMD at concentrations (5–60 µg/ml) increased the viability and proliferation of PDLSCs. The treatment with 30 and 60 µg/ml of EMD significantly elevated ALP activity, augmented mineralized nodule formation and calcium deposition, and upregulated the mRNA and protein levels of Runx-2 and osteocalcin (OCN) in the PDLSCs grown on the Ti surface. Further investigation found that EMD treatment did not change the protein levels of phosphatidylinositol-3-kinase (PI3K), p-PI3K, Akt and mTOR, but significantly upregulated the phosphorylated levels of Akt and mTOR. Collectively, these results suggest that EMD stimulation can promote the proliferation and osteogenic differentiation of PDLSCs grown on Ti surface, which is possibly associated with the activation of Akt/mTOR signaling pathway.     

The Effects of Mn2+ on the Proliferation, Osteogenic Differentiation and Adipogenic Differentiation of Primary Mouse Bone Marrow Stromal Cells

The effects of Mn²⁺ on the proliferation, osteogenic and adipogenic differentiation of BMSCs were evaluated by employing MTT, ΔΨm, cell cycle, ALP activity, collagen production, ARS and oil red O stain assays. The results indicated that Mn²⁺ decreased the viability at most concentrations for 24 h, but the viability was increased with prolonging incubation time. Mn²⁺ at the concentrations of 1?×?10^-7 and 1?×?10^-6?mol/L decreased ΔΨm in the BMSCs for 48 h. Mn²⁺ induced G2/M phase cell cycle arrest at tested concentrations. On day 7 and 10, the effect of Mn²⁺ on the osteogenic differentiation depended on concentration, but it inhibited osteogenic differentiation at all tested concentrations for 14 d. The effect of Mn²⁺ on the synthesis of collagen of BMSCs depended on concentration for 7 d, but Mn²⁺ inhibited the synthesis of collagen at all tested concentrations for 10 d. On day 14, Mn²⁺ inhibited the formation of mineralized matrix nodules of BMSCs at all tested concentrations, the inhibitory effect turned to be weaker with prolonging incubation time. Mn²⁺ promoted the adipogenic differentiation of BMSCs at all tested concentrations for 10 d, but had no effect with prolonging incubation time. These findings suggested the effects of Mn²⁺ on the proliferation, osteogenic differentiation and adipogenic differentiation of BMSCs are very complicated, concentration and incubation time are key factors for switching the biological effects of Mn²⁺ from damage to protection.     

Mg<Superscript>2+</Superscript>-induced stabilization of β-galactosidase from <Emphasis Type="Italic">Bacillus megaterium</Emphasis> and its application in the galactosylation of natural products

Objective

To improve the stability of β-galactosidase from Bacillus megaterium YZ08 (BMG) in aqueous hydrophilic solvents and promote its application in the galactosylation of natural products.

Results

The addition of 5 mM Mg²⁺ significantly enhanced the stability of BMG in aqueous hydrophilic solvents, and the half-lives of BMG in these solutions reached 56 min to 208 h, while they were only 7 min to 5.9 h without addition of Mg²⁺. Studies on the kinetic parameters in buffer solution and 30% dimethyl sulfoxide (DMSO) indicated that the affinity of BMG to 2-nitrophenyl-β-d-galactopyranoside and its catalytic efficiency (κ _cat/K _m) increased with the addition of Mg²⁺. Furthermore, the addition of Mg²⁺ facilitated galactosylation reactions in 30% DMSO and increased product conversions by 24–41% due to the reversal of the thermodynamic equilibrium of hydrolysis.

Conclusion

A convenient approach was established to improve the stability of BMG in aqueous hydrophilic solvents.

     

Reversible changes of canavalin solubility controlled by divalent cation concentration in crude sword bean extract

Canavalin is a vicilin-class (7S) storage protein found in sword bean (Canavalia gladiata). Our previous report indicated that canavalin is precipitated by the addition of 20 mM MgCl₂ to crude sword bean extract. Here, we examined the solubility changes induced by the addition of Mg²⁺ and Ca²⁺ at various concentrations. Canavalin tended to be insolubilized at relatively low concentrations of MgCl₂ (< 20 mM) and solubilized at relatively high concentrations (> 20 mM). In addition, canavalin was slightly insolubilized in the presence of NaCl. Overall, the results revealed that solubility changes are reversible and depend on the concentration of divalent cations. Therefore, we suggested a reaction scheme that describes the effects of divalent cations on the solubility of canavalin, which would facilitate the study of its physiological function and the application of canavalin in the food processing industry.     

Backbone resonance assignments for the cytoplasmic region of the Mg2+ transporter MgtE in the Mg2+-unbound state

Magnesium ion (Mg²⁺) is an essential metal element for life, and has many cellular functions, including ATP utilization, activation of enzymes, and maintenance of genomic stability. The intracellular Mg²⁺ concentration is regulated by a class of transmembrane proteins, called Mg²⁺ transporters. One of the prokaryotic Mg²⁺ transporters, MgtE, is a 450-residue protein, and functions as a dimer. We previously reported that MgtE exhibits the channel-like electrophysiological property, i.e., it permeates Mg²⁺ according to the electrochemical potential of Mg²⁺. The Mg²⁺-permeation pathway opens in response to the decrease of the intracellular Mg²⁺ concentration, while it is completely closed at the intracellular Mg²⁺ concentration of 10 mM. The crystal structures of the MgtE dimer revealed that the Mg²⁺-sensing cytoplasmic region consists of the N and CBS domains. The Mg²⁺-bound state of MgtE adopts a compact, globular conformation, which is stabilized by the coordination of a number of Mg²⁺ ions between these domains. On the other hand, in the Mg²⁺-unbound state, these domains are far apart, and fixed by the crystal packing. Therefore, structural analyses in solution were awaited, in order to characterize the Mg²⁺-dependent alteration of the MgtE structure and dynamics relevant to its gating. In this paper, we report the backbone resonance assignments of the dimer of the cytoplasmic region of the MgtE from Thermus thermophilus with a molecular weight of 60 KDa, in the Mg²⁺-unbound state.     

The acid tolerant and cold-active β-galactosidase from Lactococcus lactis strain is an attractive biocatalyst for lactose hydrolysis

The gene encoding the β-galactosidase from the dairy Lactococcus lactis IL1403 strain was cloned, sequenced and overexpressed in Escherichia coli. The purified enzyme has a tetrameric arrangement composed of four identical 120 kDa subunits. Biochemical characterization showed that it is optimally active within a wide range of temperatures from 15 to 55 °C and of pH from 6.0 to 7.5. For its maximal activity this enzyme requires only 0.8 mM Fe²⁺ and 1.6 mM Mg²⁺. Purified protein displayed a high catalytic efficiency of 102 s^?1 mM^?1 for lactose. The enzyme stability was increased by immobilization mainly at low pH (from 4.0 to 5.5) and high temperatures (55 and 60 °C). The bioconversion of lactose using the L. lactis β-galactosidase allows the production of lactose with a high bioconversion rate (98 %) within a wide range of pH and temperature.     

Production and characterization of laccase from Pleurotus ferulae in submerged fermentation

Pleurotus ferulae is a mushroom typically found in arid steppe that is distributed widely in the Junggar Basin of Xinjiang, China. In this work, laccase production by P. ferulae JM30X was optimized in terms of medium composition and culture conditions. After optimization, the highest laccase activity obtained was 6,832.86 U/L. A single isozyme with a molecular weight of 66 kDa was observed by SDS-PAGE and native-PAGE. Optimum pH and temperature were 3.0 and 50–70 °C, respectively. The best laccase substrate was ABTS, for which the Michaelis-Menten constant (K _m) and catalytic efficiency (K _cat/K _m) value for P. ferulae laccase were 0.193 mM and 2.73?×?10⁶ (mM s)^?1, respectively. The activity of purified laccase was increased by more than four-fold by Cu²⁺, Mn²⁺ and Mg²⁺, while it was completely inhibited by Fe²⁺ and Fe³⁺. The production of laccase was influenced by the initial pH and K⁺ concentration, and the activity of purified laccase was enhanced by Cu²⁺, Mn²⁺ and Mg²⁺. This Pleurotus genus laccase from P. ferulae JM30X was analyzed by MS spectrum and the results are conducive to furthering our understanding of Pleurotus genus laccases.     

Dynorphin activation of kappa opioid receptor protects against epilepsy and seizure-induced brain injury via PI3K/Akt/Nrf2/HO-1 pathway

Dynorphins act as endogenous anticonvulsants via activation of kappa opioid receptor (KOR). However, the mechanism underlying the anticonvulsant role remains elusive. This study aims to investigate whether the potential protection of KOR activation by dynorphin against epilepsy was associated with the regulation of PI3K/Akt/Nrf2/HO-1 pathway. Here, a pilocarpine-induced rat model of epilepsy and Mg²⁺-free-induced epileptiform hippocampal neurons were established. Decreased prodynorphin (PDYN) expression, suppressed PI3K/Akt pathway, and activated Nrf2/HO-1 pathway were observed in rat epileptiform hippocampal tissues and in vitro neurons. Furthermore, dynorphin activation of KOR alleviated in vitro seizure-like neuron injury via activation of PI3K/Akt/Nrf2/HO-1 pathway. Further in vivo investigation revealed that PDYN overexpression by intra-hippocampus injection of PDYN-overexpressing lentiviruses decreased hippocampal neuronal apoptosis and serum levels of inflammatory cytokines and malondialdehyde (MDA) content, and increased serum superoxide dismutase (SOD) level, in pilocarpine-induced epileptic rats. The protection of PDYN in vivo was associated with the activation of PI3K/Akt/Nrf2/HO-1 pathway. In conclusion, dynorphin activation of KOR protects against epilepsy and seizure-induced brain injury, which is associated with activation of the PI3K/Akt/Nrf2/HO-1 pathway.     

Production of glucose-6-phosphate by glucokinase coupled with an ATP regeneration system

A process of glucose-6-phosphate (G-6-P) production coupled with an adenosine triphosphate (ATP) regeneration system was constructed that utilized acetyl phosphate (ACP) via acetate kinase (ACKase). The genes glk and ack from Escherichia coli K12 were amplified and cloned into pET-28a(+), then transformed into E. coli BL21 (DE3) and the recombinant strains were named pGLK and pACK respectively. Glucokinase (glkase) in pGLK and ACKase in pACK were both overexpressed in soluble form. G-6-P was efficiently produced from glucose and ACP using a very small amount of ATP. The conversion yield was greater than 97 % when the reaction solution containing 10 mM glucose, 20 mM ACP-Na₂, 0.5 mM ATP, 5 mM Mg²⁺, 50 mM potassium phosphate buffer (pH 7.0), 4.856 U glkase and 3.632 U ACKase were put into 37 °C water bath for 1 h.