The purification and characterization of glucokinase from the thermophile <i>Bacillus stearothermophilus</i>

Goward, Christopher R.; Hartwell, R. C.; Atkinson, Tony; Scawen, Michael D.

doi:10.1042/bj2370415

Cited by 52 publications

(20 citation statements)

References 21 publications

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“…The activity of HK in the gastrocnemius muscle and liver tissues was determined as previously described by Goward et al 16 with modifications. Briefly, the activity was measured by an assay in which the oxidation of G-6-PDH produced is coupled to the reduction of nicotinamide adenine dinucleotide phosphate (NADP + ) catalyzed by G-6-PDH dehydrogenase as shown in the equation below:…”

Section: Measurement Of Hk (Ec 2 7 1 1) Activitymentioning

confidence: 99%

The Effects ofSyzygium aromaticum-Derived Oleanolic Acid on Glycogenic Enzymes in Streptozotocin-Induced Diabetic Rats

2011

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Studies indicate that the antihyperglycemic effects of Syzygium aromaticum-derived oleanolic acid (OA) are mediated in part through increased hepatic glycogen synthesis. Accordingly, this study assessed the influence of OA on the activity of glucokinase (GK) and hexokinase (HK) of skeletal muscle and liver tissues in streptozotocin (STZ)-induced diabetic rats. After 5 weeks of OA treatment, hepatic and gastrocnemius muscle glycogen concentrations and activities of GK and HK were measured spectrophotometrically in reactions where the oxidation of glucose-6-phosphate (G-6-PDH) formed was coupled to nicotinamide adenine dinucleotide phosphate (NADP + ) reduction catalyzed by G-6-PDH dehydrogenase. Rats treated with deionized water or standard hypoglycemic drugs acted as untreated and treated positive controls, respectively. STZ-induced diabetic rats exhibited depleted glycogen levels and low activities of glycogenic enzymes in muscle and hepatic tissues. OA administration restored these biochemical alterations to near normalcy. The combination of OA and insulin did not significantly alter the activities of HK and GK of STZinduced diabetic rats, suggesting that glycogen synthesis can also occur from precursors such as amino acids or fructose and lactate. The attenuation of the activities of glycogenic enzymes with concomitant increases of hepatic and muscle glycogen concentrations of STZ-induced diabetic rats provides a therapeutic strategy for diabetes treatment.

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Section: Measurement Of Hk (Ec 2 7 1 1) Activitymentioning

confidence: 99%

The Effects ofSyzygium aromaticum-Derived Oleanolic Acid on Glycogenic Enzymes in Streptozotocin-Induced Diabetic Rats

2011

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“…To test glucokinase specific activities, we prepared cell lysates from DK1622 (wild type) and MC0001 (⌬glcK) cells and then added the lysates to a colorimetric reaction mixture in which glucokinase activity is measured as the rate of reduction of NADP coupled with the oxidation of glucose-6-phosphate by glucose-6-phosphate dehydrogenase (11,16). Our results show that DK1622 lysates contain a soluble glucokinase activity which increases as a linear function of protein concentration (Fig.…”

Section: Resultsmentioning

confidence: 84%

“…Cultures were placed on ice and sonicated for 45 s (in 5-s bursts) at 5 W in a model 60 Fisher sonic dismembranator. Following microcentrifugation at 4°C to remove cellular debris (20,000 ϫ g for 10 min), lysates were kept on ice and assayed within 4 h. Glucokinase activities were determined using a colorimetric reaction, which couples the production of glucose-6-phosphate to the reduction of NADP (11,16). Our standard reaction mixture (1-ml total volume) contained 50 mM Tris (pH 9.1), 20 mM MgCl 2 , 20 mM glucose, 10 mM ATP (pH 9.1), 10 to 100 l cell lysate, and 1 U of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (Sigma).…”

Section: Methodsmentioning

confidence: 99%

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β- d -Allose Inhibits Fruiting Body Formation and Sporulation in Myxococcus xanthus

Chavira¹,

Cao²,

Lê³

et al. 2007

J Bacteriol

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Myxococcus xanthus, a gram-negative soil bacterium, responds to amino acid starvation by entering a process of multicellular development which culminates in the assembly of spore-filled fruiting bodies. Previous studies utilizing developmental inhibitors (such as methionine, lysine, or threonine) have revealed important clues about the mechanisms involved in fruiting body formation. We used Biolog phenotype microarrays to screen 384 chemicals for complete inhibition of fruiting body development in M. xanthus. Here, we report the identification of a novel inhibitor of fruiting body formation and sporulation, ␤-D-allose. ␤-D-Allose, a rare sugar, is a member of the aldohexose family and a C3 epimer of glucose. Our studies show that ␤-D-allose does not affect cell growth, viability, agglutination, or motility. However, ␤-galactosidase reporters demonstrate that genes activated between 4 and 14 h of development show significantly lower expression levels in the presence of ␤-D-allose. Furthermore, inhibition of fruiting body formation occurs only when ␤-D-allose is added to submerged cultures before 12 h of development. In competition studies, high concentrations of galactose and xylose antagonize the nonfruiting response to ␤-D-allose, while glucose is capable of partial antagonism. Finally, a magellan-4 transposon mutagenesis screen identified glcK, a putative glucokinase gene, required for ␤-D-allose-mediated inhibition of fruiting body formation. Subsequent glucokinase activity assays of the glcK mutant further supported the role of this protein in glucose phosphorylation.Although Myxococcus xanthus exists as individual vegetative rods, the myxobacteria are fundamentally social organisms capable of intercellular communication, coordinated group movements, and the formation of multicellular structures. Upon starvation, a developmental program is initiated wherein bacterial cells aggregate into dome-shaped, macroscopic structures called fruiting bodies. The signaling and genetic systems orchestrating this program have received much attention in M. xanthus research (21,22,47). In particular, investigation of exogenous compounds that inhibit fruiting body formation have provided useful starting points for identifying the signaling pathways and molecular components involved in this highly regulated process. Previous studies investigating inhibition of fruiting body formation by various amino acids have shown that methionine-mediated inhibition is associated with a dramatic reduction in the production of S-adenosylmethionine (SAM) (6, 42). SAM, a methyl donor for the M. xanthus frz chemosensory system, affects fruiting through the regulation of motility (5, 45). Lysine, threonine, leucine, and isoleucine were also found to inhibit fruiting body formation through a reduction of intracellular levels of SAM. A separate study found that glycine can block purine-induced fruiting body formation, suggesting that purine-containing compounds, such as cyclic AMP and ADP, induce fruiting through a single mechanism (38).To identif...

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“…Glucose is the most important HK substrate in most of the tissues, and the product is glucose-6-phosphate. GK is an HK isozyme, and facilitates the phosphorylation of glucose to glucose-6-phosphate [41]. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Determination of human blood glucose levels using microchip electrophoresis

et al. 2007

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Determination of human blood glucose levels using microchip electrophoresisA high-performance monitoring system for human blood glucose levels was developed using microchip electrophoresis with a plastic chip. The combination of reductive amination as glucose labeling with fluorescent 2-aminoacridone (AMAC) and glucose-borate complex formation realized the highly selective detection of glucose even in a complex matrix such as a blood sample. The migration time of a single peak, observed on an electropherogram of AMAC-labeled plasma, closely resembled that of glucose standard solution. The treatment of plasma with hexokinase or glucokinase for glucose phosphorylation resulted in a peak shift from approximately 145 to 70 s, corresponding to glucose and glucose-6-phosphate, respectively. A double-logarithm plot revealed a linear relationship between glucose concentration and fluorescence intensity in the range of 1-300 mM of glucose (r 2 = 0.9963; p ,0.01), and the detection limit was 0.92 mM. Furthermore, blood glucose concentrations estimated from the standard curves of three subjects were compared with results obtained by conventional colorimetric analysis using glucose dehydrogenase. Good correlation was observed between methods according to simple linear regression analysis (p ,0.05). The reproducibility of the assay was about 6.3-9.1% (RSD) and the within-days and between-days reproducibility were 1.6-8.4 and 5.2-7.2%, respectively. This system enables us to determine blood glucose with high sensitivity and accuracy, and will be applicable to clinical diagnosis.

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The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus

Cited by 52 publications

References 21 publications

The Effects ofSyzygium aromaticum-Derived Oleanolic Acid on Glycogenic Enzymes in Streptozotocin-Induced Diabetic Rats

The Effects ofSyzygium aromaticum-Derived Oleanolic Acid on Glycogenic Enzymes in Streptozotocin-Induced Diabetic Rats

β- d -Allose Inhibits Fruiting Body Formation and Sporulation in Myxococcus xanthus

Determination of human blood glucose levels using microchip electrophoresis

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