Studies of reversible inhibition, irreversible inhibition, and activation of alkaline phosphatase by capillary electrophoresis

Whisnant, Angela R.; Gilman, S. Douglass

doi:10.1016/s0003-2697(02)00062-3

Cited by 60 publications

(63 citation statements)

References 37 publications

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“…CE has recently emerged as a powerful tool for enzyme analysis [14][15][16], including the determination of Michaelis-Menten constants [17][18][19][20], and for enzyme inhibition assays [18][19][20][21][22] exhibiting a number of advantages over conventional methods. These include rapid separation, the ability to simultaneously discriminate between, and identify multiple components of similar structure with high resolution [23], ultralow sample volume requirements, and high-throughput by automation.…”

Section: Introductionmentioning

confidence: 99%

Development of off‐line and on‐line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

2006

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Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.

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Section: Introductionmentioning

confidence: 99%

Development of off‐line and on‐line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

2006

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“…Levamisole, L-phenylalanine and Lhomoarginine, which permit discrimination between tissue-nonspecific and tissue-specific ALPs, have been used for the clinical quantitation and identification of ALP isoenzymes for almost a century (2,24,25,30). Vanadate is an ALP inhibitor (12,17,31). Corticosterone (5 mM) inhibits rat renal ALP, but not rat liver ALP, and progesterone has no effect on rat renal or liver ALP (13).…”

Section: Results Results Results Resultsmentioning

confidence: 99%

“…L-phenylalanine and L-leucine strongly inhibit intestinal, placental and germ cell Saccharomyces cerevisiae alkaline phosphatase modulation www.bjournal.com.br study) has been reported (23). Human ALPs show different susceptibilities to inactivation or activation by 0.01 mM-0.1 M EDTA (30) and calf intestinal ALP is activated by low EDTA concentrations, 20-400 µM, and irreversibly inhibited by higher concentrations, 1 mM and above (31). Levamisole inhibition of rat liver ALP decreases with increasing levamisole concentration (Martins MJ, unpublished results).…”

Section: Discussion Discussion Discussion Discussion Discussionmentioning

confidence: 99%

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium

Fernandes

Amorim

Azevedo

et al. 2007

Braz J Med Biol Res

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Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine -186%, L-leucine -155% and L-phenylalanine -168%) and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20%, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166%) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentrationdependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

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“…Whisnant et al [20,21] developed an on-capillary inhibition assay with fluorescence detection for the enzyme alkaline phosphatase (ALP). The capillary was filled with a fluorogenic substrate, AttoPhos, and zones of inhibitor (theophylline) and enzyme were injected into the substrate-filled capillary [20].…”

Section: Continuous Engagement Emmamentioning

confidence: 99%

“…The calculated inhibition constant (K i ) was in agreement with literature values. In their next paper [21] they studied different classes of ALP inhibitors and a distinction could be made between reversible and irreversible inhibition of ALP, and ALP activation in the incapillary system.…”

Section: Continuous Engagement Emmamentioning

confidence: 99%

Advances in capillary electrophoretically mediated microanalysis

et al. 2003

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Recent developments in the use of capillary electrophoretic techniques for the in-line study of enzyme reactions and derivatization protocols are reviewed. The article is divided into two parts: (i) in-line enzyme reactions and (ii) in-line derivatization. The first part introduces electrophoretically mediated microanalysis (EMMA) and discusses and illustrates the different modes of EMMA. A literature overview is provided, starting from 1996, and the investigated enzymes are classified into two tables based on the mode of engagement (i.e., continuous or transient) of the developed EMMA-based assay. The second part starts with an introduction of the procedures and the nomenclature used in the area of in-line derivatization protocols based on EMMA. Reported derivatization procedures are discussed and classified in tables, according to the functional group that is derivatized.

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Studies of reversible inhibition, irreversible inhibition, and activation of alkaline phosphatase by capillary electrophoresis

Cited by 60 publications

References 37 publications

Development of off‐line and on‐line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Development of off‐line and on‐line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium

Advances in capillary electrophoretically mediated microanalysis

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