Inhibition Assay of Theophylline by Capillary Electrophoresis/Dynamic Frontal Analysis on the Hydrolysis of p-Nitrophenyl Phosphate with Alkaline Phosphatase

A novel inhibition assay is proposed by capillary electrophoresis/dynamic frontal analysis (CE/DFA). When a substrate of p-nitrophenyl phosphate and an inhibitor of theophylline were tandemly introduced into the capillary containing alkaline phosphatase as an enzyme, two plateau signals were detected in the electropherogram. A higher plateau is based on the CE/DFA without inhibition, and a suppressed plateau is formed under the inhibition while the substrate zone passing through the inhibitor zone. Inhibition constant was successfully determined through the two plateau heights

Kinetic Analyses of Two-Steps Enzymatic Oxidation from Hypoxanthine to Uric Acid with Xanthine Oxidase by Capillary Electrophoresis/Dynamic Frontal Analysis

Two steps of enzymatic oxidations from hypoxanthine to uric acid with xanthine oxidase (XOD) were kinetically analyzed by capillary electrophoresis/dynamic frontal analysis. When a substrate solution of hypoxanthine was introduced into a capillary with a separation buffer containing XOD, the enzymatic reaction continuously proceeded during the electrophoresis and a product of xanthine was continuously resolved from the substrate zone. A plateau signal of the product xanthine was detected based on the constant reaction rate with XOD. The plateau height was directly related with the reaction rate, and a Michaelis-Menten constant KM,HXA was successfully determined as 770±40 μmol L−1. When xanthine was used as a substrate, a slope response of uric acid was obtained because of the low concentrations of the substrate and its significant decrease. However, the Michaelis-Menten constant was successfully determined by using the initial reaction rate, and a Michaelis-Menten constant of KM,XA was determined as 85±6 μmol L−1

Interactions between p27 and p88 replicase proteins of Red clover necrotic mosaic virus play an essential role in viral RNA replication and suppression of RNA silencing via the 480-kDa viral replicase complex assembly

AbstractRed clover necrotic mosaic virus (RCNMV), a positive-sense RNA virus with a bipartite genome, encodes p27 and p88 replicase proteins that are required for viral RNA replication and suppression of RNA silencing. In this study, we indentified domains in p27 and p88 responsible for their protein–protein interactions using in vitro pull-down assays with the purified recombinant proteins. Coimmunoprecipitation analysis in combination with blue-native polyacrylamide gel electrophoresis using mutated p27 proteins showed that both p27–p27 and p27–p88 interactions are essential for the formation of the 480-kDa complex, which has RCNMV-specific RNA-dependent RNA polymerase activity. Furthermore, we found a good correlation between the accumulated levels of the 480-kDa complex and replication levels and the suppression of RNA silencing activity. Our results indicate that interactions between RCNMV replicase proteins play an essential role in viral RNA replication and in suppressing RNA silencing via the 480-kDa replicase complex assembly

GAPDH-A Facilitates Intercellular Movement of RCNMV

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process

Capillary Electrophoresis/Dynamic Frontal Analysis for the Enzyme Assay of 4-Nitrophenyl Phosphate with Alkaline Phosphatase

A substrate of 4-nitrophenyl phosphate was enzymatically hydrolyzed by alkaline phosphatase (ALP) in a capillary tube, while an injected zone of the substrate was electrophoretically migrating in the separation buffer containing the enzyme by capillary electrophoresis (CE). During CE migration of the substrate from the start time of the electrophoresis to the detection time of the substrate, the substrate was continuously hydrolyzed by ALP to form a product of 4-nitrophenolate, and a plateau signal of 4-nitrophenolate was detected as a result of the zero-order kinetic reaction. The height of the plateau signal was directly related to the reaction rate, and it was used for the determination of a Michaelis–Menten constant through Lineweaver–Burk plots. Since the plateau signal is attributed to the dynamic formation of the product by the enzymatic reaction in CE, this analysis method is named as capillary electrophoresis/dynamic frontal analysis (CE/DFA). In CE/DFA, the CE separation is included on detecting the plateau signal, and the hydrolysis product before the sample injection is resolved from the dynamically and continuously formed product. The inhibition of the enzyme with the product is also eliminated in CE/DFA by the CE separation

Kinetic analysis of the transphosphorylation with creatine kinase by pressure-assisted capillary electrophoresis/dynamic frontal analysis

Kinetic reactions of the transphosphorylation with creatine kinase (CK) were individually investigated between creatine (Cr) and creatine phosphate (CrP) by pressure-assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The transphosphorylations are reversible between Cr and CrP, and reverse reactions inevitably accompany in general batch analyses. In pCE/DFA, the kinetic reaction proceeds in a separation capillary and the product is continuously resolved from the substrate zone. Therefore, the formation rate is kept constant at the substrate zone without the reverse reaction, and the product is detected as a plateau signal. This study demonstrates the direct and individual analyses of both the forward and the backward kinetic reactions with CK by pCE/DFA. A plateau signal was detected in the pCE/DFA with ADP or ATP as one of the products on either the forward or the backward reactions. The Michaelis-Menten constants of Km,ATP (from Cr to CrP) and Km,ADP (from CrP to Cr) were successfully determined through the plateau signal. Determined values of Km,ATP and Km,ADP by pCE/DFA were smaller than the ones obtained by the pre-capillary batch analyses. The results agree with the fact that the reverse reaction is excluded in the analysis of the kinetic reactions. The proposed pCE/DFA is useful on individual analyses of both forward and backward kinetic reactions without any interference from the reverse reaction

Kinetic analyses of two-steps oxidation from L-tyrosine to L-dopaquinone with tyrosinase by capillary electrophoresis/dynamic frontal analysis

Tyrosinase catalyzes the oxidation of L-tyrosine in two stages to produce L-dopa and L-dopaquinone stepwise, and L-dopaquinone is subsequently converted to dopachrome. Most of the conventional analyses subjected only one-step reaction from L-tyrosine to L-dopa or from L-dopa to L-dopaquinone. In this study, kinetic analyses of two-steps oxidation of L-tyrosine with tyrosinase were made by capillary electrophoresis/dynamic frontal analysis (CE/DFA). When L-dopa was introduced into a capillary as a sample plug in a CE/DFA format, the enzymatic oxidation continuously occurred during the electrophoresis, and the product L-dopaquinone was subsequently converted to dopachrome which was detected as a plateau signal. A Michaelis-Menten constant of the second-step kinetic reaction, Km,Do, was determined as 0.45±0.03 mmol L−1. In the analysis of the first-step kinetic reaction from L-tyrosine to L-dopa, L-dopa was not resolved by CE/DFA because both L-tyrosine and L-dopa are electrically neutral. The L-dopa formed and co-migrated at the L-tyrosine zone was calibrated beforehand with the final product of dopachrome detected as a plateau signal. Constantly formed L-dopa was successfully detected as a plateau signal of dopachrome, and a Michaelis-Menten constant of Km,Ty was also determined as 0.061±0.009 mmol L−1 by the CE/DFA. CE/DFA is applicable to two-steps enzymatic reactions

Kinetic analysis of substrate competition in enzymatic reactions with β-D-galactosidase by capillary electrophoresis / dynamic frontal analysis

Competitive inhibition between two substrates with an enzyme is investigated by capillary electrophoresis/dynamic frontal analysis (CE/DFA). Enzymatic hydrolyses of o-nitrophenyl β-D-galactopyranoside and p-nitrophenyl β-D-galactopyranoside with β-D-galactosidase were examined as a model competitive reaction. A sample solution containing the two substrates was injected into a capillary filled with a separation buffer containing an enzyme. Enzymatic hydrolysis occurred during the electrophoresis, and the products of o-nitrophenol and p-nitrophenol were continuously formed and resolved from the sample zone. Two-steps plateau signal was detected with the two-substrate solutions based on the difference in the effective electrophoretic mobility of o-nitrophenol and p-nitrophenol. Michaelis-Menten constants and inhibition constants were determined with the plateau heights. Usefulness of CE/DFA on competitive inhibition analysis is demonstrated in this study

A Y-shaped RNA structure in the 3' untranslated region together with the trans-activator and core promoter of Red clover necrotic mosaic virus RNA2 is required for its negative-strand RNA synthesis.

Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus with a bipartite genome. RNA1 encodes N-terminally overlapping replication proteins, p27 and p88. RNA2 is replicated efficiently by the replication proteins supplied in trans, whereas RNA1 needs p88 preferentially in cis for its replication. cis-Acting elements required for RNA2 replication have been mapped to the 3' terminal stem-loop structure conserved between RNA1 and RNA2, and to the protein-coding region including the trans-activator. Here, we have identified a Y-shaped RNA structure with three-way RNA junctions predicted in the 3' untranslated region of RNA2 as a novel element required for negative-strand synthesis using an in vitro translation/replication system. We also show that, in addition to the 3' terminal core promoter, several RNA elements including the trans-activator are also required for negative-strand synthesis. Functional roles and structural requirements of these cis-acting elements in RCNMV RNA replication are discussed

Kinetic analysis of an enzymatic hydrolysis of p-nitrophenyl acetate with carboxylesterase by pressure-assisted capillary electrophoresis/dynamic frontal analysis

An enzymatic hydrolysis of p‐nitrophenyl acetate with carboxyesterase was analyzed by capillary electrophoresis/dynamic frontal analysis (CE/DFA). A plateau signal was expected with the anionic product of p‐nitrophenol by the CE/DFA applying in‐capillary reaction and the continuous CE resolution of the product from the substrate zone. However, the plateau height was not sufficient, and/or the plateau signal fluctuated and drifted. Therefore, a pressure assist was utilized in the CE/DFA to detect the product zone fast and to average the fluctuated plateau signal by mixing in a laminar flow. The plateau signal became relatively flat and its height was developed by the pressure‐assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The plateau height was used for the Michaelis‐Menten analysis, and a Michaelis‐Menten constant was determined as KM = 0.83 mmol L−1. An enzyme inhibition was also examined with bis (p‐nitrophenyl) phosphate by adding it in the separation buffer. The height of the plateau signal decreased by the inhibition, and a 50% inhibitory concentration was determined as IC50 = 0.79 μmol L−1. The values of KM and IC50 obtained in this study agreed well with the reported values. Since the proposed pCE/DFA includes electrophoretic migration of the substrate zone in a capillary, it is also noticed that the deactivation of the enzyme by ethanol on the preparation of the substrate solution can be avoided, as well as the exclusion of the inhibition by the product