Atsushi Shoji

Real-time monitoring of matrix metalloproteinase-9 collagenolytic activity with a surface plasmon resonance biosensor

We describe a simple method for real-time monitoring of matrix metalloproteinase-9 (MMP-9) collagenolytic activity for native triple helical collagen IV with a surface plasmon resonance (SPR) biosensor. The proteolytic activity of MMP-9 is measured as a decrease in the SPR signal resulting from the cleavage of collagen IV immobilized on the sensor surface. The kinetic parameters of full-length MMP-9 and its catalytic domain-catalytic constant (k(cat)), association rate constant (k(a)), and dissociation rate constant (k(d))-were estimated by the SPR method. The presence of sodium chloride and a nonionic detergent Brij-35 in a reaction solution led to the lower collagenolytic activity of MMP-9, whereas they suppressed the nonspecific interaction between MMP-9 and a cysteamine-modified chip. The comparison of kinetic parameters between MMP-9 and its catalytic domain revealed that the association constant of MMP-9 is much larger than that of the catalytic domain, suggesting that the interplay among hemopexin-like domain, fibronectin type II repeats motif, and linker region (O-glycosylated domain) plays an important role in recognizing collagen IV.

Analysis of polyphenols from hop bract region using CCC

Abstract Polyphenols derived from hop (Humulus lupulus L.) bract region (HBP) can be used as food materials, thereby preventing dental caries. Chemical details of the active substances need to be elucidated. The polyphenols from hop bract (HBP) region were purified by countercurrent chromatography (CCC). The fractions were analyzed by high‐performance size‐exclusion chromatography (HPSEC) and reversed phase high‐performance liquid chromatography (RP‐HPLC). From HBP fractions by HPSEC, some low‐molecular‐ weight polyphenols (glycosides of flavonoids, catechins, and proanthocyanidins) were identified by RP‐HPLC. However, a very hydrophilic fraction was found to have the most potent cavity‐preventive activity, but it showed no peak in its RP‐HPLC chromatogram (absence of small polyphenols). HPSEC analysis showed that the major components of this fraction were high‐molecular weight substances, which were supposed to be proanthocyanidins, consisting of approximately 22 catechin units in its structure.

Determination of Log Po/w for Catechins and Their Isomers, Oligomers, and Other Organic Compounds by Stationary Phase Controlled High‐Speed Countercurrent Chromatography

Abstract A new technique, stationary phase volume controlled high‐speed counter‐current chromatography (HSCCC), was used to determine the octanol‐water partition coefficients (P o/w) of catechins and their isomers, oligomers, and other organic compounds. The stationary phase volume in the CCC column was effectively controlled under hydrodynamic equilibrium system. The log P o/w values ranging from −1.35 to +3.60 were measured within 21 min using this new HSCCC technology. The linear relationship (correlation coefficient value, r=0.993) was observed between log P o/w values obtained by the shake‐flask method and those values by the HSCCC method. In this technique, it is possible to inject multiple samples successively into the CCC column at short intervals to measure their retention times without renewing the stationary phase volume.

Counter-current chromatographic separation of nucleic acid constituents with a hydrophilic solvent system

Nucleic acid constituents such as nucleobases, nucleosides and nucleotides were separated by counter-current chromatography using type J coil planet centrifuge. The separation was performed with a hydrophilic solvent system composed of 1-propanol/800 mM potassium phosphate buffer (pH 7.4) (1:1, v/v) by eluting the lower aqueous phase at a flow-rate of 0.5 ml/min. Eight selected nucleic acid constituents (4.0 mg, 0.5 mg of each), uridine monophosphate (UMP), adenosine monophosphate (AMP), deoxyadenosine monophosphate (dAMP), uridine, urasile, deoxy uridine, adenosine and adenine were well resolved within 160 min.

A reusable liposome array and its application to assay of growth-hormone-related peptides

We describe a reusable liposome array based on the formation of cleavable disulfide cross-links between liposomes and the surface of a glass slip. The N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-modified liposomes encapsulating a pH-sensitive fluorescence dye were immobilized on a 3-mercaptopropyltrimethoxysilane (MTS)-modified glass slip through the formation of disulfide bonds. The regeneration of a used slip was performed by the lysis of immobilized liposomes with Triton X-100 and the cleavage of disulfide bonds by reduction with TCEP, followed by immobilization of SPDP-modified liposomes. The regeneration steps did not affect the fluorescence intensity of re-immobilized liposomes. The liposome array was applied to simultaneous quantification of growth hormone related peptides, i.e., GHRF and somatostatin, in a mixture. After optimizing the assay condition, the method allowed quantification of GHRF and somatostatin in concentration ranges from 0.5 × 10−9 to 0.5 × 10−7 g/mL with detection limits of 2 × 10−10 and 3 × 10−10 g/mL, respectively.

Trypsin-loaded mesoporous silica as a sensing material for amplified detection of ATP4 − ions

In this study, we describe a new method for the detection of an analyte with amplification using enzyme-loaded, polyaminated mesoporous silica. Enzymes with different molecular sizes (trypsin, alkaline phosphatase and glucose oxidase) were tested for loading in the pores of the mesoporous materials. Among the enzymes examined, only trypsin was loaded in the pores (diameter 2.8–5 nm) of the mesoporous silica. The enzymatic activity of trypsin loaded in the pores (2.8 nm) of polyaminated MCM-41(C14) was controlled by an analyte (ATP4 − ) that binds to the polyamine sites. The formation of p-nitroaniline from a substrate, N-α-benzoyl-DL-arginine-4-nitroanilide hydrochloride, in the presence of loaded trypsin was decreased by ATP4 − in the concentration range from 1.0 to 50 μM in a 2-[4-(2-hydroxyethyl)-1-piperazyl] ethanesulphonic acid buffer solution (pH 7.4). The response was selective to ATP4 − over ADP3 − , GMP2 − and phosphate. The potential use of trypsin-loaded mesoporous silica for the design of a sensing system with amplification ability is discussed in terms of working principle, the effect of pore sizes, sensitivity, selectivity and a signal transduction factor.

Evaluation of cathepsin B activity for degrading collagen IV using a surface plasmon resonance method and circular dichroism spectroscopy

Evaluation of cathepsin B activities for degrading collagen IV and heat-denatured collagen IV (gelatin) were performed by surface plasmon resonance (SPR) and circular dichroism (CD) measurements. The optimal pH of cathepsin B activity for degrading each substrate was around 4.0. The ΔRU(15 min), which is a decrease in the SPR signal at 15 min after injection of cathepsin B, was smaller for collagen IV than for heat-denatured collagen IV owing to the presence of triple-helical conformation. An unstable nature of the triple-helical conformation of collagen IV at pH 4.0 was shown by the CD study. Degrading collagen IV by cathepsin B was facilitated owing to a local unwinding of the triple-helical conformation caused by proteolytic cleavage of the non-helical region. The concentration dependence of the initial velocity for degrading collagen IV by cathepsin B at pH 4.0 was biphasic, showing that cathepsin B at low concentration exhibits exopeptidase activity, while the enzyme at high concentration exhibits endopeptidase activity. The kinetic parameters for the exopeptidase activity of cathepsin B toward collagen IV and heat-treated collagen IV were evaluated and discussed in terms of the protease mechanism.

Monitoring of cholesterol oxidation in a lipid bilayer membrane using streptolysin O as a sensing and signal transduction element.

Streptolysin O (SLO), which recognizes sterols and forms nanopores in lipid membranes, is proposed as a sensing element for monitoring cholesterol oxidation in a lipid bilayer. The structural requirements of eight sterols for forming nanopores by SLO confirmed that a free 3-OH group in the β-configuration of sterols is required for recognition by SLO in a lipid bilayer. The extent of nanopore formation by SLO in lipid bilayers increased in the order of cholestanol<cholesterol<25-OH cholesterol and in a sterol concentration-dependent manner. The immobilization of liposomes consisting of PC, cholesterol and 4-cholesten-3-one exhibited a linear relationship between calcein permeability and the molar ratio of cholesterol and 4-cholesten-3-one. The SLO-based method was successfully applied for monitoring of cholesterol oxidase-mediated oxidation of cholesterol in a lipid bilayer. The potential of the SLO nanopore-based method for monitoring cholesterol oxidation in a lipid bilayer by other oxidative enzymes is also discussed.

An enzyme-modified capillary as a platform for simultaneous fluorometric detection of d-glucose and l- lactate

&NA; The preparation of a glass capillary pattered with lipid layers on which lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH) were regionally adsorbed and its application for simultaneous detection of D‐glucose and L‐lactate in human serum is described. A lipid layer was formed on the surface of BSA‐unabsorbed octadecyltrichlorosilane (OTS) inner wall of a glass capillary. The electrostatic charge of the lipid layer was a key factor for adsorbing the enzymes on the lipid layer. The fluorescence intensities were observed at each enzyme site in the presence of diaphorase (DIA), &bgr;‐nicotinamide‐adenine dinucleotide oxidized (NAD), resazurin, D‐glucose and L‐lactate. The fluorescence intensities at each enzyme site increased with an increase in the concentration of D‐glucose and L‐lactate=with the detection limits of 32 &mgr;M and 4.9 &mgr;M, respectively.

Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance sensor

Graphical abstract Figure. No Caption available. HighlightsInhibitory assay for degratdation of collagen IV by cathepsin was developed with a SPR sensor.CA074 and Z‐Phe‐Phe‐FMK inhibited exopeptidase activity of cathepsin B rather than endopeptidase.Leupeptin inhibited the endopeptidase activity of cathepsin B more strongly. Abstract We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki) obtained by the SPR method was CA074Me ≈ Z‐Phe‐Phe‐FMK < leupeptin. This order was different from that obtained by benzyloxycarbonyl‐Phe‐Phe‐Fluoromethylketone (Z‐Phe‐Phe‐FMK) as a peptide substrate. The comparison of Ki suggested that CA074 and Z‐Phe‐Phe‐FMK inhibited exopeptidase activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly.