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Yakov Kuzyakov - One of the best experts on this subject based on the ideXlab platform.
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calibration of 2 d soil Zymography for correct analysis of enzyme distribution
European Journal of Soil Science, 2019Co-Authors: A K Guber, Bahar S Razavi, Evgenia Blagodatskaya, Alexandra Kravchenko, Yakov KuzyakovAbstract:Soil Zymography is a new technique developed to visualize two‐dimensional distributions of enzyme activities. The method consists of incubating a membrane saturated with an enzyme‐specific fluorogenic substrate on a surface of the soil sample, followed by recording the membrane image generated by a fluorescent product (e.g. MUF: methylumbelliferone) in ultraviolet light. Despite its relative ease of use, performing Zymography involves multiple user‐made decisions that might affect the accuracy of enzyme activity estimates. Therefore, unification of the Zymography methodology is required for correct estimations and comparisons of various studies. We evaluated the following methodological aspects of the implementation of Zymography: (a) camera settings and image processing, (b) effects of evaporation and (c) calibration procedures. Camera settings (shutter speeds or exposure time) affected the intensity of background fluorescence and signal‐to‐noise ratios (SNR). However, because their combined effects varied depending on MUF concentrations, light and camera setting need to be optimized for the expected range of MUF concentrations prior to Zymography. Evaporation of MUF solution from the membrane had no effect on fluorescence. Relations between MUF concentration and intensity of fluorescence during calibrations demonstrated a saturated pattern and were strongly affected by image noise outside the optimal range (e.g. 8–14 μm MUF pixel⁻¹). We developed a new calibration approach that is based on a piecewise linear regression. The new approach accounted for specific ranges of MUF concentration and uses nonuniformly saturated membranes, reflecting the real distribution of enzyme activities in soil. The new calibration algorithm eliminated biases of the standard calibration and resulted in greater accuracy in predicting MUF concentrations. HIGHLIGHTS: We developed a new approach to calibration for 2‐D soil Zymography. The approach accounted for spatial nonuniformity of soil zymograms. Standard calibration resulted in systematic underestimation of enzyme activity. Soil Zymography requires pixel‐based calibration with nonuniformly saturated membranes.
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quantitative soil Zymography mechanisms processes of substrate and enzyme diffusion in porous media
Soil Biology & Biochemistry, 2018Co-Authors: A K Guber, Bahar S Razavi, Evgenia Blagodatskaya, Alexandra Kravchenko, Daniel Uteau, Stephan Peth, Yakov KuzyakovAbstract:Abstract Soil membrane Zymography enables 2D mapping of enzyme activities on the surface of soil samples. The method is based on diffusion of components of enzymatically-mediated reactions to/from membrane, and, thus, reflects the distribution of enzyme activities at the intact soil surface. Zymography has been already successfully implemented in numerous soil ecology applications. Here we identify two methodological aspects for further improvement and expansion of the method at micro and macro scales: first, accounting for the area of contact between the soil surface and the Zymography membranes and, second, accounting for diffusion effects during the Zymography procedure. We tested three methods, namely, laser-scanning, staining with a fluorescent product (e.g. MUF: 4-methylumbelliferone), and X-ray computed micro-tomography, for assessing the area of the soil surface in contact with the membranes. We quantified diffusion of MUF, enzymes and substrate between the substrate-saturated membrane and soil as well as diffusion processes during membrane Zymography via HP2 software. Diffusion of the substrate from the membrane and of the MUF-product to the membrane was detected, while there were no clear evidence of enzyme diffusion to/in the membrane. According to the model simulations, the enzyme activities detected via 2D Zymography probably represent only a small portion, about 20%, of the actual reactions within the soil volume that is in both direct contact and in hydrological contact with Zymography membranes. This is a result of omnidirectional diffusion of reaction products. The membrane contact with the soil surface estimated by three methods ranged from 3.4 to 36.5% further signifying that only a fraction of enzymes activity is detectable in a course of 2D soil Zymography.
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spatial distribution and catalytic mechanisms of β glucosidase activity at the root soil interface
Biology and Fertility of Soils, 2016Co-Authors: Muhammad Sanaullah, Bahar S Razavi, Evgenia Blagodatskaya, Yakov KuzyakovAbstract:We compared modifications of soil Zymography, a new in situ technique to visualize enzyme activities, based on contact of fluorgenic substrate-saturated membranes with soil either through the gel layer (gel Zymography) or without gel application (direct Zymography). We coupled Zymography with quantitative measurements of enzyme kinetics to characterize catalytic mechanisms of β-glucosidase activity at the plant-soil interface including root surface (rhizoplane), rhizosphere, and bulk soil. Direct Zymography refined and focused image resolution. The area of hotspots (i.e., spots with most intensive enzyme activity) as well as color intensity ratios estimated using direct Zymography exceeded by a factor of 2 the corresponding values obtained with gel Zymography. As determined by direct Zymography, the percentage of hotspots associated to root surfaces was 58–68 % of total hotspot area. Hotspot area comprised only 6.8 ± 0.1 % of the total area of an image and 9.0 ± 3 % of the root surface area. The intensity of β-glucosidase activity, however, was up to 20 times higher in the hotspots versus bulk soil. The contribution of rhizosphere to β-glucosidase activity of the whole image (77–82 %) was four times higher than the contribution of the root surface. Enzyme kinetic parameters indicated different enzyme systems in bulk and rhizosphere soil. Higher substrate affinity and catalytic efficiency in bulk than in rhizosphere soil suggested relative domination of microorganisms with more efficient enzyme systems in the former. Coupling direct Zymography and kinetic assays enabled mapping the two-dimensional (2D) distribution of enzyme activity at the root-soil interface and estimating the catalytic properties of root-associated and soil-associated enzymes.
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soil Zymography a novel in situ method for mapping distribution of enzyme activity in soil
Soil Biology & Biochemistry, 2013Co-Authors: Marie Spohn, Andrea Carminati, Yakov KuzyakovAbstract:Recently, there has been growing interest in the spatial distribution of microbial activity in soil; however, methods for analysis of spatial distribution of microbial activity and for localization of hotspots of enzyme activity in soil are limited. Here were present an in situ Zymography technique for localization and quantification of enzyme activities in soil by means of thin gels with embedded substrates. After incubation, the substrate remaining in the gel is colored and quantified using calibration curves and digital image analysis. So far, Zymography has mostly been used to localize enzymatic activity in electrophoresis gels and in tissue sections. In this study we developed a Zymography technique for analysis of the two-dimensional distribution of enzyme activities in soil. The technique was applied to map and quantify protease and amylase activity in the rhizosphere of lupine (Lupinus polyphyllus) grown in rhizoboxes. Highest activities, of up to 46 ng mm � 2 of the soil surface h � 1 for the protease and of up to 0.90 m gm m � 2 h � 1 for the amylase were found in close association with roots. Since Zymography is an in situ method that does not require destruction of soil structure, it likely pictures enzyme activities more realistically than standard enzyme assays. In conclusion, soil in situ Zymography offers a promising tool for mapping distributions of enzyme activities in soils in a work- and cost-efficient way.
Masahiko Ikeuchi - One of the best experts on this subject based on the ideXlab platform.
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proteases are associated with a minor fucoxanthin chlorophyll a c binding protein from the diatom chaetoceros gracilis
Biochimica et Biophysica Acta, 2012Co-Authors: Ryo Nagao, Tatsuya Tomo, Eri Noguchi, Takehiro Suzuki, Akinori Okumura, Rei Narikawa, Isao Enami, Masahiko IkeuchiAbstract:Abstract We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a / c ‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using Zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, Zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.
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proteases are associated with a minor fucoxanthin chlorophyll a c binding protein from the diatom chaetoceros gracilis
Biochimica et Biophysica Acta, 2012Co-Authors: Ryo Nagao, Tatsuya Tomo, Eri Noguchi, Takehiro Suzuki, Akinori Okumura, Rei Narikawa, Isao Enami, Masahiko IkeuchiAbstract:Abstract We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a / c ‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using Zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, Zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.
Tatsuya Tomo - One of the best experts on this subject based on the ideXlab platform.
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proteases are associated with a minor fucoxanthin chlorophyll a c binding protein from the diatom chaetoceros gracilis
Biochimica et Biophysica Acta, 2012Co-Authors: Ryo Nagao, Tatsuya Tomo, Eri Noguchi, Takehiro Suzuki, Akinori Okumura, Rei Narikawa, Isao Enami, Masahiko IkeuchiAbstract:Abstract We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a / c ‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using Zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, Zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.
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proteases are associated with a minor fucoxanthin chlorophyll a c binding protein from the diatom chaetoceros gracilis
Biochimica et Biophysica Acta, 2012Co-Authors: Ryo Nagao, Tatsuya Tomo, Eri Noguchi, Takehiro Suzuki, Akinori Okumura, Rei Narikawa, Isao Enami, Masahiko IkeuchiAbstract:Abstract We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a / c ‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using Zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, Zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.
Rei Narikawa - One of the best experts on this subject based on the ideXlab platform.
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proteases are associated with a minor fucoxanthin chlorophyll a c binding protein from the diatom chaetoceros gracilis
Biochimica et Biophysica Acta, 2012Co-Authors: Ryo Nagao, Tatsuya Tomo, Eri Noguchi, Takehiro Suzuki, Akinori Okumura, Rei Narikawa, Isao Enami, Masahiko IkeuchiAbstract:Abstract We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a / c ‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using Zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, Zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.
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proteases are associated with a minor fucoxanthin chlorophyll a c binding protein from the diatom chaetoceros gracilis
Biochimica et Biophysica Acta, 2012Co-Authors: Ryo Nagao, Tatsuya Tomo, Eri Noguchi, Takehiro Suzuki, Akinori Okumura, Rei Narikawa, Isao Enami, Masahiko IkeuchiAbstract:Abstract We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a / c ‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using Zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, Zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.
Muhammad Sanaullah - One of the best experts on this subject based on the ideXlab platform.
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spatial distribution and catalytic mechanisms of β glucosidase activity at the root soil interface
Biology and Fertility of Soils, 2016Co-Authors: Muhammad Sanaullah, Bahar S Razavi, Evgenia Blagodatskaya, Yakov KuzyakovAbstract:We compared modifications of soil Zymography, a new in situ technique to visualize enzyme activities, based on contact of fluorgenic substrate-saturated membranes with soil either through the gel layer (gel Zymography) or without gel application (direct Zymography). We coupled Zymography with quantitative measurements of enzyme kinetics to characterize catalytic mechanisms of β-glucosidase activity at the plant-soil interface including root surface (rhizoplane), rhizosphere, and bulk soil. Direct Zymography refined and focused image resolution. The area of hotspots (i.e., spots with most intensive enzyme activity) as well as color intensity ratios estimated using direct Zymography exceeded by a factor of 2 the corresponding values obtained with gel Zymography. As determined by direct Zymography, the percentage of hotspots associated to root surfaces was 58–68 % of total hotspot area. Hotspot area comprised only 6.8 ± 0.1 % of the total area of an image and 9.0 ± 3 % of the root surface area. The intensity of β-glucosidase activity, however, was up to 20 times higher in the hotspots versus bulk soil. The contribution of rhizosphere to β-glucosidase activity of the whole image (77–82 %) was four times higher than the contribution of the root surface. Enzyme kinetic parameters indicated different enzyme systems in bulk and rhizosphere soil. Higher substrate affinity and catalytic efficiency in bulk than in rhizosphere soil suggested relative domination of microorganisms with more efficient enzyme systems in the former. Coupling direct Zymography and kinetic assays enabled mapping the two-dimensional (2D) distribution of enzyme activity at the root-soil interface and estimating the catalytic properties of root-associated and soil-associated enzymes.
