in vitro measurement of &alpha;-galactosidase a and acid &alpha;-glucosidase enzyme activity

In Vitro Measurement of &alpha;-Galactosidase A and Acid &alpha;-Glucosidase Enzyme Activity

Dilute the calculated amount of each sample and pipette it into fresh 1.5-milliliter reaction tubes to obtain 0.05 micrograms of alpha-galactosidase A or 0.5 micrograms of acid alpha-glucosidase per microliter of solution. Vortex the samples for 5 seconds again, and pipette 10 microliters of this dilution and duplicate into a 96-well plate.
Start the reactions by adding 20 microliters of the respective substrate solution. For alpha-galactosidase A, add 2 millimolar 4-methylumbelliferyl-alpha-D-galactopyranoside or 4-MU-Gal in 0.06 molar phosphate citrate buffer, pH 4.7. For acid alpha-glucosidase, add 2 millimolar 4-methylumbelliferyl-alpha-D-glucopyranoside or 4-MU-Glu in 0.025 molar sodium acetate, pH 4.0.
Incubate the enzyme reactions for 1 hour in the dark at 37 degrees Celsius and 300 RPM on an orbital shaker. Then, terminate the reaction by adding 200 microliters of 1.0 molar pH 10.5 adjusted glycine sodium hydroxide buffer. Prepare a standard curve of 4-MU from a 0.01 milligram per milliliter stock according to the text protocol, and pipette 10 microliters of the solutions in duplicate into a 96-well plate.
Add 200 microliters of the 1.0 molar glycine sodium hydroxide buffer to each well in order to adjust the volume and pH. Finally, measure the enzyme activity in a fluorescence reader equipped with the appropriate filter set, and analyze the data using the appropriate software for the fluorescence reader device.

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1. Cell Harvest and &#945;-galactosidase A or Acid &#945;-glucosidase Activity Measurement
<ol>
	<li>Cell harvest&#8203;
	<ol>
		<li>On the day of the harvest, remove the cells from the incubator and aspirate the medium. Carefully wash the cells 2 times with PBS with Ca2+ and Mg2+. 
		NOTE: This step is critical because DGJ and DNJ are potent inhibitors of &#945;-galactosidase and &#945;-glucosidase, respectively, and any leftover would invalidate the test.</li>
		<li>Add 200 &#181;L of deionized water directly on top of the cells. Rinse the cells from the plate and transfer them to a 1.5 mL reaction tube.</li>
	</ol>
	</li>
</ol>
2. Homogenization by freezing and thawing
<ol>
	<li>Put the samples in an appropriate foam rack and vortex for 5 s to make the lysis more efficient. Put the samples alternating in liquid nitrogen for 10 s and in a room-temperature water bath until the thawing was complete (5 min).</li>
	<li>Repeat this procedure 5 times and then spin the samples for 5 min at 10,000 x g. Retain the supernatant and pipette in a new reaction tube.</li>
</ol>
3. Protein concentration determination using bicinchoninic acid (BCA) assay
<ol>
	<li>Prepare a fresh tube for each sample containing 40 &#181;L of deionized H2O and add 10 &#181;L of the sample. Mix solution by vortexing briefly and transfer 10 &#181;L into a cavity of a 96 well plate (each sample in triplicate). Dilute a 2 mg/mL bovine serum albumin (BSA) stock solution in deionized H2O as follows to obtain a standard curve: 50 &#181;L H2O/50 &#181;L BSA; 60 &#181;L H2O/40 &#181;L BSA; 70 &#181;L H2O/30 &#181;L BSA; 80 &#181;L H2O/20 &#181;L BSA; 90 &#181;L H2O/10 &#181;L BSA; 100 &#181;L H2O.</li>
	<li>Start the reaction by adding 200 &#181;L of BCA reagent (Reagent A and reagent B mixed at a 50:1 ratio) and incubate for 1 h in the dark at 37 &#176;C under slight agitation on an orbital shaker (300 rpm). Measure the absorbance at 560 nm in a plate reader. 
	NOTE: The samples typically contain between 1 and 1.5 &#181;g protein per &#181;L.</li>
</ol>
4. Enzyme activity measurement with artificial 4-Methylumbelliferyl substrates (4-MUG)
<ol>
	<li>Dilute the calculated amount of each sample and pipet into fresh 1.5 mL reaction tubes to obtain 0.05 (for &#945;-galactosidase A) or 0.5 (for acid &#945;-glucosidase) &#181;g protein/&#181;L solutions. Vortex the samples for 5 s again and pipet 10 &#181;L of this dilution into a 96-well plate (each sample in duplicate).</li>
	<li>Start the reaction by adding 20 &#181;L of the respective substrate solution: 
	For &#945;-galactosidase A: 2 mM 4-Methylumbelliferyl-&#945;-D-galactopyranoside (4-MU-gal) in 0.06 M phosphate citrate buffer, pH 4.7. 
	For acid &#945;-glucosidase: 2 mM 4-methylumbelliferyl &#945;-D-glucopyranoside (4-MU-glu) in 0.025 M sodium acetate, pH 4.0.</li>
	<li>Incubate the enzyme reactions for 1 h in the dark at 37 &#176;C under slight agitation on an orbital shaker (300 rpm). Terminate the reaction by the addition of 200 &#181;L of 1.0 M, pH 10.5 adjusted glycine-NaOH buffer.</li>
	<li>Prepare a standard curve of 4-methylumbelliferone (4-MU) from a 0.01 mg/mL stock as follows: 
	100 &#181;L H2O/ no 4-MU; 80 &#181;L H2O/ 20 &#181;L 4-MU; 60 &#181;L H2O/ 40 &#181;L 4-MU; 40 &#181;L H2O/ 60 &#181;L 4-MU; 20 &#181;L H2O/ 80 &#181;L 4-MU; no H2O/ 100 &#181;L 4-MU, pipet 10 &#181;L of each dilution into the 96 well plate (in duplicates) and add 200 &#181;L of the 1.0 M glycine-NaOH buffer to each well in order to adjust the volume and pH.</li>
	<li>Measure the enzyme activity in a fluorescence reader equipped with the appropriate filter set and analyze the data using the appropriate software for the fluorescence reader device. 
	NOTE: Both 4-MUG substrates are reduced to 4-MU during exposure to &#945;-galactosidase A or acid &#945;-glucosidase. Released 4-MU is a fluorochrome, which can be measured at 360 and 465 nm as the excitation and emission wavelengths, respectively, using a microplate fluorescence reader.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,06,40,41,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,04,93,60,500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x 6H2O</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,05,83,30,250</td>
			<td></td>
		</tr>
		<tr>
			<td>D (+)-Glucose monohydrate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,08,34,22,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,06,49,80,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,06,26,40,050</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293H cell line</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11631-017</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeSeal reaction tubes, 1.5mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>72,706</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan GENios Reader</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magellan data analysis software v6.6</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>N.A</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Lukas, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56550">In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease.</a> J. Vis. Exp. (2017)
This video demonstrates the in vitro measurement of enzyme activity for &#945;-galactosidase A and acid &#945;-glucosidase in samples using synthetic 4-Methylumbelliferyl substrates.

1. Cell Harvest and &#945;-galactosidase A or Acid &#945;-glucosidase Activity Measurement

	Cell harvest&#8203;
	
		On the day of the harvest, remove the ...

&#945;-galactosidase&#160; A and acid &#945;-glucosidase are lysosomal enzymes crucial for cellular metabolism.
&#945;-galactosidase A breaks down substrates such as glycolipids, while acid &#945;-glucosidase hydrolyzes glycogen.
To measure their enzyme activity in vitro, take multi-well plate wells with samples containing known concentrations of &#945;-galactosidase A and acid &#945;-glucosidase, respectively.
Add the required volume of acidic solutions of synthetic 4-Methylumbelliferyl&#160; substrates to the wells, specific fluorogenic substrates designed to mimic natural substrates for these enzymes.
Incubate in the dark.
&#945;-galactosidase A and acid &#945;-glucosidase enzymes cleave their respective substrates, releasing the fluorescent product, 4-methylumbelliferone.
Add an alkaline buffer to terminate the enzymatic reaction.
Using a microplate fluorescence reader, measure the fluorescence intensity of the 4-methylumbelliferone in the wells, indicative of its concentration, to determine the &#945;-galactosidase A and acid &#945;-glucosidase enzyme activity, respectively.

65 Görüntüleme. α-Galaktosidaz A ve Asit α-Glukozidaz Enzim Aktivitesinin İn Vitro Ölçümü

Video: α-Galaktosidaz A ve Asit α-Glukozidaz Enzim Aktivitesinin İn Vitro Ölçümü - Deney

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

Tüm fare embriyo etkinliğinde β-galaktozidaz tespiti yoluyla Gen ifadesinin izleme

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

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Gelişim Biyolojisi

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

Metabolik eşleme: Nicel enzim Cytochemistry ve Histochemistry Dehydrogenases hücre ve dokulara etkinliğini belirlemek için

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...

İmmünoloji ve Enfeksiyon

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Çok enzim Yüksek verim Genetik Enzim Eleme Sisteminin Kullanılması Eleme

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

In Vitro Measurement of α-Galactosidase A and Acid α-Glucosidase Enzyme Activity

TRANSKRIPT

In Vitro Measurement of α-Galactosidase A and Acid α-Glucosidase Enzyme Activity