Enzyme Substrates

Enzyme substrates are usually a synthetic molecule that mimics the natural substrate of the enzyme of interest but with the addition of a reporter molecule that allows for the detection of enzyme activity. Most commonly this detection occurs by the reporter molecule being in some way activated by the removal of the target molecule of the enzyme leading to an increase in signal. This reporter molecule could be a fluorescent dye that is quenched by the presence of the substrate molecule and upon cleavage becomes fluorescent again, this means that the level of fluorescence is directly related to the amount of enzyme activity. As enzyme reactions are kinetic it is possible to see measurable signal when only a small amount of enzyme is present allowing for highly sensitive assays.

Enzyme Substrates

Marker Gene Technologies, Inc. provides substrates for the following enzymes:

Interest in cellulase enzymes has increased throughout the food, fuel, detergent and clothing industries. Degradation of cellulose, a polysaccharide of D-glucose residues connected by β(1-4) glycosidic linkages that aggregates into insoluble fibrils, is especially important in plant and bacterial systems. These ultrasensitive chromogenic and fluorogenic substrates can be used to monitor cellulase activity both in vivo and in vitro.

Cellulases are a category of several enzymes that occur mainly in fungi and bacteria. Cellulases are responsible for  cellulolysis, the breakdown of cellulose and other related polysaccharides.  Cellulase acts by the hydrolysis of the 1,4-beta-D-glycosidic linkages. Cellulases produce shorter chain polysaccharides or monosaccharides from cellulose, such as glucose.

The cytochrome P-450s are a large group of monooxygenase enzymes responsible for the metabolism of toxins in the body. These enzymes, requiring NADPH as a cofactor and O2 as co-substrate, are located in the endoplasmic reticulum and are highly concentrated in the liver and small intestine. P-450 enzymes are very important for drug metabolism and drug interactions and so are medically important enzymes.

Esterases are hydrolase enzymes that split esters into an acid and an alcohol by hydrolysis. Cell permeant esterase substrates are often used for cell viability and cytotoxicity assays in a wide variety of animal cells, as well as bacteria and yeast. These substrates measure enzyme activity and cell-membrane integrity, the latter of which is required for intracellular retention of their products upon enzymatic cleavage.

β-Galactosidase, an important reporter gene encoded by lacZ, is commonly used for monitoring transfection efficiency in mammalian, yeast, and bacterial cells and identifying expression of recombinant fusion genes. E. coli β-galactosidase has a high turnover rate, hydrolyzing D-galactose from various β-galactosides, which allows for very low level detection in common cell types.

β-Glucosidase is a ubiquitous enzyme in virtually all types of cells and catalyzes the hydrolysis of terminal non-reducing residues in beta-D-glucosides with release of glucose.  β-Glucosidase  can used for monitoring cell viability in mammalian, yeast, plant or bacterial cells. Mutations within β-Glucosidase enzymes can lead to diseases such as Gaucher’s disease and has also been linked to Parkinson’s disease.

The β-glucuronidase (GUS) enzyme from E. coli has been well documented to provide desirable characteristics as a marker gene in transformed plants. The GUS reporter gene has many advantages including stable expression of E. coli GUS enzyme, no interference with normal plant metabolism, and low intrinsic GUS activity in higher plants. The enzyme is tolerant of amino-terminal additions, making it useful for study of plant organelle transport.

Glutathione transferases are multifunctional enzymes which play important roles in cellular detoxification. They protect cells from many xenobiotic and endobiotic compounds thru conjugation to glutathione, resulting in neutralization of the toxic species, and the conjugates are then further metabolized to mercapturates and excreted.

Hexosaminidase controls the hydrolysis of terminal N-acetyl-D-hexosamine residues in N-acetyl-β-D-hexosaminides.Two subunits, a and b, coded by genes HEXA and HEXB, comprise the hexosaminidases. Hex A is α and hex B is β. Absence of hexosaminidase A is the cause of Tay-Sachs disease and similar variants, while total hexosaminidase deficiency (hexosaminidase A and hexosaminidase B) occurs in Sandhoff disease.

Lactosidase is a hydrolytic enzyme (a β-galactosidase) that acts on a number of lactosides, cutting either the internal linkage in lactose at the Gal(β1-4)Glc linkage and producing glucosylated aglycones and D-galactose or removing the disaccharide in one step. Most cells also contain β-glucosidase, that can be used to complete release of the fluorophore or chromogen used in common cell assay conditions.

Lipases are a family of enzymes including cholesterol esterases and glycerol ester hydrolases that release fatty acids from triacylglycerols in a site-specific manner. Most lipases have optimum activity for the primary ester groups of triglycerides, while some lipases remove fatty acyl groups from either the C-1 or C-3 acyl positions. The substrate is typically not a single molecule, but a non-aqueous phase of aggregated lipid.

Luciferases are a class of oxidative enzymes used in bioluminescence. Luciferase converts the chemical energy of luciferin oxidation into light  through an electron transition. The reaction releases light when oxyluciferin returns to the ground state. Luciferase has been utilized as a reporter system across almost all model systems.

Peptidases and proteases are important components of cell regulation and signaling, protein activation and synthesis, and other metabolic pathways. Peptidases cleave shorter peptide bonds and are further subdivided depending on their hydrolysis sites: endopeptidases cleave internal peptide bonds while exopeptidases hydrolyze terminus residues.

Peroxidases are a group of enzymes that catalyze oxidation by the decomposition of hydrogen peroxide or an organic peroxide. The most commonly used oxidase is horseradish peroxidase (HRP), which has been coupled with sensitive fluorogenic and chromogenic substrates to quantitate intracellular hydrogen peroxide production as well as a diverse assortment of analytes including glucose, galactose, cholesterol, and uric acid.

Phosphatases are a group of enzymes that remove a phosphate group and utilize a variety of phosphate and polyphosphate esters as substrates, including phospholipids, phophorylated proteins, nucleotides, membrane structural components, and energy reservoirs. Simple phosphatases, such as alkaline and acid phosphatase, hydrolyze phosphate monoesters to an alcohol and inorganic phosphate

 

Proteases are a family of enzymes that cleave proteins and peptides at site-specific peptide linkages within their structures. They are further classified as either endopeptidases, which cleave proteins or peptides at internal peptide linkages, or exopeptidases, which can cleave the terminal amino-acid residues from peptides or proteins. 

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  1. ERLive™
    MarkerGene™ ERLive™ β-Glucosidase Assay Kit
    $220.00

    Product size: 1 kit

    This kit utilizes the sensitive substrate GlucRed to monitor beta-glucosidase activity in the Endoplasmic Reticulum (ER) of live cells.
     
    Learn More

    Product ID: M3102

  2. 5-Carboxyfluorescein di-Acetate, Methyl ester
    5-Carboxyfluorescein di-Acetate, Methyl ester
    $79.00

    Product size: 10 mg

    Brightly fluorescent, membrane permeable esterase substrate for monitoring cell viability, bacterial counts, fungal analysis or for water purity testing. Learn More

    Product ID: M0279

  3. Dibenzylfluorescein
    Dibenzylfluorescein
    $201.00

    Product size: 5 mg

    Cytochrome P450 Substrate Learn More

    Product ID: M4701

  4. p-Nitrophenyl alpha-L-arabinopyranoside
    p-Nitrophenyl alpha-L-arabinopyranoside
    $221.00

    Product size: 2.5 g

    Chromogenic Arabinosidase substrate Learn More

    Product ID: M5295

  5. L-Valine 7-amido-4-methylcoumarin
    L-Valine 7-amido-4-methylcoumarin
    $590.00

    Product size: 0.25 g

    L-Valine 7-amido-4-methylcoumarin is an AMC fluorogenic substrate Learn More

    Product ID: M5294

  6. L-Tyrosine 7-amido-4-methylcoumarin
    L-Tyrosine 7-amido-4-methylcoumarin
    $72.00

    Product size: 0.25 g

    L-Tyrosine 7-amido-4-methylcoumarin is an AMC fluorogenic substrate Learn More

    Product ID: M5293

  7. L-Tryptophan 7-amido-4-methylcoumarin hydrochloride
    L-Tryptophan 7-amido-4-methylcoumarin hydrochloride
    $120.00

    Product size: 50 mg

    L-Tryptophan 7-amido-4-methylcoumarin hydrochloride is an AMC fluorogenic substrate Learn More

    Product ID: M5292

  8. L-Threonine 7-amido-4-methylcoumarin
    L-Threonine 7-amido-4-methylcoumarin
    $353.00

    Product size: 0.25 g

    L-Threonine 7-amido-4-methylcoumarin is an AMC fluorogenic substrate Learn More

    Product ID: M5291

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