Transferases

Transferases are a class of enzymes that catalyze the transfer of specific functional groups (e.g. a methyl or glycosyl group) from a donor molecule to an acceptor molecule. They are involved in a myriad of vital cellular processes including the transfer of thiol esters, metabolism of tryptophan and the regulation of pyruvate dehydrogenase (PDH). Transferases are classified primarily based on the type of biochemical group transferred, there are 10 categories in all comprising over 400 unique enzymes.

Common classifications include:

• Methyltransferases and formyltransferase – enzymes that transfer single-carbon groups methyl (-CH3) and formyl (-CHO) groups
• Transketolase and transaldolase – enzymes that transfer ketone or aldehyde group
• Oxi aminotransferase or transaminase – enzymes that transfer nitrogenous groups
• Sulfotransferase – enzymes that transfer sulfur-containing groups

AAT Bioquest provides a series of “mix and read” transferase assay kits which are compatible with HTS liquid handling instruments, and also offers fluorogenic substrate to quantify transferase activity in solutions.

Alanine Aminotransferase

Alanine aminotransferase (ALT), also called serum glutamate pyruvate transaminase (GPT), catalyzes the reversible transfer of an ?-amino group between alanine and glutamate, and is an important enzyme in amino acid metabolism. ALT is mainly found in the liver as well as in the heart, muscle, and kidneys but at much lower levels. In healthy subjects, serum ALT levels are relatively low. However, when cells are damaged, such as during acute and chronic hepatitis, obstructive jaundice, carcinoma of liver, myocardial infarction, ALT leaks into the blood stream causing a significant increase in serum ALT levels. Therefore, determination of serum ALT levels has great clinical and diagnostic significance.

Amplite™ Fluorimetric Alanine Aminotransferase Assay

Amplite™ Fluorimetric Alanine Aminotransferase assay kit (Cat No. 13802) provides a quick and sensitive method to measure ALT concentrations in various biological samples. The product glutamate is measured by the generation of a red fluorescent product through an enzyme coupled reaction cycle. The signal can be read using a fluorescence microplate reader at Ex/Em = 530-570 / 590-600 nm (optimal Ex/Em = 540 / 590 nm). With Amplite™ Fluorimetric Alanine Aminotransferase Assay Kit, as little as 4 mU/mL ALT was detected in a 100 ?L reaction volume. The assay is robust, and can be readily adapted for a wide variety of applications.

Figure 1. ALT dose response was measured with Amplite™ Fluorimetric Alanine Aminotransferase Assay Kit (Cat No. 13802) in a 96-well black plate using a Gemini fluorescence microplate reader.

Amplite™ Colorimetric Alanine Aminotransferase Assay

The product glutamate can also be measured using Amplite™ Colorimetric Alanine Aminotransferase Assay Kit (Cat No. 13803) by the generation of a blue color product through an enzyme coupled reaction cycle. The signal can be read using an absorbance microplate reader at ?575 nm or by the absorbance ratio of A570 nm to A610 nm to increase assay sensitivity. With Amplite™ Colorimetric Alanine Aminotransferase Assay Kit, as little as 10 mU/mL ALT was detected in a 100 ?L reaction volume.

Figure 2. ALT dose response was measured with Amplite™ Colorimetric Alanine Aminotransferase Assay Kit (Cat No. 13803) in a 96-well white, clear-bottom plate using a SpectraMax microplate reader (Molecular Devices).

Aspartate Aminotransferase

Aspartate aminotransferase (AST), also called serum glutamic oxaloacetic transaminase (GOT), catalyzes the reversible transfer of an ?-amino group between aspartate and glutamate, and is an important enzyme in amino acid metabolism. AST is mainly found in tissues such as liver, heart, muscle, kidneys and brain. In healthy subjects, serum AST levels are relatively low. However, when cells are damaged, such as during acute and chronic hepatitis, obstructive jaundice, carcinoma of liver, myocardial infarction, AST leaks into the blood stream causing a significant increase in serum AST levels. Therefore, etermination of serum AST levels has great clinical and diagnostic significance.

Amplite™ Fluorimetric Aspartate Aminotransferase Assay

Amplite™ Fluorimetric Aspartate Aminotransferase (AST) Assay Kit (Cat No. 13800) provides a quick and sensitive method to measure AST concentrations in various biological samples. The product L-glutamate is measured by the generation of a red fluorescent product through an enzyme coupled reaction cycle. The signal can be read by a fluorescence microplate reader at Ex/Em = 530-570 / 590-600 nm (optimal Ex/Em = 540 / 590 nm). With Amplite™ Fluorimetric Aspartate Aminotransferase Assay Kit, as little as 2 mU/mL AST in a 100 µL reaction volume. The assay is robust, and can be readily adapted for a wide variety of applications.

Figure 3. AST dose response was measured with Amplite™ Fluorimetric Aspartate Aminotransferase Assay Kit (Cat No. 13800) in a 96-well black plate using a Gemini fluorescence microplate reader.

Amplite™ Colorimetric Aspartate Aminotransferase Assay

The product glutamate can also be measured using Amplite™ Colorimetric Aspartate Aminotransferase Assay Kit (Cat No. 13801) by the generation of a blue color product through an enzyme coupled reaction cycle. The signal can be read using an absorbance microplate reader at ?575 nm or by the absorbance ratio of A570 nm to A610 nm to increase assay sensitivity. With Amplite™ Colorimetric Aspartate Aminotransferase Assay Kit, as little as 3 mU/mL AST was detected in a 100 ?L reaction volume.

Figure 4. AST dose response was measured with Amplite™ Colorimetric Aspartate Aminotransferase Assay Kit (Cat No. 13801) in a 96-well white, clear-bottom plate using a SpectraMax microplate reader (Molecular Devices).

HDAC Activity Assay

Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from a ?-N-acetyl lysine amino acid on a histone. Deacetylation restores the positive electric charge of the lysine amino acids, which increases the histone’s affinity to the negatively charged phosphate backbone of DNA. This process generally down-regulates DNA transcription by blocking the access of transcription factors. HDAC inhibitors are being studied as a treatment for cancers.

Amplite™ Fluorimetric HDAC Activity Assay

Amplite™ Fluorimetric HDAC Activity Assay Kit (Cat No. 13601) provides a quick, convenient, and sensitive method for the detection of HDAC activity. This kit uses our non-peptide HDAC Green™ substrate that is much more sensitive than conventional peptide based HDAC substrates such as Ac-RGK(Ac)-R110, Ac-RGK(Ac)-AMC and Ac-RGK(Ac)-AFC. In addition, HDAC Green™ substrate is also much more resistant to protease hydrolysis than other commercial peptide-based HDAC substrates. Our kit can be used for measuring HDAC activity in cell lysates or HDAC inhibitor screening with cell extracts or purified enzymes. The long wavelength emission of the HDAC Green™ substrate makes the assay less interfered from compounds and cell components. HDAC activity is monitored with excitation at 490 nm and emission at 525 nm.

Figure 5. HDAC activity in HeLa nuclear extract was measured with Amplite™ Fluorimetric HDAC Activity Assay Kit (blue) was compared with Vendor X (red) and Vendor Y (green), both of which use Ac-RGK(Ac)-R110 peptide substrate. The signal/background ratio of the HDAC activity measured with Amplite™ Fluorimetric HDAC Activity Assay Kit is more than 10 times higher than those of Vendors X and Y.

Table 1. Ordering Info for Transferase Assays Products