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United States Department of Agriculture

Agricultural Research Service

Research Project: Engineering Enzymatic Redirection of Natural Crop Oil Production to Industrial Oil Production Title: Measuring phosphatidic acid phosphatase (EC 3.1.3.4) activity using two phosphomolybdate-based colorimetric methods

Authors
item Ullah, Abul
item Sethumadhavan, Kandan
item Shockey, Jay

Submitted to: Advances in Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 21, 2012
Publication Date: November 30, 2012
Citation: Ullah, A.H.J., Sethumadhavan, K., Shockey, J. 2012. Measuring phosphatidic acid phosphatase (EC 3.1.3.4) activity using two phosphomolybdate-based colorimetric methods. Advances in Biological Chemistry. 2:416-421.

Interpretive Summary: The biocatalyst, phosphatidic acid phosphatase (PAP), performs a committed step in the production of oil in the seed of oil-producing crops such as soybean, cottonseed, peanut, etc. If the biocatalyst (enzyme) functions sub-optimally because of poor recognition of the substrate or fatty acids, the oleaginous plants won’t produce oil efficiently. The enzyme works at the penultimate step of the Kennedy Pathway, which is responsible for oil synthesis in plants seeds. One of the goals of our CRIS is to engineer plants to produce oil containing polyunsaturated fatty acids or PUFA. To achieve this goal, we have to make sure that PAP could recognize and accept PUFA as a substrate for PAP. Therefore, we need to develop enzyme assay that is easy and convenient to use in the laboratory. Traditionally, radioactive fatty acids are used to carry out PAP activity, which is not user-friendly and costly. The method we report in this paper is non-radioactive and less costly. Not to mention, the health hazard created by the handling of radio-isotopes was totally eliminated.

Technical Abstract: Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), which is also known as PAP, catalyzes the dephosphorylation of phosphatidate (PtdOH) to form diacylglycerol (DAG) and inorganic phosphate. In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacylglycerol (TAG), the most common storage lipid in biological systems. Existing methods for measuring PAP activity rely on the use of radioactive phosphatidic acid. These methods are costly and cumbersome. In this report, we describe a simple assay procedure to measure released inorganic orthophosphate, which is a co-product of the PAP reaction. Each molecule of PtdOH would release one molecule of DAG and one molecule of inorganic orthophosphate (Pi) when subjected to enzymatic breakdown under optimal conditions. Given the published rates of in vitro PAP enzymatic activity from various sources, we proposed that colorimetric determination of released Pi is possible. With this view, we performed in vitro PAP activity assays using freshly isolated enzyme from bitter gourd, Momordica charantia, and measured the released Pi using two spectrophotometric methods. Both methods gave about 2.0 to 2.25 'kat per mg of protein. Thus, it is now possible to perform PAP activity assays using a simple procedure that uses nonradioactive substrates, provided the sample is dialyzed extensively to lower the intrinsic concentration of free phosphate. The kinetics data presented in this study is comparable to that of other PAP enzymes reported elsewhere, which gives credence to the notion that non-radioactive methods can be used to perform PAP activity.

Last Modified: 11/21/2014
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