Determination method for activity of phospholipase D alpha

A technology of phospholipase and activity, which is applied in the field of determination of phospholipase Dα activity, can solve the problems of physical injury to operators, high measurement sensitivity, and time-consuming operation, and achieve the effect of easy operation, high sensitivity and fast method

Inactive Publication Date: 2014-11-19
SHANGHAI UNIV
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Problems solved by technology

The detection result of thin plate chromatography is accurate, but the operation is time-consuming and easily interfered by exogenous phospholipids
The radioactive isotope labeling method has hig

Method used

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  • Determination method for activity of phospholipase D alpha
  • Determination method for activity of phospholipase D alpha
  • Determination method for activity of phospholipase D alpha

Examples

Experimental program
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experiment example 1

[0021] Additional experimental example 1 Determination of protein content

[0022] At 4°C, take 7 g of peach fruit and use HEPES buffer solution (pH 7.0, containing 0.32 mol / L sucrose, dithiothreitol, phenylmethylsulfonyl fluoride, and EGTA each 1 mmol / L) to make 20 % homogenate, centrifuged at 12,000g for 45min, and the supernatant was centrifuged at 105,000g for 1 hour. The precipitated part was the membrane component of the cell membrane, which was dissolved in 100mmol / L DMG at pH6.5 to obtain the crude enzyme extract of PLDα, and the protein was determined by the Coomassie brilliant blue method content. Take the protein content as the abscissa and the activity of phospholipase Dα as the abscissa figure 2 .

[0023] exist figure 2 Among them, BA represents the peach fruit soaked in n-butanol and stored at 4°C for 28 days, and CK represents the peach fruit without any treatment and stored at 4°C for 28 days.

[0024] From figure 2 It can be seen that in the 200μl rea...

experiment example 2

[0025] Additional experimental example 2 Ca 2+ Determination of concentration

[0026] In the 200μl reaction system, the final concentrations of calcium ions added were 0, 0.05mM, 0.1mM, 1mM, 10mM, and 50mM. The final concentration of calcium ions was taken as the abscissa, and the activity of phospholipase Dα was taken as the ordinate. image 3 .

[0027] From image 3 It can be seen that the activity of phospholipase Dα increases with Ca 2+ The increase of the concentration presents a fluctuating growth trend, when the Ca 2+ When the concentration reaches 10mM, phospholipase Dα shows the maximum activity, see image 3 . Determination of Ca in Peach Fruit Phospholipase Dα Activity 2+ The concentration is preferably 10mM.

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Abstract

The invention provides a determination method for the activity of phospholipase D alpha. The determination method for the activity of the phospholipase D alpha is a method for determining the activity of the phospholipase D alpha by using an enzyme-linked colorimetry. The technical scheme is that the determination method comprises the following steps: performing catalyzed hydrolysis on a phosphodiester bond at the tail end of phosphatidylcholine serving as a substrate by the phospholipase D alpha to generate phosphatidic acid and choline; enabling the choline to react to generate betaine and H2O2 under catalytic action of a choline oxidase; oxidizing 4-aminoantipyrine aspirin and redistilled phenol into a pink substance by the generated H2O2 under the catalytic action of peroxidase, determining the absorbance value of the pink substance, determining the corresponding choline content according to a choline standard curve which is prepared in advance, and deducing the activity of the phospholipase D alpha according to the choline content. In a reaction system with 200 microliters of the phospholipase D alpha, the protein content and the Ca<2+> concentration are preferably 10 microgramme and 10mM. The determination method is high in efficiency, convenient, quick, high in sensitivity, low in hazard and stable in results, and is suitable for simultaneously determining the activities of the phospholipase D alpha of a large number of samples.

Description

technical field [0001] The invention relates to a method for determining the activity of phospholipase Dα, in particular to the determination of the activity of phospholipase Dα by an enzyme-linked colorimetric method and the selection of key parameters in the determination process. Background technique [0002] Phospholipase D (PLD), namely phosphatidylcholine phospholipid hydrolase (EC3.1.4.4), is a general term for a class of enzymes that catalyze the hydrolysis of phosphodiester bonds and base exchange reactions. Phospholipase D was first obtained from carrot root and spinach leaf extracts by Hanahan and Chaikoff. Phospholipase D widely exists in various organisms from prokaryotic bacteria to higher animals and plants, and is the main phospholipase in plants, mainly existing on the cell membrane, and also exists in a small amount in the cytoplasm. Studies have shown that in plants, phospholipase D has physiological functions such as participating in cell lipid metaboli...

Claims

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Application Information

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IPC IPC(8): G01N21/78G01N21/31
Inventor 万嗣宝张宏宇李伟丽朱月莹
Owner SHANGHAI UNIV
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