The Activtity Determination Of Protein Phosphatase And Further Exploration On Its Detection In Gel Electrophoresis

Protein phosphorylation and dephosphorylation play quite an important biochemical role in organisms. The research work on dephosphorylation has changed from phosphatase to protein phosphotase, however, only a few protein serine/threonine phosphatases have been reported until now. Due to the lack of a simple, rapid and specific detection method, the study on protein phosphatase suffers a bottleneck. A new method based on protein reactant for detecting the enzyme activity of protein phophatase was established in this research. The natural reactant casein was used to react with protein tyrosine phophatase and the content of product, inorganic phosphate, was then detected by spectrometry to analyze the enzyme activity. The effects of various reagents on the reaction were also studied throughout the process of optimization. It was the first time to involve the natural reactant casein to determine the activity of protein tyrosine phophatase, which can significantly reduced the detection cost as well as the background noise. This method is thus more reliable and more applicable for relevant research. The effect of various methods (such as TCA and ultrafiltration) to removing the reactant protein was studied and optimized accordingly. This new method was cost-effective, rapid, sensitive, low in background noise and simple in operation.The new detection system for protein phophatase activity could be summarized as follows: a 500μL reaction volume (buffer based on the enzyme used) containing 0.2mM casein, 1~2μg phophatase was incubated in 37℃water for 10min; after adding TCA (final concentration 5%), the deposit was excluded by centrifugation with 10,000 rpm for 15min (or ultrafiltration with 7,000 rpm for 50 min); molybdate solution which occupied total volume 1/10 was added into supernatant and incubated in 37℃water for 1h and then detected by spectrometer at 700nm wavelength.In order to simplify the successive work of isolation, rapid identification and analysis of protein phosphatase, it is quite necessary to develop a simple, rapid, specific and sensitive method to isolate protein phosphatase from cytoplasm or protein complex in culture media. Our lab has established a novel phosphatase isoenzyme analysis method based on its substrate. Gel electrophoresis and a novel fluorescence-based detection dye (Pro-Q Diamond Phophoprotein Gel Stain) were employed to separate and analyze the isoenzymes which fulfilled the requirement above. This method started with the reactant casein, which was coupled with the polyacrylamide gel grains using glutaraldehyde linking method. The grains were then added into the electrophoresis gel as a component. After electrophoresis, the enzymatic reaction and a specific staining method performed in situ, the isoenzymes were isolated, negative stained and identified. In this research, we have tested the substrate's specificity and its endurance of proteinase. The coupled substrates were improved upon their actual instance.