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Detection of activity of human PDE4A expressed in yeast GL62 and inhibition of PDE inhibitors & Acetamide45

Author: WangKai
Tutor: ChenJiQiang
School: Zhejiang University
Course: Pharmacology
Keywords: Phosphodiesterase cAMP yeast p138NB Acetamide45
CLC: R965
Type: Master's thesis
Year: 2001
Downloads: 87
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Abstract

BACKGROUNDPDE is the enzyme to hydrolyze cyclic nucleotide. This enzymatic activity terminates the biologic actions of cAMP and cGMP, ubiquitous second messengers that regulate countless biologic processes. According to their substrate, characteristic and distribution PDE exists extensively in body, divided into 11 classes isozyme families, in which PDE4 is characterized by hydrolyzing cAMP, have little effect on cGMP. cGMP with low concentration could not affect the activity of PDE4, but rolipram can inhibit it selectively. Research has established that asthma is characterized by inflammation of the airways. Typically, the airway wall is infiltrated by a variety of cells including mast cells, eosinophils and T lymphocytes. These cells release a plethora of mediators including IL-4, IL-5, GM-CSF and eotaxin, which ultimately cause the histopathological changes and symptoms of asthma. It is wellknown glucocorticosteroids are the only drugs currently available that effectively impact upon the inflammation but it is also known to result in a lot of adverse reaction. New drugs are clearly required. One group of potential therapeutic agents for asthma are inhibitors of PDE4, which can negatively regulate the function of almost all pro-inflammatory and immune cells, and exert widespread anti-inflammatory activity in animal models of asthma. Therefore PDE4 is regarded as an important therapeutic target for asthma. Now PDE4A gene has been identified from the human cDNA library and expressed in the yeast cells, that affords a new opportunity for the research of PDE4 inhibitors.AIMSThe present study aims to detect the activity of PDE4A extracted from yeast GL62 which have been transfected human PDE4A gene ligated in the vector P138NB, then investigate the inhibitory effect on it by theophylline, rolipram and acetarm’de45.MATERIALS AND METHODSYeast cells were grown aerobically, in YPD medium at 30 癈, to A540 = 1.0. PDE4A expression was induced by the addition of 150uM CuSCX and protein extraction was done byY-PER after harvested at various times. Extract 20ul was added into the reaction system where there was 20 ul substrate (cAMP or cGMP) and total volume is 200 |jl in which the last concentration of substrate is 1 uM. Another 20ul extract that has been inactivated was as control. Reactions were conducted at 37 癈 for 30 minute and terminated in 100 癈, then centrifuged at 12000g for 30 minute at 4 癈 and take supernatant to HPLC to detect the remaining cAMP. Enzyme activity = [cAMP remaining (reaction) - cAMP remaining(control)]/reaction time/protein weigh(pmol/min/mg). Theophylline, Rolipram and Acetamide 45 were dissolved at lOmM by 100% DMSO and diluted into the various concentration when needed. Same experiment procedure were carried out as before. We also use SDS-PAGE to analyze the extract and use agarose electrophoresis to analyze the size of DNA extracted from GL62.RESULT1.The analysis of agarose electrophoresisThe plasmids P138NB-hPDE4A extracted from GL62 were mixed with endonuclease Xhol and Nhel, then do agarose electrophoresis. Obvious strip was observed between 2500bp and3000bp(Figl).2.The analysis of SDS-PAGEThe extract inducing at 3h and 6h show obvious strip between 62KD and 83KD, but the extract inducing Oh and Ih just show shallow strip in corresponding site (Fig2).3.The analysis of PDE4A activity on cAMP of GL62 extract The activity is concerned with inducing time. From 0 to 3 h, the activity increases gradually. But from 3 to 9 h, the activity do not show obviously change (Fig3).4.The effect of cAMP concentration on velocity of enzyme reactionThe l/[cAMP] is designated as abscissa, 1/[velocity of enzyme reaction](l/V) is ordinate and make plot as Lineweaver-Burk method. According to Michaelis equation, the Km is 17.7 + 2.6uM (Fig4).5. The hydrolysis effect of GL62 extract on cGMP and the effect of cGMP on PDE4A activity of GL62The hydrolysis activity of GL62 extract on cGMP is 8.48?1.2pmol/mg/min, while the activity on cA

Detection of activity of human PDE4A expressed in yeast GL62 and inhibition of PDE inhibitors & Acetamide45

Abstract

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