Inhibition of human phosphodiesterase 4A expressed in yeast cell GL62 by theophylline, rolipram, and acetamide-45.
OBJECTIVE
To study the inductive expression of human phosphodiesterase 4A (hPDE4A) in yeast cell GL62 and investigate the inhibitory effects of theophylline, rolipram, and acetamide-45 on PDE4A activity of the expressed product in yeast cell GL62.


METHODS
Yeast cell GL62 were transfected with human PDE4A gene cloned in the expression plasmid p138NB. Expression was induced by adding CuSO4 to a final concentration of 150 micromol/L, and the expressed product was extracted. The activity of PDE4A was detected by HPLC.


RESULTS
Yeast cell GL62 expressed a certain protein at CuSO4 150 micromol/L, the size of the expressed product was between 62 kDa and 83 kDa, the activity of PDE4A of the expressed product at 3 h was in maximum (188 23) micromol/g/min, and the Km was (17.7 2.6) micromol/L. Theophylline, rolipram, and acetamide-45 could inhibit the activity of PDE4A extracted from yeast cell GL62. The IC50 (95 % confidence limits) of theophylline, rolipram, and acetamide-45 were 1642 (989-2727), 4.58 (3.45-6.08), and 275 (170-444) micromol/L respectively.


CONCLUSIONS
PDE4A expressed in yeast cell GL62 is biologically active. Theophylline, rolipram, and acetamide-45 can inhibit the PDE4A activity. The expressed product in yeast cell GL62 may be used in the research work of PDE4 and its inhibitors.
Wang
Kai
K
Respiratory Drugs Research Laboratory of State Drugs Administration of China,Medical School of Zhejiang University, Hangzhou 310031, China.
Chen
Ji-Qiang
JQ
Chen
Zhong
Z
Chen
Jun-Chun
JC
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism*; D000081:Acetamides / Q000494:pharmacology*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D020889:Rolipram / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D013806:Theophylline / Q000494:pharmacology*; D014162:Transfection
D000081:Acetamides; D010726:Phosphodiesterase Inhibitors; C030686:acetamide; D013806:Theophylline; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517355:PDE4A protein, human; D020889:Rolipram