Human Recombinant Phosphodiesterase 4B2B Binds (R)-Rolipram at a Single Site with Two Affinities

• Published in: Biochemistry (Easton) 1997-11, Vol.36 (46), p.14250-14261
• Main Authors: Rocque, Warren J, Tian, Gaochao, Wiseman, Jeffrey S, Holmes, William D, Zajac-Thompson, Irene, Willard, Derril H, Patel, Indravaden R, Wisely, G. Bruce, Clay, William C, Kadwell, Sue H, Hoffman, Christine R, Luther, Michael A
• Format: Article
• Language: English
• Subjects: 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Binding Sites; Calorimetry; Centrifugation, Isopycnic; Cyclic Nucleotide Phosphodiesterases, Type 4; Dialysis; Humans; Isoenzymes - metabolism; Nerve Tissue Proteins - metabolism; Phosphodiesterase Inhibitors - metabolism; Pyrrolidinones - metabolism; Recombinant Proteins - metabolism; Rolipram; Time Factors
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi971112e

The interactions between (R)-rolipram and purified human recombinant low-K m, cAMP-specific phosphodiesterase (HSPDE4B2B) constructs were investigated using biochemical, kinetic, and biophysical approaches. The full-length protein (amino acids 1−564) and an N-terminal truncated protein (amino acids 81−564) exhibited high-affinity (R)-rolipram binding, whereas an N-terminal and C-terminal truncated protein (amino acids 152−528) lacked high-affinity (R)-rolipram binding. The 152−528 and 81−564 proteins had similar K m's and k cat/K m's and differed less than 4-fold compared with the 1−564 protein. (R)-Rolipram inhibition plots were biphasic for the 1−564 and 81−564 proteins and fit to two states, a high-affinity (K i = 5−10 nM) state and a low-affinity (K i = 200−400 nM) state, whereas the 152−528 protein fit to a single state (K i = 350−400 nM). The stoichiometry for high-affinity binding using a filter binding assay was found to be