Purification, characterization and analysis of rolipram inhibition of a human type-IVA cyclic AMP-specific phosphodiesterase expressed in yeast.
Analyses were done on a human type-IV cyclic AMP (cAMP) phosphodiesterase (hPDE-IVA-h6.1) expressed in an engineered strain of Saccharomyces cerevisiae. This strain (YMS6) expressed soluble PDE activity, together with an insoluble activity which was not released by re-homogenization, treatment with high-ionic-strength solutions or with the detergent Triton X-100. Pellet and soluble PDE activities were typical of type-IV PDE. They were cAMP-specific, insensitive to the addition of either cGMP (1 microM) or Ca2+/calmodulin, and inhibited by rolipram. Thermostability studies showed both activities to decay as single exponentials, indicating the presence of homogeneous PDE protein species in each fraction. Pellet PDE activity was more thermostable than the soluble enzyme. Mg2+ and Mn2+ dose-dependently increased PDE activity and reversed the inactivating effect of EDTA.h6.1 was engineered to express a C-terminal five-histidine motif (h6.1his5). This allowed purification of the PDE to apparent homogeneity in a simple two-step process involving a rolipram affinity column and a Ni2(+)-chelate column. A single monomeric protein of subunit molecular mass approximately 73 kDa and native molecular mass approximately 74 kDa resulted after a approximately 53000-fold purification. This exhibited a Km for cAMP of 8 microM, a true Vmax. of 0.8 mumol of cAMP hydrolysed/min per mg of PDE protein, a kcat. of 3702 s-1, and a value of the specificity constant kcat/Km of 4.6 x 10(8) M-1.s-1, the last implying a diffusion controlled reaction. Rolipram (Ki 0.4 soluble; 0.7 microM pellet) and 3-isobutyl-1-methylxanthine (Ki 15 soluble; 19 microM pellet) served as simple competitive inhibitors for both soluble and pellet forms of h6.1, respectively.
Wilson
M
M
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Sullivan
M
M
Brown
N
N
Houslay
M D
MD
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D000242:Cyclic AMP / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011760:Pyrrolidinones / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D020889:Rolipram; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D011760:Pyrrolidinones; D011994:Recombinant Proteins; D008345:Manganese; D004492:Edetic Acid; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D008274:Magnesium; D020889:Rolipram; D015056:1-Methyl-3-isobutylxanthine