Purification, characterization and analysis of rolipram inhibition of a human type-IVA cyclic AMP-specific phosphodiesterase expressed in yeast

Abstract

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.

Characterization of phosphodiesterase 4 in guinea‐pig macrophages: multiple activities, association states and sensitivity to selective inhibitors

“…A consistent finding of these studies was that CPPDE4α and CPPDE4β were significantly more thermolabile than the two activities present in macrophage membranes, suggesting that the association of PDE4 with macrophage membranes confers considerable thermostability. This concept is entirely consistent with results of experiments published by Houslay and colleagues, where a particulate PDE4A subtype in rat brain (Shakur et al , 1993) and yeast (Wilson et al , 1994) is significantly more thermostable than the soluble or detergent‐solubilized form of the same enzyme.…”

The Human Cyclic AMP-specific Phosphodiesterase PDE-46 (HSPDE4A4B) Expressed in Transfected COS7 Cells Occurs as Both Particulate and Cytosolic Species That Exhibit Distinct Kinetics of Inhibition by the Antidepressant Rolipram

“…This generated the reported clone h6.1 which thus bears close similarity to h-PDE1 (17). However, h-PDE1 (17) differs from h6.1 (32) in having certain base changes, which would lead to five differences in amino acids found in and around the putative catalytic region of the protein and which have been suggested (32,39) might account for the differences in rolipram inhibition kinetics exhibited by these two forms. Excluding the engineered region, the nucleotide sequence of h6.1 (32) exactly matches that which can be found in the human PDE4A genomic sequence 2 and, with one base difference, is identical to that reported for the cognate region of PDE46 (16).…”

“…From these we were able to derive K i values of ϳ1.6 M for the association of rolipram with cytosolic PDE-46 (Table I). Such a value is slightly greater than that recorded for h6.1 where, similarly, rolipram acts as a simple competitive inhibitor (32,39). Double reciprocal plots of particulate PDE activity, done at different rolipram concentrations, exhibited a common intersection on the y axis (Fig.…”

“…In contrast, to our knowledge, h-PDE1 (17) has not been shown to be expressed natively. Nevertheless, at a minimum, h-PDE1 (17) and h6.1, the species we generated (32,39) to mimic it, provide useful representations of what we might presume, from analogy with the rat PDE4A splice variants and the species met 26 RD1 (27) (Fig. 1), as the "core" human PDE4A gene product (16,27,30,31,41).…”

“…Intriguingly, however, using h6.1 expressed in S. cerevisiae, rigorous kinetic analyses served only to show that simple competitive kinetics of inhibition by rolipram ensued (39). It has been suggested (39) that the differences in inhibition between these two forms might reflect differences in the sequence of these forms at residues in and around the catalytic region.…”