Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1998-01-07
1999-07-13
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 19, 435 4, 435 76, C12Q 142, C12Q 144, C12Q 100
Patent
active
059225572
ABSTRACT:
A CHO-K1 cell line stably expressing a recombinant full-length human PDE IVa (rhPDEIVa) enzyme was established under hygromycin B selection. Inhibition of the expressed PDE IVa activity by selective PDE IV inhibitors was evaluated. The rank order of potencies of the inhibitors in elevating cAMP in the whole-cell assay was quite different from that on the soluble enzyme. The whole-cell rank order of potencies was also maintained when PKA activity ratios were measured in place of cAMP levels. When inhibition of soluble PDE IVa activity was examined in the presence of 100 mM MgCl.sub.2, the rank order of potencies of the inhibitors mirrored that obtained for the whole-cell assays (both cAMP and PKA). The observed "high-affinity" conformation of the enzyme was not dependent upon a specific cation or anion. The shift in sensitivity of the rhPDE IVa enzyme for (R)-rolipram in the presence of increased ionic strength was accompanied by an enhanced capacity of the soluble enzyme preparation to
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Billups Richard C.
Leary Louise N.
Merck & Co. , Inc.
Rose David L.
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