JBC, Vol. 251, Issue 18, 5726-5737, Sep, 1976
Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics
A. L. Pichard and W. Y. Cheung
An extract of rat liver or human platelet displayed three cyclic
3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a
continuous sucrose density gradient when assayed with millimolar adenosine
3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The
three fractions obtained from each nucleotide were not superimposable. The
molecular weights corresponding to the three activity peaks of cAMP
phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000;
and III, 140,000. In both tissues, fraction I was barely detectable when
assayed with micromolar concentrations of either nucleotide, presumably
because fraction I has low affinity for cAMP and cGMP. Any one of the three
forms upon recentrifugation on the gradient generated the others,
indicating that they were interconvertible. The multiple forms appear to
represent different aggregated states of the enzyme. The ratio of the three
forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl
cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the
formation of fraction I. Low enzyme concentration favored the equilibrium
towards fraction I, while high enzyme concentration favored fraction III.
When phosphodiesterase activities in the extract of rat liver, human
platelets, or bovine brain were examined as a function of enzyme
concentration, rectilinear rates were observed with micromolar, but not
with millimolar cAMP or cGMP. The specific activity with millimolar cAMP
was higher with low than with high protein concentrations, suggesting that
the dissociated form catalyzed the hydrolysis of cAMP faster than that of
the associated form. In contrast, the specific activity with millimolar
cGMP was lower with low than with high protein concentrations.
Supplementing the reaction mixture with bovine serum albumin to a final
constant protein concentration did not affect the activity, suggesting that
the concentration of the enzyme rather than that of extraneous proteins
affected the enzyme activity. A change in enzyme concentration affected the
kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP
phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X
10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while
a high enzyme concentration yielded a nonlinear plot, and apparent Km
values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X
10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low
enzyme concentration favored fraction I, the dissociated form, whereas a
high enzyme concentration favored fraction III, the associated form, these
kinetic constants suggest that the dissociated form exhibits a high Km and
the associated form exhibits a low Km. In contrast, a high enzyme
concentration gave a linear kinetic plot for cGMP phosphodiesterase, while
a low enzyme concentration gave a nonlinear plot...
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