Specific dinucleoside polyphosphate cleaving enzymes from chromaffin cells: a fluorimetric study

Area of Science:

• Biochemistry
• Enzymology

Background:

• Adrenal medulla cytosolic extracts contain dinucleoside polyphosphate hydrolases.
• Dinucleoside polyphosphates play roles in cellular signaling and metabolism.

Purpose of the Study:

• To characterize the properties of dinucleoside tetraphosphate (asymmetrical) hydrolase (Ap4Aase) and dinucleoside triphosphate hydrolase (Ap3Aase).
• To investigate substrate specificity and kinetic parameters of these enzymes using artificial fluorogenic substrates.

Main Methods:

• Fluorimetric assays using diethenoadenosine polyphosphates (epsilon-(ApnA)) as substrates.
• Enzyme characterization including molecular mass determination, pH optimum, and metal ion requirements.
• Kinetic analysis (Km, Ki) and substrate analog inhibition studies.
• High-performance liquid chromatography (HPLC) for kinetic parameter determination.

Main Results:

• Ap4Aase (20 kDa) has a neutral pH optimum (7.0-7.5), requires Mg2+, and preferentially hydrolyzes tetraphosphate substrates (Km for epsilon-(Ap4A) = 1.3 microM).
• Ap3Aase (30 kDa) has a neutral pH optimum (7.0-7.5), requires Mg2+ or Ca2+, and hydrolyzes triphosphate substrates (Km for epsilon-(Ap3A) = 11 microM).
• Both enzymes showed inhibition by specific substrate analogs and metal ions, with Ap4Aase being strongly inhibited by Ap4 and epsilon-Ap4.

Conclusions:

• The study elucidates key enzymatic properties of Ap4Aase and Ap3Aase from adrenal medulla.
• Characterization provides insights into their substrate specificity and regulatory mechanisms.
• Findings contribute to understanding dinucleoside polyphosphate metabolism in adrenal medulla.