Characterization of detergent-solubilized beef liver mitochondrial NAD+ glycohydrolase and its truncated hydrosoluble form.
Membrane-bound beef liver mitochondrial NAD+ glycohydrolase (NADase) was partially purified after its solubilization by either detergent or crude pancreatic lipase, steapsin. Solubilization by steapsin yielded a homogeneous water-soluble enzyme. A fluorescence assay was developed that allowed visualization of NADase activity directly within the gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular masses of the detergent- and steapsin-solubilized forms were estimated to be about 30,000 and 28,000, respectively. The small part that was cleaved by steapsin represents presumably the membrane anchor of the mitochondrial NADase, as its removal converted the enzyme from a highly hydrophobic to a hydrosoluble protein. The fluorescence staining for activity was also successfully applied to other NADases. Kinetic analyses of the two forms of solubilized mitochondrial NADase revealed that the catalytic properties were unaffected after the steapsin treatment. Neither the binding affinity of the substrate analog 1, N6-etheno-NAD+ nor the inhibition by nicotinamide differed significantly between these two forms of the enzyme. Moreover, the dependence of the enzyme activity on temperature, pH, or ionic strength was also similar for both preparations. However, activity of the detergent-solubilized but not of the truncated steapsin-solubilized enzyme was strongly dependent on the presence of bivalent metal ions such as ZN2+. These results suggest that the membrane part of the mitochondrial NAD+ glycohydrolase is not required for catalysis. It appears, however, to be of importance for the regulation of the enzyme.
Ziegler
M
M
Institute of Biochemistry, Free University Berlin, Germany. mziegler@chemie.fu-berlin.de
Jorcke
D
D
Zhang
J
J
Schneider
R
R
Klocker
H
H
Auer
B
B
Schweiger
M
M
D000818:Animals; D002417:Cattle; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008049:Lipase / Q000494:pharmacology; D008565:Membrane Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009244:NAD+ Nucleosidase / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D012995:Solubility
D003902:Detergents; D008565:Membrane Proteins; D010446:Peptide Fragments; D008049:Lipase; D009244:NAD+ Nucleosidase