Characterization of detergent-solubilized beef liver mitochondrial NAD+ glycohydrolase and its truncated hydrosoluble form

Biochemistry
M ZieglerM Schweiger

Abstract

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 s...Continue Reading

References

Jan 1, 1990·Annual Review of Physiology·R M Denton, J G McCormack
Jan 1, 1987·Annual Review of Biochemistry·E Carafoli

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Citations

Feb 21, 2002·Biochimica Et Biophysica Acta·Andrea ThieleSunna Hauschildt
Jul 24, 2002·Protein Expression and Purification·Shuang Ding WuZhengang Zhu
May 15, 2001·The EMBO Journal·A Herrero-YraolaM Ziegler
Dec 26, 2001·Proceedings of the National Academy of Sciences of the United States of America·E ZocchiC Usai
Sep 27, 2003·The European Journal of Neuroscience·Christian T ShelineDennis W Choi
Jan 8, 1999·Biochemical and Biophysical Research Communications·N Matsumura, S Tanuma
Oct 31, 2001·European Journal of Biochemistry·M PfisterS Hauschildt
Mar 11, 2000·European Journal of Biochemistry·M Ziegler
Mar 14, 2021·Nature Communications·Øyvind StrømlandMathias Ziegler
Apr 13, 2005·Biochemical and Biophysical Research Communications·Guang-Hua XieUh-Hyun Kim

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