Subcellular fractionation of bovine thyroid tissue by differential pelleting and isopycnic gradient centrifugation in a zonal rotor indicated that NAD+ glycohydrolase is predominantly located and rather uniformly distributed in the plasma membrane. Comparison of NAD+ glycohydrolase activities of intact thyroid tissue slices, functional rat thyroid cells in culture (FRTl) and their respective homogenates indicated that most if not all of the enzyme (catalytic site) is accessible to extracellular NAD+. The reaction product nicotinamide was predominantly recovered from the extracellular medium. The diazonium salt of sulphanilic acid, not penetrating into intact cells, was able to decrease the activity of intact thyroid tissue slices to the same extent as in the homogenate. Under the same conditions this reagent almost completely abolished NAD+ glycohydrolase activity associated with intact thyroid cells in culture. The triazine dye Cibacron Blue F3GA and its high-Mr derivative Blue Dextran respectively completely eliminated or caused a severe depression in the NAD+ glycohydrolase activity of FRTl cells. The enzyme could be readily solubilized from bovine thyroid membranes by detergent extraction, and was further purified by gel filtration and affinity chromatography on Blue Sepharose CL-6B. The overall procedure resulted in a 1940-fold purification (specific activity 77.6μmol of nicotinamide released/h per mg). The purified enzyme displays a Km of 0.40mm for β-NAD+, a broad pH optimum around pH7.2 (0.1 m-potassium phosphate buffer) and an apparent Mr of 120000. Nicotinamide is an inhibitor (Ki 1.9mm) of the non-competitive type. The second reaction product ADP-ribose acts as a competitive inhibitor (Ki 2.7mm). The purified enzyme splits β-NAD+, β-NADP+, β-NADH and α-NAD+ at rates in the relative proportions 1:0.75:<0.02:<0.02 and exhibits transglycosidase (pyridine-base exchange) activity. Anionic phospholipids such as phosphatidylinositol and phosphatidylserine inhibit the partially purified enzyme. A stimulating effect was observed upon the addition of histones.
Skip Nav Destination
Follow us on Twitter @Biochem_Journal
Article navigation
March 1985
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkAdvertising
Research Article|
March 01 1985
Topography, purification and characterization of thyroidal NAD+ glycohydrolase
Marc J. S. De Wolf;
Marc J. S. De Wolf
*RUCA Laboratory of Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
Search for other works by this author on:
Guido A. F. Van Dessel;
Guido A. F. Van Dessel
†UIA-Laboratory of Pathological Biochemistry, University of Antwerp, B-2020 Antwerp, Belgium
Search for other works by this author on:
Albert R. Lagrou;
Albert R. Lagrou
*RUCA Laboratory of Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
Search for other works by this author on:
Herwig J. J. Hilderson;
Herwig J. J. Hilderson
*RUCA Laboratory of Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
Search for other works by this author on:
Wilfried S. H. Dierick
Wilfried S. H. Dierick
*RUCA Laboratory of Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
†UIA-Laboratory of Pathological Biochemistry, University of Antwerp, B-2020 Antwerp, Belgium
Search for other works by this author on:
Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1985 London: The Biochemical Society
1985
Biochem J (1985) 226 (2): 415–427.
Citation
Marc J. S. De Wolf, Guido A. F. Van Dessel, Albert R. Lagrou, Herwig J. J. Hilderson, Wilfried S. H. Dierick; Topography, purification and characterization of thyroidal NAD+ glycohydrolase. Biochem J 1 March 1985; 226 (2): 415–427. doi: https://doi.org/10.1042/bj2260415
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.
Biochemical Society Member Sign in
Sign InSign in via your Institution
Sign in via your InstitutionGet Access To This Article
Follow us on Twitter @Biochem_Journal
Open Access for all
We offer compliant routes for all authors from 2025. With library support, there will be no author nor reader charges in 5 journals. Check here |
![]() View past webinars > |