Gang, Katherine Ann; (1994) Characterization and properties of ecto-5' nucleotidase in white adipose tissue. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Membrane-bound 5'-nucleotidase (5'Nase, EC 3.1.3.5) activity was measured in rat white adipose tissue by radiochemical assay in several metabolic states, including normal fed, streptozotocin-diabetes, hypothyroidism, hyperthyroidism, and fasting. A doubling in 5'Nase activity was measured in the diabetic rats as compared to the normal fed control animals, whilst no statistical differences were noted in the other metabolic states tested. Two protein bands were detected at 76kDa and 7AkDa in control and diabetic rat adipose tissue membrane samples by exposing a Western blot to a polyclonal anti-rat-ecto-5'Nase. The combined intensities of these two bands increased to the same extent in diabetics as did the membrane-bound 5'Nase activities when comparing with control values. In vivo insulin administration to the diabetic rats resulted in a reduction of membrane-bound 5'Nase activity to marginally below control activities over a period greater than 2A hours, but not exceeding 48 hours. Partial purification of rat adipose tissue membrane- bound 5'Nase was achieved by a sulphobetaine detergent solubilization of the membranes, followed by concanavalin A- Sepharose, AMP-Sepharose and Superose-12 column chromatography. Only two protein bands were visualised by silver staining when this partially purified sample was subjected to denaturing gel electrophoresis. Characterization of the partially purified 5'Nase demonstrated that its properties were similar to ecto-5'Nases purified from other tissues and species. These properties included: Km(AMP) = 11.1μM, pH optimum at pH7.5, no magnesium inhibition, competitive inhibition with Ki values for α,β-methylene-ADP in the nM range and for α,β-methylene-ATP in the μM range, IC50 = 2.3mM for dithiothreitol inhibition, and almost complete inhibition at l0μM concanavalin A. Although the rat adipose tissue membrane-bound 5'Nase was susceptible to cleavage by the glycosyl-phosphatidylinositol (GPI)-anchor cleavage enzyme, GPI-phospholipase C, a significant loss of 5'Nase activity meant that the procedure was not a viable option for use in the purification. Additionally, a factor derived from rat brain which was found to co-purify with "soluble" low-Km 5'Nase, enhanced ATP inhibition of the partially purified adipose tissue membrane-bound 5'Nase by 40-fold.

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