The cytochemical assay of NAD+ kinase activity in the follicular cells of guinea-pig thyroid gland

Abstract

A quantitative cytochemical assay for NAD+ kinase-like activity in the guinea-pig thyroid gland is described. The NADP+ produced by the activity of the kinase was used to drive the NADP+-dependent enzyme glucose-6-phosphate dehydrogenase which is endogenous to the tissue. The activity of glucose-6-phosphate dehydrogenase is greatly in excess of that of the kinase and was unaffected by the constituents of the kinase incubation medium (ATP, Mg2+ and NAD+) either alone or in combination. Kinase activity was dependent both on ATP and Mg2+, with maximal activity seen when the Mg-ATP ratio was between 1:1 and 4:1. Free ATP inhibited the activity of the enzyme. Enzyme activity was exhibited over a broad pH range (7-9) with a peak at pH 8.2. The sulphydryl-blocking agents, p-chloromercuribenzoate, iodoacetate and iodoacetamide (at 1 mM), completely abolished kinase activity but were without effect on glucose-6-phosphate dehydrogenase activity. N-ethylmaleimide and citrate (both at 1 mM) had no effect on either kinase or glucose-6-phosphate dehydrogenase activities.

Measurement of low concentrations of bovine thyrotrophin by iodide uptake and organification in porcine thyrocytes

“…The medium was removed, the cells were gently washed with Hank's balanced salt solution and assayed cytochemically for either TPO (Ealey, Henderson & Loveridge, 1984) or TD activity (Perrild, Frost & Robertson, 1984). The cells were then dried and mounted in Farrant's medium (for TD) or styrolite (for TPO) before examination and photo¬ graphy using a standard Zeiss WL research micro¬ scope.…”