Summary
A technique is described for the monitoring of enzyme-catalyzed pyridine nucleotide reduction in cryostat sections. By virtue of the native fluorescence of NADPH the kinetics of glucose 6-phosphate dehydrogenase in the mouse endocrine pancreas could be studied in a microscope fluorophotometer. The development of fluorescence was most rapid at a pH of about 8.0. Apparent K mvalues were 0.2 mM for glucose 6-phosphate and 2 mM for NADP. The reaction was enhanced by MgCl2 (optimum concentration of about 2 mM) but insensitive to KCN and oxygen. The rates of fluorescence development appeared unaffected by NADPH diffusion.
The observed K mvalues are higher than those obtained by biochemical analysis of homogenized pancreatic islets. It is suggested that this discrepancy is due to the relative morphological integrity of the cryostat section. If the present data are more representative of the properties of the enzyme in the living cell than are the values obtained with disintegrated tissue, the phosphogluconate pathway in the pancreatic β-cells could be regulated by both glucose 6-phosphate and NADP.
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Täljedal, IB. Direct fluorophotometric recording of enzyme kinetics in cryostat sections. Histochemie 21, 307–313 (1970). https://doi.org/10.1007/BF00280900
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DOI: https://doi.org/10.1007/BF00280900