Identification of 5'-adenylylimidodiphosphate-hydrolyzing enzyme activity in rabbit taste bud cells using X-ray microanalysis.
X-ray microanalysis has been used to characterize the enzyme activity hydrolyzing the ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) in taste bud cells. Rabbit foliate papillae fixed with paraformaldehyde and glutaraldehyde were incubated cytochemically with AMP-PNP as the substrate and lead ion as capture agent. The reaction product which appeared on the microvilli of taste bud cells was examined using an energy dispersive X-ray microanalyzer connected to an analytical electron microscope. The X-ray spectrum thus obtained was compared with that obtained from the product obtained from the demonstration of ATPase activity. Comparison of the phosphorus/lead ratios in the two products showed that twice as much phosphorus was released from an AMP-PNP molecule by the activity in question compared with that released from an ATP molecule by ATPase activity. This indicates that the enzyme hydrolyzes AMP-PNP into AMP and imidodiphosphate and that the enzyme is adenylate cyclase or ATP pyrophosphohydrolase, which possesses a similar hydrolytic property, but not ATPase or alkaline phosphatase, which hydrolyzes AMP-PNP into ADP-NH2 and orthophosphate. This paper provides an example of the use of X-ray microanalysis as a tool for enzyme distinction. The method is applicable to a variety of enzymes and tissues.
Asanuma
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Department of Oral Physiology, Matsumoto Dental College, Shiojiri, Japan.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000262:Adenylyl Cyclases / Q000032:analysis*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism*; D000818:Animals; D004577:Electron Probe Microanalysis; D006651:Histocytochemistry; D008297:Male; D008871:Microvilli; D011755:Pyrophosphatases / Q000032:analysis*; D011817:Rabbits; D013650:Taste Buds / Q000201:enzymology* / Q000648:ultrastructure
D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; C019757:ATP pyrophosphohydrolase; D000262:Adenylyl Cyclases