Effect of glutaraldehyde and decalcifying agents on acid phosphomonoester hydrolase activity in the enamel organ of the rat incisor: a biochemical study comparing enamel organ with liver.
Enamel organs were dissected from the labial surface of unfixed and glutaraldehyde-fixed rat incisors and assayed biochemically at pH 5.0 for acid phosphomonoester hydrolase activity using cytidine 5'-monophosphate (5'-CMP), beta-glycerophosphate (beta GP), phosphorylcholine (PC), and phosphoserine (PS) as substrates. Whole homogenates from unfixed enamel organs showed substantial enzyme activity toward 5'-CMP and beta GP, but 1/4 and 1/8 less activity toward PC and PS, respectively. Perfusion fixation with 2% glutaraldehyde resulted in a net loss of 80% of the enzyme activity toward each substrate. Lineweaver-Burk plots revealed that the fixative depressed the rate of hydrolysis of substrate (decrease in Vmax) and it also lowered the affinity of enzymes for substrate (increase in KM). Hence, fixed tissue generally required two or three times as much substrate to saturate the enzymes, but less substrate was hydrolyzed, as compared to unfixed tissue. Decalcification of fixed incisors with either formic citric acid, ethylenediaminetetraacetic acid (EDTA), or ethyleneglycoltetacetic acid (EGTA) did not further alter enzyme activity in the enamel organ as determined by Lineweaver-Burk plots, However, EDTA and EGTA were found to increase the susceptibility of fixed enzymes to inhibition by lead ions. This chelator-enhanced lead inhibition was greatest following decalcification with EGTA and using PC as substrate. Similar results were obtained for liver.[1]References
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