Identification and characterization of a specific dolichylmonophosphate phosphatase activity in bovine thyroid microsomes.
Bovine thyroid membranes are able to dephosphorylate exogenous [1-3H]DMP as well as endogenous prelabeled [32P]DMP. The kinetics, properties and specificity of the dolichylmonophosphatase activity have been studied by monitoring respectively the release of [1-3H]dolichol from [1-3H]DMP and the residual amount of [32P]DMP. The DMP-phosphatase activity is not linear with respect to time and exhibits a neutral pH optimum. There is only linearity in a narrow range of protein concentration when 0.25% Triton X-100 is included in the incubation mixture. Studying the enzymatic activity in function of protein concentration, the detergent requirement shows to be very critical. Triton X-100 is necessary for enzymatic activity with [1-3H]DMP (only 10% of enzymatic activity in the absence of detergent) although the detergent inhibits the hydrolysis of endogenous prelabeled [32P]DMP. Divalent cations are not essential for enzymatic activity, Ca2+-ions being even inhibitory. In accordance, EDTA (EGTA) is slightly stimulatory. The DMP-Pase activity is not influenced by the ionic strength of the incubation system and sulphydryl groups are not involved. NaF, VOS and VO4(3-) are strongly inhibitory. The inhibition by dolichol and PO3-4 can be explained as the result of product inhibition. An apparent Km-value of 2.5 X 10(-5) M is computed for [1-3H]DMP. Bacitracin inhibits DMP-phosphatase in contrast with other reports. Propylthiouracyl, cAMP, TSH and several other bio-effectors are without effect on the in vitro system. The specificity of the DMP-Pase activity is discussed, showing that the phosphatase is distinctly different from other phosphatases especially phosphatidic acid phosphohydrolase.
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002417:Cattle; D006728:Hormones / Q000494:pharmacology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D013961:Thyroid Gland / Q000201:enzymology*
D000900:Anti-Bacterial Agents; D006728:Hormones; D010744:Phosphoric Monoester Hydrolases; C020088:dolichyl-phosphatase