Abstract
Recent articles have confused the steady state concentration of radioactivity in N-methylphosphatidylethanolamine (PME) and N,N-dimethylphosphatidylethanolamine (PDE) with the amount of these products formed during the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). This paper clarifies this problem and reports the apparent Km values for AdoMet and pH optima for the conversion of PE to PME, PDE, and PC by rat liver microsomes. We purified AdoMet and [methyl-3H]AdoMet and measured the transfer of tritium to PME, PDE, and PC as a function of time. There was an initial lag in the formation of [3H]PC followed by linear incorporation of isotope. In contrast, labeled PME and PDE reached and maintained steady state levels within 1 to 2 min. Hence, calculations of the rate of formation of PME, PDE, and PC must take into account the subsequent conversion of PME and PDE to PC. The PE N-methyltransferase was assayed at pH 6.6, 9.2, and 10.25 and the apparent Km for AdoMet for the three methylation reactions was calculated. The formation of PME was best estimated by the dpm in PME + 1/2 dpm in PDE + 1/3 dpm in PC. The synthesis of PDE from PME was estimated from 1/2 dpm in PDE and 1/3 dpm in PC, and the formation of PC from PDE estimated by 1/3 dpm in PC. The apparent Km for AdoMet at pH 10.25 for the conversion of PE to PME was 58 microM, PME to PDE was 65 microM, and PDE to PC was 96 microM. The pH optimum for each of these methylation reactions was 10.25. This high value was not due to alkaline degradation of AdoMet or denaturation of the enzyme. The apparent Km for AdoMet was also estimated for the conversion of exogenous PME to PDE (50 microM) and exogenous PDE to PC (45 microM). Since recent studies on the methylation of PE have not taken into account the conversion of newly formed PME and PDE to PC, the results and conclusions about apparent Km values for AdoMet, pH optima, and the number of enzymes involved must be re-evaluated.
Highlights
Recent articles have confused the steady state con- (1,2)
There was an apparent K, for AdoMet of 30 p~ for thefirst reaction and 8-18 pM for
The conclusions yltransferase was assayed at pH 6.6, 9.2, and 10.25 about two enzymes in these animal systems(6-9) were based and the apparent K, for AdoMet for the three meth- on the differencesin the radioactivityassociatedwith the ylationreactionswascalculated.Theformation of intermediate products PME and PDE a sfunction of pH, on
Summary
The apparent K , for AdoMet was estimated for the conversion of exogenous PME toPDE (50 WM) and exogenous PDE to PC(45 p ~ ) .Since recent studieson livermicrosomes at the lower and higher pH, respectively. These results have been taken as argumentsfor the existence of two enzymes, one that converts PE to PME with a high affinity for AdoMet, and a second enzymethat converts PME toPDEandPC, with low affinity for AdoMet.A much the methylation of PE have not taken into account the different result has been reported for P E methyltransferase conversion of newly formed PME and PDE toPC, the activities from rat pituitary (10). In an attempt toreconcile the apparent discrepancies among the various reports, we foundconfusion by a number of workers on the calculation of the apparent K,,, for AdoMet
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