Differential phospholipid-labeling suggests two subtypes of phospholipase D in rat Leydig cells.

The aim of the present study was to compare the transphosphatidylation activity of phospholipase D (PLD) under different substrate labeling conditions, in order to investigate whether PLD in rat Leydig cells exhibited any substrate preferences for the alkyl- or acyl-form of phosphatidylcholine (PtdCho). The [3H]phosphatidylethanol formation in response to 4 beta-phorbol 12-myristate 13-acetate (PMA), sphingosine, or Ca(++)-ionophore A23187, was lower when Leydig cells were labeled with 1-O-[3H]alkyl lysoPtdCho compared with the responses when [3H]myristic acid was employed. In contrast, the results for the receptor agonists (vasopressin, bradykinin, and lysophosphatidic acid), using the two labels, showed more consistency. Thus, the PLD-activity induced by PMA, sphingosine, or A23187 has a more selective substrate range (i.e. mainly acyl-linked PtdCho) than the PLD-activity stimulated via a receptor. Our data suggests the existence of PLD isozymes that differ with respect to substrate specificity and activation mechanisms.