TY - JOUR
T1 - The subcellular localization of phospholipase D activities in rat Leydig cells
AU - Strand, Anne Mette
AU - Lauritzen, Lotte
AU - Vinggaard, Anne Marie
AU - Hansen, Harald S
PY - 1999
Y1 - 1999
N2 - Rat Leydig cells contain a phospholipase D (PLD), which can be activated by vasopressin and phorbol ester. In order to clarify which Leydig cell organelles that express PLD activity, the subcellular localization of two differently regulated PLD activities was investigated by subcellular fractionation on a 40% (v/v) self-generating Percoll gradient. PLD activities in broken cells were estimated using radiolabeled didecanoylphosphatidylcholine as a substrate. Initial experiments revealed the presence of an oleate Mg2+-activated PLD and a phosphatidylinositol 4,5-bisphosphate-activated PLD (PIP2-PLD) in the microsomal fraction of Leydig cells. The latter activity could be further stimulated by recombinant nonmyristoylated ADP ribosylating factor 1 (ARF1) plus GTPγS. The peak of oleate Mg2+ - PLD activity colocalized with the plasma membrane marker, whereas the highest specific activity of the PIP2-PLD activity was found in fractions with a slightly lower density than those containing the plasma membrane and trans-Golgi marker enzymes. In order to localize phorbol ester- stimulated PLD activity in intact Leydig cells, the cells were prelabeled with [14C]-palmitate and then stimulated for 15 min with 100 nM 4-β- phorbol-12-myristate-13-acetate (PMA) in the presence of ethanol or butanol. The PLD product [14C]-phosphatidylethanol, expressed as the percentage of total labeled phospholipids in the fraction, was slightly increased in all Percoll fractions and showed a prominent peak in the fractions containing plasma membrane, trans-Golgi, and fractions of slightly lower density. The PMA-induced formation of [14C]-phosphatidylbutanol could be inhibited dose- dependently with brefeldin A suggesting that the activation of PLD by the phorbol ester was mediated by ARF.
AB - Rat Leydig cells contain a phospholipase D (PLD), which can be activated by vasopressin and phorbol ester. In order to clarify which Leydig cell organelles that express PLD activity, the subcellular localization of two differently regulated PLD activities was investigated by subcellular fractionation on a 40% (v/v) self-generating Percoll gradient. PLD activities in broken cells were estimated using radiolabeled didecanoylphosphatidylcholine as a substrate. Initial experiments revealed the presence of an oleate Mg2+-activated PLD and a phosphatidylinositol 4,5-bisphosphate-activated PLD (PIP2-PLD) in the microsomal fraction of Leydig cells. The latter activity could be further stimulated by recombinant nonmyristoylated ADP ribosylating factor 1 (ARF1) plus GTPγS. The peak of oleate Mg2+ - PLD activity colocalized with the plasma membrane marker, whereas the highest specific activity of the PIP2-PLD activity was found in fractions with a slightly lower density than those containing the plasma membrane and trans-Golgi marker enzymes. In order to localize phorbol ester- stimulated PLD activity in intact Leydig cells, the cells were prelabeled with [14C]-palmitate and then stimulated for 15 min with 100 nM 4-β- phorbol-12-myristate-13-acetate (PMA) in the presence of ethanol or butanol. The PLD product [14C]-phosphatidylethanol, expressed as the percentage of total labeled phospholipids in the fraction, was slightly increased in all Percoll fractions and showed a prominent peak in the fractions containing plasma membrane, trans-Golgi, and fractions of slightly lower density. The PMA-induced formation of [14C]-phosphatidylbutanol could be inhibited dose- dependently with brefeldin A suggesting that the activation of PLD by the phorbol ester was mediated by ARF.
KW - ARF-stimulated phospholipase D
KW - Brefeldin A
KW - Leydig cells
KW - Magnesium
KW - Oleate-stimulated phospholipase D
KW - Pertoll
KW - Phosphatidylethanol
KW - Phosphatidylinositol 4,5- bisphosphate
KW - Subcellular fractionation
U2 - 10.1016/S0303-7207(99)00057-X
DO - 10.1016/S0303-7207(99)00057-X
M3 - Journal article
C2 - 10432228
AN - SCOPUS:0345643390
VL - 152
SP - 99
EP - 110
JO - Molecular and Cellular Endocrinology
JF - Molecular and Cellular Endocrinology
SN - 0303-7207
IS - 1-2
ER -