Biochemical Toxicology Of Human Placental High Affinity Calcium-stimulated Atpase And Calcium Transport (uptake, Ddt, Dot).

ATPase activity which is stimulated by submicromolar concentrations of Ca('2+) was identified and characterized in human placental microvillous brush border membranes. The enzyme exhibited an apparent K(,m) for free Ca('2+) of 18.3 (+OR-) 3.7 nM and a V(,max) of 233.0 (+OR-) 30.0 nmol/min/mg protein. Studies using trans-1,2-diaminocyclohexane-N,N,N',N',-tetraacetic acid (CDTA) showed this enzyme to require Mg('2+) for maximal activity. The high affinity Ca('2+)-ATPase was unaffected by up to 100 (mu)M concentrations of vanadate, but was sensitive to trifluoperazine inhibition (IC(,50) < 50 (mu)M). The enzyme was not stimulated by the addition of up to 10 (mu)g calmodulin, but this lack of effect may be related to the endogenous calmodulin content of the membrane preparation. High affinity Ca('2+)-ATPase was significantly inhibited by 1,1-Bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) and its specific homologs at 10 (mu)M concentrations. In addition to the high affinity ATPase, an ATP-dependent Ca('2+) uptake was characterized in human placental microvillous brush border membranes. Multiple freeze-thaw cycles greatly diminished uptake activity by 90%, indicating an intravesicular accumulation of Ca('2+). Kinetic studies indicate an apparent K(,m) for calcium of 0.22 (+OR-) 0.04 M and a V(,max) of 441 (+OR-) 137 pmoles/min/mg protein at 37(DEGREES)C. Ca('2+) uptake was shown to have a optimum pH of 7.2, and was unaffected by the addition of oxalate, consistent with a plasma membrane origin of uptake. The activity was temperature dependent and was significantly inhibited by trifluoperazine (IC(,50) = 100 uM) and vanadate (IC(,50) = 100 uM). DDT and its specific homologs were shown to inhibit Ca('2+) uptake, but to a lesser degree than that seen with the high affinity Ca('2+)-ATPase. Thus, human term placental brush border membranes have both a Ca('2+) transport activity and a calcium-stimulated ATPase activity with similar biochemical characteristics. It is suggested that the high affinity Ca('2+)-ATPase is the mechanism responsible for Ca('2+) uptake in this membrane fraction, which functions to maintain the low intracellular level of free Ca('2+) in placental syncytiotrophoblasts.PhDBiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127934/2/8621393.pd