|Institution:||Texas A&M University|
|Keywords:||nutrition.; Major nutrition.|
|Full text PDF:||http://hdl.handle.net/1969.1/ETD-TAMU-1997-THESIS-L866|
Cellular copper homeostasis depends on a Cu-ATPase enzyme in the membrane. Surprisingly, flask-grown BeWo cells do not display ATPase. We found Cu-ATPase is only expressed in differentiated cells such as Caco-2 cells. Since BeWo cells are not differentiated cells, we changed the cells' culture condition by growing them on porous filters or by adding sodium butyrate reagent to flask-grown cells. BeWo cells express considerable amounts of alkaline phosphatase (ALP). ALP is a marker enzyme of the plasma membrane. ALP is also the placental isozyme that correlates with the degree of differentiation of trophoblast cells. So we used ALP as a biochemical marker of cellular differentiation to test if filter-grown or butyrate-treated BeWo cells can cause greater induction of ALP activity compared with flask-grown cells. We found that the specific activity of ALP in total membrane (TM) from filter-grown cells was about 40% greater than that from flask-grown, and that from butyrate-treated was 5.7 times greater than NaCl-treated (control). In contrast, the ALP specific activity of TM from Caco-2 cells which differentiated spontaneously did not show an increase with butyrate treatment. We separated TM into a light membrane (LM) fraction (I 7/40%) and a heavy membrane (HM) fraction (40/80%) by discontinuous sucrose gradient centrifugation. The ratio of ALP specific activity of LM to HM was about 5 in flask-grown, filter-grown and butyrate-treated BeWo cells. We also applied the TM to a 9-60% sucrose gradient and collected each fraction after centrifugation. The highest ALP specific activity in butyrate-treated cells was almost ten times greater than in the untreated cells. The results of the observations suggest that either growth on filters or butyrate treatment can make BeWo cells differentiate. When we assayed ATPase specific activity in TM from differentiated BeWo cells, the activity did not show activation with nanomolar amounts of copper or inhibition with 100 gM orthovanadate. Further success in this approach may have to depend on purifying the specific cell fraction that contains the Cu-specific ATPase. Our data support the hypothesis that growth on porous filters will cause BeWo cells to differentiate.