[30] Distribution of PGE and PGF2α receptor proteins in the intracellular organelles of bovine corpora lutea

Publisher Summary Recent data from the laboratories demonstrated that a variety of intracellular organelles, as well as outer cell membranes of bovine corpora lutea, contain PGE and PGF 2α receptors. This chapter outlines the methodology and approach that can be used in obtaining data. Achieving a high degree of purity of various organelles is not only a function of good centrifugation strategy, but also critically depends on the care and handling during the procedures. To illustrate this point, one should remove organelles from the top sucrose interfaces first, not attempt to remove them to the extent that would overlap with other interfaces, and use fresh syringes and needles to remove different organelles. Purity is severely compromised despite an excellent centrifugation strategy if sufficient attention is not paid to the above and other seemingly minor handling details. The use of marker enzymes to assess the purity of subcellular organelles should be more sensitive than morphological assessment when they remain active during the isolation procedures. A prerequisite for using marker enzymes for assessment of purity is that the selected enzyme(s) should be found in only one organelle. In reality, this prerequisite may not be strictly met for all the enzymes, primarily because it is difficult to resolve unequivocally whether small amounts of enzymes found in inappropriate organelles are a reflection of low-level contamination or intrinsic.