Detailed analytical subcellular fractionation of non-pregnant porcine corpus luteum reveals peroxisomes of normal size and significant UDP-glucuronosyltransferase activity in the high-speed supernatant.
A detailed subfractionation of the non-pregnant porcine corpus luteum (CL) was performed employing differential centrifugation. Marker enzyme assays (i.e., lactate dehydrogenase for the cytosol, NADPH-cytochrome P450 reductase for the endoplasmatic reticulum, catalase (CAT) for peroxisomes, glutamate dehydrogenase for the mitochondrial matrix and acid phosphatase for lysosomes) in all subfractions obtained exhibited a pattern of distribution similar to that observed with rat liver. These subfractions should be useful in connection with many types of future studies. In disagreement with previous biochemical and morphological studies, peroxisomes (identified on the basis of catalase activity and by Western blotting of catalase and of the major peroxisomal membrane protein (PMP-70)) sedimented together with mitochondria (i.e., at 5000 x g(av) for 10 min) and not in the post-mitochondrial fraction prepared at 30,000 x g(av) for 20 min by Peterson and Stevensson. No other classical peroxisomal enzymes were detectable in the porcine ovary, raising questions concerning the function of peroxisomes in this organ. Furthermore, UDP-glucuronosyltransferase (UGT), generally considered to be an integral membrane protein anchored in the endoplasmatic reticulum, was recovered in both the cytosolic (i.e., the supernatant after centrifugation at 50,000 x g(av) for 1h) and the microsomal fraction of the porcine corpus luteum, even upon further centrifugation of the former. In contrast, UGT sediments exclusively in the microsomal fraction upon subfractionation of the liver and ovary from rat.
Boström
Malin
M
Department of Biochemistry and Biophysics, Arrhenius Laboratories for the Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden. malin@dbb.su.se
Björk
Karl
K
Nelson
B Dean
BD
DePierre
Joseph W
JW
D000818:Animals; D002374:Catalase / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003338:Corpus Luteum / Q000201:enzymology* / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D014453:Glucuronosyltransferase / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D020675:Peroxisomes / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D013552:Swine
D008565:Membrane Proteins; D002374:Catalase; D014453:Glucuronosyltransferase