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Peroxisomes are essential subcellular organelles, which appear to be derived from pre-existing organelles. This biogenetic mechanism assumes the presence of peroxisomes in either or both mammalian gametes (sperms and/or oocytes). In order to test the presence and subcellular localization of peroxisomal proteins in rat sperms and oocytes, the authors carried out fractionation and immunofluorescence experiments. The results showed that rat oocytes contain peroxisome-like structures, which were detected by indirect immunofluorescence, using an antisera against total peroxisomal proteins. In contrast, such structures were not detected in rat sperms, which appear to contain catalase localized in the cell cytosol. The results reported herein show evidence for the first time of the presence of peroxisome-like structures in mammalian oocytes, and provide evidence for the peroxisome biogenesis hypothesis, by division of pre-existing organelles.

The peroxisomal membrane appears to play a crucial role in transporting proteins into the organelle. Some human genetic disorders involving peroxisome biogenesis, such as Zellweger syndrome, may be caused by genetic defects of the import machinery located in the peroxisomal membrane. In order to characterize the proteins of the human peroxisomal membrane, we isolated peroxisomes from human liver. We obtained their membranes using various procedures and analyzed their proteins by SDS-polyacrylamide gel electrophoresis and silver staining. We compared the protein composition of peroxisomal membranes with membranes derived from mitochondria and microsomes. The main peroxisomal membrane proteins (PMPs) have apparent molecular masses of 147, 112, 95, 87, 81, 79, 74, 69(70), 53-52 (double band), 47, 45, 43, 37, 31, 28, 22, and 17 kDa. The following PMPs of 147, 112, 79, 69(70), 53-52 (double band), 47, 43, 31, 28, 22, and 17 kDa fit the criteria for integral membrane proteins. We then produced rabbit polyclonal and mouse monoclonal antibodies that recognized some human PMPs. One of these antibodies detected mainly PMP43. We used this antiserum to evaluate the presence and subcellular distribution of the PMP43 in fibroblasts derived from patients affected by Zellweger syndrome. These results represent new information about the protein composition of the human peroxisomal membrane and provide biological tools for further characterization of the human PMPs and their genes in normal and pathological conditions.

This paper shows the successful isolation of peroxisomes from human liver samples that were kept frozen at -70 degrees C. Purification of these peroxisomes was obtained by a combination of two subcellular fractionation techniques: differential centrifugation and isopycnic fractionation in Nycodenz density gradients. Peroxisome integrity was evaluated by latency measurements and by ultrastructural observation. The procedure described here may be useful for the isolation of other subcellular organelles from frozen human samples.