Liver peroxisome isolation

Figure 4. Immuuolocali/ation of peroxisomal COT. The peptide ANEDEYKKTEEI corresponding to the N-terminus of COT protein (sequence 43 54,) wa.s used to obtain the antibodies as described elsewhere. Rat liver peroxisomes were isolated in a Nycodenz cushion as described elsewhere." 10mg of rat liver peroxisomal extracts were separated by SDS/PACiE and. subjected (0 immuno-blotting using specific A43 antibodies (A) for carnitine octanoyllransferase or preimmune sera (U). Two bands corresponding to Mr of approximately 69 and 79 k Da are observed. The markers (M) were used 10 determine the approximate molecular weights of the species indicated in the figure.

Investigation of the 3-hydroxyacyl-CoA dehydrogenase activities in purified rat liver peroxisomes, using the 3-hydroxyacyl-CoAs of straight chain fatty acids, of 2-methyl-branched chain fatty acids and of trihydroxycoprostanic acid as substrates, revealed initially 5 different enzymes (named I to V)." Enzyme IV was a monomeric 78 kD protein, possessed crotonase activity, was induced by clofibrate, and was identified as the inducible multifunctional protein. Interestingly, enzyme III, a monomeric 80 kD protein, also hydrated crotonyl-CoA. In contrast to enzyme IV, enzyme III was not induced by clofibrate." This was the first indication, pubhshed in 1994, that peroxisomes contained a second multifunctional protein. It was named multifunctional protein 2 (MFP-2) (the inducible, firstly isolated protein is referred to as MFP-1). Based on its substrate spectrum, the newly identified multifunctional protein was postulated to be involved in bile acid formation. ... 

In 1990, a reeeptor that mediates the effects of PPs, the peroxisome proliferator activated reeeptor alpha, PPARa, was identified in motrse liver. The isolation of htrman PPARa arrd other isoforms of PPAR (P and y) both from roderrts and humans followed on rapidly. " In rats and mice, PPARa is highly expressed in the liver, whereas other forms such as PPARy are expressed predominantly in fatty adipose tissue and in the immune system. This tis-sue-specifie pattern of expression implies a differerrt ftmetion for the PPAR isoforms both in normal tissue homeostasis and in resportse to dmgs and toxicants. 

Purified preparations of COT from beef liver peroxisomes " and CPT-II from the inner membrane of beef liver mitochondria, the two enzymes that can be isolated in stable form, were used for the kinetic studies. Based on the kinetic patterns obtained when both substrates were varied and on product inhibition studies, we concluded that the mechanism of COT was a rapid equilibrium random one. In contrast, that of CPT-II was ordered with the CoA substrate binding first (acyl-CoA in the forward direction, CoA in the reverse) to prepare the carnitine binding site. The kinetic constants are given in Table 1. 

Thiolester hydrolases are present in most tissues and cell compartments. High concentrations are found in liver microsomes and in brown adipose tissue mitochondria and peroxisomes. Several acyl-CoA hydrolases have shown a close relationship to the nonspecific carboxylesterases EC 3.1.1.1. Thus, palmitoyl-CoA hydrolase purified from rat liver microsomes was found to be identical to esterase pI 6.2I6A (ES4 type). An acyl-CoA hydrolase was isolated that showed high similarity to esterase pI 6.1 [74a] [129] [130]. These few examples are further illustrations of the unsatisfying situation of the traditional classification of esterases. 

Species comparisons of hepatic peroxisomal proliferation have also included studies of human and non-human primate primary hepatocyte cultures. Hepatocytes isolated from Wistar-derived rats (180-220 g), male Alderley Park guinea-pigs (400-500 g), male marmosets (350-500 g) and three human liver samples (renal transplant donors) were treated with 0-0.5 mM mono(2-ethylhexyl) phthalate for 72 h (Elcombe Mitchell, 1986). While there was a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation in rat hepatocytes, no induction was observed in guinea-pig or human hepatocytes and only small non-concentration-dependent effects were observed in marmoset hepatocytes. Metabolite VI induced cyanide-insensitive palmitoyl-CoA oxidation and lauric acid hydroxylation in cultured... 

Peroxisome proliferators have also been shown to induce replicative DNA synthesis in cultured rodent hepatocytes (lARC, 1995). In contrast, several peroxisome proliferators have failed to induce replicative DNA s mthesis in human hepatocyte cultures (Doull et al., 1999). Hepatocytes were isolated from male Wistar-derived rats and from three human liver samples (liver transplantation donors) and treated with 2-ethylhexanoic acid and some other peroxisome proliferators for 72 h (Elcombe et al, 1996). While 2-ethylhexanoic acid induced replicative DNA s5mthesis in cultured rat hepatocytes, no effect was observed in human hepatocytes. Hepatocytes were isolated from male Fischer 344 rats and three humans and treated in culture with 250-2000 pM mono(2-ethylhexyl) phthalate (Hasmall et al, 1999). Increased peroxisomal (O-oxi-dation (at 250-750 pM), replicative DNA s mthesis (at 500-1000 pM), and inhibition of apoptosis (at 250-1000 pM) were observed in rat hepatocytes. None of these parameters was affected by mono(2-ethylhexyl) phthalate in human hepatocytes. 

The feeding of an EPA-free source of supplementary DHA (from algae) to human volunteers indicated a metabolic retroconversion of DHA to EPA (Conquer and Holub, 1996, 1997). Previous animal and in vitro studies in isolated rat liver cells have demonstrated that DHA can be retroconverted to EPA, and that this retroconversion is a peroxisomal function (Schlenk et al., 1969 Gronn et al., 1991). Studies in isolated rat liver cells by Schlenk et al., 1969 have also indicated that the resultant EPA can be chain-elongated to DPA (22 5n-3) for subsequent esterification into cellular lipids. An acyl-CoA oxidase has been identified as the enzyme responsible for the chain shortening of DHA in the peroxisomal beta-oxidation of PUFA in human fibroblasts (Christensen et al., 1993). The aforementioned in vivo human studies have estimated the extent of retroconversion of DHA to EPA to be approximately 10% (Conquer and Holub, 1996, 1997). 

Gronn, M., Christensen, E., Hagve, T.A. and Christophersen, B.O. (1991) Peroxisomal retro-conversion of docosahexaenoic acid (22 6(n-3)) to eicosapentaenoic acid (20 5(n-3)) studied in isolated rat liver cells. Biochim. Biophys. Acta. 1081 85-91. 

Staple et al. showed that the conversion of 3a,7a,12a,24-tetrahydroxy-5/8-cholestanoic acid into cholic acid (cf. Fig. 3) can occur in rat liver microsomes or in cytosolic fractions fortified with NAD" or NADP and that propionic acid is released [42,43]. Pedersen and Gustafsson showed recently that the peroxisomal fraction had a high capacity to convert 3a,7a,12a-trihydroxy-5 8-cholestanoic acid into cholic acid [150]. Later, Kase et al. found that the peroxisomal fraction was more active than the microsomal and the mitochondrial fractions and that 3a,7a,12a-24-tetrahydroxy-5j8-cholestanoic acid was an intermediate in the conversion [151]. Thus, some of the previous contradictory results may be explained by varying degrees of contamination of the microsomal and mitochondrial fractions with peroxisomes. In the work by Kase et al. it was shown that the over-all conversion of 3a,7a,12a-trihydroxy-5 -cholestanoic acid into cholic acid in the peroxisomes was absolutely dependent upon the presence of Mg ", CoA, ATP and NAD. The reaction was stimulated by FAD, by cytosolic protein, by microsomal protein and by bovine serum albumin. It is possible that the stimulatory effect of the microsomes and cytosol was imspecific and due to the increased protein concentration per se. The stimulatory effect of FAD was taken as evidence that 3a,7a,12a-tri-hydroxy-5yS-cholestanoyl-CoA oxidase is a FAD-containing protein. There was a lag phase in the reaction, possibly due to the activation step, and it was suggested that the activation was rate limiting. Also in this case, it was not possible to isolate a A -unsaturated intermediate in the reaction. The participation of a desaturase and a hydratase was proved by the incorporation of from H20 into 3a,7a,12a,24-te-trahydroxy-5/8-cholestanoic acid (Bjorkhem, Kase and Pedersen, unpublished study). 

Mannaerts, G.P., Etebeer, L.J., Thomas, J. DeShepper, P.J. 1979. J. Biol. Chem., 254, 4585-4595. Mitochondrial and peroxisomal fatty acid oxidation in liver homogenates and isolated hepatocytes from control and clofibrate treated rats. 

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