A preparative method for the isolation of peroxisomes from the liver of normal, untreated rats is described. The peroxisome-enriched "light mitochondrial" fraction is layered on a 30% Nycodenz (5-[N-2,3-dihydroxypropylacetamido]-2,4,6-triiodo-N,N'-bis[2, 3-dihydroxypropyl]isophthalamide) solution containing 1 mM tetrasodium EDTA and then centrifuged in an angular rotor for 1 h at 130,000gavg. Peroxisomes are sedimented to the bottom leaving other organelles at the top of the tube. On the basis of morphological and biochemical studies, it is found that the peroxisomes (marker-enzymes catalase and urate oxidase) obtained in this method are not contaminated with lysosomes (marker-enzyme acid phosphatase) and contained very few mitochondria (marker-enzyme succinate-cytochrome c reductase) and microsomal vesicles (marker-enzyme glucose-6-phosphatase).

An all phosphorodithioate oligodeoxyribonucleotide (PS2; 17-mer) complementary to the coding region of the rabbit beta-globin mRNA was compared with the normal (PO2) and phosphorothioate (POS) oligonucleotide of the same size and sequence with respect to physicochemical properties and antisense activity in cell-free systems. The melting temperature (Tm) of the PS2-cDNA duplex was reduced by 17 degrees C relative to the PO2-cDNA duplex, compared to 11 degrees C for the POS-cDNA duplex, suggesting a decreased stability of the duplex with an increasing sulfur substitution. Like the POS-derivative, the PS2 oligonucleotide is quite stable against exonucleases, but these modified oligonucleotides showed different stability towards endonucleases and also towards different sub-cellular fractions of MCF-7 cells. During in vitro protein binding studies, the PS2 oligonucleotide showed similar binding (10-20%) to that of the PO2 oligonucleotide, while the POS oligonucleotide bound 60%. In cell-free translation, the PS2 oligonucleotide produced slightly higher specific translation inhibition of rabbit beta-globin mRNA compared to that of the PO2 oligonucleotide, and this was true only at concentration below 2 mM. The POS-derivative, except at 10 mM concentration, always showed higher translation arrest of the rabbit beta-globin mRNA compared to that of the other two oligonucleotides. The present study suggests that the PS2 oligonucleotide offers very little advantage over the POS oligonucleotide for use as an antisense analog.

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On subcellular fractionation, the enzyme acyl/alkyl dihydroxyacetone phosphate (DHAP) reductase (EC 1.1.1.101) in guinea pig and rat liver was found to be present in both the light mitochondrial (L) and microsomal fractions. By using metrizamide density gradient centrifugation, it was shown that the alkyl DHAP reductase activity in the "L" fraction is localized mainly in peroxisomes. From the distribution of the marker enzymes it was calculated that about two-thirds of the liver reductase activity is in the peroxisomes and the rest in the microsomes. The properties of this enzyme in peroxisomes and microsomes are similar with respect to heat inactivation, pH optima, sensitivity to trypsin, and inhibition by NADP+ and acyl CoA. The enzyme activity in the peroxisomes and microsomes from mouse liver is increased to the same extent by chronically feeding the animals clofibrate, a hypolipidemic drug. The kinetic properties of this enzyme in these two different organelles are also similar. From these results it is concluded that the same enzyme is present in two different subcellular compartments of liver.

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To assess the usefulness of computer-assisted modeling of mRNA as an aid in design of antisense DNA, the efficiency of inhibition of translation of rabbit beta-globin mRNA by various antisense sequences was compared with calculated structures of the mRNA. The model obtained by consideration of 30 lowest-energy computer-simulated structures is consistent with the high accessibility of the AUG initiation codon region known from digestion with nucleases and with previous antisense inhibition studies reported in the literature. Additional antisense inhibition data were obtained with 20-mer phosphorothioate oligonucleotides, targeted to regions of beta-globin mRNA differing moderately in their degree of participation in intramolecular folding. The efficiency of translation arrest by the oligonucleotides in cell-free expression systems (wheat germ extract and rabbit reticulocyte lysate) was obtained by measuring incorporation of [35S]methionine into total protein, and corrected for sequence-nonspecific inhibition using brome mosaic virus mRNA. In the presence of RNase H (wheat germ system), the inhibitory activity of the oligonucleotides showed correlation with the calculated secondary structure of mRNA, in particular at low oligonucleotide-to-mRNA ratios (correlation coefficient, 0.95). No correlation was observed in the reticulocyte lysate system, in which the inhibition is mediated by translational arrest.

A detailed comparison was made of the concentration dependence of translation inhibition by phosphorothioate and phosphodiester oligodeoxynucleotides of the same anti-beta-globin sequence in cell-free systems using beta-globin mRNA and unrelated mRNAs as controls. The results confirm that at low concentrations the phosphorothioate oligomer is more potent as an antisense compound, while at higher concentration (greater than 4 microM) it exhibits more nonspecific inhibition than the phosphodiester oligomer for RNase H-mediated translation inhibition.