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We purified catalase-2 of the nematode Caenorhabditis elegans and identified peroxisomes in this organism. The peroxisomes of C. elegans were not detectable by cytochemical staining using 3, 3'-diaminobenzidine, a commonly used method depending on the peroxidase activity of peroxisomal catalase at pH 9 in which genuine peroxidases are inactive. The cDNA sequences of C. elegans predict two catalases very similar to each other throughout the molecule, except for the short C-terminal sequence; catalase-2 (500 residues long) carries a peroxisomal targeting signal 1-like sequence (Ser-His-Ile), whereas catalase-1 does not. The catalase purified to near homogeneity from the homogenate of C. elegans cells consisted of a subunit of 57 kDa and was specifically recognized by anti-(catalase-2) serum but not by anti-(catalase-1) serum. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected catalase-2 inside vesicles judged to be peroxisomes using morphological criteria. The purified enzyme (220 kDa) was tetrameric, similar to many catalases from various sources, but exhibited unique pH optima for catalase (pH 6) and peroxidase (pH 4) activities; the latter value is unusually low and explains why the peroxidase activity was undetectable using the standard alkaline diaminobenzidine-staining method. These results indicate that catalase-2 is peroxisomal and verify that it can be used as a marker enzyme for C. elegans peroxisomes.

We examined the expression and localization of type-II 3-oxoacyl-CoA thiolase in the nematode Caenorhabditis elegans. Type-II thiolase acts on 3-oxoacyl-CoA esters with a methyl group at the alpha carbon, whereas conventional thiolases do not. Mammalian type-II thiolase, which is also termed sterol carrier protein x (SCPx) or SCP2/3-oxoacyl-CoA thiolase, is located in the peroxisomes and involved in phytanic acid degradation and most probably in bile acid synthesis. The nematode enzyme lacks the SCP2 domain, which carries the peroxisomal-targeting signal, but produces bile acids in a cell-free system. Northern and Western blot analyses demonstrated that C. elegans expressed type-II thiolase throughout its life cycle, especially during the larval stages, and that the expression was significantly enhanced by the addition of clofibrate at 5 mM or more to the culture medium. Whole-mount in situ hybridization and immunostaining of L4 larvae revealed that the enzyme was mainly expressed in intestinal cells, which are multifunctional like many of the cell types in C. elegans. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected the enzyme in the matrix of peroxisomes. These results indicate the fundamental homology between mammalian SCPx and the nematode enzyme regardless of whether the SCP2 part is fused, suggesting their common physiological roles.

We cloned a full-length cDNA of the nematode Caenorhabditis elegans that encodes a 44-kDa protein (P-44, 412 residues) similar to sterol carrier protein x (SCPx). Mammalian SCPx is a bipartite protein: its 404-residue N-terminal and 143-residue C-terminal domains are similar to 3-ketoacyl-CoA thiolase and identical to the precursor of sterol carrier protein 2 (SCP2; also termed non-specific lipid-transfer protein), respectively. P-44 has 56% sequence identity to the thiolase domain of SCPx but lacks the SCP2 sequence. Northern blot analysis revealed only a single mRNA species of 1.4 kb, which agrees well with the length of the cDNA (1371 bp), making it improbable that alternative splicing produces an SCPx-like fusion protein. The sequence similarities of P-44 to conventional thiolases are lesser than that to SCPx. Purified recombinant P-44 cleaved long-chain 3-ketoacyl-CoAs (C(8-16)) in a thiolytic manner by the ping-pong bi-bi reaction mechanism. The inhibition of P-44 by acetyl-CoA was competitive with CoA and non-competitive with 3-ketooctanoyl-CoA. This pattern of inhibition is shared with SCPx but not with conventional 3-ketoacyl-CoA thiolase, which is inhibited uncompetitively with respect to 3-ketoacyl-CoA. From these results, we concluded that nematode P-44 and mammalian SCPx constitute a second isoform of thiolase, which we propose to term type-II 3-ketoacyl-CoA thiolase.

We have sequenced the nucleotides of the gene POX18 that encodes PXP-18, a major peroxisomal polypeptide inducible by oleic acid in the yeast Candida tropicalis. POX18 had a single open reading frame of 127 amino acids. Some 33% of the amino acid sequence of the predicted basic polypeptide (13,805 Da), was identical to that of the nonspecific lipid-transfer protein (sterol carrier protein 2) from rat liver. PXP-18, purified to near homogeneity from isolated peroxisomes, had an amino-terminal sequence identical to that of the predicted polypeptide except for the initiator methionine, and had nonspecific lipid-transfer activity comparable to that of its mammalian equivalents. Unexpectedly, PXP-18 lacked the cysteine residue thought to be essential for the activity of this protein in mammals. RNA blot analysis showed that the POX18 gene was expressed exclusively in cells grown on oleic acid, suggesting that PXP-18 has a role in the beta-oxidation of long-chain fatty acids. PXP-18 modulated acyl-coenzyme A oxidase activity at low pH.

A clone harbouring the genomic DNA sequence for the peroxisomal catalase of an n-alkane-utilizable yeast, Candida tropicalis, has been isolated by the hybrid-selection method and confirmed with a probe of catalase partial cDNA. Nucleotide sequence analysis of the cloned DNA disclosed that the gene fragment coding for catalase had a length of 1455 base pairs (corresponding to 485 amino acids; m = 54937 Da), and that the size of this enzyme was the smallest among all catalases reported hitherto. No intervening sequence was found in this coding region and some portions coincided with the amino acid sequences obtained from the analysis of the purified catalase. The comparison with three peroxisomal catalases from rat liver, bovine liver and human kidney, and one cytosolic catalase from Saccharomyces cerevisiae has revealed that catalase from C. tropicalis was more homologous to the peroxisomal enzymes than to the cytosolic one. C. tropicalis used the codons of the high-expression type. Amino acid residues were all conserved at the active and heme-binding sites. In the N and C-terminal regions there was no characteristic signal sequence or consensus sequence. However, a noticeable region, which can be discriminated between peroxisomal and cytosolic catalases, was proposed.

The level of acetyl-coenzyme-A carboxylase activity in Candida lipolytica undergoes large variations depending upon the carbon source on which the yeast is grown. Cells grown on n-alkanes or fatty acids exhibit a lower activity level than do cells grown on glucose. Among the n-alkanes and fatty acids tested, n-heptadecane, n-octadecane, oleic acid and linoleic acid reduce the enzyme activity to the lowest levels, which are 16-18% of the activity level in glucose-grown cells. Immunochemical titrations and Ouchterlony double-diffusion analysis with specific antibody as well as kinetic studies have indicated that the observed decrease in the level of acetyl-CoA carboxylase activity is due to a reduction in the cellular content of the enzyme. Furthermore, isotopic leucine incorporation studies with the use of the immunoprecipitation technique have demonstrated that the relative rate of synthesis of the enzyme in oleic-acid-grown cells is diminished to 12% of that in glucose-grown cells. Evidence has also been obtained to support the view that the enzyme in this yeast is not degraded at a rate high enough to contribute to the marked decrease in the cellular content of the enzyme. Thus, it is concluded that the reduction in acetyl-CoA carboxylase content in fatty-acid-grown cells is due to diminished synthesis of the enzyme.

Acyl-coenzyme-A synthetase I from Candida lipolytica has been purified to homogeneity as evidenced by polyacrylamide gel electrophoresis in the presence and absence of dodecylsulfate as well as by Ouchterlony double-diffusion analysis. The purification procedure involves resolution of cellular particles with Triton X-100 and chromatography on phosphocellulose, Blue-Sepharose and Sephadex G-100. The purified enzyme exhibits a specific activity of 20--24 U/mg protein at 25 degree C, which is about 100-fold higher than those of long-chain acyl-CoA synthetases hitherto reported. The molecular weight of the enzyme has been estimated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate to be approximately 84 000. The enzyme is specific for fatty acids with 14--18 carbon atoms regardless of the degree of unsaturation. Studies with the use of specific antibody to acyl-CoA synthetase I have indicated that this enzyme is immunochemically distinguishable from acyl-CoA synthetase II.

Studies have been made on the subcellular localization of two long-chain acyl-coenzyme-A synthetases as well as glycerolphosphate acyltransferase and the acyl-CoA-oxidizing system in Candida lipolytica grown on oleic acid. Acyl-CoA synthetase I is distributed among different subcellular fractions, including microsomes and mitochondria where glycerolphosphate acyltransferase is located. On the other hand, acyl-CoA synthetase II is localized in microbodies where the acyl-CoA-oxidizing system is located. These results support our previous conclusion that acyl-CoA synthetase I is responsible for the production of acyl-CoA to be utilized for the synthesis of cellular lipids, while acyl-CoA synthetase II provides acyl-CoA that is exclusively degraded via beta-oxidation.

Messenger RNA from Candida lipolytica directs the synthesis of complete acetyl-coenzyme-A carboxylase in the mRNA-dependent reticulocyte lysate cell-free translation system. The identity of the translation product is evidenced by the following results: first, it is immunoprecipitated with antibody to acetyl-CoA carboxylase and competes with authentic acetyl-CoA carboxylase for binding to the antibody; secondly, it co-migrates with authentic acetyl-CoA carboxylase (Mr 230000) upon dodecylsulfate/polyacrylamide gel electrophoresis; finally, the peptide fragments formed by its partial proteolysis with papain or alpha-chymotrypsin are identical with those formed from authentic acetyl-CoA carboxylase. With the use of this assay system, it has been demonstrated that the level of acetyl-CoA carboxylase mRNA activity in C. lipolytica cells decreases with increasing concentrations of oleic acid in culture medium and that the changes in the mRNA activity parallel those in the cellular level of acetyl-CoA carboxylase. This finding, in conjunction with our previous study, indicates that the diminished synthesis of acetyl-CoA carboxylase in cells grown in the presence of fatty acid is due to a reduced level of the mRNA coding for the enzyme.

Acetyl-coenzyme-A carboxylase has been isolated in homogeneous form from Candida lipolytica. The homogeneity of the enzyme preparation is evidenced by analytical ultracentrifugation, dodecyl-sulfate-polyacrylamide gel electrophoresis and Ouchterlony double-diffusion analysis. The purified enzyme exhibits a specific activity of 8.0 U/mg protein at 25 degrees C and contains 1 mol biotin/263000 g protein. The sedimentation coefficient (S20,W) of the enzyme is 18 S. It has been shown by dodecyl-sulfate-polyacrylamide gel electrophoresis that the enzyme possesses only one kind of subunit with a molecular weight of 230000. This finding, together with the biotin content, indicates that the C. lipolytica enzyme has a highly integrated subunit structure. The C. lipolytica enzyme is very labile, but is stabilized by glycerol. The enzyme is markedly activated by poly(ethyleneglycol), the activation being due principally to a decrease in the Km values for substrates. Even in the presence of this activator, the Km value for acetyl-CoA of the C. lipolytica enzyme is much higher than that of the enzyme from Saccharomyces cerevisiae and animal tissues. The C. lipolytica enzyme, unlike the enzyme from animal tissues, is not activated by citrate.