Immunological detection of alkaline-diaminobenzidine-negativeperoxisomes of the nematode Caenorhabditis elegans purification and unique pH optima of peroxisomal catalase.
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.
Togo
S H
SH
Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, Japan.
Maebuchi
M
M
Yokota
S
S
Bun-Ya
M
M
Kawahara
A
A
Kamiryo
T
T
D015100:3,3'-Diaminobenzidine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000648:ultrastructure*; D002374:Catalase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera; D008970:Molecular Weight; D020675:Peroxisomes / Q000201:enzymology / Q000276:immunology*
D007106:Immune Sera; D015100:3,3'-Diaminobenzidine; D002374:Catalase