Abstract
Catalase (EC 1.11.1.6) was purified to near homogeneity from isolated megagametophytes of germinated loblolly pine (Pinus taeda L.) seeds, and monospecific antibodies were elicited in rabbits. Following a procedure that involved acetone extraction, (NH4)2SO4 fractionation, and four chromatographic steps (i.e. DE-52 cellulose, Superdex-200, hydroxylapatite, and phenyl-Sepharose CL-4B), catalase was purified about 140-fold to a final specific activity of 2215 mmol min-1 mg-1 of protein. Cotton isocitrate lyase antibodies were used, and protein immunoblots revealed that the resolution on hydroxylapatite and phenyl-Sepharose allowed for the complete separation of catalase from contaminating isocitrate lyase. The molecular masses of the native enzyme and its subunit are 235 and 59 kD, respectively, indicating that the pine holoenzyme is a homotetramer. Loblolly pine catalase exists as multiple isoforms. When megagametophytes taken 7 d after imbibition at 30[deg]C were extracted, subjected to nondenaturing isoelectric focusing, and stained for catalase activity, at least four catalase isoforms were observed, including one dominant form with an isoelectric point of 6.87. Purified pine catalase is not a glycoprotein and has a ratio of absorbance at 208 nm to absorbance at 405 nm of 1.5. When probed with loblolly pine catalase antibodies, protein blots of cell-free extracts from megagametophytes of mature, stratified, and germinated loblolly pine seeds, the megagametophyte glyoxysomal fraction, and purified loblolly pine catalase all revealed one immunoreactive 59-kD polypeptide. This indicates that no detectable change in the enzyme's monomeric molecular mass occurs during seed stratification and germination, early seedling growth, and purification.
Full Text
The Full Text of this article is available as a PDF (2.4 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bürk R. R., Eschenbruch M., Leuthard P., Steck G. Sensitive detection of proteins and peptides in polyacrylamide gels after formaldehyde fixation. Methods Enzymol. 1983;91:247–254. doi: 10.1016/s0076-6879(83)91021-2. [DOI] [PubMed] [Google Scholar]
- Clare D. A., Duong M. N., Darr D., Archibald F., Fridovich I. Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase. Anal Biochem. 1984 Aug 1;140(2):532–537. doi: 10.1016/0003-2697(84)90204-5. [DOI] [PubMed] [Google Scholar]
- Cooper T. G., Beevers H. Mitochondria and glyoxysomes from castor bean endosperm. Enzyme constitutents and catalytic capacity. J Biol Chem. 1969 Jul 10;244(13):3507–3513. [PubMed] [Google Scholar]
- Frevert J., Kindl H. Plant microbody proteins. Purification and glycoprotein nature of glyoxysomal isocitrate lyase from cucumber cotyledons. Eur J Biochem. 1978 Dec 1;92(1):35–43. doi: 10.1111/j.1432-1033.1978.tb12720.x. [DOI] [PubMed] [Google Scholar]
- Havir E. A., McHale N. A. Purification and characterization of an isozyme of catalase with enhanced-peroxidatic activity from leaves of Nicotiana sylvestris. Arch Biochem Biophys. 1990 Dec;283(2):491–495. doi: 10.1016/0003-9861(90)90672-l. [DOI] [PubMed] [Google Scholar]
- Havir E. A., McHale N. A. Regulation of Catalase Activity in Leaves of Nicotiana sylvestris by High CO(2). Plant Physiol. 1989 Mar;89(3):952–957. doi: 10.1104/pp.89.3.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunce C. M., Trelease R. N. Heterogeneity of catalase in maturing and germinated cotton seeds. Plant Physiol. 1986 Aug;81(4):1134–1139. doi: 10.1104/pp.81.4.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Köller W., Kindl H. Glyoxylate cycle enzymes of the glyoxysomal membrane from cucumber cotyledons. Arch Biochem Biophys. 1977 May;181(1):236–248. doi: 10.1016/0003-9861(77)90502-1. [DOI] [PubMed] [Google Scholar]
- Maeshima M., Beevers H. Purification and properties of glyoxysomal lipase from castor bean. Plant Physiol. 1985 Oct;79(2):489–493. doi: 10.1104/pp.79.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mörikofer-Zwez S., Cantz M., Kaufmann H., von Wartburg J. P., Aebi H. Heterogeneity of erythrocyte catalase. Correlations between sulfhydryl group content, chromatographic and electrophoretic properties. Eur J Biochem. 1969 Nov;11(1):49–57. doi: 10.1111/j.1432-1033.1969.tb00737.x. [DOI] [PubMed] [Google Scholar]
- Ni W., Trelease R. N. Post-Transcriptional Regulation of Catalase Isozyme Expression in Cotton Seeds. Plant Cell. 1991 Jul;3(7):737–744. doi: 10.1105/tpc.3.7.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scandalios J. G. The antioxidant enzyme genes Cat and Sod of maize: regulation, functional significance, and molecular biology. Isozymes Curr Top Biol Med Res. 1987;14:19–44. [PubMed] [Google Scholar]
- Trelease R. N., Hermerath C. A., Turley R. B., Kunce C. M. Cottonseed malate synthase : purification and immunochemical characterization. Plant Physiol. 1987 Aug;84(4):1343–1349. doi: 10.1104/pp.84.4.1343. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vanni P., Giachetti E., Pinzauti G., McFadden B. A. Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp Biochem Physiol B. 1990;95(3):431–458. doi: 10.1016/0305-0491(90)90002-b. [DOI] [PubMed] [Google Scholar]
- Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
- Yamaguchi J., Nishimura M., Akazawa T. Purification and characterization of heme-containing low-activity form of catalase from greening pumpkin cotyledons. Eur J Biochem. 1986 Sep 1;159(2):315–322. doi: 10.1111/j.1432-1033.1986.tb09870.x. [DOI] [PubMed] [Google Scholar]
- Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem. 1969 Jul;30(1):148–152. doi: 10.1016/0003-2697(69)90383-2. [DOI] [PubMed] [Google Scholar]