TITLE Purification and characterization of isoforms of b-N-acetylhexosaminidase from mungbean seedlings
AUTHOR Yi-Ching CHEN
Graduate Institute of Bioscience and Biotechnology, National Ocean University, Keelung 202, Taiwan
Wei-Liang LIU
Department of Life Science, Fu-Jen Catholic University, HsinChuang, Taipei 242, Taiwan
Hui-Ching HSU
Institute of Botany, Academia Sinica, Nankang, Taipei 115, Taiwan
Yung-An LEE
Department of Life Science, Fu-Jen Catholic University, HsinChuang, Taipei 242, Taiwan
Ching-San CHEN
Institute of Botany, Academia Sinica, Nankang, Taipei 115, Taiwan
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ABSTRACT Three isoforms of b-N-acetylhexosaminidase (b-NAHA), named b-NAHAs I, II and III, were isolated from six-day-old etiolated mungbean (Vigna radiata) seedlings. b-NAHA I was purified to apparent homogeneity by a procedure involving Con A-Sepharose chromatography, chromatofocusing, and gel filtration. b-NAHAs II and III were highly purified. b-NAHAs I, II and III had molecular masses of 135,127 and 110 kDa, respectively. b-NAHA I was dissociated into a single 67 kDa protein band. II was dissociated into two protein bands corresponding to 60 and 48 kDa, and III was dissociated into a single 48 kDa protein band in SDS-polyacrylamide gel electrophoresis. The results suggest that isoforms I and III are homodimeric enzymes, each comprising two identical subunits with molecular masses of 67 kDa and 48 kDa, respectively, while isoform II is a heterodimeric enzyme, comprising two non-identical subunits with molecular masses of 60 kDa and 48 kDa. All the enzymes were active against para- nitrophenyl-b-N-acetylglucosaminide (PNP-b-N-acetylglucosaminide) and PNP-b-N-galactosaminide. The enzymes were inhibited by 5, 5'-dithiobis (2-nitrobenzoic acid)(DTNB), Ag+, Hg2+, and N, N'-diacetylchitobiose. Km values for isoforms I, II and III were 0.67 mM, 1.04 mM and 1.76 mM, respectively, using PNP-b-N-acetylglucosaminide as a substrate. These three isoforms had acidic pI values (I, 6.3; II, 6.1; and III, 5.9). Their optimal pH in the reaction towards PNP-b-N-acetylglucosaminide was 5.4, 4.7 and 5.7, and optimal temperatures were 65°C, 65°C and 50°C for isoforms I, II and III, respectively.
KEYWORD b-N-acetylhexosaminidase; Enzyme purification; Germination; Oligomeric structure; Vigna radiata;
ARTICLE INFO Botanical Bulletin of Academia Sinica, Volume 45 Number 4 October 2004, page 275-283, 9 pages
PUBLISHER Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China