Studies on the N-acetyl-β-d-hexosaminidase B from germinating Lupinus luteus L. seeds I. Purification and characterization

I. Pócsi, L. Kiss, Pál Nánási

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18 Citations (Scopus)

Abstract

The N-acetyl-β-d-hexosaminidase B of germinating Lupinus luteus L. seeds (McFarlane et al. (1984) Phytochemistry 23, 2431-2433) was partially purified with a six-step purification procedure following extraction. This enzyme consists of one protein chain (Mr 69000, as determined by SDS-PAGE and 62500, as obtained by gel filtration on Bio-Gel P-60 Gel) and has a neutral isoelectric point (pI = 7.05, as determined by chromatofocusing). Moreover, it was found to be very sensitive to low ionic strength, especially in the presence of different gels based on Sephadex. Considering the substrate specificity, the enzyme splits both p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucosaminide and -galactosaminide substrates, but lacks N,N′-diacetylchitobiase activity. A new mixed-substrate procedure was developed and is presented here to demonstrate that a common active site is responsible for the splitting of both synthetic substrates.

Original languageEnglish
Pages (from-to)110-118
Number of pages9
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1039
Issue number1
DOIs
Publication statusPublished - May 31 1990

Fingerprint

Hexosaminidase B
Lupinus
Purification
Seed
Seeds
Gels
Substrates
Acetylglucosaminidase
Isoelectric Point
Enzymes
Substrate Specificity
Osmolar Concentration
Gel Chromatography
Polyacrylamide Gel Electrophoresis
Catalytic Domain
Ionic strength
Proteins

Keywords

  • Enzyme purification
  • Hexosaminidase B

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

Cite this

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abstract = "The N-acetyl-β-d-hexosaminidase B of germinating Lupinus luteus L. seeds (McFarlane et al. (1984) Phytochemistry 23, 2431-2433) was partially purified with a six-step purification procedure following extraction. This enzyme consists of one protein chain (Mr 69000, as determined by SDS-PAGE and 62500, as obtained by gel filtration on Bio-Gel P-60 Gel) and has a neutral isoelectric point (pI = 7.05, as determined by chromatofocusing). Moreover, it was found to be very sensitive to low ionic strength, especially in the presence of different gels based on Sephadex. Considering the substrate specificity, the enzyme splits both p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucosaminide and -galactosaminide substrates, but lacks N,N′-diacetylchitobiase activity. A new mixed-substrate procedure was developed and is presented here to demonstrate that a common active site is responsible for the splitting of both synthetic substrates.",
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AU - Kiss, L.

AU - Nánási, Pál

PY - 1990/5/31

Y1 - 1990/5/31

N2 - The N-acetyl-β-d-hexosaminidase B of germinating Lupinus luteus L. seeds (McFarlane et al. (1984) Phytochemistry 23, 2431-2433) was partially purified with a six-step purification procedure following extraction. This enzyme consists of one protein chain (Mr 69000, as determined by SDS-PAGE and 62500, as obtained by gel filtration on Bio-Gel P-60 Gel) and has a neutral isoelectric point (pI = 7.05, as determined by chromatofocusing). Moreover, it was found to be very sensitive to low ionic strength, especially in the presence of different gels based on Sephadex. Considering the substrate specificity, the enzyme splits both p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucosaminide and -galactosaminide substrates, but lacks N,N′-diacetylchitobiase activity. A new mixed-substrate procedure was developed and is presented here to demonstrate that a common active site is responsible for the splitting of both synthetic substrates.

AB - The N-acetyl-β-d-hexosaminidase B of germinating Lupinus luteus L. seeds (McFarlane et al. (1984) Phytochemistry 23, 2431-2433) was partially purified with a six-step purification procedure following extraction. This enzyme consists of one protein chain (Mr 69000, as determined by SDS-PAGE and 62500, as obtained by gel filtration on Bio-Gel P-60 Gel) and has a neutral isoelectric point (pI = 7.05, as determined by chromatofocusing). Moreover, it was found to be very sensitive to low ionic strength, especially in the presence of different gels based on Sephadex. Considering the substrate specificity, the enzyme splits both p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucosaminide and -galactosaminide substrates, but lacks N,N′-diacetylchitobiase activity. A new mixed-substrate procedure was developed and is presented here to demonstrate that a common active site is responsible for the splitting of both synthetic substrates.

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