On the active site of β-hexosaminidase from latex of Ficus glabrata

Orlacchio, Aldo; Maffei, C.; Emiliani, Carla; Reinosa, J

doi:10.1016/s0031-9422(00)84872-4

Cited by 9 publications

(6 citation statements)

References 22 publications

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“…pNP-GlcNAc was used at 0.3 to 5.0 mM and pNP-GalNAc at 0.15 to 2.5 mm. The Km values, given in Table II, range from 0.27 to 1.71 mm and are in line with those reported from ,BNAHAs from other plant sources (1,6,14,19,23,25,27,32). j3-NAHA-IA was inhibited by concentrations of pNP-GlcNAc above 3.5 mM, but none of the other isozymes were.…”

Section: Hanes Plots [S]/v Versus [S]supporting

confidence: 87%

“…Like ,3-NAHAs described from pinto beans (1), fenugreek cotyledons (4), wheat aleurone (6), castor beans ( 14), jack beans (19), lupin seeds (21), Ficus latex (25), and cotton seeds (32), the #-NAHA isozymes from pea will release p-nitrophenol from both pNP-GlcNAc and pNP-GalNAc, hence the general name off,-N-acetylhexosaminidase. None of the pea isozymes hydrolyzed p-aminophenyl-N-acetyl-f3-i-glucosaminide or p-nitrophenyl-Nacetyl-l-thio-f3-i-glucosaminide when aliquots of enzyme were incubated with 2 mM substrate at pH 4.5 at 30°C for 1 h.…”

Section: Resultsmentioning

confidence: 99%

“…The thiol compounds alone had no effect on enzyme activity. ,B-NAHAs from pinto bean (1), fenugreek (5), wheat germ (6), jack bean (19), Ficus latex (25), and cotton (32) are also strongly inhibited by HgC92, while the castor bean enzyme is not (14). A number of proteins destined for protein bodies, such as 7S globulins and many lectins, are glycosylated (16).…”

Section: Hanes Plots [S]/v Versus [S]mentioning

confidence: 99%

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Isozymes of β-N-Acetylhexosaminidase from Pea Seeds (Pisum sativum L.)

Harley¹,

Beevers²

1987

Plant Physiol.

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Four isozymes of O-N-acetylhexosaminidase (#-NAHA) from pea seeds (Pisum sativum L.) have been separated, with one, designated f-NAHA-II, purified to apparent homogeneity by means of an affinity column constructed by ligating p-aminophenyl-N-acetyl-#-D-thioglucosaminide to Affi-Gel 202. The other three isozymes have been separated and purified 500-to 1750-fold by chromatography on Concanavalin ASepharose, Zn2" charged immobilized metal affinity chromatography, hydrophobic chromatography, and ion exchange chromatography on CMSephadex. All four isozymes are located in the protein bodies of the cotyledons. The molecular weight of each isozyme is 210,000. ,-NAHA-II is composed of two heterogenous subunits. The subunits are not held together by disulfide bonds, but sulfhydryl groups are important for catalysis. All four isozymes release p-nitrophenol from both p-nitrophenyl-N-acetyl-j%D-glucosaminide and p-nitrophenyl-N-acetyl-t-D-ga- ' Abbreviations: #-NAHA, f3-N-acetylhexosaminidase; 2ME, 2-mercaptoethanol; bis, N,N'-methylene-bis-acrylamide; CM, carboxymethyl; Con A, concanavalin A; IMA, immobilized metal affinity; PHA, phytohemagglutinin; pNP-GlcNAc, p-nitrophenyl-N-acetyl-(3-D-glucosaminide; pNP-GalNAc, p-nitrophenyl-N-acetyl-,B-D-galactosaminide. PA) were surface sterilized by stirring for 10 min in a 10% (v/v) solution of commercial bleach. The seeds were rinsed thoroughly with water and imbibed 20 h in aerated water. The imbibed seeds were washed once with 500 ml of water, once with 500 ml of 0.5 M NaCl, once with 500 ml of 0.1 M NaCl, 10 mM Na4EDTA, and three times with 500 ml water. All subsequent operations were performed at 4°C. The washed seeds were homogenized in 400 ml of 50 mm Na-phosphate (pH 7.0) in a Waring Blendor for 30 s. The homogenate was squeezed through four layers of cheesecloth and centrifuged for 20 min at 16,000g. The pH of the supernatant was adjusted to 5 by the dropwise addition of 1 M citric acid and the precipitate that formed was removed by centrifugation (20 min at 16,000g). The supernatant was neutralized to pH 7.0 by the dropwise addition of 2 M Tris. Solid (NH4)2SO4 was added to the neutralized supernatant, and the material that precipitated between 30 and 70% saturation (176 and 472 g/l) (13) was collected by centrifugation (20 min at 16

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Section: Hanes Plots [S]/v Versus [S]supporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Hanes Plots [S]/v Versus [S]mentioning

confidence: 99%

See 1 more Smart Citation

Isozymes of β-N-Acetylhexosaminidase from Pea Seeds (Pisum sativum L.)

Harley¹,

Beevers²

1987

Plant Physiol.

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“…The β-HexNAc'ase from rice seeds released p-nitrophenol from both pNP-GlcNAc and pNP-GalNAc (Table 2). This is the same as β-HexNAc'ases that are described from pinto beans (Agrawal and Bahl, 1972), fenugreek cotyledons (Bouquelet and Spik, 1978), wheat aleurone (Carratu et al, 1985), castor beans (Harley and Beevers, 1985), jack beans (Li and Li, 1970), lupin seeds (McFarlane et al, 1984), Ficus latex (Orlacchio et al, 1985), and cotton seeds (Yi, 1981). The ability of the enzyme to utilize a number of potential substrates was assessed in McIlvaine's buffer (pH 5.0).…”

Section: Substrate Specificitymentioning

confidence: 96%

Purification and Characterization of β-N-Acetylhexosaminidase from Rice Seeds

Jin

Jo²,

Kim³

et al. 2002

BMB Reports

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N-Acetyl-beta-D-hexosaminidase (beta-HexNAc'ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sativa L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions (Fsub1;- F7sub7) by CM-Sephadex chromatography. Among them, F6 was further purified to homogeneity with a 13.0% yield and 123.3 purification-fold. The molecular mass was estimated to be about 52 kDa on SDS-PAGE and 37.4 kDa on Sephacryl S- 300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNPGalNAc) as substrates, which are typical properties of beta-HexNAc'ase. The ratio of the pNP-GlcNAc'ase activity to the pNP-GalNAc'ase activity was 4.0. However, it could not hydrolyze chitin, chitosan, pNP-beta-glucopyranoside, or pNP-beta-galactopyranoside. The enzyme showed K(M), V(max) and K(cat) for pNP-GlcNAc of 1.65mM, 79.49mM min(1), and 4.79 x 10(6) min(1), respectively. The comparison of kinetic values for pNPGlcNAc and pNP-GalNAc revealed that the two enzyme activities are associated with a single binding site. The purified enzyme exhibited optimum pH and temperature for pNPGlcNAc of 5.0 and 50 degrees C, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and 20-40 degrees C. The enzyme activity was completely inhibited at a concentration of 0.1 mM HgCl(2) and AgNO(3), suggesting that the intact thiol group is essential for activity. Chloramine T completely inhibited the activity, indicating the possible involvement of methionines in the mechanism of the enzyme.

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“…Not much information is available on the activation energy of plant b-NAHA-catalysed reactions. The activation energies of b-NAHA from pinto beans (Agrawal & Bahl, 1968) and the latex of Ficus glabrata (Orlacchio, Maffel, Emiliani, & Reinosa, 1985) for the catalysis of pNP-b-GlcNAc hydrolysis were found to be 9.8 and 13.2 kcal/mol, respectively.…”

Section: Activation Energymentioning

confidence: 99%