Isozymes of β-<i>N</i>-Acetylhexosaminidase from Pea Seeds (<i>Pisum sativum</i> L.)

Harley, Suzanne M.; Beevers, Leonard

doi:10.1104/pp.85.4.1118

Cited by 14 publications

(4 citation statements)

References 13 publications

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“…The β-HexNAc'ase isozymes also have different hydrolysis ratios towards pNP-GlcNAc and pNP-GalNAc, ranging from 1.25 to 18.0 for the four isoenzymes from fenugreek (Bouquelet and Spik, 1976), 1.9 to 4.8 for the three from lupin (McFarlane et al, 1984), and 4.0-12.9 for the four from pea seeds (Harley and Beevers, 1987). It is reported that the activity ratio of pNP-GlcNAc'ase to pNP-GalNAc'ase is pH dependent due to different pH optima for the substrates Harley and Beevers, 1987). Temperature dependence The optimal temperature of the enzyme towards pNP-GlcNAc and pNP-GalNAc were 50 o C and 55 o C, respectively (Fig.…”

Section: Substrate Specificitymentioning

confidence: 99%

“…These values are comparable with those for the lupin enzyme (Póci et al, 1990). The K m of the enzyme for pNP-GlcNAc from various other plant sources varied from 40 µM to 1.13 mM (Bouquelet and Spik, 1978;Chang et al, 1998;Giordani et al, 1992) and from 40 µM to 1.71 mM for pNP-GalNAc (Bouquelet and Spik, 1978;Harley and Beevers, 1987;Choi and Gross, 1994). It was reported that pNP-GlcNAc and pNP-GalNAc are bound to a single active site (Choi and Gross, 1994;Li and Li, 1970).…”

Section: Effect Of Substrate Concentrationmentioning

confidence: 99%

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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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Section: Substrate Specificitymentioning

confidence: 99%

Section: Effect Of Substrate Concentrationmentioning

confidence: 99%

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

Jin

Jo²,

Kim³

et al. 2002

BMB Reports

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“…Binding protein was isolated from untreated (lane 10) or protease-treated (lane 11) CCVs by affinity chromatography of CHAPS-extracted membrane proteins on the proaleurain column. Antibodies specific for plant Asn-linked oligosaccharides were purified from rabbit anti-pea P-N-acetylhexosaminidase antiserum (26) by chromatography on a pineapple stem bromelain-agarose affinity column (S. M. Harley and L.B., unpublished data). Because antibodies against the polypeptide portions of these two enzymes do not crossreact, only antibodies specific for plant oligosaccharides were retained on the column and then eluted for use in these experiments.…”

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confidence: 99%

Purification and initial characterization of a potential plant vacuolar targeting receptor.

Kirsch

Paris

Butler

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

228

259

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Clathrin-coated vesicles are known to be involved in the transport ofproteins from the Golgi to the vacuole in plant cells. The mechanisms by which proteins are directed into this pathway are not known. Here we identify an integral membrane protein of -80 kDa, extracted from clathrin-coated vesicles of developing pea (Pisum saivum L.) cotyledons, that bound at neutral pH to an affinity column prepared with the N-terminal targeting determinant of the vacuolar thiol protease, proaleurain, and eluted when the pH was lowered to 4. The protein was not retained on a control column prepared with the N-terminal sequence of a homologous, secreted thiol protease, endopeptidase B. The 80-kDa protein also accumulated in a membrane fraction that is less dense than clathrin-coated vesicles. In vitro studies demonstrated a binding constant of 37 nM between the =80-kDa protein and the proaleurain targeting determinant. A peptide with a vacuolar targeting determinant from prosporamin weakly competed for binding to the "80-kDa protein, while a peptide carrying a single amino acid substitution known to abolish prosporamin vacuolar targeting had no measurable binding affinity for the protein. The binding protein is a glycoprotein with a transmembrane orientation in which the C terminus is exposed to the cytoplasm. The binding domain is located in the N-terminal luminal portion of the protein. These properties of the binding protein are consistent with the function of a receptor that would select proteins in the trans-Golgi for sorting to clathrin-coated vesicles and delivery to the vacuole.Vacuoles are acidic compartments occupying up to 80% of the volume of mature plant cells. In addition to functioning in the maintenance ofturgor and as a depository for such solutes as amino acids, sugars, organic acids, and mineral salts, the vacuoles also contain hydrolytic enzymes and, at certain stages of plant development, may serve as sites of accumulation of storage proteins (1). The accumulation of hydrolytic enzymes has been used as evidence to consider that the vacuole is a lytic compartment in plants analogous to the lysosome of the mammalian cell (2). Because of the presence of hydrolytic enzymes and the accumulation of reserve proteins, the vacuole/protein body is a useful system in which to study protein targeting in plants (3, 4). As in the mammalian lysosome system, soluble vacuolar proteins are synthesized in the endoplasmic reticulum and progress through the Golgi apparatus and clathrin-coated vesicles (CCVs) (5, 6) prior to accumulating in the vacuole. Although some soluble vacuolar proteins are glycosylated, N-linked oligosaccharides have no role in sorting glycoproteins to the vacuole (3, 4, 7). There is an accumulating body of information indicating that targeting of soluble proteins to the vacuole is mediated by determinants that reside in the polypeptide (3, 4, 7). To date, N-terminal (8-11) and C-terminal targeting determinant sequences (12, 13) have been identified; internal peptide sequences also appear to be in...

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“…Electrophoresis and Immunoblotting SDS-PAGE was performed using a discontinuous buffer system (19) and previously described acrylamide ratios (9). The proteins were visualized by silver staining (3).…”

Section: Isolation Of Coated Vesiclesmentioning

confidence: 99%

Coated Vesicles Are Involved in the Transport of Storage Proteins during Seed Development in Pisum sativum L

Harley

Beevers²

1989

Plant Physiol.

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During seed development, various storage proteins and hydrolases accumulate in specialized storage vacuoles, the protein bodies, via an elaborate intracellular transport system involving the rough endoplasmic reticulum, the Golgi apparatus, and transit vesicles. Clathrin-coated vesicles, similar to those which transport lysosomal proteins to lysosomes, an organelle analogous to the vacuole, in animal cells, could be involved in this intracellular transport mechanism. Clathrin-coated vesicles have been isolated from cotyledons of developing pea (Pisum sativum L.) seeds at the time of rapid protein accumulation and analyzed for the presence of protein body constituents. A 23,000 M, polypeptide, corresponding to pea lectin precursor, was found associated with the vesicles, as determined by immunoblotting. The lectin precursor was apparently sequestered within the vesicles, as the polypeptide was only susceptible to proteolysis if detergents were included in the digestion buffer. A number of glycosidase activities, including a-mannosidase, a-galactosidase, and f8-Nacetylhexosaminidase, were also associated with the vesicles. Thus, it appears that clathrin-coated vesicles are involved in the intracellular transport of storage proteins during seed development.

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

Cited by 14 publications

References 13 publications

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

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

Purification and initial characterization of a potential plant vacuolar targeting receptor.

Coated Vesicles Are Involved in the Transport of Storage Proteins during Seed Development in Pisum sativum L

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