Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1 I. Purification and some enzymatic properties

Masaki, Takeharu; Tanabe, Manabu; Nakamura, Keiji; Soejima, Mitsuhiro

doi:10.1016/0005-2744(81)90106-6

Cited by 168 publications

(60 citation statements)

References 16 publications

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“…Enzymatic Digestion and Chemical Cleavage-Reversedphase HPLC-purified lectin (2-5 nmol) was digested in 100 mM Tris-HCl buffer containing 2 M urea, pH 9.0, with lysyl endopeptidase (30) at an enzyme-to-substrate molar ratio of 1:300 or in 50 mM Tris-HCl buffer containing 2 M urea, pH 8.5, with endoproteinase Asp-N at an enzyme-to-substrate molar ratio of 1:20, at 37°C for 18 h. The lectin (2 nmol) was chemically cleaved at the methionyl bonds with 2% CNBr in 70% (v/v) formic acid at 25°C for 18 h in the dark by the method of Gross (31). The peptide with the amino-terminal blocking group was treated with the deblocking aminopeptidase N-acetyl deblocking aminopeptidase in 50 mM N-ethylmorpholine/acetic acid buffer containing 0.1 mM CoCl 2 , pH 7.5, at 37°C for 20 h, according to the manufacturer's instructions (32).…”

Section: Methodsmentioning

confidence: 99%

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells

Fujii

Dohmae

Takio

et al. 2012

Journal of Biological Chemistry

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Background: Studies on the diversity of carbohydrate-binding proteins (lectins) are important in glycobiology. Results: A lectin having a novel primary structure was isolated from a mussel and found to have a globotriose-dependent cytotoxicity on Burkitt lymphoma cells. Conclusion: A new primary structure quite distinct from known lectin is described. Significance: Discovery of similar lectin structures from vertebrates will lead to progress in medical sciences.

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Section: Methodsmentioning

confidence: 99%

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells

Fujii

Dohmae

Takio

et al. 2012

Journal of Biological Chemistry

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“…The 81-kD protein band was cut out from the polyacrylamide gel and treatecl with a lysyl endopeptidase, Acromobacter protease I (Masaki et al, 1981). The resulting peptides were separated by reverse-phase HPLC and the sequences of the major peptides were analyzed (Kawasaki et al, 1990).…”

Section: Peptide Sequence Analysismentioning

confidence: 99%

Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin

et al. 1995

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enf-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbifa maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular m a s of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81 -kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The K,,, value for copalyl pyrophosphate was 0.35 p~, and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and BaZ+ inhibited the activity.ent-Kaurene is an important intermediate in GA biosynthesis and is synthesized from GGPP via CPP (Fig. 1). These steps are catalyzed by KSA and KSB, respectively (Coolbaugh, 1983). Other terpenoids, such as carotenoids and phytol, also have GGPP as a precursor, whereas CPP is a precursor of macrocyclic diterpenes. Hence, KSA and KSB are important enzymes in the early stage of GA biosynthesis (Coolbaugh, 1983; Chung and Coolbaugh, 1986;Graebe, 1987). Duncan and West (1981) separated KSA and KSB from wild cucumber (Mavah macrocarpus L.) using a quaternalyaminoethyl column and suggested that the conversion of GGPP to ent-kaurene is catalyzed by the two different enzymes. These authors showed that KSA and KSB associated with each other during ent-kaurene synthesis and that KSB preferentially utilized endogenous CPP This study was performed through special coordination funds of the Science and Technology Agency of the Japanese government, and the Shorai Foundation for Science and Technology.* Corresponding author; e-mail ykamiya@postman.riken.go.jp; fax 81-48 -462-4691.produced by KSA rather than exogenous CPP. Both KSA and KSB were suggested to be localized in plastids (Aach et al., 1995). Sun et al. (1992) cloned the GAZ locus of Arabidopsis by genomic subtraction. Recently, Sun and Kamiya (1994) showed that the GA1 locus encodes KSA. A putative KSA has also been cloned from maize by transposon tagging (Bensen et al., 1995). Castor bean (Xicinus communis) produces kaurene and the related diterpenes beyerene, trachylobane, and sandaracopimaradiene, which are formed by alternative cyclization of the common intermediate CPP, and KSB was partially purified (Spickett et al., 199...

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“…The eluted fractions of affinity chromatography were collected, precipitated by 10% trichloroacetic acid, and subjected to SDS-PAGE. A Coomassie Brilliant Blue-stained band of 90 kDa was cut out and digested with Acromobacter protease I (API; a gift from Dr. Masaki, Ibaraki University) (14). The resulting peptides were separated by reverse phase high pressure liquid chromatography on tandemly connected DEAE-5PW (1 ϫ 20 mm; Tosoh, Tokyo, Japan) and Capcel Pak C 18 UG120 (1 ϫ 50 mm; Shiseido, Tokyo, Japan) columns with a 0 -80% gradient of acetonitrile in 0.1% trifluoroacetic acid.…”

Section: Methodsmentioning

confidence: 99%

Type F Scavenger Receptor SREC-I Interacts with Advillin, a Member of the Gelsolin/Villin Family, and Induces Neurite-like Outgrowth

Shibata

Ishii

Koizumi

et al. 2004

Journal of Biological Chemistry

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The scavenger receptor expressed by endothelial cells (SREC) was isolated from a human endothelial cell line and consists of two isoforms named SREC-I and -II. Both isoforms have no significant homology to other types of scavenger receptors. They contain 10 repeats of epidermal growth factor-like cysteine-rich motifs in the extracellular domains and have unusually long C-terminal cytoplasmic domains with Ser/Pro-rich regions. The extracellular domain of SREC-I binds modified low density lipoprotein and mediates a homophilic SREC-I/ SREC-I or heterophilic SREC-I/SREC-II trans-interaction. However, the significance of large Ser/Pro-rich cytoplasmic domains of SRECs is not clear. Here, we found that when SREC-I was overexpressed in murine fibroblastic L cells, neurite-like outgrowth was induced, indicating that the receptor can lead to changes in cell morphology. The SREC-I-mediated morphological change required the cytoplasmic domain of the protein, and we identified advillin, a member of the gelsolin/villin family of actin regulatory proteins, as a protein binding to this domain. Reduction of advillin expression in L cells by RNAi led to the absence of the described SREC-I-induced morphological changes, indicating that advillin is a prerequisite for the change. Finally, we demonstrated that SREC-I and advillin were co-expressed and interacted with each other in dorsal root ganglion neurons during embryonic development and that overexpression of both SREC-I and advillin in cultured Neuro-2a cells induced long process formation. These results suggest that the interaction of SREC-I and advillin are involved in the development of dorsal root ganglion neurons by inducing the described morphological changes.Scavenger receptors are defined by their ability to bind and metabolize modified low density lipoproteins (LDLs), 1 such as acetylated LDL (AcLDL) and oxidized LDL (OxLDL), and have been regarded as relevant in the pathogenesis of atherosclerosis (1, 2). Mammalian cells have several different classes of scavenger receptors, and their relative contributions to lipid metabolism in pathophysiological conditions, such as atherosclerosis, are the subject of intense investigation (2).Endothelial cells express several distinct scavenger receptors, such as SR-BI (3-5), LOX-1 (6), and FEEl-1/stabilin-1 (7). We cloned a novel scavenger receptor from a DNA library prepared from human umbilical vein endothelial cells employing expression cloning and termed it SREC (scavenger receptor expressed by endothelial cells)-I (8). Subsequently, we also succeeded in cloning a homologous protein, SREC-II, by a data base search (9). These two receptors are now classified as type F scavenger receptors (2, 9).Both SREC-I and -II have no significant homology to other types of scavenger receptors. They contain 10 repeats of epidermal growth factor-like cysteine-rich motifs in their extracellular domains and unusually long C-terminal cytoplasmic domains with Ser/Pro-rich regions (8, 9). SREC-I mediates the binding and degradation of AcLDL and O...

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Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1 I. Purification and some enzymatic properties

Cited by 168 publications

References 16 publications

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells

Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin

Type F Scavenger Receptor SREC-I Interacts with Advillin, a Member of the Gelsolin/Villin Family, and Induces Neurite-like Outgrowth

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