Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye

Satoshi Toyoshima, Naoki Matsumoto, Peng Wang, Hiroko Inoue, Tohru Yoshioka, Yoshiki Hotta, Toshiaki Osawa

    研究成果: Article

    38 引用 (Scopus)

    抄録

    To examine whether the norpA (no receptor potential A) gene encodes a phosphoinositide-specific phospholipase C (PLC) in the eye of Drosophila, a major PLC in the extract from normal Drosophila heads, which was absent in the extract from norpA mutant heads, and purified and its partial amino acid sequences were determined. The purification of the major PLC in KC1 extract from normal Drosophila heads was achieved by sequential column chromatography on DEAE-Sepharose CL-6B, Mono Q, Superose 12, Mono S, second Mono S, and second Mono Q, followed by column chromatography on Superose 12 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 98,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). Interestingly, the calcium and pH requirements for activation of the crude enzyme (KCl extract) were quite different from those of partially purified enzyme (active fraction from second Mono Q column). The maximal activity for PIP2 hydrolysis was observed at calcium concentrations between 10-7 and 10-5 M for both the crude and partially purified enzymes. On the other hand, the activity for PI hydrolysis of the crude enzyme increased with increasing calcium concentrations, while that of the partially purified enzyme reached a maximum at calcium concentrations between 10-6 and 10-4 M, and decreased at millimollar concentration. The pH dependences for PI hydrolysis of the crude enzyme and the partially purified enzyme were similar. The crude enzyme hydrolyzed PIP2 over a broad pH range from 6 to 8.5, while the activity of the partially purified enzyme monotonously increased with increasing pH. The partial amino acid sequences were determined by treating the purified enzyme with endopeptidase Lys-C; the resultant peptide fragments were purified on a high performance liquid chromatography-reverse phase column and then sequenced with sequencer. The obtained sequences were found to be a part of the deduced amino acid sequences of cDNA which was suggested to be norpA gene (Bloomquist, B. T., Shortridge, R. S., Schneuwly, S., Perdew, M., Monell, C., Steller, H., Rubin, G., and Pak, W. L. (1989) Cell 54, 723-733).

    元の言語English
    ページ(範囲)14842-14848
    ページ数7
    ジャーナルJournal of Biological Chemistry
    265
    発行部数25
    出版物ステータスPublished - 1990 9 5

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    Phosphoinositide Phospholipase C
    Type C Phospholipases
    Phosphatidylinositols
    Drosophila
    Purification
    Amino Acid Sequence
    Amino Acids
    Enzymes
    Calcium
    Hydrolysis
    Column chromatography
    Head
    Sodium Dodecyl Sulfate
    Electrophoresis
    Chromatography
    Polyacrylamide Gel Electrophoresis
    Sodium Cholate
    Genes
    Peptide Fragments
    Enzyme Activation

    ASJC Scopus subject areas

    • Biochemistry

    これを引用

    Toyoshima, S., Matsumoto, N., Wang, P., Inoue, H., Yoshioka, T., Hotta, Y., & Osawa, T. (1990). Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye. Journal of Biological Chemistry, 265(25), 14842-14848.

    Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye. / Toyoshima, Satoshi; Matsumoto, Naoki; Wang, Peng; Inoue, Hiroko; Yoshioka, Tohru; Hotta, Yoshiki; Osawa, Toshiaki.

    :: Journal of Biological Chemistry, 巻 265, 番号 25, 05.09.1990, p. 14842-14848.

    研究成果: Article

    Toyoshima, S, Matsumoto, N, Wang, P, Inoue, H, Yoshioka, T, Hotta, Y & Osawa, T 1990, 'Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye', Journal of Biological Chemistry, 巻. 265, 番号 25, pp. 14842-14848.
    Toyoshima S, Matsumoto N, Wang P, Inoue H, Yoshioka T, Hotta Y その他. Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye. Journal of Biological Chemistry. 1990 9 5;265(25):14842-14848.
    Toyoshima, Satoshi ; Matsumoto, Naoki ; Wang, Peng ; Inoue, Hiroko ; Yoshioka, Tohru ; Hotta, Yoshiki ; Osawa, Toshiaki. / Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye. :: Journal of Biological Chemistry. 1990 ; 巻 265, 番号 25. pp. 14842-14848.
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    title = "Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye",
    abstract = "To examine whether the norpA (no receptor potential A) gene encodes a phosphoinositide-specific phospholipase C (PLC) in the eye of Drosophila, a major PLC in the extract from normal Drosophila heads, which was absent in the extract from norpA mutant heads, and purified and its partial amino acid sequences were determined. The purification of the major PLC in KC1 extract from normal Drosophila heads was achieved by sequential column chromatography on DEAE-Sepharose CL-6B, Mono Q, Superose 12, Mono S, second Mono S, and second Mono Q, followed by column chromatography on Superose 12 in the presence of 1{\%} sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 98,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). Interestingly, the calcium and pH requirements for activation of the crude enzyme (KCl extract) were quite different from those of partially purified enzyme (active fraction from second Mono Q column). The maximal activity for PIP2 hydrolysis was observed at calcium concentrations between 10-7 and 10-5 M for both the crude and partially purified enzymes. On the other hand, the activity for PI hydrolysis of the crude enzyme increased with increasing calcium concentrations, while that of the partially purified enzyme reached a maximum at calcium concentrations between 10-6 and 10-4 M, and decreased at millimollar concentration. The pH dependences for PI hydrolysis of the crude enzyme and the partially purified enzyme were similar. The crude enzyme hydrolyzed PIP2 over a broad pH range from 6 to 8.5, while the activity of the partially purified enzyme monotonously increased with increasing pH. The partial amino acid sequences were determined by treating the purified enzyme with endopeptidase Lys-C; the resultant peptide fragments were purified on a high performance liquid chromatography-reverse phase column and then sequenced with sequencer. The obtained sequences were found to be a part of the deduced amino acid sequences of cDNA which was suggested to be norpA gene (Bloomquist, B. T., Shortridge, R. S., Schneuwly, S., Perdew, M., Monell, C., Steller, H., Rubin, G., and Pak, W. L. (1989) Cell 54, 723-733).",
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    T1 - Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye

    AU - Toyoshima, Satoshi

    AU - Matsumoto, Naoki

    AU - Wang, Peng

    AU - Inoue, Hiroko

    AU - Yoshioka, Tohru

    AU - Hotta, Yoshiki

    AU - Osawa, Toshiaki

    PY - 1990/9/5

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    N2 - To examine whether the norpA (no receptor potential A) gene encodes a phosphoinositide-specific phospholipase C (PLC) in the eye of Drosophila, a major PLC in the extract from normal Drosophila heads, which was absent in the extract from norpA mutant heads, and purified and its partial amino acid sequences were determined. The purification of the major PLC in KC1 extract from normal Drosophila heads was achieved by sequential column chromatography on DEAE-Sepharose CL-6B, Mono Q, Superose 12, Mono S, second Mono S, and second Mono Q, followed by column chromatography on Superose 12 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 98,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). Interestingly, the calcium and pH requirements for activation of the crude enzyme (KCl extract) were quite different from those of partially purified enzyme (active fraction from second Mono Q column). The maximal activity for PIP2 hydrolysis was observed at calcium concentrations between 10-7 and 10-5 M for both the crude and partially purified enzymes. On the other hand, the activity for PI hydrolysis of the crude enzyme increased with increasing calcium concentrations, while that of the partially purified enzyme reached a maximum at calcium concentrations between 10-6 and 10-4 M, and decreased at millimollar concentration. The pH dependences for PI hydrolysis of the crude enzyme and the partially purified enzyme were similar. The crude enzyme hydrolyzed PIP2 over a broad pH range from 6 to 8.5, while the activity of the partially purified enzyme monotonously increased with increasing pH. The partial amino acid sequences were determined by treating the purified enzyme with endopeptidase Lys-C; the resultant peptide fragments were purified on a high performance liquid chromatography-reverse phase column and then sequenced with sequencer. The obtained sequences were found to be a part of the deduced amino acid sequences of cDNA which was suggested to be norpA gene (Bloomquist, B. T., Shortridge, R. S., Schneuwly, S., Perdew, M., Monell, C., Steller, H., Rubin, G., and Pak, W. L. (1989) Cell 54, 723-733).

    AB - To examine whether the norpA (no receptor potential A) gene encodes a phosphoinositide-specific phospholipase C (PLC) in the eye of Drosophila, a major PLC in the extract from normal Drosophila heads, which was absent in the extract from norpA mutant heads, and purified and its partial amino acid sequences were determined. The purification of the major PLC in KC1 extract from normal Drosophila heads was achieved by sequential column chromatography on DEAE-Sepharose CL-6B, Mono Q, Superose 12, Mono S, second Mono S, and second Mono Q, followed by column chromatography on Superose 12 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 98,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). Interestingly, the calcium and pH requirements for activation of the crude enzyme (KCl extract) were quite different from those of partially purified enzyme (active fraction from second Mono Q column). The maximal activity for PIP2 hydrolysis was observed at calcium concentrations between 10-7 and 10-5 M for both the crude and partially purified enzymes. On the other hand, the activity for PI hydrolysis of the crude enzyme increased with increasing calcium concentrations, while that of the partially purified enzyme reached a maximum at calcium concentrations between 10-6 and 10-4 M, and decreased at millimollar concentration. The pH dependences for PI hydrolysis of the crude enzyme and the partially purified enzyme were similar. The crude enzyme hydrolyzed PIP2 over a broad pH range from 6 to 8.5, while the activity of the partially purified enzyme monotonously increased with increasing pH. The partial amino acid sequences were determined by treating the purified enzyme with endopeptidase Lys-C; the resultant peptide fragments were purified on a high performance liquid chromatography-reverse phase column and then sequenced with sequencer. The obtained sequences were found to be a part of the deduced amino acid sequences of cDNA which was suggested to be norpA gene (Bloomquist, B. T., Shortridge, R. S., Schneuwly, S., Perdew, M., Monell, C., Steller, H., Rubin, G., and Pak, W. L. (1989) Cell 54, 723-733).

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