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J. Biol. Chem., Vol. 265, Issue 25, 14842-14848, 09, 1990
S Toyoshima, N Matsumoto, P Wang, H Inoue, T Yoshioka, Y Hotta and T Osawa
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 KCl
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.
Purification and partial amino acid sequences of phosphoinositide- specific phospholipase C of Drosophila eye
Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
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