An 18.5 kDa protein from the amebocyte of Limulus polyphemus, homologous to the previously described amebocyte aggregation factor, expresses alternative phospholipase A2 activity.
A protein expressing phospholipase A2 activity was purified from the granular amebocyte of the horseshoe crab, Limulus polyphemus by cation-exchange, size-exclusion chromatography and semi-preparative reverse-phase-high pressure liquid chromatography (RP-HPLC). The protein had an apparent mass of 17.7 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), but a more accurate estimate of 18.5 kDa was assigned by electrospray ionization-mass spectrometry (ESI-MS). A partial sequence of this protein demonstrated total sequence homology with an 18.5 kDa protein with cell aggregating properties from Limulus reported by Fujii et al. [J. Biol. Chem. 267:22452.]. In these studies, the Limulus protein demonstrated a positive cross-reaction to polyclonal anti-human recombinant phospholipase A2 (group II, 14 kDa). The protein did not display a significant loss of biological activity after boiling, but all enzymatic activity was lost after boiling in the presence of the reducing agent betamercaptoethanol (beta-mercaptoethanol). The Limulus protein was inhibited by manoalide, a covalent irreversible phospholipase A2 inhibitor, in a dose-dependent fashion with 50% inhibition occurring at a concentration of 0.48 microM. The Limulus protein displayed no activity in a triglyceride lipase assay. These studies characterize an alternative phospholipase A2 activity for the previously described 18.5 kDa protein from the L. polyphemus amebocyte.
MacPherson
J C
JC
Department of Ecology, Evolution antd Marine Biology, UCSB, Santa Barbara, CA 93106, USA. macphej@ccf.org
Jacobs
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D015153:Blotting, Western; D002412:Cations; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005470:Fluorometry; D006384:Hemagglutination; D006737:Horseshoe Crabs; D006358:Hot Temperature; D006801:Humans; D008049:Lipase / Q000378:metabolism; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013729:Terpenes / Q000494:pharmacology; D013997:Time Factors
D060829:Arthropod Proteins; D002412:Cations; D015815:Cell Adhesion Molecules; D004220:Disulfides; D010726:Phosphodiesterase Inhibitors; D011994:Recombinant Proteins; D019163:Reducing Agents; D013729:Terpenes; C013065:cell aggregation factors; D008623:Mercaptoethanol; C045673:manoalide; D008049:Lipase; D010741:Phospholipases A; D054467:Phospholipases A2