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Abstract
We report the cloning and characterization of a novel membrane-bound, calcium-independent PLA2, named cPLA2-gamma. The sequence encodes a 541-amino acid protein containing a domain with significant homology to the catalytic domain of the 85-kDa cPLA2 (cPLA2-alpha). cPLA2-gamma does not contain the regulatory calcium-dependent lipid binding (CaLB) domain found in cPLA2-alpha. However, cPLA2-gamma does contain two consensus motifs for lipid modification, a prenylation motif (-CCLA) at the C terminus and a myristoylation site at the N terminus. We present evidence that the isoprenoid precursor [3H]mevalonolactone is incorporated into the prenylation motif of cPLA2-gamma. Interestingly, cPLA2-gamma demonstrates a preference for arachidonic acid at the sn-2 position of phosphatidylcholine as compared with palmitic acid. cPLA2-gamma encodes a 3-kilobase message, which is highly expressed in heart and skeletal muscle, suggesting a specific role in these tissues. Identification of cPLA2-gamma reveals a newly defined family of phospholipases A2 with homology to cPLA2-alpha.
Highlights
Phospholipases A2 (PLA2)1 are a diverse group of enzymes that hydrolyze the sn-2 fatty acids from phospholipids and play a role in a wide range of physiological functions
The spacer region separating these two domains corresponds to an area in cPLA2-␣ considered to be an exposed hinge region containing many protease-accessible sites, as well as the MAP kinase activation site Ser-505 [6]. cPLA2-␥ contains a sequence that is similar to the lipase consensus sequence, GLSGS, in cPLA2-␣, which has been found to be critical for cPLA2-␣ activity [7]
We have identified a novel 60.9-kDa calcium-independent phospholipase A2, which we termed cPLA2-␥. cPLA2-␥ contains 28.7% overall sequence identity with cPLA2-␣ and was identified by searching the expressed sequence tag (EST) data base for related proteins
Summary
Cell Culture and Antibodies—COS cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 50 units/ml penicillin, 50 g/ml streptomycin, and 1 mM glutamine. The resulted clone, named pED⌬C-cPLA2-␥WT, was sequenced to confirm the desired C-terminal coding sequence. The resulted clone, named pED⌬C-cPLA2-␥SSLA, was confirmed by sequencing to contain the desired C-terminal coding sequence. The restriction fragment (EcoRI-EcoRI) containing the entire coding sequence was isolated from pED⌬C-cPLA2-␥WT and ligated to a controlled expression vector pHTOP. Lysis buffer was added to the lysate to 1 ml to reduce the SDS to 0.1%, incubated for another 30 min, and centrifuged 1 h at 100,000 ϫ g, 4 °C. The supernatant was immunoprecipitated with 10 l of anti-cPLA2-␥ antibody, 44282, or anti-Ras antibody (Upstate Biotechnology) for 3 h at 4 °C, incubated with protein A-Sepharose (Amersham Pharmacia Biotech) saturated with 10% bovine serum albumin for 30 min. Proteins were transferred to a nitrocellulose filter (Novex), immunoblotted for cPLA2-␥ with 44284 antibody, and detected by ECL (Amersham)
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