Cytryńska Małgorzata, Zdybicka-Barabas Agnieszka, Jakubowicz Teresa
Department of Invertebrate Immunology, Institute of Biology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland.
J Insect Physiol. 2006 Jul;52(7):744-53. doi: 10.1016/j.jinsphys.2006.04.002. Epub 2006 Apr 25.
Protein kinase A (PKA) activity was detected in the fat body of Galleria mellonella larvae by a non-radioactive method using a specific peptide substrate-kemptide. The enzyme activity was stimulated by cAMP and its analogues: BzcMP, 8-Chl-cAMP and 8-Br-cAMP in concentrations of 1-4muM. Cyclic GMP was not effective in PKA activation. A two-fold increase in PKA activity was detected in the fat body of G. mellonella LPS-challenged larvae. Selective, membrane-permeable PKA inhibitors, H89 and Rp-8-Br-cAMPS, inhibited protein kinase A activity in the fat body of G. mellonella larvae in vitro and in vivo. The inhibition of PKA activity in vivo was correlated with a considerable lowering of haemolymph antibacterial activity and a decrease in lysozyme content in the fat body of immune challenged larvae. The use of phospho-motif antibodies recognising PKA phosphorylation consensus site allowed identification of four potential PKA phosphorylation substrates of 79, 45, 40 and 36kDa in G. mellonella fat body.
采用非放射性方法，使用特异性肽底物-肯普肽（kemptide）检测了大蜡螟幼虫脂肪体中的蛋白激酶A（PKA）活性。该酶活性受到环磷酸腺苷（cAMP）及其类似物BzcMP、8-氯-cAMP和8-溴-cAMP的刺激，浓度为1-4μM。环磷酸鸟苷（cGMP）对PKA激活无效。在受到脂多糖（LPS）攻击的大蜡螟幼虫脂肪体中，检测到PKA活性增加了两倍。选择性的、可透过细胞膜的PKA抑制剂H89和Rp-8-溴-cAMPS在体外和体内均抑制了大蜡螟幼虫脂肪体中的蛋白激酶A活性。体内PKA活性的抑制与免疫攻击幼虫血淋巴抗菌活性的显著降低以及脂肪体中溶菌酶含量的减少相关。使用识别PKA磷酸化共有位点的磷酸化基序抗体，在大蜡螟脂肪体中鉴定出了四种潜在的PKA磷酸化底物，分子量分别为79、45、40和36 kDa。
