Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Phospholipase A(2) and related biochemistry in fat body of the tobacco hornworm, Manduca sexta
Abstract
This dissertation presents the results of research into phospholipase A$\sb2$ (PLA$\sb2$) and related biochemistry in fat body of the tobacco hornworm, Manduca sexta. PLA$\sb2$ activity from 11,750 x g supernatants produced from fat body homogenates was sensitive to substrate concentration, protein concentration, pH, temperature and denaturation. Optimal activity was observed under the following conditions: 4 $\mu$M substrate(phosphatidylcholine (1-palmitoyl, 2-arachidonyl (arachidonyl-1-$\sp{14}$C))) concentration, pH 8.0-9.0, and an incubation temperature of 40$\sp\circ$C. This activity was independent of calcium concentration. The PLA$\sb2$ activity in 11,750 x g supernatant from fat body homogenate is predominantly associated with the cytosol as at least 64% of PLA$\sb2$ activity was recovered in the supernatant fraction of homogenates centrifuged at 100,000 x g. This cytosolic PLA$\sb2$ activity was calcium-independent and inhibited in a dose-dependent manner by the PLA$\sb2$-specific inhibitor oleyloxyethyl phosphorylcholine (OOPC), with IC$\sb{50}$ of 5-50 $\mu$M. PLA$\sb2$ activity in whole fat body tissue, 11,750 x g supernatant, 100,000 x g pellet or 100,000 x g supernatant was unaffected during 8 weeks storage at $-$80$\sp\circ$C. Size exclusion chromatography of 100,000 x g supernatant indicated that the PLA$\sb2$ has a molecular weight of 44 $\pm$ 2.2 kDa. The most active fraction displayed a distinct 45.5 kDa protein band in SDS polyacrylamide gel electrophoresis analysis. Fatty acids were primarily incorporated into PLs and triacylglycerols (TGs) in in vivo, time dependent incorporation studies of radioactive 20:4n-6 into fat body lipids. Incorporation of radioactive 18:1n-9 into fat body lipids was greatest in TGs. Both radioactive 20:4n-6 and 18:1n-9 were redistributed among fat body PL classes in a time dependent manner. Both fatty acids were primarily ($>$80%) incorporated into the sn-2 position of fat body PLs. The data indicate that a cytosolic PLA$\sb2$ that is capable of hydrolyzing 20:4n-6 is present in fat body of Manduca sexta.
Subject Area
Entomology|Biochemistry|Animal diseases
Recommended Citation
Uscian, John Michael, "Phospholipase A(2) and related biochemistry in fat body of the tobacco hornworm, Manduca sexta" (1994). ETD collection for University of Nebraska-Lincoln. AAI9516593.
https://digitalcommons.unl.edu/dissertations/AAI9516593