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The CoA-independent transacylase in PAF biosynthesis: tissue distribution and molecular species selectivity
M L Blank1, Z L Smith, V Fitzgerald
1Medical Sciences Division, Oak Ridge Institute for Science and Education, TN 37831-0117.
Abstract:
Microsomal membranes from six different rat tissues (spleen, lung, kidney, brain, testis, and liver) were found to possess CoA-independent transacylase activity that could both acylate lyso-[3H]PAF (1-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine) and then deacylate the 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine product via the transacylation of added exogenous 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine. Platelet-activating factor (1-[3H]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) was produced when acetyl-CoA was added to the spleen microsomes during generation of lyso-[3H]PAF by the transacylases. More extensive studies with subcellular fractions from spleen revealed that, in addition to microsomes, the transacylase activities were also present in the 15,000 x g membrane fraction but not in the cytosol. Analysis of molecular species of 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine before and after addition of 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine as the acyl acceptor demonstrated a high selectivity for polyunsaturated fatty acids (> 3 double bonds/acyl group) in both the acylation and deacylation processes that occurred in testicular microsomal membranes. The transfer of acyl groups by the transacylase appeared to be equally effective for either arachidonic or docosapentaenoic(n - 6) fatty acids, whereas linoleic and oleic fatty acids were not transferred from 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine following the addition of 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine. Similar experiments with the membrane fraction of undifferentiated HL-60 cells showed that arachidonic acid supplementation of intact cells enhanced both the CoA-independent transacylation of lyso-[3H]PAF and the subsequent deacylation of 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine caused by addition of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. Differentiation of the HL-60 cells into a neutrophil-like form had no effect on the transacylase activity. Our results indicate the PAF-related transacylase is widely distributed among tissues and, although highly selective for polyunsaturated acyl groups, does not discriminate selectively among the polyunsaturates.
Insights
This study identifies a CoA-independent transacylase in rat tissues that modifies platelet-activating factor (PAF) and related phospholipids. This enzyme shows a preference for polyunsaturated fatty acids, suggesting a role in lipid metabolism.
Area of Science:
- Biochemistry
- Cell Biology
- Lipid Metabolism
Background:
- Platelet-activating factor (PAF) is a potent lipid mediator involved in inflammation and other biological processes.
- The synthesis and metabolism of PAF involve complex enzymatic pathways, including acylation and deacylation steps.
- Transacylases play a crucial role in lipid remodeling, but their specific roles in PAF metabolism are not fully elucidated.
Purpose of the Study:
- To investigate the presence and characteristics of CoA-independent transacylase activity in various rat tissues.
- To determine the substrate specificity and acyl group preference of this transacylase.
- To explore the role of this enzyme in the metabolism of lyso-PAF and related phospholipids.
Main Methods:
- Microsomal membranes and subcellular fractions from six rat tissues were isolated.
- CoA-independent transacylase activity was assayed using lyso-[3H]PAF as an acyl acceptor and 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine as an acyl donor.
- Molecular species analysis of acylated products was performed using techniques like HPLC.
- Experiments were conducted with HL-60 cell membranes to assess the impact of arachidonic acid supplementation and differentiation.
Main Results:
- A CoA-independent transacylase activity was detected in microsomal membranes from spleen, lung, kidney, brain, testis, and liver.
- This enzyme could acylate lyso-[3H]PAF and subsequently deacylate the product by transferring acyl groups to 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine.
- Testicular microsomal membranes exhibited high selectivity for polyunsaturated fatty acids (PUFAs) in both acylation and deacylation.
- Arachidonic acid and docosapentaenoic acid were effectively transferred, while linoleic and oleic acids were not.
- Arachidonic acid supplementation in HL-60 cells enhanced transacylase activity, but cell differentiation did not alter it.
Conclusions:
- A widely distributed CoA-independent transacylase exists in rat tissues, involved in PAF-related lipid metabolism.
- The enzyme demonstrates a strong preference for PUFAs, particularly in acylation and deacylation processes.
- This transacylase may play a significant role in regulating the composition and function of cellular lipids, especially those containing PUFAs.