PubMed · 3320683
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L Huang, J Connor, C Y Wang. 1987. pH-sensitive immunoliposomes.. https://doi.org/10.1016/0076-6879(87)49046-0
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This study investigates the importance of thioester-linked acyl groups in lung surfactant protein C (SP-C) in facilitating interactions with phospholipids that yield functionally important surface active behaviors. Native SP-C, palmitoylated at cysteine residues at positions 5 and 6, was isolated from bovine lung surfactant by liquid chromatography. Deacylated SP-C (dSP-C), unchanged in composition and sequence from SP-C but having a decreased alpha-helical content in films with dipalmitoyl phosphatidylcholine (DPPC) of 52 versus 70%, was obtained by treatment with 0.1 M sodium carbonate buffer at pH 10. Surface activity was studied for SP-C and dSP-C combined with column-purified phospholipids (PPL) from calf lung surfactant or with synthetic phospholipids (DPPC or a synthetic phospholipid mixture (SPL) containing 50:35:15, DPPC:egg phosphatidylcholine:egg phosphatidylglycerol). Interfacial measurements included surface pressure time adsorption isotherms for dispersed surfactants with diffusion minimized, dynamic surface pressure area isotherms and respreading for films in the Wilhelmy balance, and overall surface tension lowering at physiologic cycling rate in oscillating bubble experiments. Dispersions of PPL:SP-C and SPL:SP-C rapidly adsorbed to high equilibrium surface pressures of 47-48 mN/m, significantly better than corresponding dispersions containing dSP-C. The adsorption of PPL:dSP-C was essentially unchanged from that of PPL alone, and the adsorption of SPL:dSP-C was improved only slightly over SPL alone. In Wilhelmy balance studies, dynamic respreading was significantly improved over phospholipids alone in films of SP-C plus PPL, SPL, or DPPC. Respreading was improved less markedly by dSP-C in corresponding films with SPL or DPPC and not at all in films with PPL. Maximum surface pressures were also higher in cycled films of SP-C versus dSP-C combined with PPL or SPL. In bubble experiments (37 degrees C, 20 cycles/min), dispersions of PPL:SP-C and SPL:SP-C reached low minimum surface tensions of <1 and 5 mN/m, respectively, whereas PPL:dSP-C and SPL:dSP-C only reached minima of approximately 20 mN/m as did PPL and SPL alone. Acylation in SP-C is crucial for its interactions with phospholipids over the full spectrum of adsorption and dynamic surface behaviors important for lung surfactant.
The reacylation of lysophospholipids back to their parent molecules is important for attaining the appropriate fatty acyl composition in many phospholipids and for preventing the accumulation of arrhythmia generating lysophospholipids in the heart. In this study, we report the presence of an active acyltransferase activity for lysophosphatidylglycerol reacylation to phosphatidylglycerol in rat heart membrane preparations. The activity of acyl-Coenzyme A:1-acylglycerophosphorylglycerol acyltransferase in rat heart subcellular fractions was in the order of microsomal > mitochondrial > cytosol. The activity in the membrane fractions were characterized and found to have a pH optimum in the alkaline range. However, significant enzyme activity was observed at physiological pH. With oleoyl-Coenzyme A as substrate, the microsomal activity had a preference for lysophosphatidylglycerol substrates in the order of myristoyl > palmitoyl > oleoyl > stearoyl. The apparent K(m) values for 1-palmitoylglycerophosphorylglycerol and oleoyl-Coenzyme A were 9.4 and 7.1 microM, respectively. In contrast, the mitochondrial activity had a preference for lysophosphatidylglycerol substrates in the order of oleoyl > myristoyl = stearoyl = palmitoyl. The apparent K(m) values for 1-oleoylglycerophosphorylglycerol and oleoyl-Coenzyme A were 17.8 and 18.0 microM, respectively. Both membrane activities were heat labile as pre-incubation at 55 degrees C for 1 min completely abolished the activity. However, pre-incubation at 50 degrees C resulted in different profiles of inactivation in both microsomal and mitochondrial fractions. Both membrane activities were inhibited by high concentrations of lysophosphatidylglycerol and affected to a similar extent by various detergents. To demonstrate whether reacylation of lysophosphatidylglycerol to phosphatidylglycerol occurred in vivo, isolated rat hearts were perfused for 60 min in the Langendorff mode with 0.1 microM 1-palmitoylglycerophosphoryl[14C]glycerol bound to albumin. 1-Palmitoylglycerophosphoryl[14C]glycerol was readily taken up by the isolated perfused rat heart and significant synthesis of phosphatidyl[14C]glycerol was observed. The findings indicate the presence of an acyl-Coenzyme A:1-acylglycerophosphorylglycerol acyltransferase activity in the rat heart subcellular membranes which is capable of catalyzing lysophosphatidylglycerol acylation to phosphatidylglycerol in vitro and in vivo.
Neutral lipid storage disease (NLSD) is an autosomal recessive disorder in which excess triacylglycerol (TG) accumulates in most cells. Although it has been hypothesized that the TG accumulation is caused by a functional defect in cytosolic lipase activity, we were able to expose TG hydrolysis in NLSD cells by using triacsin C, an inhibitor of acyl-CoA synthetase that blocks the reincorporation of hydrolyzed fatty acids into glycerolipids. Our data suggest that TG lipolysis in NLSD cells is masked by rapid TG resynthesis, occurring because released acylglycerols cannot be used for phospholipid synthesis. In uptake studies, triacsin C blocked the incorporation of [3H]glycerol into glycerolipids, incorporation of [14C]oleate into TG, but not incorporation of [14C]oleate into phospholipid. Thus, the drug inhibited both de novo synthesis of glycerolipids via the glycerol-3-phosphate pathway and the synthesis of TG from diacylglycerol. The drug did not appear to block reacylation of lysophospholipids. Triacsin C caused a loss of about 60% of the TG mass from both NLSD and oleate-loaded control cells. Rates of TG lipolysis were similar in NLSD cells and oleate-loaded control cells labeled with [6-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoic acid or labeled with [14C]oleate or [3H]glycerol and chased in the presence of triacsin C. During a 96-h chase, [14C]oleate reincorporation into the different phospholipid species increased only in control cells. Similar results were observed when NLSD, and control cells were chased after labeling with [3H]glycerol. These data strongly suggest that normal human fibroblasts mobilize stored TG for phospholipid synthesis and that recycling to PC occurs via a TG-derived mono- or diacylglycerol intermediate. Normal recycling to phosphatidylethanolamine may primarily involve TG-derived acyl groups rather than an acylglycerol precursor. NLSD cells appear to have a block in this recycling pathway with the result that both hydrolyzed fatty acids and the acylglycerol backbone are re-esterified to form TG. Because the NLSD phenotype includes ichthyosis, fatty liver, myopathy, cardiomyopathy, and mental retardation, the recycling pathway appears to be critical for the normal function of skin, liver, muscle, heart, and the central nervous system.