| dc.description.abstract | A great variety of lipids are found in biological membranes, and the relationship between lipid composition and membrane function poses major unsolved problems in membrane biology. However, many membrane?bound enzymes are known to require phospholipids for activity, and the regulation of many membrane?bound enzyme activities by phospholipids has been reported.
Palmitoyl?CoA synthetase, in various organisms, has been reported to be a membrane?bound enzyme. Earlier work done in our laboratory showed that the palmitoyl?CoA synthetase from the brush?border?free particulate fraction of the chicken intestinal mucosa required the presence of lysophosphatidylcholine (LPC) and a detergent like Triton X?100 for maximal activity, in addition to the cofactors ATP, CoA, and Mg²?. It was of interest to study the effect of LPC and Triton X?100, if any, on this enzyme in other tissues of chicken and to understand the mechanism of activation of the enzyme by LPC and Triton X?100.
Palmitoyl?CoA synthetase activities from liver and intestinal mucosa were activated by LPC and Triton X?100, whereas that from adipose tissue was activated only by LPC and not by Triton X?100. In contrast, palmitoyl?CoA synthetase(s) from heart and brain were inhibited by the two compounds, showing that the effect of Triton X?100 and LPC on the enzyme(s) of various tissues in chicken is not a general one.
Since liver was used extensively in most of the earlier work on palmitoyl?CoA synthetase, it was selected for detailed studies. A subcellular fractionation showed that a high percentage of the overall activity was present in the microsomal fraction of the chicken liver homogenate. The kinetic properties of the chicken liver microsomal enzyme, with particular emphasis on the mechanism of activation of the enzyme by LPC and Triton X?100, were studied. The activation was found not to be due to a general detergent effect or due to solubilisation of the enzyme. The requirement of the enzyme for LPC was found to be absolutely specific. As LPC had no effect on the Km value of the enzyme for palmitate and Triton X?100 doubled the Km value, the possibility that LPC and/or Triton X?100 may increase the affinity of the enzyme for palmitate was ruled out.
It also appeared possible that LPC and/or Triton X?100 may lower the energy of activation of the reaction, i.e., conversion of palmitate to palmitoyl?CoA. To study this possibility, the temperature?dependence of the enzyme was examined and the energies of activation calculated. A biphasic Arrhenius plot of activity was obtained, and the biphasic nature of the plot was found to be abolished by the addition of Triton X?100 or LPC to the assay mixture. The activation energy calculations indicated that LPC and/or Triton X?100 could not be activating the enzyme by lowering the energy of activation of the reaction.
Membrane phospholipids are known to undergo a phase transition at a particular temperature, the transition temperature (Tc), and many membrane?bound enzymes are also known to exhibit breaks in their Arrhenius plots of activities. The break temperature has been shown, in many cases, to correspond to Tc. To study the phase transition in chicken liver microsomal phospholipids, Nuclear Magnetic Resonance (NMR) technique has been used in the present investigations. It was found that the break in the Arrhenius plot of enzyme activity corresponded to the completion of the phase change in the microsomal phospholipids, suggesting a regulation of the enzyme by membrane phospholipids in vivo.
In order to understand the mechanism of activation of the enzyme by LPC and/or Triton X?100 better, it was decided to purify the enzyme from the microsomal membranes. The chicken liver microsomal palmitoyl?CoA synthetase appears to be an intrinsic protein, as indicated by attempts to solubilise it by sonication, high?salt concentration, detergents, etc. The enzyme was solubilised using Triton X?100 and was purified using Sephadex G?50 column chromatography and AMP?Sepharose affinity chromatography.
The partially purified enzyme preparation contained two proteins, as shown by polyacrylamide gel electrophoresis. The properties of this purified enzyme were studied and compared to the properties of the membrane?bound microsomal enzyme. Even though the properties of the purified enzyme were grossly similar to those of the microsomal enzyme, it differed from the membrane?bound form in some aspects such as pH optimum, Km values for ATP and CoA, metal?ion requirement, and stability during preincubation.
The purified enzyme also required LPC and Triton X?100 for activity. Both Triton X?100 and LPC increased the maximum velocity (Vmax) of the reaction. However, the Km of the enzyme for palmitate was not affected by LPC, even though it was doubled by Triton X?100. The Arrhenius plot of enzyme activity exhibited a straight?line graph in the absence of Triton X?100 or LPC in the assay mixture, and the activation energy of the reaction was unaffected by the addition of LPC, while a slight increase was obtained in the presence of Triton X?100. Based on the results, a possible conformational change in the enzyme protein has been postulated to account for the activation, and the physiological significance of the effect of LPC on palmitoyl?CoA synthetase has been discussed. A possible mechanism for palmitate activation has also been proposed based on the effect of the cofactors ATP, CoA, and Mg²? on the loss of activity during preincubation of the enzyme. | |
