The Gram-negative bacterium 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 can live on long chain fatty acids as its sole carbon source. However, the rate of fatty acid uptake was reduced after starvation (Mangroo, 1992). In this study, it was found that this starvation effect of reducing oleate uptake rate could only be observed in the initial minute post starvation. The starvation effect was not due to the depletion of its substrates ATP or reduced coenzyme A. Neither was the effect caused by a lowered acyl-CoA synthetase level. This reduction in oleate uptake rate can be reversed by incubation in oleate. It has been reported that lactate activated oleate uptake in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 (Mangroo and Gerber, 1993). In the present study, it was discovered that when [9, 10-³H]oleate was used as the radioactive tracer in uptake assay, the majority of the radioactivity effluxed from the cells. It was found that lactate did not have an effect on the overall oleate uptake. Its apparent activation on oleate uptake was due to the reduction in the efflux rate of radioactive probes, instead of increasing the uptake rate of oleate. Acyl-CoA synthetase in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 has always been described as a protein of around 45 kDa (Kameda and Nunn, 1981; Kameda 𝘦𝘵 𝘢𝘭., 1986). However, the 𝘧𝘢𝘥𝘋 gene that encodes the 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 acyl-GoA synthetase predicts a 62 kDa protein (Black 𝘦𝘵 𝘢𝘭., 1992; Fulda 𝘦𝘵 𝘢𝘭., 1994). This new form of acyl-CoA synthetase has been partially purified. This form, which has a size of around 62 kDa, can be converted to its 45 kDa counterpart in a Triton X-1 00 and temperature dependent manner. This induced processing of the 62 kDa enzyme was found to be inhibited by oleate, a natural substrate of acyl-CoA synthetase. The two forms of acyl-CoA synthetase have also been shown to be immunologically related. A much simpler purification scheme was developed for the 45 kDa acyl-CoA synthetase using the membrane bound form of acyl-CoA synthetase. The selective extraction of the 62 kDa acyl-CoA synthetase from the membrane, together with the induced processing into the 45 kDa form has provided the basis for this comparatively simple process. The availability of the expression system made the low yield associated with this simple process acceptable. Using oleate as substrate, the Kₘ and Vₘₐₓ for the 45 kDa acyl-CoA synthetase were determined to be 85 ± 18μM and 1550 ± 165 nmole/min/mg protein respectively, whereas the Kₘ and Vₘₐₓ for the 62 kDa enzyme were determined to be 38 ± 12μM and 633 ± 79 nmole/min/mg protein respectively. This suggested that the 62 kDa enzyme has higher affinity towards oleate than its 45 kDa counterpart under standard assay conditions while the ratio of Vₘₐₓ /Kₘ remained relatively constant. Evidence suggesting Triton X-1 00 activates acyl-CoA synthetase by providing a surface for catalysis was presented. This interaction between acyl-CoA synthetase and the Triton X-1 00 I oleate mixed micelle could be affected by the surface charge of the mixed micelle. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22950 |
| Date | 09 1900 |
| Creators | Cheng, Oscar |
| Contributors | Gerber, G. E., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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