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    The purification and immunological isolation of ATP citrate lyase from rat liver : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, New Zealand
    (Massey University, 1986) Walker, Brett Keith
    ATP CITRATE LYASE (E.C 4.1.3.8) has been purified from rat hepato­cyte cytoplasm by a combination of existing published procedures. The final purification method produced homogeneous ATPCL with specific activity of 10-16 units/mg. Antibodies were raised in rabbits against purified ATPCL eluted from reactive Blue Sepharose CL-68 or DEAE anion exchange column. The purified antibodies were tested for their specificity for ATPCL. This was accomplished by Ouchterlony double diffusion analysis and also by disruption of antibody-antigen complexes and visualizing the gene­ rated protein bands on detergent gels. The equivalence point of the purified antibody was determined by immunotitration with both purified enzyme and crude extract. The equi­valence point was later confirmed by immunotitration of radiolabelled proteins. Antibodies were then used to immunochemically isolate and quantitate the amount of (35-S) methionine or (14-C) radiolabelled ATPCL in the cytosolic fraction of rat liver. Pulse labelling of rat liver proteins in vivo and then precipitation of radiolabelled proteins demonstrated that the purified antibodies precipitated proteins other than just the ATPCL subunit. The amount of ATPCL present in the cytosolic fraction could be calculated after immu­ noprecipitation and excision of radiolabelled ATPCL subunition SOS-PAGE. The proportion of ATPCL protein to the total TCA precipitable protein could then be calculated since the immunoprecipitation was carried out under conditions of antibody excess. Radiolabelled ATPCL was then immunoprecipitated from the cytosolic fractions of rats that had been subjected to different nutritional regimes. The results of this set of experiments showed that induction of ATPCL activity resulted from an increase in immunologically reactive protein. Increasing amounts of radiolabelled immunoreactive ATPCL protein could be precipitated by antibodies as the enzyme was induced. Induction of ATPCL activity resulted from increased rate of synthesis or decreased rate of degradation of immunoreactive protein and not from the activation of pre-existing enzyme protein.
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    Isolation and characterisation of the 5' region sequence for the bovine ATP-citrate lyase gene : a thesis presented in fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 2001) Tong, Xingzhang
    ATP-citrate lyase (ACL) is one of the major lipogenic enzymes. It catalyzes the synthesis of acetyl-CoA from citrate in the cytosol. This is the first committed step towards the conversion of carbohydrate precursors into fatty acids. Acetyl-CoA serves as the major precursor for lipogenesis and cholestogenesis. Examination of this pathway shows that the rate of fatty acid synthesis from glucose is dependent on the activity of ACL. In rats the activity of this enzyme can be increased by feeding high carbohydrate diet and reduced to low levels by fasting. These changes are regulated at the transcriptional level. The ruminant provides a good model to study the regulation of expression of ACL. The levels of this enzyme are high in young ruminants, but fall to very low levels once a functional rumen is developed. In adult ruminants, acetyl-CoA for fatty acid synthesis is produced directly from acetate formed by microbial fermentation in the rumen and carried to the peripheral tissues. The down-regulation of this enzyme can be reversed by the administration of glucogenic precursors by a route that bypasses their fermentation to volatile fatty acids in the rumen. An understanding of the regulation of expression of ACL in the adult ruminant and a comparison with monogastric animals will provide significant new information about the regulation of the conversion of carbohydrate into fat. A probe containing exon 2 to exon 3 of the rat ACL gene was prepared. Its specificity to bovine genomic DNA was verified and the probe was then used to screen a bovine λ genomic library. A 17 kb clone was isolated. The restriction map of this clone was determined with several enzymes. A part of this clone (9490 base pairs) was sequenced and shown to consist of a 3 kb promoter region and doenstream seqence as far as intron 3 of bovine ACL. The transcription start sites were determined by 5'RACE. Several important features of this gene were discovered by computer analysis of the sequence. Two key transcription factor binding sites were found in the promoter region. This work provided a solid basis for further investigation towards elucidating the mechanism of the transcriptional regulation of bovine ACL and the process of lipogenesis.
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    Isolation of 5' regulatory sequences for ruminant ATP citrate lyase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1998) Sanders, Rebecca Jane
    ATP citrate lyase is an essential enzyme in the pathway for conversion of glucose to fatty acids in mammalian tissues. The enzyme catalyses the cleavage of cytosolic citrate to acetyl CoA and oxaloacetate in an ATP-dependent reaction. The sequence of the cDNAs for both rat and human ATP citrate lyase have been published and have 96.3% identity at the amino acid level. This high level of identity may also extend to other mammals, including ruminants. The ruminant presents a unique system in which to study the regulation of ATP citrate lyase as levels of expression of the enzyme change during the development of a functional rumen. An analysis of the 5'-regulatory region of ruminant ATP citrate lyase will be important in determining factors that contribute to the developmental regulation of this enzyme in ruminants. In order to analyse the 5'-regulatory region of ruminant ATP citrate lyase, a probe was constructed with which to screen an amplified bovine genomic library. The probe was produced by cloning a 282 bp PCR product containing rat ATP citrate lyase exon II sequence, amplified from rat genomic DNA. This clone was sequenced to verify that it contained rat ATP citrate lyase exon II sequence. This probe was then used for northern and Southern blotting, and for screening an amplified bovine genomic library. Northern blotting of rat and lamb total RNA showed that the probe hybridised with rat RNA, but not with lamb RNA. Conditions for hybridisation were not optimised, as hybridisation between the probe and rat RNA was not as specific as expected. The quality of RNA used for preparing the northern blots could have also affected the specificity of hybridisation. Southern blotting experiments were also inconclusive, as the hybridisation signals seen were not specific. However the probe was shown to hybridise to rat and human genomic DNA. Screening of the bovine genomic library was unsuccessful, but once conditions for hybridisation are optimised, then the probe could be used to rescreen the amplified bovine genomic library, and isolate a clone containing the 5'-regulatory sequences for bovine ATP citrate lyase.