Molecular properties of carnitine palmitoyltransferases I and II from rat liver mitochondria

• Main Author: Marshall, Myles C. B.
• Published: University of Edinburgh 1993
• Subjects: 572.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.657347

CPT I was localised in the outer mitochondrial membrane and then partially purified. The protein was digested with restriction endonucleases and the fragments N-terminally sequenced and used to generate oligonucleotides for cDNA library screening and polymerase chain reaction (PCR). The partially purified protein was also used to generate polyclonal antibodies, which were used to screen λgt11 cDNA libraries. A positive clone was isolated from the λgt11 cDNA library using affinity purified serum. When sequenced, the clone was identified as long chain fatty acid CoA synthase. CPT II was purified from rat liver mitochondrial inner membranes and n-terminally sequenced. The purified protein was used to generate polyclonal antibodies. Oligonucleotides were generated to the cDNA encoding CPT II reported by Woeltje <I>et al</I> (1990b) and used to PCR up a 300 bp fragment, which when cloned and sequenced was identified as the C-terminus of CPT II. This fragment was then used unsuccessfully to screen λgt10, λgt11 and λgt11-stretch cDNA libraries, in order to find a full length clone. Using the information generated by this thesis along with other recent publications a possible topology of CPT I in the rat liver mitochondrial outer membrane was elucidated.