UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Expression and characterisation of a novel cyclic nucleotide phosphodiesterase Type lA from dog heart

Sidhu, Ravinder; (2003) Expression and characterisation of a novel cyclic nucleotide phosphodiesterase Type lA from dog heart. Doctoral thesis (Ph.D), UCL (University College London). Green open access

[thumbnail of Expression_and_characterisatio.pdf] Text
Expression_and_characterisatio.pdf

Download (29MB)

Abstract

Cyclic nucleotide phosphodiesterase Type 1A belongs to a family of intracellular enzymes involved in the modulation of the key second messengers cAMP and cGMP. There are eleven members in the superfamily of PDE enzymes (PDE1 - PDE11), all exhibiting tissue-specific distribution. The PDE1 enzymes are found mainly in the brain, lungs, heart and vascular smooth muscle with smaller amounts present in inflammatory B lymphocytes and macrophages. In this study, the expression of dog heart PDE1 A1 was investigated using cDNA previously produced by Clapham and Wilderspin (2001). This cDNA had not been explored for the expression of a functional protein so it formed the starting point of the present work. The experimental work comprised of expression studies to identify suitable host/vector combinations followed by detailed biochemical characterisation of the recombinant enzyme for comparison with the native enzyme. The dog has been used extensively as a model for the investigation of cardiovascular function in the pharmacological analysis of PDE inhibitors but there is little information regardingthe characterisation of PDE enzymes in this species so the present study extends the information regarding characterisation of PDE1A enzymes in the dog. For the first part of the work, the full-length dog heart PDE1A1 cDNA was cloned into pPICZαC for secreted expression in the Pichia pastoris expression system. Culture media from P. pastoris cells transformed with the construct pPICZαC-PDE1 A1 showed no PDE activity. Full-length and N-terminal truncated PDE1 A 1 enzymes were then expressed in E. coli using the expression vectors pGEX-3X and pTrcH isA to generate the enzymes GST-PDE1 A1 and H is6-PDE1 A1N-trunc respectively. The majority of the recombinant protein was sequestered into insoluble inclusion bodies and exhibited extensive proteolytic degradation in both cases. The full-length as well as the N- and/or C-terminal truncated dog heart PDE1 A1 enzymes were then produced using the Semliki Forest virus (SFV) system. Recombinant dog heart PDE1A1 was successfully produced in a soluble, active form using the SFV system. The results of the present study revealed that only the full-length and the N-terminal truncated constructs had PDE activity. The successful expression of dog heart PDE1A1 enzymes in the SFV system represents the first report of the expression of PDE1 enzymes in this expression system. The Km (1.99μM) for the full length enzyme, for cAMP, was comparable to the native enzyme (1.2μM), while the Km for cGMP was higher for the recombinant enzyme (12.55μM) compared to the native enzyme (0.53μM). Inhibitor studies on the recombinant enzymes showed that the enzymes were sensitive to the PDE1-selective inhibitor vinpocetine as well as the PDE1/5-selective inhibitor zaprinast while being insensitive to the PDE2- and PDE3-inhibitors EHNA and amrinone respectively. However, the sensitivity of the recombinant enzymes to the selective inhibitors, vinpocetine and zaprinast, was reduced compared to the native enzyme with sensitivity to zaprinast reduced by approximately 18-fold. There was also a particularly notable difference between the recombinant enzymes produced in the present study and the native enzyme. This was the unexpected sensitivity that the recombinant enzymes had to the archetypical PDE4 inhibitor, rolipram, with the full-length PDE1A1 having an IC50 of 0.2μM. The findings of the present study have implications in the interpretation of data obtained for recombinant PDE1 enzymes particularly with regards to evaluation of PDE1 as well as PDE4 inhibitors for clinical use since PDE1 enzymes are often found in tissues having PDE4 enzymes. The results also indicate that rolipram may not be as selective as first reported.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Expression and characterisation of a novel cyclic nucleotide phosphodiesterase Type lA from dog heart
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Cyclic; Dog; Heart; Novel; Nucleotide; Phosphodiesterase
URI: https://discovery.ucl.ac.uk/id/eprint/10097628
Downloads since deposit
57Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item