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CTP: Choline Phosphate Cytidylyltransferase in human lung

CTP: Choline Phosphate Cytidylyltransferase in human lung
CTP: Choline Phosphate Cytidylyltransferase in human lung
Human lung cytidylyltransferase was found associated with both 'soluble' (S100) and membrane-rich particulate (P100) fractions of Tris-buffered saline homogenates. S100 enzyme activities in 15 - 16 week fetal and adult human lungs represented a constant proportion of overall recovery, (66.8 4.8% vs 66.1 7.5%, means standard error). A lack of support for the regulatory translocation of human lung cytidylyltransferase at these extremes of development was unable to rule out a transient change in distribution near term. Rat lung P100 enzyme increased from 31% to 40% between d18 gestation and term, d22, but a concomitant increase in total.

S100 cytidylyltransferase, measured in the presence of the lipid activator PG, questioned the physiological significance of the apparent translocation.

Cytidylyltransferase from human and rat lung S100 were resolved into a high molecular weight H form (> 106 daltons) and a lower molecular weight L form (~200,000 daltons). Incubation of S100 at 37oC for 2 hours yielded insoluble, protein-rich aggregates which were strongly associated with rat H form cytidylyltransferase, while less strongly with the human H form. The principal 43,000 dalton, protein in these aggregates was identified as a cytoplasmic actin on the basis of its properties, amino acid composition and western blot analysis. The association of H form cytidylyltransferase with cytoskeletal F-actin containing fractions in vitro was disrupted by the detergent CHAPS, which was also able to release a portion of P100 enzyme. Separation of human S100 H and L form enzyme, by gel filtration or ultracentrifugation, revealed the presence of latent cytidylyltransferase, often as high as 3 fold, which questioned activity determinations in fresh S100. Within the framework of an emerging concept of a highly ordered aqueous cytoplasm, the incorporation of these results suggested that a portion of human lung cytidylyltransferase might by cytoskeletally bound in vivo, as has been described for many enzymes or enzyme systems.

The use of conventional purification techniques, including affinity chromatography, with a view to testing these ideas in defined systems, met with little success. Low yields or highly unstable enzyme characterised many individual steps, especially where cytidylyltransferase was separated from F-actin enriched fractions. A number of triazine dyes screened as pseudoaffinity ligands revealed a rapid inhibition with Procion Green H-4G and a partial protection with MgCTP. Sepharose CL4B-immobilised Green H-4G bound cytidylyltransferase, but MgCTP was unable to effect elution. Increasing ionic strength eluted some activity but also inhibited enzyme irreversibly, while CHAPS at 1% released a maximum of only 18% bound enzyme and SDS PAGE revealed a relatively non-specific binding. The use of dye-affinity matrix offered the potential of a useful purification step with partially purified enzyme if suitable elution conditions could be devised.

Hunt, Alan Nigel
6f35b7c2-560c-4b24-baff-c88990aedc38
Hunt, Alan Nigel
6f35b7c2-560c-4b24-baff-c88990aedc38
Postle, Anthony
0fa17988-b4a0-4cdc-819a-9ae15c5dad66

Hunt, Alan Nigel (1988) CTP: Choline Phosphate Cytidylyltransferase in human lung. University of Southampton, Medicine, Doctoral Thesis, 206pp.

Record type: Thesis (Doctoral)

Abstract

Human lung cytidylyltransferase was found associated with both 'soluble' (S100) and membrane-rich particulate (P100) fractions of Tris-buffered saline homogenates. S100 enzyme activities in 15 - 16 week fetal and adult human lungs represented a constant proportion of overall recovery, (66.8 4.8% vs 66.1 7.5%, means standard error). A lack of support for the regulatory translocation of human lung cytidylyltransferase at these extremes of development was unable to rule out a transient change in distribution near term. Rat lung P100 enzyme increased from 31% to 40% between d18 gestation and term, d22, but a concomitant increase in total.

S100 cytidylyltransferase, measured in the presence of the lipid activator PG, questioned the physiological significance of the apparent translocation.

Cytidylyltransferase from human and rat lung S100 were resolved into a high molecular weight H form (> 106 daltons) and a lower molecular weight L form (~200,000 daltons). Incubation of S100 at 37oC for 2 hours yielded insoluble, protein-rich aggregates which were strongly associated with rat H form cytidylyltransferase, while less strongly with the human H form. The principal 43,000 dalton, protein in these aggregates was identified as a cytoplasmic actin on the basis of its properties, amino acid composition and western blot analysis. The association of H form cytidylyltransferase with cytoskeletal F-actin containing fractions in vitro was disrupted by the detergent CHAPS, which was also able to release a portion of P100 enzyme. Separation of human S100 H and L form enzyme, by gel filtration or ultracentrifugation, revealed the presence of latent cytidylyltransferase, often as high as 3 fold, which questioned activity determinations in fresh S100. Within the framework of an emerging concept of a highly ordered aqueous cytoplasm, the incorporation of these results suggested that a portion of human lung cytidylyltransferase might by cytoskeletally bound in vivo, as has been described for many enzymes or enzyme systems.

The use of conventional purification techniques, including affinity chromatography, with a view to testing these ideas in defined systems, met with little success. Low yields or highly unstable enzyme characterised many individual steps, especially where cytidylyltransferase was separated from F-actin enriched fractions. A number of triazine dyes screened as pseudoaffinity ligands revealed a rapid inhibition with Procion Green H-4G and a partial protection with MgCTP. Sepharose CL4B-immobilised Green H-4G bound cytidylyltransferase, but MgCTP was unable to effect elution. Increasing ionic strength eluted some activity but also inhibited enzyme irreversibly, while CHAPS at 1% released a maximum of only 18% bound enzyme and SDS PAGE revealed a relatively non-specific binding. The use of dye-affinity matrix offered the potential of a useful purification step with partially purified enzyme if suitable elution conditions could be devised.

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Published date: 1988
Organisations: University of Southampton, Faculty of Medicine

Identifiers

Local EPrints ID: 361174
URI: https://eprints.soton.ac.uk/id/eprint/361174
PURE UUID: 72605dbe-7be6-4d86-9d56-efe955314767
ORCID for Anthony Postle: ORCID iD orcid.org/0000-0001-7361-0756

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Date deposited: 14 Jan 2014 15:37
Last modified: 06 Jun 2018 13:20

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Contributors

Author: Alan Nigel Hunt
Thesis advisor: Anthony Postle ORCID iD

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