Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338674
Title: Protein phosphorylation in cyanobacterial light-harvesting complexes
Author: Tsai, Jeng-Wen
ISNI:       0000 0001 3536 9359
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1996
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Abstract:
Protein kinase activities have been demonstrated in the thylakoid membranes of higher plants and in cyanobacteria, furthermore, their role in state-transitions has been widely accepted. Several proteins in cell-free extracts of Synechocystis sp. PCC 6803 were found to be phosphorylated in vitro and the existence of protein kinase activities was thus demonstrated. Total membranes, obtained by pelleting the cell-free extracts, were subjected to sucrose density gradient centrifugation to separate the thylakoid membranes. The thylaoid membranes were found in the 50 to 60% sucrose region. Two blue bands located in the 30% sucrose fraction contained phycobiliproteins. In vitro phosphorylation analysis showed that several proteins were strongly phosphorylated, especially an 18 kDa protein, which occurred in both the phycobiliprotein-containing fractions of a sucrose density gradient and the thylakoid membranes was strongly phosphorylated. Digestion of the sucrose density gradient- and SDS-PAGE-purified, labelled 18 kDa protein with Staphylococcal V8 protease yielded a labelled 9 kDa fragment, which was the N-terminal part of the 18 kDa protein. The 18 kDa protein was tentatively identified as P-phycocyanin by comparison of the N-terminal amino acid sequence of the 9 kDa polypeptide against proteins in the protein databases. The phosphoamino acid of P- phycocyanin was identified as phosphoserine by acid hydrolysis of SDS-PAGE-purified P-phycocyanin. However, which particular serine residue(s) which is modified is still to be determined. Besides protein kinase activities, phosphatase activities were also demonstrated by pulse-chase reactions. Phosphorylation and dephosphorylation of p-phycocyanin occurred simultaneously and were insensitive to classical inhibitors of protein kinases and phosphatases. P-phycocyanin, p-phycocyanin kinase and the phosphatase co-occurred and could not be separated by anion exchange chromatography and gel filtration chromatography, suggesting that P-phycocyanin and p-phycocyanin kinase were located in a macromolecular complex. Dénaturation of the P-phycocyanin complex by urea showed that P-phycocyanin kinase activities could be denatured and renatured. However, P-phycocyanin kinase activity could not be restored after separation of the denatured P- phycocyanin complex by anion exchange chromatography in the presence of urea. Phosphorylation and dephosphorylation of p-phycocyanin were light-independent in vitro. Spectroscopy showed that the absorption spectra of P-phycocyanin before and after phosphorylation were not significantly altered. However, phosphorylation of P- phycocyanin resulted in fluorescence quenching, suggesting there was a loss of excitation energy, which might be in the form of non-radiative dissipation. Screening of cosmid library containing Synechocystis sp. PCC 6803 genome showed that a cosmid clone contained Synechocystis sp. PCC 6803 cpcB gene, encoding P-phycocyanin. The predicted amino acid sequence deduced from the Synechocystis sp. PCC 6803 cpcB gene provided further evidence that the 18 kDa protein was p- phycocyanin.
Supervisor: Not available Sponsor: University of Warwick ; Rotary Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.338674  DOI: Not available
Keywords: QK Botany ; QR Microbiology
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