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Title: Protein phosphorylation in Rhodomicrobium vannielii
Author: Turner, Andrew Michael
ISNI:       0000 0001 3538 0099
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1987
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Rm. vannielii was shown to contain phosphopolypeptides, the synthesis of which varied through the growth curve. Phosphoamino acid analysis demonstrated that the predominant phosphoamino acid was phosphoserine, though phosphotyrosine and phosphothreonine were also detected. The major phosphopolypeptide in the cell was shown to be the RuBisCO large subunit. Phosphorylation of this polypeptide was shown to be growth stage-dependent, commencing around mid log phase. The role of RuBisCO large subunit phosphorylation is as yet undetermined. A second phosphopolypeptide of Mr 12,700 was tentatively identified as a light harvesting chlorophyll binding protein. Analysis of phosphopolypeptides from swarmer and chain cells revealed that there were no detectable phosphopolypeptides in the undifferentiating swarmer cell. Analysis of phosphorylation during differentiation revealed that the accumulation of phosphopolypeptides did not become detectable until 1 hour into differentiation and the phosphorylation of most polypeptides appeared to reach a maximum rate by 2 hours into differentiation. In addition, two phosphopolypeptides specific to the end, and later stages of differentiation were observed. Investigations in to protein kinase activities in cell-free extracts established that the conditions under which the experiments were carried out had to be extremely carefully controlled. On examining the effects of Mg2+ and Ca2+ on the pattern of phosphopolypeptides obtained from cell-free extracts it was found that both cations could influence the phosphopolypeptide patterns produced and that the effects were concentration-dependent in a way that supported the suggestion that there were multiple protein kinases active in the extracts. In particular, Ca2+ was found to prevent the phosphorylation of a Mr 47,000 phosphopolypeptide the phosphorylation of which was Mg2+-dependent. The addition of chlorpromazine (a calmodulin antagonist) instead of Ca2+ produced the same effect leading to the suggestion that there may be a calmodulin-like molecule in Em- vannielii that exerts its effect on protein kinases through an interaction with Mg2+ rather than Ca2+. Other explanations are also put forward. As has been found in other studies on protein phosphorylation in prokaryotes, the resemblance between the proteins observed to be phosphorylated in vivo and in vitro was slight (see section 1.4). Examination of protein kinase activities through differentiation revealed that the dark-incubated swarmer cell contained potentially active protein kinases despite there being no detectable phosphorylation of polypeptides in vivo. This suggests that protein kinases are present in the inhibited swarmer cell, but inactive.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry ; QP Physiology ; QR Microbiology