Báo cáo khoa học: Differential susceptibility of Plasmodium falciparum versus yeast and mammalian enolases to dissociation into active monomers

Differential Susceptibility of Plasmodium Falciparum Versus Yeast and Mammalian Enolases to Dissociation into Active Monomers

Tác giả: Ipsita Pal-Bhowmick, Sadagopan Krishnan and Gotam K. Jarori

Lĩnh vực: Biological Sciences

Nội dung tài liệu:

Nghiên cứu này tập trung vào việc khám phá khả năng phân ly các enolase dị hình (dimeric) thành các đơn phân (monomeric) có hoạt tính từ các sinh vật khác nhau, bao gồm Plasmodium falciparum, nấm men và cơ thỏ. Các phương pháp hóa học và vật lý đã được sử dụng để đạt được sự phân ly này. Nghiên cứu so sánh đặc tính động học và cấu trúc của các dạng đơn phân và dị hình của enolase tái tổ hợp từ P. falciparum, cho thấy những khác biệt đáng kể về hoạt tính riêng, khả năng ức chế bởi muối, sự bất hoạt và độ bền nhiệt. Quan trọng hơn, nghiên cứu đã xác định sự khác biệt trong cách các loài enolase khác nhau phản ứng với các yếu tố phân ly, đặc biệt là imidazole và Mg(II). Sự khác biệt về độ ổn định của giao diện dị hình enolase P. falciparum so với enolase động vật có vú có thể được khai thác để phân ly có chọn lọc enzyme của ký sinh trùng thành dạng đơn phân kém hiệu quả về mặt xúc tác và kém bền nhiệt. Điều này cho thấy enolase có thể là một mục tiêu điều trị mới cho bệnh sốt rét.

Mục lục chi tiết:

• Keywords
• Correspondence
• (Received 7 December 2006, revised 16 January 2007, accepted 12 February 2007)
• doi:10.1111/j.1742-4658.2007.05738.x
• In the past, several unsuccessful attempts have been made…
• Enolase (EC 4.2.1.11) is a glycolytic enzyme…
• Abbreviations
• 1932
• FEBS Journal 274 (2007) 1932-1945© 2007 The Authors Journal compilation 2007 FEBS
• I. Pal-Bhowmick et al.
• Dissociation of enolase into active monomers
• out. Hydrostatic pressure, which is viewed as a gentle…
• Results
• Imidazole-induced dissociation of r-Pfen into active monomers
• Like most enolases, r-Pfen is a homodimer of two 50 kDa subunits [24]…
• Fig. 1. Imidazole-induced dissociation of r-Pfen. (A) Superdex-75 gel filtration chromatograms…
• favored the dissociation of r-Pfen. Figure 1C shows a gel filtration chromatogram…
• Fig. 2. Imidazole-induced dissociation of Yen and RMen. Samples were in 50 mm sodium phosphate…
• 1934
• FEBS Journal 274 (2007) 1932-1945© 2007 The Authors Journal compilation 2007 FEBS
• pH-induced dissociation of enolases
• The effect of pH on the oligomeric state of enolases was examined…
• Activity and reassociation of MpH and Mim
• Measurements of enzyme activity in monomeric and dimeric fractions showed that Mim…
• Fig. 4. Reaction progress curves for the monomeric r-Pfen-catalyzed reaction…
• MpH from Yen and RMen also showed nonlinear reaction progress curves…
• Comparison of kinetic properties of monomeric (Mim) and dimeric forms of r-Pfen
• As Mim did not reassociate rapidly into dimers, this form of the enzyme was used…
• Fig. 5. (A) Comparison of monomer and dimer activity with varying concentrations of phosphoenolpyruvate…
• inactivated at 50 °C in ≤150 min (Fig. 5B). Thus, subunit-subunit interface interactions…
• As enolases from different organisms are known to differ…
• Fig. 6. Effect of NaCl, KCI and KBr on the activity of monomeric (Mim) (■) and dimeric () r-Pfen…
• Comparison of CD and fluorescence spectra of monomeric and dimeric enolase
• The effect of the loss of subunit-subunit interface interactions…
• CDNN program for secondary structure analysis [44] gave helix 36.6%…
• Fluorescence emission spectra of the monomic, dimeric and denatured states…
• Fig. 7. Comparison of CD and fluorescence spectra of monomeric (MPH) and dimeric forms of r-Pfen and Yen…
• results presented here unequivocally establish that both H+ and imidazole are able…
• An examination of the enolase intersubunit interface showed that it is stabilized…
• 1940
• FEBS Journal 274 (2007) 1932-1945© 2007 The Authors Journal compilation 2007 FEBS
• Fig. 8. Intersubunit interface hydrogen bond involving His191 in enolase crystal structures…
• length (as measured between two non-hydrogen atoms…
• enolase interface using PP server, which identified Trp56 in Yen…
• Although r-Pfen is highly homologous to mammalian enolases…
• The comparison of various properties between the dimeric and monomeric forms…
• Materials and methods
• Materials
• Hexahistidine-tagged r-Pfen was purified as described previously [24]…
• Gel filtration chromatography
• Gel filtration chromatography was performed with a Superdex-75 prep grade column…
• Enzyme activity assay
• Enolase activity was measured by a continuous spectrophotometric assay…
• 1942
• FEBS Journal 274 (2007) 1932-1945© 2007 The Authors Journal compilation 2007 FEBS
• from phosphoenolpyruvate at 240 nm using a Perkin-Elmer (Waltham, MA, USA) lambda 40…
• Fluorescence and CD spectra
• The fluorescence measurements were carried out by using a SPEX Fluorolog FL1T11…
• CD studies were carried out using a Jasco J-810 spectropolarimeter…
• Acknowledgements
• We would like to thank Mr Hardeep K. Vora for help with some of the experiments…
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