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Characterization of Inosine triphosphate pyrophosphatase, an important protein involved in purine metabolism
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
2015 (English)Independent thesis Basic level (degree of Bachelor), 10,5 credits / 16 HE creditsStudent thesis
Abstract [en]

The enzyme inosine triphosphate pyrophosphatase (ITPase) is responsible for controlling the levels of the by-products guanosine monophosphate (GMP) and adenosine monophosphate (AMP) through their precursor inosine monophosphate (IMP). ). Human ITPase consists of a 194-amino acid homodimer which relies upon either an Mg2+ ion or a Mn2+ ion for catalytic activity, and orthologs of this protein have been found in many different organisms.

The purpose of this project was to try out methods learned throughout the education and to use this knowledge to gather new data about the human protein inosine triphosphate pyrophosphatase (ITPase). The protein was expressed in BL21/DE3 cells from a pre-made vector. Experiments performed during this project include secondary- and tertiary stability measurements, tryptophan fluorescence spectra, binding curve and thermic stability to ITPase with ANS and methotrexate.

The Tm-value of human ITPase was examined with Trp-Fluorescence, ANS-fluorescence and Near-UV and Far-UV circular dichroism (CD). The stability of ITPase monitored by Near-UV as well as Far-UV coincides, indicating that secondary- and tertiary-unfolding occur simultaneously without any intermediates.

The results of Trp-fluorescence showed that the tryptophans were already exposed and thus it did not yield a reliable result. The binding properties of ANS and MTX to ITPase were also examined.

Place, publisher, year, edition, pages
2015. , 30 p.
Keyword [en]
Inosine triphosphate pyrophosphatase, acute lymfoblastic leukemia, circular dichroism, fluorescence, methotrexate, protein stability
National Category
Other Chemistry Topics
URN: urn:nbn:se:liu:diva-118076ISRN: LITH-IFM-G-EX--15/2989—SEOAI: diva2:813855
Subject / course
Chemical Biology
Available from: 2015-05-25 Created: 2015-05-20 Last updated: 2015-05-25Bibliographically approved

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