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Publikation

Schilling, S.; Hoffmann, T.; Rosche, F.; Manhart, S.; Wasternack, C.; Demuth, H.-U.; Heterologous Expression and Characterization of Human Glutaminyl Cyclase: Evidence for a Disulfide Bond with Importance for Catalytic Activity Biochemistry 41, 10849-10857, (2002) DOI: 10.1021/bi0260381

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions. CD spectral analysis indicated a high α-helical content, which contrasts with plant QC from Carica papaya. The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularly in E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain a disulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistently inactivated by treatment with 15 mM DTT, whereas deglycosylation had no effect on enzymatic activity. Analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of the protein upon treatment with DTT. In terms of the different enzymatic properties, the consequences of QC expression in different environments are discussed.
Bücher und Buchkapitel

Kohlmann, M.; Kuntzsch, A.; Wasternack, C.; Feussner, I.; Effect of Jasmonic Acid Methyl Ester on Enzymes of Lipoxygenase Pathway in Barley Leaves 339-340, (1998)

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Bücher und Buchkapitel

Feussner, I.; Balkenhohl, T. J.; Porzel, A.; Kühn, H.; Wasternack, C.; Structural Elucidation of Oxygenated Triacylglycerols in Cucumber and Sunflower Cotyledons 57-58, (1998)

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Bücher und Buchkapitel

Feussner, I.; Blée, E.; Weichert, H.; Rousset, C.; Wasternack, C.; Fatty Acid Catabolism at the Lipid Body Membrane of Germinating Cucumber Cotyledons 311-313, (1998)

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Bücher und Buchkapitel

Balkenhohl, T.; Kühn, H.; Wasternack, C.; Feussner, I.; A Lipase Specific for Esterified Oxygenated Polyenoic Fatty Acids in Lipid Bodies of Cucumber Cotyledons 320-322, (1998)

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Bücher und Buchkapitel

Bachmann, A.; Kohlmann, M.; Wasternack, C.; Feussner, I.; Oxylipins in Sorbitol-Stressed Barley Leaf Segments 288-290, (1998)

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