Mild Chemical Deglycosylation of Horseradish Peroxidase Yields a Fully Active, Homogeneous Enzyme

Tams, Jeppe Wegener; Welinder, Karen G.

doi:10.1006/abio.1995.1313

Cited by 72 publications

(56 citation statements)

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“…Glycosylation appears to have a potential to modulate the physical properties of proteins without compromising enzymatic activity [25,38,[45][46][47][48]. This makes controlled glycosylation an interesting avenue for the engineering and formulation of pharmaceutical peptides and industrial enzymes.…”

Section: Discussionmentioning

confidence: 99%

Glycoprotein-surfactant interactions: A calorimetric and spectroscopic investigation of the phytase-SDS system

Bagger

Hoffmann

Fuglsang

et al. 2007

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 AbstractThe interactions of sodium dodecyl sulfate (SDS) and two glyco-variants of the enzyme phytase from Peniophora lycii were investigated. One variant (Phy) was heavily glycosylated while the other (dgPhy) was enzymatically deglycosylated. Effects at 24°C of titrating SDS to Phy and dgPhy were studied by Isothermal Titration Calorimetry (ITC) and Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. Comparisons of results for the two variants were used to elucidate glycan-surfactant interrelationships.The CD spectra suggested that both the native and the SDS-denatured states of the two variants were mutually similar, and hence that the denaturation process was structurally equivalent for the two glyco-variants.The denatured state was far from fully unfolded and probably retained a substantial content of native-like structure. Furthermore, it was found that the glycans brought about only a small increase in the resistance towards SDS induced denaturation. The SDS concentration required to denature half of the protein molecules differed less than 1 mM for the two variants.The affinity for SDS of both variants was unusually low. The amount of bound SDS (w/w) at different stages of the binding isotherm was 3-10 times lower than that reported for the most previously investigated globular proteins. Analysis of the relative affinity of the glycan and peptide moieties suggested that the carbohydrates bind much less surfactant. At saturation, glycans adsorbed about half as much SDS (in g/g) as the peptide moiety of Phy and about five times less than average proteins.

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Section: Discussionmentioning

confidence: 99%

Glycoprotein-surfactant interactions: A calorimetric and spectroscopic investigation of the phytase-SDS system

Bagger

Hoffmann

Fuglsang

et al. 2007

Biophysical Chemistry

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“…Deglycosylation of ZePrx33.44 and ZePrx34.70 was performed as described by Tams and Welinder (1995), with minor modifications. Phenol (8.5 mL), heated at 50°C, was mixed with 1,000 mL TFMS and 40 mg dry powder protein and incubated at 220°C for 2 h in an O 2 -free atmosphere.…”

Section: Assay Of Peroxidase Activity Determination Of Kinetic Propementioning

confidence: 99%

Cloning and Molecular Characterization of the Basic Peroxidase Isoenzyme from Zinnia elegans, an Enzyme Involved in Lignin Biosynthesis

et al. 2005

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The major basic peroxidase from Zinnia elegans (ZePrx) suspension cell cultures was purified and cloned, and its properties and organ expression were characterized. The ZePrx was composed of two isoforms with a M r (determined by matrix-assisted laser-desorption ionization time of flight) of 34,700 (ZePrx34.70) and a M r of 33,440 (ZePrx33.44). Both isoforms showed absorption maxima at 403 (Soret band), 500, and 640 nm, suggesting that both are high-spin ferric secretory class III peroxidases. M r differences between them were due to the glycan moieties, and were confirmed from the total similarity of the N-terminal sequences (LSTTFYDTT) and by the 99.9% similarity of the tryptic fragment fingerprints obtained by reverse-phase nano-liquid chromatography. Four full-length cDNAs coding for these peroxidases were cloned. They only differ in the 5#-untranslated region. These differences probably indicate different ways in mRNA transport, stability, and regulation. According to the k cat and apparent K m RH values shown by both peroxidases for the three monolignols, sinapyl alcohol was the best substrate, the endwise polymerization of sinapyl alcohol by both ZePrxs yielding highly polymerized lignins with polymerization degrees $87. Western blots using anti-ZePrx34.70 IgGs showed that ZePrx33.44 was expressed in tracheary elements, roots, and hypocotyls, while ZePrx34.70 was only expressed in roots and young hypocotyls. None of the ZePrx isoforms was significantly expressed in either leaves or cotyledons. A neighbor-joining tree constructed for the four full-length cDNAs suggests that the four putative paralogous genes encoding the four cDNAs result from duplication of a previously duplicated ancestral gene, as may be deduced from the conserved nature and conserved position of the introns.

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“…The presence of N-glycosylations is important in ensuring protein solubility 178,179 . The The expression of the IR described in the literature contains many different constructs, from L1-CR-L2 to the entire ectodomain 50,63,171,172 , however the L1 domain has not been expressed by itself.…”

Section: Resultsmentioning

confidence: 99%

Insulin analogues for insulin receptor studies and medical applications

Watson¹,

Jiráček²,

Žáková³

et al. 2012

Acta Cryst Sect A

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The structure of insulin molecule was determined by Dorothy Hodgkin in 1969.Subsequently, it has been established that insulin must rearrange upon binding to its receptor (Insulin Receptor -IR). However, all known structures of the hormone depict its storage or inactive form. It has been shown that some residues, key for IR binding, are buried inside the insulin molecule and must be exposed for an efficient insulin-IR complex These results provide a wealth of information about the active form of insulin, they provide important tools towards the first insulin-IR complex, and deliver novel insulin analogues.3

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Mild Chemical Deglycosylation of Horseradish Peroxidase Yields a Fully Active, Homogeneous Enzyme

Cited by 72 publications

References 0 publications

Glycoprotein-surfactant interactions: A calorimetric and spectroscopic investigation of the phytase-SDS system

Glycoprotein-surfactant interactions: A calorimetric and spectroscopic investigation of the phytase-SDS system

Cloning and Molecular Characterization of the Basic Peroxidase Isoenzyme from Zinnia elegans, an Enzyme Involved in Lignin Biosynthesis

Insulin analogues for insulin receptor studies and medical applications

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