A Fluorogenic Heparan Sulfate Disaccharide for the Measurement of Heparanase Activity

2020 ◽  
Author(s):  
Liang Wu ◽  
Norbert Wimmer ◽  
Vito Ferro ◽  
Gideon J. Davies
Keyword(s):  
Enzyme Activity ◽  
Heparan Sulfate ◽  
Crystal Structures ◽  
Rapid Assessment ◽  
High Sensitivity ◽  
Inhibition Kinetics ◽  
Biochemical Studies ◽  
Enzyme Turnover ◽  
Medical Interest ◽  
Slow Turnover

<p>The endo-β-glucuronidase heparanase mediates mammalian heparan sulfate catabolism, and is of considerable medical interest due to its prominent role in cancer aggression and metastasis. Biochemical studies of heparanase are currently hampered by a lack of suitable chromogenic or fluorogenic assay substrates, instead relying on lengthy multistep procedures to measure activity. Herein, we demonstrate that N’,6-O’-bis-sulfated 4-methylumbelliferyl heparan sulfate disaccharide is a competent fluorogenic heparanase substrate. Despite somewhat slow turnover, the high sensitivity of 4-methylumbelliferyl fluorescence provides a wide signal window that enables both enzyme turnover and inhibition kinetics measurements. Crystal structures with heparanase also provide the first ever observation of a substrate in an activated <sup>1</sup>S<sub>3</sub> conformation, highlighting previously unknown interactions involved in turnover. Our results pave the way for the design of further improved HPSE substrates that may enable rapid assessment of enzyme activity, which in turn will drive development of new heparanase inhibitors for therapeutic use.</p>

A Fluorogenic Heparan Sulfate Disaccharide for the Measurement of Heparanase Activity

2020 ◽  
Author(s):  
Liang Wu ◽  
Norbert Wimmer ◽  
Vito Ferro ◽  
Gideon J. Davies
Keyword(s):  
Enzyme Activity ◽  
Heparan Sulfate ◽  
Crystal Structures ◽  
Rapid Assessment ◽  
High Sensitivity ◽  
Inhibition Kinetics ◽  
Biochemical Studies ◽  
Enzyme Turnover ◽  
Medical Interest ◽  
Slow Turnover

<p>The endo-β-glucuronidase heparanase mediates mammalian heparan sulfate catabolism, and is of considerable medical interest due to its prominent role in cancer aggression and metastasis. Biochemical studies of heparanase are currently hampered by a lack of suitable chromogenic or fluorogenic assay substrates, instead relying on lengthy multistep procedures to measure activity. Herein, we demonstrate that N’,6-O’-bis-sulfated 4-methylumbelliferyl heparan sulfate disaccharide is a competent fluorogenic heparanase substrate. Despite somewhat slow turnover, the high sensitivity of 4-methylumbelliferyl fluorescence provides a wide signal window that enables both enzyme turnover and inhibition kinetics measurements. Crystal structures with heparanase also provide the first ever observation of a substrate in an activated <sup>1</sup>S<sub>3</sub> conformation, highlighting previously unknown interactions involved in turnover. Our results pave the way for the design of further improved HPSE substrates that may enable rapid assessment of enzyme activity, which in turn will drive development of new heparanase inhibitors for therapeutic use.</p>

Phosphatase systems in Fasciolopsis buski Lankester, 1857 and Gastrodiscus aegyptiacus Cobbold, 1876

Parasitology ◽  
1973 ◽  
Vol 67 (2) ◽  
pp. 197-204 ◽  
Author(s):  
Madan M. Goil
Keyword(s):  
Enzyme Activity ◽  
Tartaric Acid ◽  
Maximum Activity ◽  
Nitrophenyl Phosphate ◽  
Biochemical Studies ◽  
Phosphate Hydrolysis ◽  
Fasciolopsis Buski ◽  
Enzyme I ◽  
Inhibited Enzyme ◽  
Acid Phosphomonoesterase

Biochemical studies on the non-specific phosphomonoesterases have demonstrated the presence of acid phosphomonoesterase with maximum activity at pH 4·0 in Gastrodiscus aegyptiacus (enzyme I) and at pH 4·5 in the case of Fasdolopsis buski (enzyme II). The Km for ρ-nitrophenyl phosphate hydrolysis was 0·66 mM for enzyme I and 1·1 mM for enzyme II. Different concentrations of fluoride, arsenate, tartrate, tartaric acid, cysteine and copper brought about inhibition of both enzymes and magnesium, iodoaeetate, iodoacetamide and EDTA had no influence on either enzyme activity. Cobalt activated both enzymes while zinc inhibited enzyme I and strongly stimulated enzyme II.

scholarly journals Intraoperative localization and preservation of reading in ventral occipitotemporal cortex

2021 ◽  
Author(s):  
Oscar Woolnough ◽  
Kathryn M Snyder ◽  
Cale W Morse ◽  
Meredith J McCarty ◽  
Samden D Lhatoo ◽  
...  
Keyword(s):  
Rapid Assessment ◽  
High Sensitivity ◽  
Word Form ◽  
Gamma Activation ◽  
Reading Deficits ◽  
Visual Word Form Area ◽  
Cortical Areas ◽  
Eloquent Cortex ◽  
Occipitotemporal Cortex ◽  
Intraoperative Localization

Resective surgery in language-dominant ventral occipitotemporal cortex (vOTC) carries the risk of causing impairment to reading. As it is not on the lateral surface, it is not easily accessible for intraoperative mapping and extensive stimulation mapping can be time consuming. Here we assess the feasibility of using task-based electrocorticography (ECoG) recordings intraoperatively to help guide stimulation mapping of reading in vOTC. In 11 patients undergoing extraoperative, intracranial seizure mapping we recorded induced broadband gamma activation (70 - 150 Hz) during a visual category localizer. Word-responsive cortex localized in this manner showed a high sensitivity (72%) to stimulation-induced reading deficits, and the confluence of ECoG and stimulation positive sites appears to demarcate the visual word form area. In two additional patients, with pathologies necessitating resections in language-dominant vOTC, task-based functional mapping was performed intraoperatively using subdural ECoG, alongside direct cortical stimulation. Cortical areas critical for reading were mapped and successfully preserved, while enabling pathological tissue to be completely removed. Data collection is possible in <3 minutes and initial intraoperative data analysis takes <3 minutes, allowing for rapid assessment of broad areas of cortex. Eloquent cortex in ventral visual cortex can be rapidly mapped intraoperatively using ECoG. This method acts to guide high-probability targets for stimulation, with limited patient participation, and can be used to avoid iatrogenic dyslexia following surgery.

A fluorescent G-quadruplex probe for the assay of base excision repair enzyme activity

2015 ◽  
Vol 51 (72) ◽  
pp. 13744-13747 ◽  
Author(s):  
Chang Yeol Lee ◽  
Ki Soo Park ◽  
Hyun Gyu Park
Keyword(s):  
Enzyme Activity ◽  
Base Excision Repair ◽  
Fluorescence Enhancement ◽  
Excision Repair ◽  
High Sensitivity ◽  
Repair Enzyme ◽  
Quadruplex Structure ◽  
G Quadruplex ◽  
Base Excision ◽  
Novel Strategy

A G-quadruplex probe incorporating 2-AP is utilized to develop a novel strategy to accurately determine UDG activity. The excision reaction promoted by UDG is designed to trigger the formation of G-quadruplex structure with significant fluorescence enhancement of 2-AP within the probe. By employing this strategy, UDG activity can be reliably determined with high sensitivity and specificity.

Genetic and Biochemical Studies on the Conversion of Dihydroflavonols to Flavonols in Flowers of Petunia hybrida

1986 ◽  
Vol 41 (1-2) ◽  
pp. 179-186 ◽  
Author(s):  
G. Forkmann ◽  
P. de Vlaming ◽  
R. Spribille ◽  
H. Wiering ◽  
A. W. Schram
Keyword(s):  
Enzyme Activity ◽  
Petunia Hybrida ◽  
Soluble Enzyme ◽  
Flower Buds ◽  
Ph Optimum ◽  
Enzyme Preparations ◽  
Biochemical Studies ◽  
High Enzyme ◽  
Substantial Correlation ◽  
High Enzyme Activity

Abstract Soluble enzyme preparations from flower buds of Petunia hybrida catalyzed the conversion of dihydroflavonols to flavonols. Dihydrokaempferol and dihydroquercetin were readily converted to the respective flavonols, whereas dihydromyricetin was a poor substrate. The reaction required 2-oxoglutarate, ascorbate and Fe2+ as cofactors and had a pH optimum at about 6.5. In the presence of the dominant allele Fl, high enzyme activity for flavonol formation was found, whereas in enzyme preparations from flower buds of recessive genotypes (fl/fl) only low enzyme activity could be observed. A substantial correlation was found between enzyme activity for flavonol formation and the flavonol content of buds and flowers during development.

scholarly journals QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling

2003 ◽  
Vol 162 (2) ◽  
pp. 341-351 ◽  
Author(s):  
Xingbin Ai ◽  
Anh-Tri Do ◽  
Olga Lozynska ◽  
Marion Kusche-Gullberg ◽  
Ulf Lindahl ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Wnt Signaling ◽  
Present Model ◽  
Receptor Activation ◽  
Heparan Sulfate Proteoglycans ◽  
Biochemical Studies ◽  
Frizzled Receptors ◽  
Wnt Signal ◽  
Function Cell

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state “catch or present” model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS–Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.

scholarly journals Freeze substituted tissue in 5'-nucleotidase histochemistry. Comparative histochemical and biochemical investigations.

1979 ◽  
Vol 27 (12) ◽  
pp. 1582-1587 ◽  
Author(s):  
K Klaushofer ◽  
H von Mayersbach
Keyword(s):  
Enzyme Activity ◽  
Sensitivity And Specificity ◽  
Rat Liver ◽  
Freeze Substitution ◽  
High Sensitivity ◽  
Histochemical Demonstration ◽  
Frozen Sections ◽  
Enzyme Localization ◽  
Fresh Frozen ◽  
Preparation Techniques

The suitability of freeze-substitution in n-butanol and paraffin embedding of tissues for the histochemical demonstration of 5'-nucleotidase was investigated and compared with commonly used preparation techniques, such as fresh frozen sections and cryostate sections of cold formalin and glutaraldehyde-fixed rat liver. The influences of each step of the preparation techniques on the enzyme activity were controlled by a quantitative radiochemical assay. Freeze substitution was revealed to be superior to the commonly used preparation techniques with respect to: 1) high sensitivity and specificity of the histochemical 5'-nucleotidase reaction (this is based on the fact that incubation media with very low lead concentrations (0,6 mM/1) can be used); 2) excellent morphological appearance of the tissues showing cytological details of enzyme localization; 3) unlimited storage of the tissue materials and ease of sectioning.

scholarly journals Biochemical studies on the activity of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase from Streptomyces clavuligerus

1992 ◽  
Vol 283 (3) ◽  
pp. 691-698 ◽  
Author(s):  
J Zhang ◽  
S Wolfe ◽  
A L Demain
Keyword(s):  
Enzyme Activity ◽  
High Temperature ◽  
Bioactive Compounds ◽  
Streptomyces Clavuligerus ◽  
Synthetase Activity ◽  
Enzyme Specificity ◽  
Acidic Ph ◽  
Reaction Parameters ◽  
Biochemical Studies ◽  
Isopenicillin N

The enzyme activity of purified delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from Streptomyces clavuligerus was studied biochemically. The dependence of ACV synthetase activity on reaction parameters, including substrates, cofactors, temperature and pH, were determined, resulting in a substantially increased enzyme activity. The activity is very labile to high temperature and is also unstable at acidic pH. The enzyme specificity is strict towards L-alpha-aminoadipate, but rather loose with respect to L-valine; certain modifications of L-cysteine can also be tolerated. Some unnatural tripeptides synthesized by ACV synthetase can be converted into bioactive compounds by isopenicillin N synthase. The only nutrient found to negatively affect ACV synthetase activity is phosphate, but various compounds such as thiol-blocking reagents and ATP-utilization products (AMP and pyrophosphate) are inhibitory to the enzyme.

scholarly journals Crystal Structures of the Heparan Sulfate-binding Domain of Follistatin

2003 ◽  
Vol 278 (41) ◽  
pp. 39969-39977 ◽  
Author(s):  
C. Axel Innis ◽  
Marko Hyvönen
Keyword(s):  
Heparan Sulfate ◽  
Crystal Structures ◽  
Binding Domain
Sign in / Sign up

Export Citation Format

Share Document