scholarly journals Enzymatic Synthesis of a Fluorogenic Reporter Substrate and a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation

2019 ◽  
Author(s):  
Maxim Soroko ◽  
David Kwan
Keyword(s):  
Enzyme Activity ◽  
Enzymatic Synthesis ◽  
Enzyme Assay ◽  
Enzyme Reaction ◽  
Complex Type ◽  
The Core ◽  
Fluorogenic Probe ◽  
Tissue Lysate ◽  
High Throughput Assay ◽  
Core Fucosylation

We report a straight-forward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core-fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of <i>N</i>-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequential coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and very specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.

scholarly journals Enzymatic Synthesis of a Fluorogenic Reporter Substrate and a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation

2019 ◽  
Author(s):  
Maxim Soroko ◽  
David Kwan
Keyword(s):  
Enzyme Activity ◽  
Enzymatic Synthesis ◽  
Enzyme Assay ◽  
Enzyme Reaction ◽  
Complex Type ◽  
The Core ◽  
Fluorogenic Probe ◽  
Tissue Lysate ◽  
High Throughput Assay ◽  
Core Fucosylation

We report a straight-forward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core-fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of <i>N</i>-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequential coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and very specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.

Reappraisal of the uroporphyrinogen I synthase assay, and a proposed modified method.

1980 ◽  
Vol 26 (9) ◽  
pp. 1346-1347 ◽  
Author(s):  
B R Chamberlain ◽  
J E Buttery
Keyword(s):  
Enzyme Activity ◽  
Enzyme Assay ◽  
Enzyme Reaction ◽  
Slit Width ◽  
Lag Phase ◽  
Excitation Wavelength ◽  
Blood Specimen ◽  
Modified Method ◽  
Triton X

Abstract Problems encountered in the uroporphyrinogen I synthase assay were found to be due to the Triton reagent, slit-width and excitation wavelength settings of the spectrophotofluorometer, and leg-phase in the enzyme reaction. Of Triton X-100 from four suppliers, only three were found suitable. Differences in enzyme activity owing to slit-width adjustments were resolved by measuring the fluorescence of the sample (uroporphyrin) at 405 nm and the standard (coproporphyrin) at 401 nm. The lag-phase in the activated-enzyme assay is incorporated in the 30-min preincubation of the blood specimen in our modified method, thus giving higher enzyme activity than found with the original assay. The range for our method is 7.3--15.8 nmol s-1 L-1 (mean = 10.9; SD = 2.07).

scholarly journals Appropriate aglycone modification significantly expands the glycan substrate acceptability of α1,6-fucosyltransferase (FUT8)

2021 ◽  
Author(s):  
Roushu Zhang ◽  
Qiang Yang ◽  
Bhargavi M Boruah ◽  
Guanghui Zong ◽  
Chao Li ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Turnover Rate ◽  
Human Neutrophils ◽  
Complex Type ◽  
The Core ◽  
Km Value ◽  
High Mannose ◽  
Overall Efficiency ◽  
Core Fucosylation ◽  
Lysosomal Proteins

The α1,6-fucosyltransferase, FUT8, is the sole enzyme catalyzing the core-fucosylation of N-glycoproteins in mammalian systems. Previous studies using free N-glycans as acceptor substrates indicated that a terminal β1,2-GlcNAc moiety on the Man-α1,3-Man arm of N-glycan substrates is required for efficient FUT8-catalyzed core-fucosylation. In contrast, we recently demonstrated that, in a proper protein context, FUT8 could also fucosylate Man5GlcNAc2 without a GlcNAc at the non-reducing end. We describe here a further study of the substrate specificity of FUT8 using a range of N-glycans containing different aglycones. We found that FUT8 could fucosylate most of high-mannose and complex-type N-glycans, including highly branched N-glycans from chicken ovalbumin, when the aglycone moiety is modified with a 9-fluorenylmethyloxycarbonyl (Fmoc) moiety or in a suitable peptide/protein context, even if they lack the terminal GlcNAc moiety on the Man-α1,3-Man arm. FUT8 could also fucosylate paucimannose structures when they are on glycoprotein substrates.  Such core-fucosylated paucimannosylation is a prominent feature of lysosomal proteins of human neutrophils and several types of cancers. We also found that sialylation of N-glycans significantly reduced their activity as substrate of FUT8. Kinetic analysis demonstrated that Fmoc aglycone modification could either improve the turnover rate or decrease the Km value depending on the nature of the substrates, thus significantly enhancing the overall efficiency of FUT8 catalyzed fucosylation. Our results indicate that an appropriate aglycone context of N-glycans could significantly broaden the acceptor substrate specificity of FUT8 beyond what has previously been thought.

scholarly journals Decreased rate of ketone-body oxidation and decreased activity of d-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats

1986 ◽  
Vol 240 (1) ◽  
pp. 49-56 ◽  
Author(s):  
L Grinblat ◽  
L F Pacheco Bolaños ◽  
A O Stoppani
Keyword(s):  
Enzyme Activity ◽  
Enzyme Reaction ◽  
Diabetic Rats ◽  
Heart Mitochondria ◽  
Maximal Velocity ◽  
Coa Transferase ◽  
Slow Adaptation ◽  
Significant Difference ◽  
Metabolic Conditions ◽  
Onset Of Diabetes

Heart mitochondria from chronically diabetic rats (‘diabetic mitochondria’), in metabolic State 3, oxidized 3-hydroxybutyrate and acetoacetate at a relatively slow rate, as compared with mitochondria from normal rats (‘normal mitochondria’). No significant differences were observed, however, with pyruvate or L-glutamate plus L-malate as substrates. Diabetic mitochondria also showed decreased 3-hydroxybutyrate dehydrogenase and succinyl-CoA: 3-oxoacid CoA-transferase activities, but cytochrome content and NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and acetoacetyl-CoA thiolase activities proved normal. The decrease of 3-hydroxybutyrate dehydrogenase activity was observed in diabetic mitochondria subjected to different disruption procedures, namely freeze-thawing, sonication or hypoosmotic treatment, between pH 7.5 and 8.5, at temperatures in the range 6-36 degrees C, and in the presence of L-cysteine. Determination of the kinetic parameters of the enzyme reaction in diabetic mitochondria revealed diminution of maximal velocity (Vmax) as its outstanding feature. The decrease in 3-hydroxybutyrate dehydrogenase in diabetic mitochondria was a slow-developing effect, which reached full expression 2-3 months after the onset of diabetes; 1 week after onset, no significant difference between enzyme activity in diabetic and normal mitochondria could be established. Insulin administration to chronically diabetic rats for 2 weeks resulted in limited recovery of enzyme activity. G.l.c. analysis of fatty acid composition and measurement of diphenylhexatriene fluorescence anisotropy failed to reveal significant differences between diabetic and normal mitochondria. The Arrhenius-plot characteristics for 3-hydroxybutyrate dehydrogenase in membranes of diabetic and normal mitochondria were similar. It is assumed that the variation of the assayed enzymes in diabetic mitochondria results from a slow adaptation to the metabolic conditions resulting from diabetes, rather than to insulin deficiency itself.

scholarly journals Extracellular α-Galactosidase from Trichoderma sp. (WF-3): Optimization of Enzyme Production and Biochemical Characterization

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Aishwarya Singh Chauhan ◽  
Arunesh Kumar ◽  
Nikhat J. Siddiqi ◽  
B. Sharma
Keyword(s):  
Enzyme Activity ◽  
Enzyme Assay ◽  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Culture Conditions ◽  
Significant Loss ◽  
Trichoderma Sp ◽  
Rate Of Reaction ◽  
Higher Temperature ◽  
Substrate Concentrations

Trichoderma spp. have been reported earlier for their excellent capacity of secreting extracellular α-galactosidase. This communication focuses on the optimization of culture conditions for optimal production of enzyme and its characterization. The evaluation of the effects of different enzyme assay parameters such as stability, pH, temperature, substrate concentrations, and incubation time on enzyme activity has been made. The most suitable buffer for enzyme assay was found to be citrate phosphate buffer (50 mM, pH 6.0) for optimal enzyme activity. This enzyme was fairly stable at higher temperature as it exhibited 72% activity at 60°C. The enzyme when incubated at room temperature up to two hours did not show any significant loss in activity. It followed Michaelis-Menten curve and showed direct relationship with varying substrate concentrations. Higher substrate concentration was not inhibitory to enzyme activity. The apparent Michaelis-Menten constant (Km), maximum rate of reaction (Vmax), Kcat, and catalytic efficiency values for this enzyme were calculated from the Lineweaver-Burk double reciprocal plot and were found to be 0.5 mM, 10 mM/s, 1.30 U mg−1, and 2.33 U mg−1 mM−1, respectively. This information would be helpful in understanding the biophysical and biochemical characteristics of extracellular α-galactosidase from other microbial sources.

scholarly journals Analysis of site and structure specific core fucosylation in liver disease progression using exoglycosidase-assisted data-independent LC-MS/MS

2020 ◽  
Author(s):  
Miloslav Sanda ◽  
Jaeil Ahn ◽  
Petr Kozlik ◽  
Radoslav Goldman
Keyword(s):  
Liver Disease ◽  
Disease Progression ◽  
Serum Proteins ◽  
Receptor Activation ◽  
Biological Macromolecules ◽  
The Core ◽  
Liver Disease Progression ◽  
Living Organisms ◽  
Protein Attachment ◽  
Core Fucosylation

ABSTRACTCarbohydrates form one of the major groups of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response, and receptor activation are regulated by glycosylation. Fucosylation of proteins regulates such processes and is associated with various diseases including autoimmunity and cancer. Mass spectrometry efficiently identifies structures of fucosylated glycans or sites of core fucosylated N-glycopeptides but quantification of the glycopeptides remains less explored. We performed experiments that facilitate quantitative analysis of the core fucosylation of proteins with partial structural resolution of the glycans and we present results of the mass spectrometric SWATH-type DIA analysis of relative abundances of the core fucosylated glycoforms of 45 glycopeptides derived from 18 serum proteins in liver disease of different etiologies. Our results show that a combination of soft fragmentation with exoglycosidases is efficient at the assignment and quantification of the core fucosylated N-glycoforms at specific sites of protein attachment. In addition, our results show that disease-associated changes in core fucosylation are peptide-dependent and further differ by branching of the core fucosylated glycans. Further studies are needed to verify whether tri- and tetra-antennary core fucosylated glycopeptides could be used as markers of liver disease progression.

QUANTITATIVE STUDIES ON ISOLATED PANCREATIC ISLETS OF MAMMALS

1963 ◽  
Vol 42 (4) ◽  
pp. 615-624 ◽  
Author(s):  
Claes Hellerström ◽  
Bo Hellman
Keyword(s):  
Enzyme Activity ◽  
B Cells ◽  
Enzyme Level ◽  
Enzyme Reaction ◽  
Histochemical Staining ◽  
Pancreatic Tissue ◽  
Free Access ◽  
Isolated Pancreatic Islets ◽  
Insulin Synthesis ◽  
Dipeptidase Activity

ABSTRACT Microtitrimetric assays of dipeptidase activity were performed in isolated pancreatic islet tissue from mice. Considerable enzyme activity was found in both the endocrine and exocrine pancreas of normal mice, the enzyme level of the exocrine parenchyma being significantly higher. In obesehyperglycaemic mice with free access to food, isolated islets of Langerhans had a much higher enzyme activity than in normal animals. The increased islet dipeptidase activity in the obese-hyperglycaemic animals may, at least in part, be accounted for by their higher proportion of B cells. The intense insulin synthesis and renewal of B cells in these animals have been considered as alternative explanations. Histochemical staining for leucine aminopeptidase revealed a moderate enzyme reaction in both the endocrine and exocrine pancreatic tissue of normal and obese-hyperglycaemic mice.

scholarly journals ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

1963 ◽  
Vol 19 (2) ◽  
pp. 337-347 ◽  
Author(s):  
M. A. Epstein ◽  
S. J. Holt
Keyword(s):  
Enzyme Activity ◽  
Hela Cells ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Enzyme Reaction ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Simplex Virus ◽  
Vacuolar Membranes

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.

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