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 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.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

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 Differential effects of non-ionic detergents on microsomal and sarcolemmal adenylate cyclase in cardiac muscle

1978 ◽  
Vol 175 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Prakash V. Sulakhe ◽  
Njanoor Narayanan
Keyword(s):  
Enzyme Activity ◽  
Sarcoplasmic Reticulum ◽  
Adenylate Cyclase ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Protein Ratio ◽  
Guinea Pig Heart ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Ionic Detergent ◽  
Triton X

1. About 4 and 23% of the homogenate adenylate cyclase activity was recovered in the microsomal and sarcolemmal fractions isolated from guinea-pig heart ventricles. 2. Cardiac microsomal adenylate cyclase activity [basal as well as p[NH]ppG (guanyl-5′-yl imidodiphosphate)- and NaF-stimulated] was increased over 2-fold in the presence of Lubrol-PX (0.01–0.1%). 3. The sarcolemmal enzyme, however, showed concentration-dependent inhibition caused by the detergent under all assay conditions, except when p[NH]ppG was included in the assay. In the latter case, the detergent (0.01–0.02%) caused a modest increase (30–45%) in enzyme activity. 4. Another non-ionic detergent, Triton X-100, also stimulated the microsomal cyclase and inhibited the sarcolemmal enzyme. 5. With either membrane fraction, Lubrol-PX solubilized the enzyme when the detergent/membrane protein ratio was 2.5 (μmol of detergent/mg of protein). 6. The findings with homogenate and a washed particulate fraction resembled those obtained with sarcolemma, and those with isolated sarcoplasmic reticulum resembled those with microsomal preparations. 7. p[NH]ppG, and to some extent NaF, protected the detergent-induced inactivation of the enzyme observed at higher detergent concentrations (0.5% Lubrol-PX and 0.05–0.5% Triton X-100). 8. In the absence of detergents, p[NH]ppG increased the basal enzyme activity about 2-fold in microsomal fractions, but did not appreciably stimulate the sarcolemmal enzyme. Isoproterenol, on the other hand, increased the sarcolemmal enzyme activity (>2-fold) in the presence of p[NH]ppG and caused only moderate stimulation (31%) of the microsomal enzyme under these conditions. 9. These findings support the view that, although the bulk of adenylate cyclase resides in heart sarcolemma (plasma membrane), the microsomal activity cannot be accounted for solely by contamination of the microsomal fraction with sarcolemma, as has been suggested by others [Besch, Jones & Watanabe (1976) Circ. Res.39, 586–595; Engelhard, Plut & Storm (1976) Biochim. Biophys. Acta451, 48–61]. Further, the results of this study show that cardiac sarcoplasmic-reticulum membranes possess this enzyme.

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.

IMMOBILIZATION OF AMYLASE PRODUCING BACTERIA (BACILLUS SUBTILIS) FROM SOIL OF WESTERN HIMALAYAN REGION SOLAN, HIMACHAL PRADESH, INDIA

2021 ◽  
pp. 109-115
Author(s):  
ARUN KUMAR ◽  
POONAM KUMARI ◽  
KASAHUN GUDETA ◽  
JM JULKA
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Physicochemical Properties ◽  
Bacterial Strain ◽  
Enzyme Activities ◽  
Enzyme Assay ◽  
Himachal Pradesh ◽  
Genus Bacillus ◽  
Western Himalayan Region

Objective: The paper aimed to immobilize amylase producing bacterial strain on a suitable matrix and characterization of its physicochemical properties so that much amount of amylase could be produced to be applied in different industries. Methods: Bacterial colonies were sub-cultured from samples collected from soil in freshly prepared dishes containing starch agar by dot method using sterile inoculating needles from which five different bacteria belonged to genus Bacillus were isolated and assigned as A1, A2, A3, A4, and A5. Results: It was found that A1 displayed the highest enzyme activity of 17.89 IU/ml with enzyme assay of 0.83 mg/ml and the bacterium was identified to be Bacillus subtilis. A5 displayed 10.13 IU/ml with protein contents of 0.11 mg/ml indicated that A1 possess the highest enzyme activities which were categorized under Bacillus and protein contents and A5 showed less amount of enzyme activities and protein contents as compared to other. Conclusion: The bacteria which were produced much amount of enzyme activities identified as Bacillus subtilis and recommended and have been recommended to be cultured for the production of amylase enzyme.

scholarly journals The Relative Merits and Applicability of Kinetic and Fixed-Incubation Methods of Enzyme Assay in Clinical Enzymology

1972 ◽  
Vol 18 (12) ◽  
pp. 1449-1454 ◽  
Author(s):  
D W Moss
Keyword(s):  
Enzyme Activity ◽  
Continuous Monitoring ◽  
Enzyme Assay ◽  
Kinetic Method ◽  
Reaction Progress ◽  
Kinetic Principle ◽  
Clinical Enzymology

Abstract The considerations are discussed that make continuous monitoring of reaction-progress curves superior to fixed-incubation methods in the determination of enzyme activity. Provided that they are used with caution and their limitations are appreciated, fixed-incubation methods continue to fulfill a useful, though diminishing, role in clinical enzymology because of their adaptability to existing patterns of automation. The introduction of suitable mechanized equipment will favor the eventual complete adoption of the kinetic method. However, the use of such equipment should not be at the expense of th important characteristics of the kinetic principle.

Sign in / Sign up

Export Citation Format

Share Document