Developments in Methodology of Enzyme Assays

2015 ◽  
pp. 73-86
Author(s):  
Hans Ulrich Bergmeyer
Keyword(s):  
Enzyme Assays

scholarly journals Microbial Respiration and Enzyme Activity Downstream from a Phosphorus Source in the Everglades, Florida, USA

Land ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 696
Author(s):  
Sanku Dattamudi ◽  
Saoli Chanda ◽  
Leonard J. Scinto
Keyword(s):  
Nutrient Dynamics ◽  
Microbial Respiration ◽  
Organic Matter Content ◽  
Physicochemical Analysis ◽  
Matter Content ◽  
Enzyme Assays ◽  
Co2 Efflux ◽  
Soil Organic Matter Content ◽  
Phosphorus Source ◽  
P Distribution

Northeast Shark River Slough (NESS), lying at the northeastern perimeter of Everglades National Park (ENP), Florida, USA, has been subjected to years of hydrologic modifications. Construction of the Tamiami Trail (US 41) in 1928 connected the east and west coasts of SE Florida and essentially created a hydrological barrier to southern sheet flow into ENP. Recently, a series of bridges were constructed to elevate a portion of Tamiami Trail, allow more water to flow under the bridges, and attempt to restore the ecological balance in the NESS and ENP. This project was conducted to determine aspects of soil physiochemistry and microbial dynamics in the NESS. We evaluated microbial respiration and enzyme assays as indicators of nutrient dynamics in NESS soils. Soil cores were collected from sites at certain distances from the inflow (near canal, NC (0–150 m); midway, M (150–600 m); and far from canal, FC (600–1200 m)). Soil slurries were incubated and assayed for CO2 emission and β-glucoside (MUFC) or phosphatase (MUFP) activity in concert with physicochemical analysis. Significantly higher TP contents at NC (2.45 times) and M (1.52 times) sites than FC sites indicated an uneven P distribution downstream from the source canal. The highest soil organic matter content (84%) contents were observed at M sites, which was due to higher vegetation biomass observed at those sites. Consequently, CO2 efflux was greater at M sites (average 2.72 µmoles g dw−1 h−1) than the other two sites. We also found that amendments of glucose increased CO2 efflux from all soils, whereas the addition of phosphorus did not. The results indicate that microbial respiration downstream of inflows in the NESS is not limited by P, but more so by the availability of labile C.

Monosaccharide Biosynthesis Pathways Database

Glycobiology ◽  
2021 ◽  
Author(s):  
Jaya Srivastava ◽  
P Sunthar ◽  
Petety V Balaji
Keyword(s):  
Expression Profiling ◽  
Experimental Studies ◽  
Structural Complexity ◽  
Evolutionary Relationships ◽  
Enzyme Assays ◽  
Biosynthesis Pathway ◽  
Knock Out ◽  
Set Up ◽  
Function Relationship ◽  
Natural Glycans

Abstract A distinctive feature of glycans vis-à-vis proteins and nucleic acids is its structural complexity which arises from the huge repertoire of monosaccharides, isomeric linkages and branching. A very large number of monosaccharides have so far been discovered in natural glycans. Experimentally, pathways for the biosynthesis have been characterized completely for 55 monosaccharides and partially for a few more. However, there is no single platform which provides information about monosaccharide biosynthesis pathways and associated enzymes We have gathered 572 experimentally characterized enzymes of 66 biosynthesis pathways from literature and set up a first of its kind database called the Monosaccharide Biosynthesis Pathways Database http://www.bio.iitb.ac.in/mbpd/). Annotations such as the reaction catalysed, substrate specificity, biosynthesis pathway and PubMed IDs are provided for all the enzymes in the database. Sequence homologs of the experimentally characterized enzymes found in nearly 13,000 completely sequenced genomes from Bacteria and Archaea have also been included in the database. This platform will help in the deduction of evolutionary relationships among enzymes such as aminotransferases, nucleotidyltransferases, acetyltransferases and SDR family enzymes. It can also facilitate experimental studies such as direct enzyme assays to validate putative annotations, establish structure–function relationship, expression profiling to determine the function, determine the phenotypic consequences of gene knock-out/knock-in and complementation studies.

scholarly journals Sulfoquinovose metabolism in marine algae

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Sabine Scholz ◽  
Manuel Serif ◽  
David Schleheck ◽  
Martin D.J. Sayer ◽  
Alasdair M. Cook ◽  
...  
Keyword(s):  
High Performance ◽  
Marine Algae ◽  
Model Organisms ◽  
Enzyme Assays ◽  
Laser Desorption Ionization ◽  
Genome Sequences ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Global Biogeochemical Cycles ◽  
Algal Extracts

Abstract This study aimed to survey algal model organisms, covering phylogenetically representative and ecologically relevant taxa. Reports about the occurrence of sulfonates (particularly sulfoquinovose, taurine, and isethionate) in marine algae are scarce, and their likely relevance in global biogeochemical cycles and ecosystem functioning is poorly known. Using both field-collected seaweeds from NW Scotland and cultured strains, a combination of enzyme assays, high-performance liquid chromatography and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry was used to detect key sulfonates in algal extracts. This was complemented by bioinformatics, mining the publicly available genome sequences of algal models. The results confirm the widespread presence of sulfonates and their biosynthetic pathways in macro- and microalgae. However, it is also clear that catabolic pathways, if present, must be different from those documented from the bacterial systems since no complete cluster of gene homologues of key genes could be detected in algal genomes.

scholarly journals Activation of short-chain ketones and isopropanol in sulfate-reducing bacteria

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jasmin Frey ◽  
Sophie Kaßner ◽  
Dieter Spiteller ◽  
Mario Mergelsberg ◽  
Matthias Boll ◽  
...  
Keyword(s):  
Sulfate Reducing Bacteria ◽  
Short Chain ◽  
Enzyme Assays ◽  
Protein Patterns ◽  
Sulfate Reducing ◽  
Protein Gel ◽  
Branched Chain ◽  
Reducing Bacteria ◽  
Coenzyme B ◽  
Nitrate Reducing Bacteria

Abstract Background Degradation of acetone by aerobic and nitrate-reducing bacteria can proceed via carboxylation to acetoacetate and subsequent thiolytic cleavage to two acetyl residues. A different strategy was identified in the sulfate-reducing bacterium Desulfococcus biacutus that involves formylation of acetone to 2-hydroxyisobutyryl-CoA. Results Utilization of short-chain ketones (acetone, butanone, 2-pentanone and 3-pentanone) and isopropanol by the sulfate reducer Desulfosarcina cetonica was investigated by differential proteome analyses and enzyme assays. Two-dimensional protein gel electrophoresis indicated that D. cetonica during growth with acetone expresses enzymes homologous to those described for Desulfococcus biacutus: a thiamine diphosphate (TDP)-requiring enzyme, two subunits of a B12-dependent mutase, and a NAD+-dependent dehydrogenase. Total proteomics of cell-free extracts confirmed these results and identified several additional ketone-inducible proteins. Acetone is activated, most likely mediated by the TDP-dependent enzyme, to a branched-chain CoA-ester, 2-hydroxyisobutyryl-CoA. This compound is linearized to 3-hydroxybutyryl-CoA by a coenzyme B12-dependent mutase followed by oxidation to acetoacetyl-CoA by a dehydrogenase. Proteomic analysis of isopropanol- and butanone-grown cells revealed the expression of a set of enzymes identical to that expressed during growth with acetone. Enzyme assays with cell-free extract of isopropanol- and butanone-grown cells support a B12-dependent isomerization. After growth with 2-pentanone or 3-pentanone, similar protein patterns were observed in cell-free extracts as those found after growth with acetone. Conclusions According to these results, butanone and isopropanol, as well as the two pentanone isomers, are degraded by the same enzymes that are used also in acetone degradation. Our results indicate that the degradation of several short-chain ketones appears to be initiated by TDP-dependent formylation in sulfate-reducing bacteria.

Magnetic Particle-Based Enzyme Assays and Immunoassays for Microcystins: From Colorimetric to Electrochemical Detection.

2012 ◽  
Vol 47 (1) ◽  
pp. 471-478 ◽  
Author(s):  
Laia Reverté ◽  
Diana Garibo ◽  
Cintia Flores ◽  
Jorge Diogène ◽  
Josep Caixach ◽  
...  
Keyword(s):  
Electrochemical Detection ◽  
Magnetic Particle ◽  
Enzyme Assays

Abstract 657: Increased Urinary Angiotensin Converting Enzyme 2 (ACE2) In Type 2 Diabetic Patients

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Sridevi Gutta ◽  
Nadja Grobe ◽  
Hassan Osman ◽  
Mohammad Saklayen ◽  
Khalid M Elased
Keyword(s):  
Mass Spectrometry ◽  
Medical Center ◽  
Diabetic Patients ◽  
Enzyme Assays ◽  
Renal Tubules ◽  
Type 2 Diabetic Patients ◽  
Major Health ◽  
Angiotensin Converting Enzyme 2 ◽  
Noninvasive Biomarker

Diabetes and its associated chronic kidney disease (CKD) is a major health burden and there is an urgent need for new sensitive biomarkers to detect and monitor the progression of CKD. Albuminuria is still the gold standard for the evaluation of kidney function. However, its sensitivity and reliability have recently been questioned. ACE2 is highly expressed in renal tubules and has been shown to be shed in the urine of diabetic patients with CKD. The aim of the study was to investigate whether urinary ACE2 is increased in diabetic patients with CKD before the onset of microalbuminuria. Participants were recruited from Dayton VA Medical Center (Dayton, OH, USA). Baseline urinary albumin creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) were determined three months before initiation of the study in non-diabetic patients (UACR <30 mg/g, eGFR=97.40±16 ml/min/1.73 m 2 ), and in diabetic patients with normoalbuminuria (UACR <30 mg/g, eGFR=83.08±17 ml/min/1.73 m 2 ), microalbuminuria (UACR = 30-300 mg/g, eGFR=47.13±23 ml/min/1.73 m 2 ), and macroalbuminuria (UACR >300 mg/g, eGFR=39.68±20 ml/min/1.73 m 2 ). Using fluorogenic and mass spectrometry-based enzyme assays, we measured urinary and plasma ACE2 activity in patients. Urinary ACE2 activity was significantly increased in diabetic patients with normoalbuminuria (0.58±0.2 nmol/hr/mg creatinine), microalbuminuria (1.19 ±0.5 nmol/hr/mg creatinine), and macroalbuminuria (2.265±0.4 nmol/hr/mg creatinine) compared with non-diabetic controls (0.06 ± 0.02 nmols/hr/mg creatinine) (p<0.0001). These results were confirmed by detecting the ACE2 product Ang-(1-7) ( m/z 899) in incubations of urine samples with the natural substrate Ang II ( m/z 1046) using mass spectrometry-based enzyme assays. In addition, urinary ACE2 expression was significantly increased in diabetic patients as determined by western blot analysis (p<0.05). Plasma ACE2 activity was not detectable in control and diabetic patients. In conclusion, urinary ACE2 is increased in diabetic patients with CKD which suggests that urinary ACE2 could be used as an early, noninvasive biomarker for diabetic nephropathy before the onset of microalbuminuria.

Sign in / Sign up

Export Citation Format

Share Document