Zymography for Picogram Detection of Lipase and Esterase Activities.
Ontology highlight
ABSTRACT:
SUBMITTER: Ng AMJ
PROVIDER: S-EPMC7998489 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Fluorogenic enzyme probes go from a dark to a bright state following hydrolysis and can provide a sensitive, real-time readout of enzyme activity. They are useful for examining enzymatic activity in bacteria, including the human pathogen Mycobacterium tuberculosis. Herein, we describe two fluorogenic esterase probes derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). These probes offer enhanced optical properties compared to existing esterase probes because the hydrolysis product, DDAO, excites above 600 nm while retaining a good quantum yield (ϕ=0.40). We validated both probes with a panel of commercially available enzymes alongside known resorufin- and fluorescein-derived esterase substrates. Furthermore, we used these probes to reveal esterase activity in protein gel-resolved mycobacterial lysates. These probes represent new tools for esterase detection and characterization and should find use in a variety of applications.
| S-EPMC5096734 | biostudies-literature
Project description:The objective of the present study was to analyze serum protein complexes and detect serum esterase activities using nongradient blue native polyacrylamide gel electrophoresis (BN-PAGE). For analysis of potential protein complexes, serum from rat was used. Results demonstrate that a total of 8 gel bands could be clearly distinguished after Coomassie blue staining, and serum albumin could be isolated nearly as a pure protein. Moreover, proteins in these bands were identified by electrospray mass spectrometry and low-energy collision induced dissociation (CID)-MS/MS peptide sequencing and the existence of serum dihydrolipoamide dehydrogenase (DLDH) was confirmed. For studies of in-gel detection of esterase activities, serum from rat, mouse, and human was used. In-gel staining of esterase activity was achieved by the use of either ?-naphthylacetate or ?-naphthylacetate in the presence of Fast blue BB salt. There were three bands exhibiting esterase activities in the serum of both rat and mouse. In contrast, there was only one band showing esterase activity staining in the human serum. When serum samples were treated with varying concentrations of urea, esterase activity staining was abolished for all the bands except the one containing esterase 1 (Es1) protein that is known to be a single polypeptide enzyme, indicating that majority of these esterases were protein complexes or multimeric proteins. We also identified the human serum esterase as butyrylcholinesterase following isolation and partial purification using ammonium sulfate fractioning and ion exchange column chromatographies. Where applicable, demonstrations of the gel-based method for measuring serum esterase activities under physiological or pathophysiological conditions were illustrated. Results of the present study demonstrate that nongradient BN-PAGE can serve as a feasible analytical tool for proteomic and enzymatic analysis of serum proteins.
| S-EPMC3034814 | biostudies-literature
Project description:In an effort to explore the feasibility of converting a lipase into an esterase by modifying the lid region, we designed and characterized two novel Rhizopus chinensis lipase variants by lid swapping. The substrate specificity of an R. chinensis lipase was successfully modified toward water-soluble substrates, that is, turned into an esterase, by replacing the hydrophobic lid with a hydrophilic lid from ferulic acid esterase from Aspergillus niger Meanwhile, as a comparison, the lid of R. chinensis lipase was replaced by a hydrophobic lid from Rhizomucor miehei lipase, which did not alter its substrate specificity but led to a 5.4-fold higher catalytic efficiency (k*cat/K*m) toward p-nitrophenyl laurate. Based on the analysis of structure-function relationships, it suggests that the amphipathic nature of the lid is very important for the substrate specificity. This study provides new insight into the structural basis of lipase specificities and a way to tune the substrate preference of lipases.
| S-EPMC4031936 | biostudies-literature
Project description:Superparamagnetic iron-oxide nanoparticles can be used in medical applications like vascular or targeted imaging. Magnetic particle imaging (MPI) is a promising tomographic imaging technique that allows visualizing the 3D nanoparticle distribution concentration in a non-invasive manner. The two main strengths of MPI are high temporal resolution and high sensitivity. While the first has been proven in the assessment of dynamic processes like cardiac imaging, it is unknown how far the detection limit of MPI can be lowered. Within this work, we will present a highly sensitive gradiometric receive-coil unit combined with a noise-matching network tailored for the imaging of mice. The setup is capable of detecting 5 ng of iron in-vitro with an acquisition time of 2.14 sec. In terms of iron concentration we are able to detect 156 μg/L marking the lowest value that has been reported for an MPI scanner so far. In-vivo MPI mouse images of a 512 ng bolus and a 21.5 ms acquisition time allow for capturing the flow of an intravenously injected tracer through the heart of a mouse. Since it has been rather difficult to compare detection limits across MPI publications we propose guidelines to improve the comparability of future MPI studies.
| S-EPMC5537232 | biostudies-literature
Project description:Digital droplet assays-in which biological samples are compartmentalized into millions of femtoliter-volume droplets and interrogated individually-have generated enormous enthusiasm for their ability to detect biomarkers with single-molecule sensitivity. These assays have untapped potential for point-of-care diagnostics but are currently mainly confined to laboratory settings, due to the instrumentation necessary to serially generate, control, and measure tens of millions of droplets/compartments. To address this challenge, we developed an optofluidic platform that miniaturizes digital assays into a mobile format by parallelizing their operation. This technology is based on three key innovations: (i) the integration and parallel operation of a hundred droplet generators onto a single chip that operates >100× faster than a single droplet generator, (ii) the fluorescence detection of droplets at >100× faster than conventional in-flow detection using time domain-encoded mobile phone imaging, and (iii) the integration of on-chip delay lines and sample processing to allow serum-to-answer device operation. To demonstrate the power of this approach, we performed a duplex digital ELISA. We characterized the performance of this assay by first using spiked recombinant proteins in a complex media (FBS) and measured a limit of detection, 0.004 pg/mL (300 aM), a 1,000× improvement over standard ELISA and matching that of the existing laboratory-based gold standard digital ELISA system. We additionally measured endogenous GM-CSF and IL6 in human serum from n = 14 human subjects using our mobile duplex assay, and showed excellent agreement with the gold standard system ([Formula: see text]).
| S-EPMC6410864 | biostudies-literature
Project description:Human diet is undergoing a shift towards plant-based diet as a sustainable source of protein compared to animal-derived protein. In this study, cholesterol esterase (CEase) and pancreatic lipase (PL) inhibitory activities of amaranth protein hydrolysates (APHs) were studied. Bromelain, chymotrypsin, and actinase E were used for generating APHs at 2, 4 & 6 h of hydrolysis. Higher PL inhibiting potential were observed in bromelain-derived APHs (IC50 = 0.38-0.66 mg/mL) in comparison to intact amaranth proteins (IC50 = 3.93 mg/mL). Bromelain-4 h hydrolysates (AB4) demonstrated significant inhibitory potential for both CEase (IC50 = 0.47 mg/mL) and PL (IC50 = 0.48 mg/mL) activity. Peptide identification in AB-4 hydrolysate revealed that among 17 bioactive peptides, three peptides (FPFPPTLGY, FGAPR, and FPFVPAPT) were predicted as potential PL inhibitors and only one peptide (FPFVPAPT) was predicted as CEase inhibitor based on the number of substrate binding sites on active site of the enzymes. This is the first study providing insights into amaranth protein derived bioactive peptide possessing CEase and LIP inhibitory potential.
| S-EPMC8633574 | biostudies-literature
Project description:A number of intracellular lipase/esterase have been reported in adipose tissue either by functional assays of activity or through proteomic analysis. In the current work, we have studied the relative expression level of 12 members of the lipase/esterase family that are found in white adipose tissue. We found that the relative mRNA levels of ATGL and HSL are the most abundant, being 2-3 fold greater than TGH or ADPN; whereas other intracellular neutral lipase/esterases were expressed at substantially lower levels. High fat feeding did not alter the mRNA expression levels of most lipase/esterases, but did reduce CGI-58 and WBSCR21. Likewise, rosiglitazone treatment did not alter the mRNA expression levels of most lipase/esterases, but did increase ATGL, TGH, CGI-58 and WBSCR21, while reducing ADPN. WAT from HSL-/- mice showed no compensatory increase in any lipase/esterases, rather mRNA levels of most lipase/esterases were reduced. In contrast, BAT from HSL-/- mice showed an increase in ATGL expression, as well as a decrease in ES-1, APEH and WBSCR21. Analysis of the immunoreactive protein levels of some of the lipases confirmed the results seen with mRNA. In conclusion, these data highlight the complexity of the regulation of the expression of intracellular neutral lipase/esterases involved in lipolysis.
| S-EPMC1933526 | biostudies-literature
Project description:The hydrophobic cuticle covers the surface of the most aerial organs of land plants. The barley mutant eceriferum-zv (cer-zv), which is hypersensitive to drought, is unable to accumulate a sufficient quantity of cutin in its leaf cuticle. The mutated locus has been mapped to a 0.02 cM segment in the pericentromeric region of chromosome 4H. As a map-based cloning approach to isolate the gene was therefore considered unlikely to be feasible, a comparison was instead made between the transcriptomes of the mutant and the wild type. In conjunction with extant genomic information, on the basis of predicted functionality, only two genes were considered likely to encode a product associated with cutin formation. When eight independent cer-zv mutant alleles were resequenced with respect to the two candidate genes, it was confirmed that the gene underlying the mutation in each allele encodes a Gly-Asp-Ser-Leu (GDSL)-motif esterase/acyltransferase/lipase. The gene was transcribed in the epidermis, and its product was exclusively deposited in cell wall at the boundary of the cuticle in the leaf elongation zone, coinciding with the major site of cutin deposition. CER-ZV is speculated to function in the deposition of cutin polymer. Its homologs were found in green algae, moss, and euphyllophytes, indicating that it is highly conserved in plant kingdom.
| S-EPMC6508521 | biostudies-literature
Project description:The esterase and depsidase activities of tannase have been examined by kinetic methods. Although the esterase/depsidase ratio of tannase may be varied by cultural methods and isolation procedures, evidence has been obtained to show that tannase, esterase and depsidase are enzymes with low specificities capable of hydrolysing both esters and depsides of gallic acid.
| S-EPMC1264952 | biostudies-other
Project description:GDSL-type esterase/lipase proteins (GELPs) belong to the SGNH hydrolase superfamily and contain a conserved GDSL motif at their N-terminus. GELPs are widely distributed in nature, from microbes to plants, and play crucial roles in growth and development, stress responses and pathogen defense. However, the identification and functional analysis of GELP genes are hardly explored in soybean. This study describes the identification of 194 GELP genes in the soybean genome and their phylogenetic classification into 11 subfamilies (A-K). GmGELP genes are disproportionally distributed on 20 soybean chromosomes. Large-scale WGD/segmental duplication events contribute greatly to the expansion of the soybean GDSL gene family. The Ka/Ks ratios of more than 70% of duplicated gene pairs ranged from 0.1-0.3, indicating that most GmGELP genes were under purifying selection pressure. Gene structure analysis indicate that more than 74% of GmGELP genes are interrupted by 4 introns and composed of 5 exons in their coding regions, and closer homologous genes in the phylogenetic tree often have similar exon-intron organization. Further statistics revealed that approximately 56% of subfamily K members contain more than 4 introns, and about 28% of subfamily I members consist of less than 4 introns. For this reason, the two subfamilies were used to simulate intron gain and loss events, respectively. Furthermore, a new model of intron position distribution was established in current study to explore whether the evolution of multi-gene families resulted from the diversity of gene structure. Finally, RNA-seq data were used to investigate the expression profiles of GmGELP gene under different tissues and multiple abiotic stress treatments. Subsequently, 7 stress-responsive GmGELP genes were selected to verify their expression levels by RT-qPCR, the results were consistent with RNA-seq data. Among 7 GmGELP genes, GmGELP28 was selected for further study owing to clear responses to drought, salt and ABA treatments. Transgenic Arabidopsis thaliana and soybean plants showed drought and salt tolerant phenotype. Overexpression of GmGELP28 resulted in the changes of several physiological indicators, which allowed plants to adapt adverse conditions. In all, GmGELP28 is a potential candidate gene for improving the salinity and drought tolerance of soybean.
| S-EPMC7332888 | biostudies-literature