scholarly journals Activity- and Enrichment-Based Metaproteomics Insights into Active Urease from the Rumen Microbiota of Cattle

2022 ◽  
Vol 23 (2) ◽  
pp. 817
Author(s):  
Xiaoyin Zhang ◽  
Zhanbo Xiong ◽  
Ming Li ◽  
Nan Zheng ◽  
Shengguo Zhao ◽  
...  
Keyword(s):  
Urease Activity ◽  
Protein Extraction ◽  
Protein Structures ◽  
Gel Filtration ◽  
Optimization Method ◽  
Utilization Efficiency ◽  
Protein Database ◽  
Nitrogen Emissions ◽  
Rumen Microbiota ◽  
Highly Active

Regulation of microbial urease activity plays a crucial role in improving the utilization efficiency of urea and reducing nitrogen emissions to the environment for ruminant animals. Dealing with the diversity of microbial urease and identifying highly active urease as the target is the key for future regulation. However, the identification of active urease in the rumen is currently limited due to large numbers of uncultured microorganisms. In the present study, we describe an activity- and enrichment-based metaproteomic analysis as an approach for the discovery of highly active urease from the rumen microbiota of cattle. We conducted an optimization method of protein extraction and purification to obtain higher urease activity protein. Cryomilling was the best choice among the six applied protein extraction methods (ultrasonication, bead beating, cryomilling, high-pressure press, freeze-thawing, and protein extraction kit) for obtaining protein with high urease activity. The extracted protein by cryomilling was further enriched through gel filtration chromatography to obtain the fraction with the highest urease activity. Then, by using SDS-PAGE, the gel band including urease was excised and analyzed using LC-MS/MS, searching against a metagenome-derived protein database. Finally, we identified six microbial active ureases from 2225 rumen proteins, and the identified ureases were homologous to those of Fibrobacter and Treponema. Moreover, by comparing the 3D protein structures of the identified ureases and known ureases, we found that the residues in the β-turn of flap regions were nonconserved, which might be crucial in influencing the flexibility of flap regions and urease activity. In conclusion, the active urease from rumen microbes was identified by the approach of activity- and enrichment-based metaproteomics, which provides the target for designing a novel efficient urease inhibitor to regulate rumen microbial urease activity.

scholarly journals A chromatographic preparation of ox growth hormone

1966 ◽  
Vol 100 (3) ◽  
pp. 593-600 ◽  
Author(s):  
M Wallis ◽  
HBF Dixon
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Gel Electrophoresis ◽  
Anterior Pituitary ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Starch Gel Electrophoresis ◽  
Highly Active ◽  
Cross Linkage ◽  
Starch Gel

1. A method is described for the chromatographic preparation of ox growth hormone. It involves chromatography of an extract of anterior pituitary lobes on DEAE-cellulose, followed by rechromatography on a dextran gel of low cross-linkage (Sephadex G-100). 2. The product is highly active in growth-hormone assays, and is obtained in good yield. It was homogeneous by several criteria, but showed some heterogeneity on starch-gel electrophoresis. 3. The molecular weight of the hormone was estimated from its behaviour on gel-filtration columns under various conditions. Evidence that the hormone may dissociate into sub-units under some conditions is presented.

Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose

1976 ◽  
Vol 54 (2) ◽  
pp. 120-129 ◽  
Author(s):  
W. S. Rickert ◽  
P. A. McBride-Warren
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Affinity Column ◽  
Mucor Miehei ◽  
Highly Active ◽  
Elution Buffer ◽  
Turbidimetric Assay ◽  
Ph Range

The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5–8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65 000 and 52 000 were isolated, the 65 000 molecular weight species apparently representing a protomer of concanavalin A (24 000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity.At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including α-methyl D-glucoside and NaCl in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 °C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor rennin did not react with concanavalin A and were not bound to the affinity column.

TOXOHORMONE IN BACTERIA-FREE TUMORS

1966 ◽  
Vol 44 (8) ◽  
pp. 1069-1087 ◽  
Author(s):  
J. C. Nixon ◽  
B. Zinman
Keyword(s):  
Gel Filtration ◽  
Human Kidney ◽  
Metastatic Carcinoma ◽  
Exchange Chromatography ◽  
Human Spleen ◽  
Normal Tissues ◽  
Highly Active ◽  
Normal Human Kidney ◽  
Tumor Tissues ◽  
Normal Human

Toxohormone was extracted from bacteria-free human tumors and normal tissues, and assayed for activity by measuring the decrease in serum iron levels of rats 12 hours after injection of the extracts. In contrast with the findings of others, the results of the present study demonstrated that active toxohormone could be isolated from bacteria-free tumor tissues. Bacteria-free normal human kidney and spleen also yielded active toxohormone extracts, whereas extracts of normal human- and rat-skeletal muscle and rat liver had no activity.Four active toxohormone extracts were purified by ion-exchange chromatography followed by gel filtration. Human leukemic spleen, metastatic carcinoma of the cecum, and normal human spleen and kidney yielded several highly active purified fractions.

scholarly journals Beyond History: The List of The Most Well Studied Human Protein Structures

2020 ◽  
Author(s):  
Zhenlu Li ◽  
Matthias Buck
Keyword(s):  
Protein Structures ◽  
Protein Sequences ◽  
Human Protein ◽  
Current Status ◽  
Protein Database ◽  
X Ray ◽  
X Ray Crystallography ◽  
Protein Biophysics ◽  
The Relationship ◽  
Past Trend

Of 20,000 or so canonical human protein sequences, as of July 2020, 6,747 proteins have had their full or partial medium to high-resolution structures determined by x-ray crystallography or other methods. Which of these proteins dominate the protein database (the PDB) and why? In this paper, we list the 272 top protein structures based on the number of their PDB depositions. This set of proteins accounts for more than 40% of all available human PDB entries and represent past trend and current status for protein science. We briefly discuss the relationship which some of the prominent protein structures have with protein biophysics research and mention their relevance to human diseases. The information may inspire researchers who are new to protein science, but it also provides a year 2020 snap-shot for the state of protein science.

scholarly journals Biochemical and structural insights into how amino acids regulate pyruvate kinase muscle isoform 2

2020 ◽  
Vol 295 (16) ◽  
pp. 5390-5403 ◽  
Author(s):  
Suparno Nandi ◽  
Mishtu Dey
Keyword(s):  
Pyruvate Kinase ◽  
Cancer Metabolism ◽  
Gel Filtration ◽  
Binding Pocket ◽  
Glycolytic Enzyme ◽  
X Ray Crystallography ◽  
Highly Active ◽  
Attractive Therapeutic Target ◽  
Fluorescence Binding ◽  
Small Molecule Modulators

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various stimuli allosterically regulate PKM2 by cycling it between highly active and less active states. Several small molecules activate PKM2 by binding to its intersubunit interface. Serine and cysteine serve as an activator and inhibitor of PKM2, respectively, by binding to its amino acid (AA)-binding pocket, which therefore represents a potential druggable site. Despite binding similarly to PKM2, how cysteine and serine differentially regulate this enzyme remains elusive. Using kinetic analyses, fluorescence binding, X-ray crystallography, and gel filtration experiments with asparagine, aspartate, and valine as PKM2 ligands, we examined whether the differences in the side-chain polarity of these AAs trigger distinct allosteric responses in PKM2. We found that Asn (polar) and Asp (charged) activate PKM2 and that Val (hydrophobic) inhibits it. The results also indicate that both Asn and Asp can restore the activity of Val-inhibited PKM2. AA-bound crystal structures of PKM2 displayed distinctive interactions within the binding pocket, causing unique allosteric effects in the enzyme. These structure-function analyses of AA-mediated PKM2 regulation shed light on the chemical requirements in the development of mechanism-based small-molecule modulators targeting the AA-binding pocket of PKM2 and provide broader insights into the regulatory mechanisms of complex allosteric enzymes.

scholarly journals Analysis of leukotriene B4 metabolism in human promyelocytic HL-60 cells

1991 ◽  
Vol 279 (1) ◽  
pp. 283-288 ◽  
Author(s):  
S Kasimir ◽  
W Schönfeld ◽  
R A Hilger ◽  
W König
Keyword(s):  
Phorbol Myristate Acetate ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Leukotriene B4 ◽  
Dimethyl Sulphoxide ◽  
Lipid Mediator ◽  
Myristate Acetate ◽  
Ph Optimum ◽  
Highly Active ◽  
Undifferentiated State

We previously reported that human alveolar macrophages rapidly metabolize the chemotactic active lipid mediator leukotriene B4 (LTB4) into the dihydro-LTB4 by reduction of one of the conjugated double bonds. We herein report that human HL-60 cells (a myeloid precursor which can be differentiated into granulocyte- as well as monocyte-like cells by dimethyl sulphoxide or phorbol myristate acetate) express a highly active LTB4 reductase in the undifferentiated state. Differentiation by dimethyl sulphoxide (1.3%) along the granulocyte lineage, as confirmed by light microscopy, conversion of NitroBlue Tetrazolium into formazan, failed to induce a substantial capacity for omega-oxidation of LTB4; this reaction is exclusively found in mature granulocytes. Studies with the cell homogenate of undifferentiated HL-60 cells indicated that the activity of the enzyme depends on the presence of NADPH, Ca2+ and Mg2+, with a pH optimum of 7.5 at 37 degrees C. The enzyme was not released into the supernatant after stimulation of HL-60 cells with phorbol myristate acetate (100 ng) or Ca2+ ionophore (7.5 microM). Subcellular fractionation revealed evidence that the LTB4 reductase is located within the membrane fraction. Purification of the enzyme by gel filtration and gel electrophoresis suggests an apparent molecular mass of 40 kDa.

scholarly journals Calculation model and bearing capacity optimization method for the soil settlement between piles in geosynthetic-reinforced pile-supported embankments based on the membrane effect

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256190
Author(s):  
Zhen Liu ◽  
Aobo Zhang ◽  
Jiangping Xu ◽  
Cuiying Zhou ◽  
Lihai Zhang
Keyword(s):  
Bearing Capacity ◽  
Soft Soil ◽  
Field Tests ◽  
Road Construction ◽  
Optimization Method ◽  
Calculation Model ◽  
Utilization Efficiency ◽  
Project Cost ◽  
Pile Spacing ◽  
Membrane Effect

The geosynthetic-reinforced pile-supported embankment (GRPSE) system has been widely used in road construction on soft soil. However, the application of the GRPSE system is often restricted by its high-cost. The reason is that they are designed for bearing control as defined in the past. During the construction process, the pile spacing is reduced to meet the requirements for the embankment bearing capacity and settlement. These factors cause the membrane effect to not be exploited. As a result, the utilization efficiency of the bearing capacity of the soil between the piles is low and the project cost is high. Therefore, in order to solve the problem of insufficient bearing capacity of soil between piles, we established a settlement calculation model of soil between piles based on membrane effect. The model considers the relationship between the geosynthetic reinforcement (GR) and the pile spacing. Based on the obtained model, a method for optimizing the soil bearing capacity of GRPSEs is proposed. By controlling the settlement of soil between piles, the bearing capacity of soil between piles and the membrane effect of embankment can be fully utilized. Therefore, the project cost can be reduced. Finally, the method is applied to field tests for comparison. The results show that the method is reasonable and applicable. This method can effectively exploit the membrane effect and improve the utilization efficiency of the bearing capacity of the soil between piles. An economical and reasonable arrangement scheme for the piles and GR was obtained. This scheme can not only ensure the safety of the project, but also fully utilize the bearing capacity of the soil between the piles and provide a new method for engineering design.

scholarly journals Time and Area Efficient 2-D DWT using Multiplier-less Canonic Signed Digit Technique

2019 ◽  
Vol 8 (4) ◽  
pp. 5425-5429
Keyword(s):  
Optimization Method ◽  
Test System ◽  
Utilization Efficiency ◽  
Cycle Period ◽  
Arithmetic Complexity ◽  
Power Efficient ◽  
Canonic Signed Digit ◽  
Memory Reduction ◽  
Memory Complexity ◽  
Current Engineering

A few models have been recommended for proficient VLSI usage of 2-D DWT for constant applications. It is disc overed that multipliers devour more chip zone and expands multifaceted nature of the DWT design. Multiplier-less equipment usage approach gives an answer for diminish chip region, lower equipment intricacy and higher throughput of calculation of the DWT design.The proposed design outline is (i) priority must be given for memory complexity optimization over the arithmetic complexity optimization or reduction of cycle period and (ii) memory utilization efficiency to be considered ahead of memory reduction due to design complexity of memory optimization method. Based on the proposed design outline four separate design approaches and concurrent architectures are presented in this thesis for area-delay and power efficient realization of multilevel 2-D DWT.In this theory a multiplier-less VLSI engineering is proposed utilizing new dispersed number juggling calculation named CSD. We show that CSD is an effective engineering with adders as the principle part and free of ROM, duplication, and subtraction. The proposed design utilizing CSD gives less postponement and least number of cut thought about the current engineering. The reenactment was performed utilizing XILINX 14.1i and ModelSim test system.

scholarly journals Accumulation of malto-oligosaccharides in the syncytiotrophoblastic cells of first-trimester human placentas

1981 ◽  
Vol 200 (1) ◽  
pp. 93-98 ◽  
Author(s):  
S J Fisher ◽  
R A Laine
Keyword(s):  
Gel Filtration ◽  
First Trimester ◽  
Structural Elements ◽  
Trophoblastic Cells ◽  
Highly Active ◽  
A Cell ◽  
Liver Homogenates ◽  
High Concentrations ◽  
Very High

A cell-surface microvillar fraction that was isolated from the syncytiotrophoblastic cells of first-trimester human placentas was found to contain very high concentrations (890 +/- 32 microgram of hexose/mg of protein) of a class of low-molecular-weight oligosaccharides that were comprised entirely of glucose. T.l.c. and gel filtration showed that the saccharides contained from one to six glucose residues. The structures of the most prominent members of the series, a tetra- and a tri-saccharide, were determined. The anomeric configuration of the glucose residues was alpha, and methylation linkage analysis gave terminal and 4-linked hexose residues. These malto-oligosaccharides contained one reducing terminus per molecule, indicating that they were free and not bound to other structural elements of the cells. Within the placenta they appeared to be concentrated in the first-trimester trophoblastic cells, since crude membrane and particulate fractions isolated from either term trophoblastic cells or cultured placental fibroblasts did not contain detectable amounts of glucose oligomers. This series of oligosaccharides was similar to the products that are formed when glycogen is degraded by alpha-amylase in liver homogenates and may be indicative of a similar, highly active enzymic reaction closely associated with the brush border of the syncytiotrophoblastic cells of the first-trimester human placenta. Although the role of these oligosaccharides remains obscure they are probably involved in foetal metabolism.

Water Balance Optimization of Non-Traditional Water Resources Utilization in Green Building Based on Landscape Water Regulation Function

2012 ◽  
Vol 170-173 ◽  
pp. 2329-2334
Author(s):  
Hong Xiang Chai ◽  
Ke Deng ◽  
Fang Zhao
Keyword(s):  
Water Resources ◽  
Supply And Demand ◽  
Green Building ◽  
Optimization Method ◽  
Utilization Efficiency ◽  
Western China ◽  
Water Regulation ◽  
Landscape Water ◽  
Regulation Function ◽  
Water Resources Utilization

According to the extremely uneven situation of monthly rainfall in China, in order to both improve utilization efficiency of non-traditional water resources and realize economy applicable in green residential districts, an optimization method of monthly water dynamic balance of non-traditional water resources utilization in green building based on landscape water regulation function was put forward. The optimization method was made full use of large capacity of landscape water regulation function, combined with monthly water consumption law between supply and demand of non-traditional water resources in districts. And this method was applied in a green residential demonstration district in western China.

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