THE PROTEOLYTIC ENZYMES OF MICROORGANISMS: VI. THE SEPARATION OF PROTEASES FROM MORTIERELLA RENISPORA DIXON-STEWART BY ZONE ELECTROPHORESIS

L. R. Wetter

doi:10.1139/y54-003

THE PROTEOLYTIC ENZYMES OF MICROORGANISMS: VI. THE SEPARATION OF PROTEASES FROM MORTIERELLA RENISPORA DIXON-STEWART BY ZONE ELECTROPHORESIS

Canadian Journal of Biochemistry and Physiology ◽

10.1139/o54-003 ◽

1954 ◽

Vol 32 (1) ◽

pp. 20-26 ◽

Cited By ~ 3

Author(s):

L. R. Wetter

Keyword(s):

Filter Paper ◽

Active Component ◽

Culture Medium ◽

Moving Boundary ◽

Proteolytic Enzymes ◽

Potato Starch ◽

Paper Electrophoresis ◽

Active Components ◽

Zone Electrophoresis ◽

The Difference

Moving-boundary electrophoresis indicated that a protease concentrate obtained from the culture medium of Mortierella renispora Dixon-Stewart (PRL 26) was made up of a number of proteins. Filter-paper electrophoresis demonstrated that two of the proteins were capable of hydrolyzing denatured hemoglobin. One active component had a negative mobility, the other a positive mobility in phosphate buffer pH 6.8, ionic strength 0.1. Because of the difference in electrical properties it was possible to separate the two active components by zone electrophoresis. Though yields were low when filter paper was employed, the use of potato starch as the supporting medium resulted in excellent recoveries.

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Cholesterol in Serum and Lipoprotein Fractions

Clinical Chemistry ◽

10.1093/clinchem/2.3.145 ◽

1956 ◽

Vol 2 (3) ◽

pp. 145-159 ◽

Cited By ~ 187

Author(s):

Joseph T Anderson ◽

Ancel Keys

Keyword(s):

Human Serum ◽

Total Cholesterol ◽

Filter Paper ◽

Room Temperature ◽

Paper Electrophoresis ◽

Cholesterol Content ◽

Intraindividual Variability ◽

Detailed Data ◽

The Stability ◽

Source Of Error

Abstract 1. Methods are described for the separation, by paper electrophoresis and by cold ethanol, of α- and β-lipoproteins in 0.1 ml. of serum, with subsequent analysis of cholesterol in the separated portions. 2. It is shown that both methods of separation yield separated fractions containing substantially the same amounts of cholesterol. 3. Detailed data are given on the errors of measurement for total cholesterol and for cholesterol in the separated lipoprotein fractions. 4. Studies are reported on the stability of cholesterol in stored serum and on paper electrophoresis strips. It is shown that simple drying on filter paper causes no change in cholesterol content and yields a product that is stable for many weeks at ordinary room temperature. 5. The sources of variability in human serum cholesterol values are examined and it is shown that spontaneous intraindividual variability is a much greater source of error than the errors of measurement with these methods.

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Extracellular Proteome Analysis Shows the Abundance of Histidine Kinase Sensor Protein, DNA Helicase, Putative Lipoprotein Containing Peptidase M75 Domain and Peptidase C39 Domain Protein in Leptospira interrogans Grown in EMJH Medium

Pathogens ◽

10.3390/pathogens10070852 ◽

2021 ◽

Vol 10 (7) ◽

pp. 852

Author(s):

Abhijit Sarma ◽

Dhandapani Gunasekaran ◽

Devasahayam Arokia Balaya Rex ◽

Thoduvayil Sikha ◽

Homen Phukan ◽

...

Keyword(s):

Culture Medium ◽

Proteome Analysis ◽

Dna Helicase ◽

Extracellular Proteins ◽

Surface Proteins ◽

Secretory Proteins ◽

Extracellular Proteome ◽

Cell Pellet ◽

The Difference ◽

Leptospira Species

Leptospirosis is a re-emerging form of zoonosis that is caused by the spirochete pathogen Leptospira. Extracellular proteins play critical roles in the pathogenicity and survival of this pathogen in the host and environment. Extraction and analysis of extracellular proteins is a difficult task due to the abundance of enrichments like serum and bovine serum albumin in the culture medium, as is distinguishing them from the cellular proteins that may reach the analyte during extraction. In this study, extracellular proteins were separated as secretory proteins from the culture supernatant and surface proteins were separated during the washing of the cell pellet. The proteins identified were sorted based on the proportion of the cellular fractions and the extracellular fractions. The results showed the identification of 56 extracellular proteins, out of which 19 were exclusively extracellular. For those proteins, the difference in quantity with respect to their presence within the cell was found to be up to 1770-fold. Further, bioinformatics analysis elucidated characteristics and functions of the identified proteins. Orthologs of extracellular proteins in various Leptospira species were found to be closely related among different pathogenic forms. In addition to the identification of extracellular proteins, this study put forward a method for the extraction and identification of extracellular proteins.

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Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22041834 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1834

Author(s):

Tomoko Okada ◽

Toshihiko Ogura

Keyword(s):

Mammalian Cells ◽

Culture Medium ◽

Related Gene ◽

Autophagosome Formation ◽

Intact Cells ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

The Difference ◽

Related Proteins ◽

Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

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Step width, spacing, and resolution in gradient elution moving boundary electrophoresis. Part 1. Theory and comparison with zone electrophoresis

Electrophoresis ◽

10.1002/elps.201000334 ◽

2010 ◽

Vol 31 (22) ◽

pp. 3650-3657 ◽

Cited By ~ 11

Author(s):

David Ross

Keyword(s):

Moving Boundary ◽

Gradient Elution ◽

Step Width ◽

Zone Electrophoresis

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Localization of Protein-bound Radioactive Iodine by Filter Paper Electrophoresis

Science ◽

10.1126/science.115.2997.626 ◽

1952 ◽

Vol 115 (2997) ◽

pp. 626-627 ◽

Cited By ~ 43

Author(s):

F. Larson ◽

W. P. Deiss ◽

E. C. Albright

Keyword(s):

Filter Paper ◽

Radioactive Iodine ◽

Paper Electrophoresis

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Global metabolomic and lipidomic analysis reveals the potential mechanisms of hemolysis effect of Ophiopogonin D and Ophiopogonin D' in vivo

Chinese Medicine ◽

10.1186/s13020-020-00412-z ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Huan-Hua Xu ◽

Zhen-Hong Jiang ◽

Cong-Shu Huang ◽

Yu-Ting Sun ◽

Long-Long Xu ◽

...

Keyword(s):

Chemical Properties ◽

Principal Component ◽

Partial Least Square ◽

Least Square ◽

Practical Significance ◽

Plasma Metabolites ◽

Active Components ◽

The Difference

Abstract Background OPD and OPD' are the two main active components of Ophiopogon japonicas in Shenmai injection (SMI). Being isomers of each other, they are supposed to have similar pharmacological activities, but the actual situation is complicated. The difference of hemolytic behavior between OPD and OPD' in vivo and in vitro was discovered and reported by our group for the first time. In vitro, only OPD' showed hemolysis reaction, while in vivo, both OPD and OPD' caused hemolysis. In vitro, the primary cause of hemolysis has been confirmed to be related to the difference between physical and chemical properties of OPD and OPD'. In vivo, although there is a possible explanation for this phenomenon, the one is that OPD is bio-transformed into OPD' or its analogues in vivo, the other one is that both OPD and OPD' were metabolized into more activated forms for hemolysis. However, the mechanism of hemolysis in vivo is still unclear, especially the existing literature are still difficult to explain why OPD shows the inconsistent hemolysis behavior in vivo and in vitro. Therefore, the study of hemolysis of OPD and OPD' in vivo is of great practical significance in response to the increase of adverse events of SMI. Methods Aiming at the hemolysis in vivo, this manuscript adopted untargeted metabolomics and lipidomics technology to preliminarily explore the changes of plasma metabolites and lipids of OPD- and OPD'-treated rats. Metabolomics and lipidomics analyses were performed on ultra-high performance liquid chromatography (UPLC) system tandem with different mass spectrometers (MS) and different columns respectively. Multivariate statistical approaches such as principal component analysis (PCA) and orthogonal partial least square-discriminant analysis (OPLS-DA) were applied to screen the differential metabolites and lipids. Results Both OPD and OPD' groups experienced hemolysis, Changes in endogenous differential metabolites and differential lipids, enrichment of differential metabolic pathways, and correlation analysis of differential metabolites and lipids all indicated that the causes of hemolysis by OPD and OPD' were closely related to the interference of phospholipid metabolism. Conclusions This study provided a comprehensive description of metabolomics and lipidomics changes between OPD- and OPD'-treated rats, it would add to the knowledge base of the field, which also provided scientific guidance for the subsequent mechanism research. However, the underlying mechanism require further research.

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