Determination of the molecular weights of ribonuclease isozymes in a cell-free crude extract of Dictyostelium discoideum, by activity-staining of gels after SDS-PAGE

Author(s):  
Saburo Uchiyama ◽  
Kazumasa Isobe ◽  
Shin-ichi Nagai
Parasitology ◽  
2004 ◽  
Vol 128 (5) ◽  
pp. 541-548 ◽  
Author(s):  
A. PARAMÁ ◽  
R. IGLESIAS ◽  
M. F. ÁLVAREZ ◽  
J. LEIRO ◽  
F. M. UBEIRA ◽  
...  

This study investigated protease activities in a crude extract andin vitroexcretion/secretion (E/S) products ofPhilasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS–PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30–63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptidep-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobinin vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology ofPhilasterides dicentrarchi.


BioTechniques ◽  
1997 ◽  
Vol 23 (1) ◽  
pp. 60-62 ◽  
Author(s):  
Martin Bourbonnière ◽  
Masoud Shekarabi ◽  
Josephine Nalbantoglu

2005 ◽  
Vol 51 ◽  
pp. 9-14
Author(s):  
Snezhana Palchevska ◽  
Velibor Tasik ◽  
Petar Korneti ◽  
Georgi Shestakov ◽  
Svetlana Tsekovska

The pattern of urinary proteins in healthy full-term neonates was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), coupled with determination of few parameters related to urinary protein excretion. Twenty healthy full-term neonates were included in the investigation. Five urine samples from each subject were collected on days 3, 7, 14, 21 and 28 after birth. Determination of total proteins was performed using turbidimetric method with sulfosalicylic acid. Urinary creatinine concentration was determined by Jaffe method. Urinary proteins were separated by horizontal gradient SDS-PAGE according to Görg. The highest values for total urinary proteins and for protein/creatinine ratio were detected in urine samples excreted on days 3 and 7 after birth. Three types of SDS-PAG electrophoretic profiles were observed. The first type of electrophoretic profile was characterized by the presence of proteins of mixed glomerular and tubular origin with molecular weights from 10 to 160 kDa. Typical for the second type of electrophoretic profile was the presence of two faint fractions with molecular weights of 78 and 90 kDa and several intensive low molecular weight fractions (14-67 kDa). In the third type of electrophoretic profile only low molecular weight proteins (10-67 kDa) were detected in all five urine samples. These findings express the transitory immaturity of the glomerular filter and tubular protein reabsorbing system of the newborn kidney. Apparently, the tubular protein handling normalizes later than the glomerular filtration of proteins.


2018 ◽  
Vol 14 (1) ◽  
pp. 72
Author(s):  
Zusfahair Zusfahair ◽  
Dian Riana Ningsih ◽  
Amin Fatoni ◽  
Darul Santri Pertiwi

<p>Urease merupakam enzim yang digunakan dalam hidrolisis urea menjadi amoniak dan asam bikarbonat dan telah banyak digunakan dalam proses industri. Tujuan penelitian adalah isolasi dan pemurnian urease dari kacang panjang serta karakterisasinya. Penelitian dimulai dengan melakukan perkecambahan biji kacang panjang selama 8 hari. Kecambah biji kacang panjang selanjutnya diekstraksi dengan menggunakan buffer fosfat pH 7 dan dipisahkan menggunakan sentrifugasi sehingga diperoleh ekstrak kasar urease. Ekstrak kasar urease selanjutnya difraksinasi menggunakan aseton pada tingkat konsentrasi 20, 40, 60 dan 80%. Fraksi yang mempunyai aktivitas spesifik paling tinggi selanjutnya dianalisis menggunakan metode SDS-PAGE untuk menentukan berat molekulnya dan dikarakterisasi lanjut meliputi: pengaruh suhu, pH, konsentrasi substrat dan penambahan ion logam terhadap aktivitas urease. Aktivitas urease ditentukan dengan metode Nessler. Hasil penelitian menunjukkan aktivitas spesifik urease dari kacang panjang paling tinggi ditemukan pada fraksi aseton (FA) 20. Hasil analisis berat molekul dengan metode SDS-PAGE diperoleh beberapa pita protein yang diduga berukuran sekitar 25 KDa dan 17 KDa. Kondisi optimum dari aktivitas urease diperoleh pada suhu 30 ºC, pH 7 dan konsentrasi urea 16,6 mM dengan nilai aktivitas 407,62 U/mL. EDTA dan ion logam dalam CaCl<sub>2</sub>, NaCl, NiCl<sub>2</sub> dan CuCl<sub>2 </sub>pada variasi konsentrasi 10<sup>-3</sup>, 10<sup>-4 </sup> dan 10<sup>-5 </sup>M merupakan inhibitor urease FA 20 dari kacang panjang.</p><p><strong><span lang="EN-US">Partial Purification and Characterization of Urease from Asparagus Bean (<em>Vigna unguiculata subsp sesquipedalis </em>L<em>.</em>). </span></strong><span lang="EN-US">Urease is an enzyme used in urea hydrolysis to ammonia and bicarbonate acid and has been widely used in industrial processes. The study focused on isolation and purification of urease from asparagus beans and its characterization. The study was started with germination of asparagus beans for 8 days. Germinated asparagus beans were further extracted using phosphate buffer pH 7 and separated by centrifugation to obtain a crude extract of urease. The crude extract of urease was further fractionated using acetone at concentrations of 20, 40, 60 and 80%. The fraction with highest specific activity was then </span><span>analyzed using SDS-PAGE method to determine its molecule weight and characterized further including the influence of </span><span lang="EN-US">temperature, pH, substrate concentration, and metal ion addition to urease activity. The urease activity was determined by the Nessler̕ s method. The results showed that the specific activity of urease from asparagus beans was found with highest activity in fraction of acetone (FA) 20. Analytical result using SDS-PAGE method was obtained some protein bands having molecular weights about 25 KD and 17 KDa. The optimum conditions of urease activity was obtained at 30 °C, pH 7, incubation time 20 min and urea concentration 16.6 mM with activity value 407.62 U/mL. EDTA and </span><span>metal ions contained in </span><span lang="EN-US">CaCl<sub>2</sub>, NaCl, NiCl<sub>2</sub> and CuCl<sub>2</sub> at concentrations of 10<sup>-3</sup>, 10<sup>-4</sup> and 10<sup>-5</sup> M were FA 20 urease inhibitors.</span></p>


Author(s):  
Henry S. Slayter

Electron microscopic methods have been applied increasingly during the past fifteen years, to problems in structural molecular biology. Used in conjunction with physical chemical methods and/or Fourier methods of analysis, they constitute powerful tools for determining sizes, shapes and modes of aggregation of biopolymers with molecular weights greater than 50, 000. However, the application of the e.m. to the determination of very fine structure approaching the limit of instrumental resolving power in biological systems has not been productive, due to various difficulties such as the destructive effects of dehydration, damage to the specimen by the electron beam, and lack of adequate and specific contrast. One of the most satisfactory methods for contrasting individual macromolecules involves the deposition of heavy metal vapor upon the specimen. We have investigated this process, and present here what we believe to be the more important considerations for optimizing it. Results of the application of these methods to several biological systems including muscle proteins, fibrinogen, ribosomes and chromatin will be discussed.


2020 ◽  
Vol 2020 ◽  
Author(s):  
Firew Admasu

Abstract: The study were conducted at Dilla University, College of Natural Sciences, Biological Sciences laboratories. Background: Ethiopia is a country with many ethnic groups, cultures and beliefs which in turn have contributed to the high diversity of traditional health care knowledge and practices of traditional medicine from local growth plants, animals and minerals for various physical and mental disorders of human and livestock population that passed from generation to generation for centuries. Medicinal plants contributors to pharmaceutical, agricultural and food industries in the world. The use of medicinal plants in the industrialized societies has been traced to extraction and development of several drugs used in order to heel some diseases having inhibiting effect against pathogenic microorganism. Objective: The main objective of this study was Extraction and Phytochemicals determination of traditional medicinal plants for anti microbial susceptibility test. Methodology: The extraction and identification of some phytochemicals crude compound which used for antimicrobial susceptibility test from plant sample such as Ocimum lamiifolium (OL), Croton maerosth (Cm) and Ruta chalepesis (RC) were conducted. Plant samples are collected, powdered using mortal and pistil and extracted using ethanol and some susceptibility tests were performed to identify some phytochemicals compound. Result: The main result of Antimicrobial activity test showed that the crude extract of OL has the highest zone of inhibition. The highest yield of crude extract (38.21%) was obtained from Croton maerosth (CM) which followed by Ruta chalepesis (RC) (32.43%). However, the lowest yield (28.37%) was obtained from Oscpmum lamifolium (OL). Conclusion: Traditional Medicine is used by many people to managing numerous conditions; it’s accessible and effective on antimicrobial activity. Therefore, it plays a significant role by reducing life-threatening ailments of people and other animals.


1988 ◽  
Vol 53 (8) ◽  
pp. 1735-1744 ◽  
Author(s):  
Jitka Horská ◽  
Jaroslav Stejskal ◽  
Pavel Kratochvíl ◽  
Aubrey D. Jenkins ◽  
Eugenia Tsartolia ◽  
...  

An attempt was made to prepare well-defined graft copolymers by the coupling reaction between acyl chloride groups located along the backbone chain and monohydroxy-terminated grafts prepared separately. The molecular weights and the parameters of heterogeneity in chemical composition of the products were determined by light scattering and osmometry. The determination of molecular characteristics revealed that the degree of grafting was low. The results therefore could not be confronted with a statistical model at this stage. The problems encountered in the synthesis, e.g., gel formation, and the data relating to the soluble products are discussed.


1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.


Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1244
Author(s):  
Malik Adil Nawaz ◽  
Tanoj Kumar Singh ◽  
Regine Stockmann ◽  
Hema Jegasothy ◽  
Roman Buckow

The objective of this research was to develop a model faba bean drink with a high concentration of protein (>4% w/w). The protein molecular weights and frequency for both faba and soy were assessed using SDS-PAGE. Results showed similarities in the protein molecular weight of both faba and soy (mainly 11S globulin ~Glycinin and 7S globulin ~β-conglycinin). Thus, faba can be considered as a potential soy replica in plant-based milk beverages. Oil-in-water emulsions (5–8% w/w available protein) were prepared using faba bean protein concentrate (FPC), 1% sunflower oil, and 0.2% sunflower lecithin. These emulsions were used as model beverages and were further investigated for UHT processibility, stability, and physicochemical properties. The physicochemical properties of emulsions at various processing stages viz., coarse emulsification, homogenisation, and UHT, were measured. An increase in the protein concentration and thermal treatment resulted in an increased oil droplet size, coalescence and flocculation, and protein aggregation. Lower protein concentrations viz., 5–6%, showed greater negative ζ-potential, and thereby, high dispersibility through enhanced electrostatic repulsions than those of higher concentrations (7–8%). Furthermore, an increase in protein concentration and UHT treatment resulted in an increased creaming index. In total, 21 different volatile compounds were detected and quantified, representing different chemical classes, namely alcohols, aldehydes, ketones, esters, furan, and acids. These volatiles have major consequences for the overall flavour chemistry of the model beverage product. Overall, this study showed the potential for application of faba bean as a protein source in UHT-treated legume-based beverages and identified areas for further development.


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