Proteolytic digestion of erythrocytes, resealed ghosts, and isolated membranes

Biochemistry ◽  
1972 ◽  
Vol 11 (15) ◽  
pp. 2897-2903 ◽  
Author(s):  
Richard B. Triplett ◽  
Kermit L. Carraway
Keyword(s):  
Proteolytic Digestion ◽  
Resealed Ghosts

scholarly journals Isolation of human synovial-fluid hyaluronate by density-gradient ultracentrifugation and evaluation of its protein content

1972 ◽  
Vol 126 (5) ◽  
pp. 1073-1080 ◽  
Author(s):  
Irwin Scher ◽  
David Hamerman
Keyword(s):  
Synovial Fluid ◽  
Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Proteolytic Digestion ◽  
Peptide Fragments ◽  
Guanidinium Chloride ◽  
Caesium Chloride ◽  
Normal Human ◽  
Protein Moiety ◽  
Human Synovial Fluid

1. A compound of hyaluronate and protein, called hyaluronate–protein was isolated from pooled human synovial fluids by caesium chloride density-gradient ultracentrifugation. 2. The isolated hyaluronate–protein was labelled with [125I]iodide and the following studies were done. (a) Ultracentrifugation in caesium chloride showed that the protein moiety (125I counts) and hyaluronate (hexuronate) sedimented together in the middle of the gradient. (b) The labelled hyaluronate–protein was treated with trypsin, and ultracentrifugation showed that peptide fragments (125I counts) were dispersed throughout the gradient, indicating proteolytic digestion. Hyaluronate sedimented in the middle of the gradient. (c) The labelled hyaluronate–protein was digested with streptococcal hyaluronidase, and ultracentrifugation showed that hyaluronate fragments were dispersed throughout the gradient, indicating digestion of the polysaccharide. The protein moiety, without attached hyaluronate, now sedimented at the top of the gradient. (d) Ultracentrifugation of labelled hyaluronate–protein in 4m-guanidinium chloride showed that protein and hyaluronate sedimented together. 3. These studies confirm that hyaluronate is combined with a small quantity of protein in normal human synovial fluid. A mild method for the rapid isolation of hyaluronate–protein in good yield is described.

scholarly journals Human Erythrocytes and Resealed Ghosts

1974 ◽  
Vol 249 (15) ◽  
pp. 5004-5007
Author(s):  
James V. Staros ◽  
Boyd E. Haley ◽  
Frederic M. Richards
Keyword(s):  
Human Erythrocytes ◽  
Resealed Ghosts

scholarly journals Human plasma fibronectin as a substrate for human urokinase

1989 ◽  
Vol 262 (2) ◽  
pp. 529-534 ◽  
Author(s):  
L I Gold ◽  
R Schwimmer ◽  
J P Quigley
Keyword(s):  
Extracellular Matrix ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Early Event ◽  
Plasma Fibronectin ◽  
Proteolytic Digestion ◽  
Enzyme Substrate ◽  
Tumour Cell Invasion ◽  
Substrate Ratio ◽  
Dose Dependent

An early event in malignant transformation is the increased expression of proteases, such as plasminogen activator, which can degrade surrounding extracellular matrices, thereby conferring an advantage for tumour cell invasion and metastasis. The present studies provide evidence that plasma fibronectin (Fn), which is a component of the extracellular matrix, is a direct substrate for the plasminogen activator urokinase (UK). Human plasma Fn was incubated with human UK under plasminogen-free conditions. Fn cleavage was both time- and dose-dependent and was evident within 30 min. The proteolytic digestion was limited and complete within 12 h at an enzyme/substrate ratio of 1:20. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native dimeric 440 kDa structure of Fn with the concomitant appearance of three proteolytic fragments of 210, 200 and 25 kDa. Since two large fragments of similar size to the 220 kDa monomeric chains of Fn were obtained following proteolysis, it is proposed that UK cleaves Fn at two sites, one towards the N-terminal and one close to the C-terminal, but N-terminal to its interchain disulphide bonds. These studies suggest that the local proteolytic digestion and release of Fn from the extracellular matrix by tumour cells possessing high levels of UK may involve the direct proteolytic breakdown of Fn by UK.

scholarly journals Proteolytic Digestion of Serum Cardiac Troponin I as Marker of Ischemic Severity

2018 ◽  
Vol 3 (3) ◽  
pp. 450-455 ◽  
Author(s):  
Somaya Zahran ◽  
Vivian P Figueiredo ◽  
Michelle M Graham ◽  
Richard Schulz ◽  
Peter M Hwang
Keyword(s):  
Cardiac Troponin ◽  
Troponin I ◽  
Thrombus Formation ◽  
Cardiac Troponin I ◽  
Total Serum ◽  
Proteolytic Degradation ◽  
Troponin Level ◽  
Proteolytic Digestion ◽  
Severity Of Injury ◽  
Clinically Significant

Abstract Background The serum troponin assay is the biochemical gold standard for detecting myocardial infarction (MI). A major diagnostic issue is that some believe troponin levels can rise with reversible injury, in the absence of radiologically detectable infarct. Hypothesis Because cell death activates intracellular proteases, troponin released by irreversible infarct will be more proteolyzed than that released by milder processes. Our goal was to quantify proteolytic digestion of cardiac troponin I in patients with varying degrees of myocardial injury. Methods Serum or plasma samples from 29 patients with cardiac troponin I elevations were analyzed for proteolytic degradation, using 3 different sandwich ELISAs designed to specifically detect the N-terminal, core, or C-terminal regions of cardiac troponin I. Results As predicted, the degree of proteolytic digestion increased with increasing severity of injury, as estimated by the total troponin level, and this trend was more pronounced for C-terminal (vs N-terminal) degradation. The highest degree of proteolytic digestion was observed in patients with ST-elevation MI; the least, in type 2 MI (supply–demand ischemia rather than acute thrombus formation). Conclusions The proteolytic degradation pattern of cardiac troponin I may be a better indicator of clinically significant MI than total serum troponin level. Distinguishing between intact and degraded forms of troponin may be useful for (a) identifying those patients with clinically significant infarct in need of revascularization, (b) monitoring intracellular proteolysis as a possible target for therapeutic intervention, and (c) providing an impetus for standardizing the epitopes used in the troponin I assay.

Adenovirus coded deoxyribonucleic acid binding protein. Isolation, physical properties, and effects of proteolytic digestion

Biochemistry ◽  
1980 ◽  
Vol 19 (12) ◽  
pp. 2802-2810 ◽  
Author(s):  
Norman M. Schechter ◽  
William Davies ◽  
Carl W. Anderson
Keyword(s):  
Physical Properties ◽  
Deoxyribonucleic Acid ◽  
Binding Protein ◽  
Proteolytic Digestion ◽  
Protein Isolation ◽  
Acid Binding Protein

Rapid proteolytic digestion and peptide separation using monolithic enzyme microreactor coupled with capillary electrophoresis

2019 ◽  
Vol 165 ◽  
pp. 129-134 ◽  
Author(s):  
Mengxia Cheng ◽  
Rong Wang ◽  
Baofang Zhang ◽  
Zhenkun Mao ◽  
Zilin Chen
Keyword(s):  
Capillary Electrophoresis ◽  
Proteolytic Digestion ◽  
Peptide Separation

N -terminal guanidinylation of the cyclic 1,4-ureido-deltorphin analogues: the synthesis, receptor binding studies, and resistance to proteolytic digestion

2015 ◽  
Vol 21 (6) ◽  
pp. 467-475 ◽  
Author(s):  
Krzysztof Bańkowski ◽  
Olga M. Michalak ◽  
Anna Leśniak ◽  
Katarzyna E. Filip ◽  
Piotr Cmoch ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Proteolytic Digestion ◽  
Binding Studies

scholarly journals Oligomerization of a membrane protein correlates with its retention in the Golgi complex

1993 ◽  
Vol 122 (6) ◽  
pp. 1185-1196 ◽  
Author(s):  
OA Weisz ◽  
AM Swift ◽  
CE Machamer
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Cytochalasin D ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Proteolytic Digestion ◽  
Golgi Transport ◽  
Large Oligomer ◽  
Membrane Spanning ◽  
Lumenal Domain

The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS-sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.

Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry

2011 ◽  
Vol 415 (2) ◽  
pp. 212-214 ◽  
Author(s):  
Guillaume Chevreux ◽  
Nolwenn Tilly ◽  
Nicolas Bihoreau
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibodies ◽  
Electrospray Mass Spectrometry ◽  
Proteolytic Digestion ◽  
Fast Analysis
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