scholarly journals Resistance of the peptidyltransferase centre of rabbit ribosomes to attack by nucleases and proteinases

1980 ◽  
Vol 190 (1) ◽  
pp. 199-214 ◽  
Author(s):  
R A Cox ◽  
S Kotecha
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein S ◽  
High Salt ◽  
Protein Fractions ◽  
Marginal Effect ◽  
Trypsin Treatment ◽  
Salt Shock ◽  
Split Protein ◽  
Core Particles

Larger ribosomal subparticles (L-subparticles) of rabbit ribosomes were treated with either ribonucleases (I or T1) or proteinases (trypsin or chymotrypsin), and their capacity to function in poly(U)-directed polyphenylalanine synthesis and in the puromycin reaction was investigated. The effects of pretreatment of L-subparticles on the reconstruction of active subparticles from core-particles derived by treatment with 2.75 M-NH4Cl/69 mM-MgCl2 and split-protein fractions were also examined. The protein moiety of proteinase-treated L-subparticles was analysed by one-dimensional sodium dodecyl sulphate/polyacrylamide- and two-dimensional polyacrylamide-gel electrophoresis. The introduction of 16–100 scissions in the RNA moiety had no effect on the activity of the L-subparticles in polyphenylalanine synthesis, and there was no effect on the stability of L-subparticles to high-salt shock treatment and a marginal effect on the reconstruction of L-subparticles from high-salt-shock core-particles and split-protein fractions. In contrast, L-subparticles treated with low amounts of trypsin (0.56 ng of trypsin/microgram of L-subparticle) were inactive in polyphenylalanine synthesis, and their capacity to function in partial-reconstruction experiments was diminished. Activity in the puromycin reaction was increased by 70% as a result of trypsin treatment (280 ng of trypsin/microgram of L-subparticle). At least two of the acidic proteins implicated in the translocation function were not affected by trypsin treatment. Trypsin-treated L-subparticles had lost their capacity to bind the smaller ribosomal subparticle (S-subparticle). The protein(s) needed for S-subparticle binding were shown to be present in high-salt-shock cores. At least six proteins associated with the core-particles were attack during trypsin treatment of L-subparticles. An examination of L-subparticles isolated from trypsin-treated polyribosomes showed that the amount of trypsin necessary to decrease the activity of the subparticle by 50% was about twice that needed in the treatment of L-subparticles alone. The largest protein of rabbit L-subparticles (approx. 51 000 daltons) was cleaved in a stepwise fashion by trypsin to fragments of approx. 40 000 daltons. This protein was also cleaved by chymotrypsin.

scholarly journals Protein synthesis by hybrid ribosomes reconstructed from rabbit reticulocyte ribosomal core-particles and amphibian or fungal split-proteins

1980 ◽  
Vol 186 (3) ◽  
pp. 861-872 ◽  
Author(s):  
R A Cox ◽  
P Greenwell
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Fractions ◽  
Molecular Architecture ◽  
Reductive Methylation ◽  
Active Centre ◽  
Split Protein ◽  
Cytoplasmic Ribosomes ◽  
Core Particles

It was shown that high-salt (2.75 M-NH4Cl/69mM-MgCl2) shock treatment at 0 degrees C of the larger subparticles (L-subparticles) of rabbit, Xenopus laevis and Neurospora crassa cytoplasmic ribosomes yielded split-protein fractions that were not only functionally equivalent but also interchangeable. Thus, although the remaining core-particles were inactive in both the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis, activity was restored when they were combined with either homologous or heterologous split-protein fractions. This technique was used to prepare active hybrid L-subparticles, e.g. rabbit cores/N. crassa split-proteins, and also active hybrid ribosomes, e.g. rabbit smaller subparticle/X. laevis core-particle/rabbit split-proteins. Rabbit and X. laevis split-protein fractions labelled with 14C by reductive methylation with [14C]formaldehyde and sodium cyanoborohydride were both shown to bind to rabbit core-particles in approximate correlation with the degree of re-activation. The split-protein fractions of rabbit and X. laevis L-subparticles were analysed by two-dimensional and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights (measured in sodium dodecyl sulphate gels) of the split-proteins of rabbit and X. laevis L-subparticles were found to be similar. These results demonstrate that the peptidyltransferase active centre of cytoplasmic ribosomes of eukaryotes has components that are interchangeable over a wide evolutionary range. Evidently the essential molecular architecture of the active centre is highly conserved.

COMPARISON OF ALLERGENIC PROTEINS OF SEA SNAIL (CERITHIDEA OBTUSA) AND FRESHWATER SNAIL (POMACEA CANALICULATA)

2016 ◽  
Vol 78 (11) ◽  
Author(s):  
Rosmilah Misnan ◽  
Norazlin Salahudin Abdul Aziz ◽  
Zailatul Hani Mohd Yadzir ◽  
Noormalin Abdullah ◽  
Faizal Bakhtiar ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Freshwater Snail ◽  
Protein Profiling ◽  
Protein Fractions ◽  
Pomacea Canaliculata ◽  
Market Demand ◽  
Allergenic Proteins ◽  
Major Allergens ◽  
Minor Allergens

In Malaysian and certain Asian countries, snail has high market demand and popular to the local people as food. However, snail is also frequently reported as one of the worst food allergens, dominated by severe symptoms such as asthma and anaphylactic shock. Thus, the aims of this study is to determine the allergenicity of two species of edible snails; the local sea snail, Cerithidea obtusa and the freshwater snail Pomacea canaliculata. Snail extracts were prepared from the snail flesh and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine their protein profiling. Allergenic proteins were detected by immunoblotting test using sera from 10 snail-allergic patients. The snails contain 31 to 34 protein fractions between 11 to >250 kDa. The prominent bands were seen at 33, 42, 74 and 250 kDa. Immunoblotting detected 15 and 16 allergenic proteins in C. obtusa and P. canaliculata, respectively. Three protein fractions at 30, 33 and 42 kDa were identified as the major allergens of C. obtusa, while six major allergens at 30, 33, 42, 74, 124 and 218 kDa were detected in P. canaliculata. Various minor allergens were also detected in both snails. This study indicated that numerous proteins of C. obtusa and P. canaliculata were allergenic. Thus, combined allergen extracts of both snails are essential to be included in diagnosis of snail allergy among local allergic patients.  

scholarly journals Expression and structure of CD22 in acute leukemia

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1480-1486 ◽  
Author(s):  
DR Boue ◽  
TW LeBien
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Lymphoblastic Leukemia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Surface Expression ◽  
Immunofluorescent Staining ◽  
Reactive Protein ◽  
Bona Fide

Abstract The purpose of this study was to examine the expression and structure of CD22 in B cell precursor acute lymphoblastic leukemia (BCP-ALL), acute myeloid leukemia (AML), and T cell acute lymphoblastic leukemia (T-ALL). By using immunofluorescence microscopy and flow cytometry we observed that CD22 is expressed not only in the cytoplasm (as previously reported) but also on the cell surface of virtually all (15/16) BCP-ALL examined. CD22 that was biosynthetically labeled with 35S-cysteine and immunoprecipitated from the uncommon cytoplasmic CD22- positive/surface CD22-negative BCP-ALL cells was analyzed by single- dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our results indicated that the cytoplasmic form of CD22 comigrated with 125I/lactoperoxidase-labeled surface CD22. Therefore, cytoplasmic CD22 is probably a pool of fully processed glycoprotein. We also observed unusual cases of AML (approximately 20%) that expressed cytoplasmic CD22 based on immunofluorescent staining; however, biosynthetic labeling and immunoprecipitation revealed an apparently cross-reactive protein(s) of approximately 250 to 300 kd in AML cells. No T-ALL cell lines examined expressed either cytoplasmic or surface CD22. Thus, cytoplasmic and surface expression of bona fide CD22 appears restricted to B cells, which suggests that this molecule subserves a function unique to B cells.

scholarly journals Isolation and Identification of Protein Spike (Protein-S) in Field Isolates of Infectious Bronchitis Virus

2021 ◽  
Vol 4 (1) ◽  
pp. 104
Author(s):  
Jola Rahmahani ◽  
Dewanggi Kristi Sasmi Pradhita ◽  
Nanik Sianita Widjaja ◽  
Suwarno Suwarno
Keyword(s):  
Infectious Bronchitis Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Field Isolate ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Spike Protein ◽  
Structural Protein ◽  
Isolation And Identification ◽  
Infectious Bronchitis ◽  
Sds Page

Infectious Bronchitis (IB) is disease causes great economic impact across nations. Spike protein (protein-S) is one of structural protein of Infectious Bronchitis Virus (IBV) located on enveloped of the virion. Molecular characterization of IBV could be conducted use Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to reveal the protein weight then confirmed through Immuno-Blotting assay. These methods were conducted to know the molecule weight of field isolate compared to seed vaccine. The results of this study indicate the weight of protein-S of field isolate I-147/11 were 168.21 kDa which was same to protein-S of seed vaccine Massachusset.

scholarly journals forked proteins are components of fiber bundles present in developing bristles of Drosophila melanogaster.

Genetics ◽  
1994 ◽  
Vol 136 (1) ◽  
pp. 173-182 ◽  
Author(s):  
N S Petersen ◽  
D H Lankenau ◽  
H K Mitchell ◽  
P Young ◽  
V G Corces
Keyword(s):  
Drosophila Melanogaster ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Fiber Bundles ◽  
Alternative Promoters ◽  
Wild Type ◽  
Pupal Development ◽  
Coding Regions ◽  
Cytoplasmic Structures

Abstract The forked (f) gene of Drosophila melanogaster encodes six different transcripts 6.4, 5.6, 5.4, 2.5, 1.9, and 1.1 kb long. These transcripts arise by the use of alternative promoters. A polyclonal antibody raised against a domain common to all of the forked-encoded products has been used to identify forked proteins on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and in Drosophila pupal tissues. The antibody stains fiber bundles present in bristle cells for about 15 hr during normal pupal development. Electron microscopy shows that these fibers are present from 40 to 53 hr in bristles of wild-type flies but are absent in the null f36a mutant. The forked protein(s) thus appear to be an essential part of the bristle fibers. The phenotype of the f36a mutation can be rescued by a 13-kb fragment of the forked locus containing the coding regions for the 2.5, 1.9, and 1.1-kb transcripts, suggesting that the proteins encoded by the three large forked RNAs are dispensable during bristle development. Increasing the copy number of a P[w+,f+] construct containing the 13-kb fragment induces a hypermorphic bristle phenotype whose severity correlates with the number of copies of P[w+,f+] present. These results indicate that alterations in the ratios among the forked proteins, or between forked products and other components of the fiber, result in abnormal assembly of the fibrillar cytoplasmic structures necessary for bristle morphogenesis.

scholarly journals Expression and structure of CD22 in acute leukemia

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1480-1486
Author(s):  
DR Boue ◽  
TW LeBien
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Lymphoblastic Leukemia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Surface Expression ◽  
Immunofluorescent Staining ◽  
Reactive Protein ◽  
Bona Fide

The purpose of this study was to examine the expression and structure of CD22 in B cell precursor acute lymphoblastic leukemia (BCP-ALL), acute myeloid leukemia (AML), and T cell acute lymphoblastic leukemia (T-ALL). By using immunofluorescence microscopy and flow cytometry we observed that CD22 is expressed not only in the cytoplasm (as previously reported) but also on the cell surface of virtually all (15/16) BCP-ALL examined. CD22 that was biosynthetically labeled with 35S-cysteine and immunoprecipitated from the uncommon cytoplasmic CD22- positive/surface CD22-negative BCP-ALL cells was analyzed by single- dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our results indicated that the cytoplasmic form of CD22 comigrated with 125I/lactoperoxidase-labeled surface CD22. Therefore, cytoplasmic CD22 is probably a pool of fully processed glycoprotein. We also observed unusual cases of AML (approximately 20%) that expressed cytoplasmic CD22 based on immunofluorescent staining; however, biosynthetic labeling and immunoprecipitation revealed an apparently cross-reactive protein(s) of approximately 250 to 300 kd in AML cells. No T-ALL cell lines examined expressed either cytoplasmic or surface CD22. Thus, cytoplasmic and surface expression of bona fide CD22 appears restricted to B cells, which suggests that this molecule subserves a function unique to B cells.

scholarly journals Isolation of an abnormal protein C molecule from the plasma of a patient with thrombotic diathesis

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 940-946
Author(s):  
EM Faioni ◽  
CT Esmon ◽  
NL Esmon ◽  
PM Mannucci
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Protein C ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Activated Protein C ◽  
Protein S ◽  
Abnormal Protein ◽  
Sds Page ◽  
Before And After

Protein C has been purified from the plasma of a patient with thrombotic diathesis. Both before and after isolation, the protein showed reduced capacity to hydrolyze synthetic substrates and to anticoagulate plasma. Proteolysis with the soluble thrombin- thrombomodulin complex proceeded normally and to completion as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Approximately one-third of the protein is functional, indicating a heterozygous defect. Indirect studies suggest that the abnormal component can bind to protein S and phospholipids. Both forms of activated protein C can also incorporate radiolabeled diisopropylfluorophosphate.

A Sensitive Peroxidase Staining Immunoblotting Method for Measuring Total Protein S in Human Plasma

1993 ◽  
Vol 69 (04) ◽  
pp. 331-334 ◽  
Author(s):  
Xiangan Li ◽  
Kaoru Hatanaka ◽  
Ling Guo ◽  
Motoo Tsushima ◽  
Yukihiko Kitamura ◽  
...  
Keyword(s):  
Human Plasma ◽  
Total Protein ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Protein S ◽  
Polyacrylamide Gel ◽  
Free Form

SummaryIn plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added tonormal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.

scholarly journals Influence of glycation and pepsin hydrolysis on immunoreactivity of albumin/globulin fraction of herbicide resistant wheat line

2009 ◽  
Vol 27 (No. 5) ◽  
pp. 320-329 ◽  
Author(s):  
A. Nagy ◽  
K. Marciniak-Darmochwał ◽  
S. Krawczuk ◽  
D. Mierzejewska ◽  
H. Kostyra ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Fractions ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wheat Line ◽  
Wheat Proteins ◽  
Herbicide Resistant ◽  
Before And After ◽  
Immunogenic Properties ◽  
Enzymatic Glycosylation

The aim of this study was to investigate the influence of non-enzymatic glycosylation on the immunogenic properties of soluble wheat proteins. Albumin/globulin fractions of herbicide resistant wheat line were non-enzymatically glycosylated using glucose for seven days at 37°C. The changes in their structures and immunoreactivity were then determined. The protein fractions were also hydrolysed with pepsin to determine the resistance to digestion. Albumin/globulin fractions before and after non-enzymatic glycosylation were analysed using <i>o</i>-phthaldialdehyde method and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The immunoreactivity of the protein fractions was determined using enzyme-linked immunosorbent assay methods, as well as affinity chromatography. The soluble wheat proteins showed smaller amounts of available α-amino groups after non-enzymatic glycosylation, and were stronger immunogens after glycation, but their antigenicity was not been affected significantly. However, pepsin hydrolysis of wheat proteins decreased their immunoreactivity.

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