scholarly journals Reorientation of membrane polypeptides during erythrocyte maturation

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 803-806 ◽  
Author(s):  
RW Allen ◽  
BA Hoover
Keyword(s):  
Outer Membrane ◽  
Messenger Rna ◽  
Membrane Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
In Vitro Translation ◽  
Antigenic Determinants ◽  
Red Cell ◽  
Surface Absorption ◽  
Intact Cells

Abstract Messenger RNA extracted from the erythroleukemic cell line K562 was translated in vitro and the translation products reacted with an antiserum raised against human erythrocyte ghosts. Polypeptides immunoprecipitated by the antiserum were characterized by SDS- polyacrylamide gel electrophoresis and fluorography. The antiserum immunoprecipitated polypeptides with nominal molecular weights of 37,000 (p37), 20,000 (p20), 19,000 (p19), 18,000 (p18), 14,000 (p14), 13,000 (p13), and 11,000 (p11) daltons. Since the antiserum was raised against antigenic determinants present on both the inner and outer surface of the red cell membrane, differential absorption of antiserum with intact red cells, or ghosts, was used to localize the translation products to the inner or outer membrane surface. Absorption was also used to determine if any of the immunoprecipitated translation products represented membrane markers for the erythroid lineage. Absorption of the antiserum with red cell ghosts removed all antibodies reacting with in vitro translation products. Absorption with intact cells from various lineages removed anti-p20 antibodies and did not absorb anti- p19 or anti-p18 antibodies. Absorption with intact cells from all lineages except mature erythrocytes absorbed anti-p37, anti-p14, and anti-p13 antibodies, suggesting that these antigens are expressed on the outer membrane surface. Mature erythrocytes were incapable of absorbing these antibody populations, suggesting a lineage-specific reorientation of these antigens in the membrane during erythropoiesis.

Reorientation of membrane polypeptides during erythrocyte maturation

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 803-806
Author(s):  
RW Allen ◽  
BA Hoover
Keyword(s):  
Outer Membrane ◽  
Messenger Rna ◽  
Membrane Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
In Vitro Translation ◽  
Antigenic Determinants ◽  
Red Cell ◽  
Surface Absorption ◽  
Intact Cells

Messenger RNA extracted from the erythroleukemic cell line K562 was translated in vitro and the translation products reacted with an antiserum raised against human erythrocyte ghosts. Polypeptides immunoprecipitated by the antiserum were characterized by SDS- polyacrylamide gel electrophoresis and fluorography. The antiserum immunoprecipitated polypeptides with nominal molecular weights of 37,000 (p37), 20,000 (p20), 19,000 (p19), 18,000 (p18), 14,000 (p14), 13,000 (p13), and 11,000 (p11) daltons. Since the antiserum was raised against antigenic determinants present on both the inner and outer surface of the red cell membrane, differential absorption of antiserum with intact red cells, or ghosts, was used to localize the translation products to the inner or outer membrane surface. Absorption was also used to determine if any of the immunoprecipitated translation products represented membrane markers for the erythroid lineage. Absorption of the antiserum with red cell ghosts removed all antibodies reacting with in vitro translation products. Absorption with intact cells from various lineages removed anti-p20 antibodies and did not absorb anti- p19 or anti-p18 antibodies. Absorption with intact cells from all lineages except mature erythrocytes absorbed anti-p37, anti-p14, and anti-p13 antibodies, suggesting that these antigens are expressed on the outer membrane surface. Mature erythrocytes were incapable of absorbing these antibody populations, suggesting a lineage-specific reorientation of these antigens in the membrane during erythropoiesis.

scholarly journals Erythrocyte membrane protein band 3: its biosynthesis and incorporation into membranes.

1981 ◽  
Vol 91 (3) ◽  
pp. 637-646 ◽  
Author(s):  
E Sabban ◽  
V Marchesi ◽  
M Adesnik ◽  
D D Sabatini
Keyword(s):  
Plasma Membrane ◽  
Electrophoretic Mobility ◽  
Messenger Rna ◽  
Transmembrane Protein ◽  
Band 3 ◽  
In Vitro Translation ◽  
Intact Cells ◽  
Amino Terminal ◽  
In Cells

Band 3, a transmembrane protein that provides the anion channel of the erythrocyte plasma membrane, crosses the membrane more than once and has a large amino terminal segment exposes on the cytoplasmic side of the membrane. The biosynthesis of band 3 and the process of its incorporation into membranes were studied in vivo in erythroid spleen cells of anemic mice and in vitro in protein synthesizing cell-free systems programmed with polysomes and messenger RNA (mRNA). In intact cells newly synthesized band 3 is rapidly incorporated into intracellular membranes where it is glycosylated and it is subsequently transferred to the plasma membrane where it becomes sensitive to digestion by exogenous chymotrypsin. The appearance of band 3 in the cell surface is not contingent upon its glycosylation because it proceeds efficiently in cells treated with tunicamycin. The site of synthesis of band 3 in bound polysomes was established directly by in vitro translation experiments with purified polysomes or with mRNA extracted from them. The band-3 polypeptide synthesized in an mRNA-dependent system had the same electrophoretic mobility as that synthesized in cells treated with tunicamycin. When microsomal membranes were present during translation, the in vitro synthesized band-3 polypeptide was cotranslationally glycosylated and inserted into the membranes. This was inferred from the facts that when synthesis was carried out in the presence of membranes the product had a lower electrophoretic mobility and showed partial resistance to protease digestion. Our observations indicate that the primary translation product of band-3 mRNA is not proteolytically processed either co- or posttranslationally. It is, therefore, proposed that the incorporation of band 3 into the endoplasmic reticulum (ER) membrane is initiated by a permanent insertion signal. To account for the cytoplasmic exposure of the amino terminus of the polypeptide we suggest that this signal is located within the interior of the polypeptide. a mechanism that explains the final transmembrane disposition of band 3 in the plasma membrane as resulting from the mode of its incorporation into the ER is presented.

scholarly journals In vitro translation of avian vitellogenin messenger RNA.

1977 ◽  
Vol 252 (22) ◽  
pp. 8320-8327 ◽  
Author(s):  
J.I. Gordon ◽  
R.G. Deeley ◽  
A.T. Burns ◽  
B.M. Paterson ◽  
J.L. Christmann ◽  
...  
Keyword(s):  
Messenger Rna ◽  
In Vitro Translation

In vitro translation of human prostatic acid phosphatase mRNA and processing of the translation products by microsomal membranes and endoglycosidase H

1987 ◽  
Vol 65 (10) ◽  
pp. 921-924 ◽  
Author(s):  
Gilles Paradis ◽  
Jean Y. Dubé ◽  
Pierre Chapdelaine ◽  
Roland R. Tremblay
Keyword(s):  
Acid Phosphatase ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Prostatic Acid Phosphatase ◽  
In Vitro Translation ◽  
Acid Phosphatases ◽  
Major Band ◽  
Microsomal Membranes ◽  
Endoglycosidase H

Poly(A)+ RNA was isolated from human prostatic tissue and translated in vitro in a rabbit reticulocyte lysate translation assay. Acid phosphatase labeled with [35S]methionine was immunoprecipitated with an antibody against seminal plasma acid phosphatase. Two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitate, followed by fluorography, revealed the presence of two spots (one major and one minor), both having a molecular mass of 43 kilodaltons (kDa) and an isoelectric point higher than mature acid phosphatase. Addition of canine pancreatic membranes to the translation assay resulted in the formation of four immunoprecipitable spots with molecular masses ranging from 43 to 49 kDa on one-dimensional gels. These spots probably represent acid phosphatases containing one to four core sugar groups, since after the addition of endoglycosidase H the molecular mass heterogeneity was abolished and we observed only one major band with a molecular mass (41 kDa) slightly lower than the ones of the primary translation product. These results suggest that human prostatic acid phosphatases are synthesized as two 43-kDa preproteins, which are further processed to 41-kDa proteins by removal of their signal peptide. Heterogeneity of the native protein arises mostly from glycosylation at four sites and not from differences in the amino acid sequence of the various forms.

scholarly journals The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA.

1986 ◽  
Vol 6 (12) ◽  
pp. 4478-4485 ◽  
Author(s):  
A T Garber ◽  
J Segall
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Protein ◽  
Polyacrylamide Gel ◽  
In Vitro Translation ◽  
Specific Gene ◽  
Alpha Cells ◽  
Two Dimensional ◽  
Isoelectric Ph

The SPS4 gene of Saccharomyces cerevisiae, a sporulation-specific gene identified previously in a differential hybridization screen of a genomic yeast DNA library, has been characterized further. The protein encoded by this gene was inferred from its nucleotide sequence to be 38,600 daltons with an isoelectric pH of 8.2. Consistent with this, two-dimensional polyacrylamide gel electrophoresis of the in vitro translation products of RNA purified by hybridization with the cloned SPS4 DNA indicated that the SPS4 gene product is a 39-kilodalton, basic protein. This protein was found to be identical in size and charge to a major, sporulation-specific protein identified in a two-dimensional polyacrylamide gel electrophoretic comparison of the in vitro translation products of total RNA from sporulating MATa/MAT alpha cells and asporogenous MAT alpha/MAT alpha cells. A MATa/MAT alpha strain homozygous for a partial deletion of the SPS4 gene appeared, however, to be unaffected in its ability to form viable ascospores.

scholarly journals Catecholamine-Modulated Novel Surface-Exposed Adhesin LIC20035 ofLeptospiraspp. Binds Host Extracellular Matrix Components and Is Recognized by the Host during Infection

2017 ◽  
Vol 84 (6) ◽  
Author(s):  
Karukriti Kaushik Ghosh ◽  
Aman Prakash ◽  
Vinayagamurthy Balamurugan ◽  
Manish Kumar
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Membrane Surface ◽  
Outer Membrane Proteins ◽  
Extracellular Matrices ◽  
Content Type ◽  
Extracellular Matrix Components ◽  
Gene Transcripts ◽  
Host Tissues

ABSTRACTIn this study, the effect of the host stress hormone catecholamine onLeptospiragene transcripts encoding outer membrane proteins was investigated. There was no impact of catecholamine supplementation on thein vitrogrowth pattern ofLeptospira interrogans; however, 7 genes out of 41 were differentially transcribed, and the effect was reversed to the basal level in the presence of the antagonist propranolol. Comprehensive analysis of one of the differentially regulated proteins, LIC20035 (in serovar Copenhageni)/LB047 (in serovar Lai) (due to catecholamine supplementation), revealed immunogenicity and ability to adhere to host extracellular matrices. Protease accessibility assay and phase partition of integral membrane proteins ofLeptospirashowed LIC20035/LB047 to be an outer membrane surface-exposed protein. The recombinant LIC20035 protein can be serologically detected using human/bovine sera positive for leptospirosis. Moreover, the recombinant LIC20035 can bind to diverse host extracellular matrices, with a higher affinity toward collagen and chondroitin sulfate.IMPORTANCELeptospirosis is a neglected tropical disease of global importance. This study aimed to identify outer membrane proteins of pathogenicLeptospiraresponding to host chemical signals like catecholamines, with the potential to serve as virulence factors, new serodiagnostic antigens, and vaccine candidates. This study mimicked the plausible means by whichLeptospiraduring infection and hormonal stress intercepts host catecholamines to disseminate in host tissues.

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