scholarly journals Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells.

1983 ◽  
Vol 96 (2) ◽  
pp. 338-346 ◽  
Author(s):  
T McGarry ◽  
R Hough ◽  
S Rogers ◽  
M Rechsteiner
Keyword(s):  
Hela Cells ◽  
Immunoglobulin G ◽  
Protein Modification ◽  
Hinge Region ◽  
Myeloma Protein ◽  
Thin Sections ◽  
Intracellular Degradation ◽  
Rate Limiting Step ◽  
Rabbit Igg ◽  
Intact Rabbit

Intact rabbit immunoglobulin G molecules (IgGs) and their papain or pepsin fragments were radio-iodinated and injected into HeLa cells. Whole IgGs, Fab2, and Fc fragments were degraded with half-lives of 60-90 h, whereas half-lives of Fab fragments were 110 h. These results indicate that proteolytic cleavage in the hinge region of the IgG molecule is not the rate-limiting step in its intracellular degradation. The hingeless human myeloma protein, Mcg, was degraded at the same rate as bulk human IgG, providing further evidence that the proteolytically susceptible hinge region is not important for intracellular degradation of IgG molecules. SDS acrylamide gel analysis of injected rabbit IgG molecules revealed that heavy and light chains were degraded at the same rate. Injected rabbit IgGs and rabbit IgG fragments were also examined on isoelectric focusing gels. Fab, Fab2, and Fc fragments were degraded without any correlation with respect to isoelectric point. Positively charged rabbit IgGs disappeared more rapidly than their negative counterparts, contrary to the trend reported for normal intracellular proteins. The isoelectric points of two mouse monoclonal antibodies were essentially unchanged after injection into HeLa cells, suggesting that the altered isoelectric profile observed for intact rabbit IgG resulted from degradation and not protein modification. The intracellular distributions of IgG fragments and intact rabbit IgG molecules were determined by autoradiography of thin sections through injected cells. Intact IgG molecules were excluded from HeLa nuclei whereas both Fab and Fc fragments readily entered them. Thus, for some proteins, entry into the nuclear compartment is determined primarily by size.

scholarly journals Leporid immunoglobulin G shows evidence of strong selective pressure on the hinge and CH3 domains

Open Biology ◽  
2014 ◽  
Vol 4 (9) ◽  
pp. 140088 ◽  
Author(s):  
Ana Pinheiro ◽  
Jenny M. Woof ◽  
Tereza Almeida ◽  
Joana Abrantes ◽  
Paulo C. Alves ◽  
...  
Keyword(s):  
Positive Selection ◽  
Immunoglobulin G ◽  
Half Life ◽  
Selective Pressure ◽  
Hinge Region ◽  
European Rabbit ◽  
Rabbit Igg ◽  
Immunological Research ◽  
Constant Domains ◽  
Positively Selected Sites

Immunoglobulin G (IgG) is the predominant serum immunoglobulin and has the longest serum half-life of all the antibody classes. The European rabbit IgG has been of significant importance in immunological research, and is therefore well characterized. However, the IgG of other leporids has been disregarded. To evaluate the evolution of this gene in leporids, we sequenced the complete IGHG for six other genera: Bunolagus , Brachylagus , Lepus , Pentalagus , Romerolagus and Sylvilagus . The newly sequenced leporid IGHG gene has an organization and structure similar to that of the European rabbit IgG. A gradient in leporid IgG constant domain diversity was observed, with the CH1 being the most conserved and the CH3 the most variable domain. Positive selection was found to be acting on all constant domains, but with a greater incidence in the CH3 domain, where a cluster of three positively selected sites was identified. In the hinge region, only three polymorphic positions were observed. The same hinge length was observed for all leporids. Unlike the variation observed for the European rabbit, all 11 Lepus species studied share exactly the same hinge motif, suggesting its maintenance as a result of an advantageous structure or conformation.

scholarly journals The controlling effect of carbohydrate in human, rabbit and bovine immunoglobulin G on proteolysis by papain

1969 ◽  
Vol 111 (4) ◽  
pp. 473-478 ◽  
Author(s):  
R. B. Payne
Keyword(s):  
Immunoglobulin G ◽  
Polypeptide Chain ◽  
Initial Rate ◽  
Hinge Region ◽  
Fc Fragment ◽  
Rabbit Igg ◽  
Initial Cleavage ◽  
Bovine Immunoglobulin ◽  
Hydrolysis Of ◽  
Papain Digestion

About two-thirds of the hexose of human and rabbit immunoglobulin G (IgG) was located in the Fc fragment and one-third in the ‘hinge’ region of the γ (heavy) polypeptide chain at the junction of the Fab and Fc fragments. In contrast, bovine IgG contained more hexose in the ‘hinge’ region than in the Fc fragment. The initial cleavage of susceptible IgG molecules into Fab and Fc fragments by papain under the conditions given by Porter (1959) had reached completion after digestion for 2hr., though bovine IgG was digested somewhat more slowly than human or rabbit IgG. The release of ‘hinge’ peptides from human and rabbit IgG had also reached completion by 2hr., but was slower from bovine IgG and continued for several hours longer. Since bovine IgG molecules contained on the average a greater amount of hexose in the ‘hinge’ region, carbohydrate on this part of the γ-chain may influence not only the initial rate of enzymic hydrolysis into Fab and Fc fragments, but also, and to a greater extent, the rate of further limited hydrolysis of the N-terminal regions of the Fc fragment. The presence of carbohydrate in the ‘hinge’ region does not appear to account for the resistance of some IgG molecules to papain digestion and of some Fc fragments to N-terminal degradation.

scholarly journals Electron Microscopic Immunogold Localization of Salivary Mucins MG1 and MG2 in Human Submandibular and Sublingual Glands

2003 ◽  
Vol 51 (1) ◽  
pp. 69-79 ◽  
Author(s):  
Marco Piludu ◽  
Sean A. Rayment ◽  
Bing Liu ◽  
Gwynneth D. Offner ◽  
Frank G. Oppenheim ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Expression Patterns ◽  
Cell Types ◽  
Mucous Cells ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Dense Cores ◽  
Duct Cells ◽  
Rabbit Igg ◽  
Lowicryl K4m

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.

scholarly journals Post-embedding colloidal gold immunolocalization of laminin to the lamina rara interna, lamina densa, and lamina rara externa of renal glomerular basement membranes.

1986 ◽  
Vol 34 (7) ◽  
pp. 847-853 ◽  
Author(s):  
D R Abrahamson
Keyword(s):  
Colloidal Gold ◽  
Basement Membranes ◽  
Lamina Densa ◽  
Thin Sections ◽  
Gold Particles ◽  
Rabbit Igg ◽  
Gold Immunolocalization ◽  
Lowicryl K4m

Ultrastructural distribution of laminin within renal glomerular (GBM) and tubular basement membranes (TBM) was investigated using post-embedding immunolocalization with colloidal gold. Rat kidneys were fixed with 4% formaldehyde and embedded at 4 degrees C in Lowicryl K4M medium. Thin sections were then sequentially treated with affinity-purified rabbit anti-laminin IgG and anti-rabbit IgG conjugated to 10 nm diameter colloidal gold. Gold bound specifically to the GBM and TBM with particle densities of 690/micron2 and 731/micron2, respectively. In the GBM, the number of gold particles bound/micron2 of lamina densa greater than lamina rara externa greater than lamina rara interna. Closely similar binding patterns were found when kidneys were fixed with 0.5% glutaraldehyde plus 3% formaldehyde and embedded at 60 degrees C in L.R. White resin, but slightly less gold bound to sections overall than that seen with formaldehyde alone and Lowicryl. Taken together, these results illustrate that anti-laminin IgG, whether applied to fixed sections in vitro or introduced in vivo, bound to the lamina rara interna, lamina densa, and lamina rara externa of the GBM and throughout the TBM.

scholarly journals ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

1963 ◽  
Vol 19 (2) ◽  
pp. 337-347 ◽  
Author(s):  
M. A. Epstein ◽  
S. J. Holt
Keyword(s):  
Enzyme Activity ◽  
Hela Cells ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Enzyme Reaction ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Simplex Virus ◽  
Vacuolar Membranes

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.

HeLa cells form focal contacts that are not fibronectin dependent

1984 ◽  
Vol 66 (1) ◽  
pp. 133-145
Author(s):  
J. Morgan ◽  
D. Garrod
Keyword(s):  
Hela Cell ◽  
Hela Cells ◽  
Immunoglobulin G ◽  
Actin Microfilament ◽  
Focal Contacts ◽  
Microfilament Bundles ◽  
High Concentrations ◽  
The Relationship ◽  
Cells Cultured

HeLa cells cultured on glass substrata produce numerous prominent focal contacts, which reside at the termini of actin microfilament bundles. However, very few of the cells stain for fibronectin with specific anti-fibronectin antibody. Moreover, the cells form focal contacts in fibronectin-depleted medium, in the presence of high concentrations of anti-fibronectin immunoglobulin G and in the presence of monensin. No fibronectin synthesis can be detected by [35S]methionine-labelling and immunoprecipitation. The possibility that HeLa cell focal contacts are independent of fibronectin in their formation is discussed in relation to the controversy about the relationship between fibronectin and focal contacts.

THE EFFECT OF ENZYMATIC TREATMENT OF RABBIT PLATELETS ON THE BINDING OF IMMUNOGLOBULIN G TO THE SURFACE

1987 ◽  
Author(s):  
R M Evans ◽  
M A Packham
Keyword(s):  
Immunoglobulin G ◽  
Rabbit Serum ◽  
Membrane Glycoproteins ◽  
Plasmin Activity ◽  
Platelet Suspension ◽  
Platelet Survival ◽  
Igg Binding ◽  
Rabbit Igg ◽  
Rabbit Platelets

As shown previously, cleavage of surface glycoproteins on the platelet membrane shortens platelet survival. Since immunoglobulins have been implicated in the condition of idiopathic thrombocytopenic purpura in which patients have an elevated level of platelet-associated immunoglobulin G (I g G) and shortened platelet survival, we measured the binding of IgG to rabbit platelets that had been treated with neuraminidase or plasmin, to determine whether increased IgG binding to these platelets might be the signal that leads to their accelerated clearance. Rabbit platelets were washed and resuspended in calcium-free Tyrode-albumin solution (1.0 x 106/ μL), pH 6.5, and then incubated with either neuraminidase (0.1 U/mL at 22°C) or plasmin (activity: 1.6 ymol BAEE/min/mL platelet suspension at 37¼C) for 30 min. The platelets were then centrifuged and resuspended in heat-inactivated rabbit serum for 30 min to allow IgG to bind to the surface. After additional washing steps to remove non-specifically bound IgG, the amount Qt IgG that associated with the platelets was measured using an 125I-labeled goat anti-rabbit IgG binding assay. Binding data were analyzed by plotting the calculated fg of IgG bound/platelet versus the amount of 125-1 goat anti-rabbit IgG added. The binding curves for both neuraminidase and plasmin indicated significantly increased amounts of specifically bound IgG compared to control platelets. At a concentration of 30 μg/mL of labeled goat antirabbit IgG added, the amount of label associated with untreated rabbit platelets had plateaued at 0.36± 0.11 fg/platelet whereas the amount associated with neuraminidase-treated platelets was 5 times greater and with pi asmin-treated platelets, 1.5 times greater, and neither had plateaued. The results obtained with plasmin are of particular interest since this proteolytic enzyme shortens platelet survival and is present in vivo during thromboembolic events. These results indicate that IgG binds specifically to the surface of rabbit platelets following enzymatic alterations of the membrane glycoproteins and therefore may represent at least one mechanism that determines which platelets are cleared by the reticuloendothelial system.

scholarly journals Differential reduction of interchain disulphide bonds of mouse immunoglobulin G

1968 ◽  
Vol 107 (6) ◽  
pp. 823-828 ◽  
Author(s):  
Alan R. Williamson ◽  
Brigitte A. Askonas
Keyword(s):  
Heavy Chain ◽  
Immunoglobulin G ◽  
Polyacrylamide Gel Electrophoresis ◽  
Partial Reduction ◽  
Free Light Chains ◽  
Myeloma Protein ◽  
Heavy Chains ◽  
Disulphide Bonds ◽  
Rabbit Immunoglobulin ◽  
Mouse Immunoglobulin

The relative lability of the interchain disulphide bonds of mouse G2a-myeloma protein 5563 was studied as a function of 2-mercaptoethanol concentration. Analysis of partial-reduction mixtures by polyacrylamide-gel electrophoresis and microdensitometry showed that the disulphide bonds between light and heavy chains are much more susceptible to reduction than the bonds between heavy chains. At a low concentration of 2-mercaptoethanol (10mm) the major dissociable products of mouse immunoglobulin G are heavy-chain dimers and free light chains. These findings contrast with the reported behaviour of rabbit immunoglobulin G, for which the lability of inter-heavy-chain bonds was found to exceed that of the bonds linking light and heavy chains (Hong & Nisonoff, 1965); the relative stability of rabbit immunoglobulin G interchain bonds was confirmed in the present study. Examination of human immunoglobulin G and an immunoglobulin G (γ2) of guinea pig showed that at least in the majority of molecules, as with mouse immunoglobulin G, the disulphide bonds between light and heavy chains are more susceptible to reduction than the inter-heavy-chain bonds.

scholarly journals The recombination of dimers of immunoglobulin peptide chains

1970 ◽  
Vol 118 (5) ◽  
pp. 703-712 ◽  
Author(s):  
G. T. Stevenson ◽  
K. J. Dorrington
Keyword(s):  
Immunoglobulin G ◽  
Sodium Acetate ◽  
Optical Rotatory Dispersion ◽  
Acetate Buffer ◽  
Light Chains ◽  
Sodium Acetate Buffer ◽  
Covalent Bonds ◽  
Myeloma Protein ◽  
Disulphide Bonds ◽  
Antigenic Properties

1. Both the γ and light peptide chains of human pooled and myeloma immunoglobulin G can be prepared as non-aggregating dimers at pH5.4 in 4mm-sodium acetate buffer. The dimeric state is maintained by non-covalent bonds, since the formation of interchain disulphide bonds was prevented by alkylation of the thiol groups. In the case of the light chains there is some evidence that the dimers are in equilibrium with a small amount of monomer. 2. When such dimers of the γ and light chains are mixed at pH5.4 in 4mm-sodium acetate buffer they combine rapidly, yielding a product that resembles the original immunoglobulin G in its physicochemical and antigenic properties. However, the original optical rotatory dispersion spectrum was regained only with the homogeneous myeloma protein. The recombined pooled immunoglobulin G had a spectrum slightly different from the original, suggesting that at least some of the recombinant molecules had not regained native conformations. 3. Dimers of γ chains stabilized by interchain disulphide bonds were able to recombine with light chains. However, light chains stabilized in the dimeric state by interchain disulphide bonds would not combine with γ chains. 4. The chains of rabbit immunoglobulin G behave similarly to the human chains in this system, apart from the alkylated light chains showing clearer evidence of monomeric components.

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