scholarly journals One cell–one immunoglobin. Origin of limited heterogeneity of myeloma proteins

1970 ◽  
Vol 116 (2) ◽  
pp. 241-248 ◽  
Author(s):  
Z. L. Awdeh ◽  
A. R. Williamson ◽  
Brigitte A. Askonas
Keyword(s):  
Isoelectric Focusing ◽  
Light Chain ◽  
Time Intervals ◽  
Myeloma Protein ◽  
Heavy Chains ◽  
Serum Component ◽  
Isoelectric Points ◽  
Ph Range ◽  
A Charge

Plasma-cell tumour 5563 forms a single molecular species of immunoglobulin IgG2a, i.e. one variant of heavy chain and one variant of light chain. The molecules formed are labile and undergo alterations in charge properties, which rapidly lead to heterogeneity of the myeloma protein after synthesis. The single immunoglobulin species originally formed is found only after the shortest time-intervals tested, i.e. 10min incubation. Two types of changes in charge properties take place: (1) The originally formed protein (component o) is converted via an intermediate o′ into the most basic form of 5563 myeloma protein found in serum (component a). Charge differences between these components are suppressed at pH8.9, but can be studied by chromatography at pH6.5 or by analysis of isoelectric points by isoelectric focusing in polyacrylamide gel. The conversion of components o and o′ into component a apparently commences soon after assembly of the molecules and proceeds to completion extracellularly. (2) The second type of charge difference that distinguishes components a, b, c and d is exhibited over the pH range 6.0–8.9, but not at acid pH, and has been studied by electrophoresis at pH8.9, by chromatography and by isoelectric focusing. Conversion of component a into components b, c, d and e is only partial so that all five components can be found at decreasing concentrations in serum. Both types of charge alteration can be effected in vitro in the presence of serum, with optimum pH8.0. None of the charge differences could be attributed to the secretion process, since a component with the same isoelectric point as component o was found in secreted myeloma protein (1h). We have found no evidence to support the idea that the first type of change from component o to component a is due to ring formation of N-terminal [14C]glutamine into pyrrolid-2-one-5-carboxylic acid; however, our findings do not exclude this process happening very rapidly to a precursor of component o, possibly the polypeptide chain during or immediately after synthesis. In studying this point we noted that not only the heavy chains but also the κ-type light chain of mouse 5563 myeloma protein have a blocked N-terminus.

An Atypical Human Immunoglobulin G with Deletions in both Heavy and Light Chains. Studies of the Conformation and the In Vitro Recombination of the Isolated Subunits

1974 ◽  
Vol 52 (7) ◽  
pp. 610-619 ◽  
Author(s):  
M. E. Percy ◽  
K. J. Dorrington
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Hypervariable Region ◽  
Light Chains ◽  
Constant Region ◽  
Myeloma Protein ◽  
Variable Regions ◽  
Heavy Chains ◽  
Amino Terminal

Both the light and gamma (heavy) chains of IgG(Sac) contain extensive deletions in their variable regions. The deletion in the light chain is internal (residues 18–88), whereas the deletion in the heavy chain is amino-terminal (residues 1–102). The hypervariable region just preceding the beginning of the constant region in other heavy chains (residues 103–115) is amino-terminal in heavy chain(Sac). In 4 mM acetate, pH 5.4, heavy chain(Sac) is dimeric like normal gamma chains, whereas light chain(Sac) is monomeric. Isolated light and heavy chains of IgG(Sac) recombine in vitro with each other and also with the heavy and light chains from a typical human IgG1-K myeloma protein, but not in a fashion entirely typical of other human gamma and light chains. These studies support the concept that non-covalent forces between the variable regions of the light and heavy chains are important in the assembly of the immunoglobulin molecule; and in view of the weak interaction between the constant region of light chain and heavy chain observed previously, our data suggest that there are points of contact between the hypervariable region of the gamma chain (residues 103–115) and the variable region of the light chain.

Effects of androgens on bioactivity and immunoreactivity of pituitary FSH in GnRH antagonist-treated male rats

1990 ◽  
Vol 122 (2) ◽  
pp. 168-174 ◽  
Author(s):  
Om P. Sharma ◽  
Shafiq A. Khan ◽  
Gerhard F. Weinbauer ◽  
Mohammed Arslan ◽  
Eberhard Nieschlag
Keyword(s):  
Isoelectric Focusing ◽  
Gnrh Antagonist ◽  
Molecular Composition ◽  
Male Rats ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Combined Administration ◽  
Ph Range ◽  
Fsh Bioactivity

Abstract The effects of androgens on the bioactivity and molecular composition of pituitary FSH were examined in intact and GnRH antagonist-suppressed male rats. Eight groups of adult Sprague-Dawley rats were subjected to the following treatments: antagonist (75 μg/day by osmotic minipumps; sc), testosterone-filled Silastic implants (3×5 cm, sc), dihydrotestosterone-filled Silastic implants (3×5 cm, sc), E2 benzoate (15 μg/day, sc), and combined administration of antagonist with either steroid for 3 weeks. At the end of the treatment period, pituitaries were dissected out and homogenised. FSH content was determined in the pituitary extracts by an in vitro bioassay and a radioimmunoassay. Individual pituitary extracts from rats treated with vehicle, testosterone and testosterone + antagonist were subjected to isoelectric-focusing on sucrose density gradients performed in the pH range from 3.5 to 7.0. Individual isoelectric-focusing fractions (100-120) were analysed for bioactive and immunoreactive FSH. Treatment with antagonist, E2 or antagonist + E2 caused a significant decrease in pituitary FSH, whereas testosterone and dihydrotesterone alone or in combination with antagonist prevented the decrease in pituitary FSH. The effects of all treatments on both bioactive and immunoreactive FSH were similar. Testosterone treatment not only maintained FSH synthesis but also altered the molecular composition of pituitary FSH. Following treatment with testosterone there was a shift of maximal FSH bioactivity to the more acidic pH range. On the other hand, less bioactivity was recovered than corresponding immunoreactivity in the higher pH region, resulting in significantly reduced ratios of bioactivity to immunoreactivity of FSH. No significant differences were found in the isoelectric-focusing profiles or bioactivity to immunoreactivity ratios of pituitary FSH in animals treated with testosterone alone or in combination with antagonist. The results demonstrate that testosterone not only maintained the synthesis of both bioactive and immunoreactive FSH in male rats, but also influences the molecular composition of pituitary FSH. These effects of testosterone on pituitary FSH appear not to be mediated through hypothalamic GnRH.

Biosynthesis of immunoglobulins on polyribosomes and assembly of the IgG molecule

1966 ◽  
Vol 166 (1003) ◽  
pp. 232-243 ◽  
Keyword(s):  
Light Chain ◽  
Gradient Centrifugation ◽  
Intermediate Stage ◽  
Light Chains ◽  
Pulse Labelling ◽  
Myeloma Protein ◽  
Heavy Chains ◽  
Specific Antisera ◽  
Independent Synthesis ◽  
Small Pool

Immunoglobulin G formation was studied using as model system an ascitic form of the murine plasmacytom 5563. Following pulse labelling of the cells with 3 H-leucine, polyribosomes were fractionated on sucrose gradients. By the use of antisera specific for various parts of the IgG molecule, nascent heavy and light chains were detected on distinct polyribosomes of different size. Polyribosomes carrying heavy chain determinants were present in clusters with maximum sedimentation constants of around 300 S.; a much lower proportion of radioactivity was detectable as light chain determinants on polyribosomes up to 200 S. This observation is consistent with the independent synthesis of each chain as one polypeptide unit. Release of light chains appears to be an intermediate stage in the assembly of the IgG molecule. After pulse labelling of cells soluble IgG determinants were analysed by sucrose gradient centrifugation and precipitation with specific antisera. Only the light chains were released into a small pool which may control the release of heavy chains from polyribosomes. The radioactivity of the light chain pool reached a maximum at 10 min, whereas that of whole myeloma protein increased linearly with time. These results fit the interpretation that light chains form a small, rapidly turning over, pool before being incorporated into whole IgG molecules.

A New Class of Hexavalent Bispecific Antibodies and Immunocytokines with Enhanced Pharmacokinetics and Improved Efficacy in Vivo.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2451-2451
Author(s):  
Edmund A Rossi ◽  
Thomas M Cardillo ◽  
David M Goldenberg ◽  
Chien-Hsing Chang
Keyword(s):  
Light Chain ◽  
Fusion Proteins ◽  
Low Dose ◽  
Molecular Size ◽  
Bispecific Antibodies ◽  
Heavy Chains ◽  
New Class ◽  
Anti Cd20

Abstract Abstract 2451 Background. Biomedical research is trending to the development of increasingly more sophisticated antibody-based biologics, such as bispecific antibodies, immunocytokines and antibody-drug conjugates. Compared to traditional mAbs, development of more complex, and less natural, fusion proteins are challenged by problems with yield, stability, toxicity, immunogenicity and Pk. Previously, we reported potent anti-lymphoma activity for both anti-CD22/CD20 bispecific hexavalent antibodies (bsHexAbs; Rossi et al., Blood 2009; 113: 6161–6171) and immunocytokines comprising anti-CD20 mAb and tetrameric IFNα2b (IgG-IFN; Rossi et al., Blood 2009;114:3864-71). For each class of immunoconjugate that were produced with the Dock-and-Lock (DNL) method using an IgG module having an AD2 peptide fused at the C-terminal end of the Fc, we identified Pk and in vivo stability as potentially limiting parameters. Methods. Using the DNL method, we generated a new class of IgG modules, which have an AD2 peptide fused at the C-terminal end of the kappa light chain and were used to produce Ck-based (indicated by *) bsHexAbs and IgG-IFNα2b, for comparison with homologous Fc-based constructs. The Ck-based immunocytokine 20*-2b, has a similar molecular size and composition to its Fc-based homolog, 20-2b, each comprising the humanized anti-CD20 mAb, veltuzumab, and 4 IFNα2b groups that are fused at the C-terminal ends of the light or heavy chains, respectively. The Ck-based bsHexAb 22*-(20)-(20) and its Fc-based homolog 22-(20)-(20), each comprise the humanized anti-CD22 mAb, epratuzumab, and 4 Fabs of veltuzumab, which are fused at the C-terminal ends of the light and heavy chains, respectively. Results. The Ck-based constructs exhibited superior Pk (longer T1/2) in mice and rabbits, with either IV or SC injection (Table 1). Although the bsHexAbs and IgG-IFN are considerably stable in sera, analysis of Pk samples indicated that some dissociation occurs in vivo, presumably by intracellular processing. The in vivo dissociation rate for 20*-2b (0.18%/h) was 5.4-fold slower than 20-2b (0.97%/h). Similarly, 22*-(20)-(20) (0.19%/h) was more stable in vivo than 22-(20)-(20) (0.55%/h). Fc effector functions were markedly enhanced for the Ck-based constructs. Where 20*-2b induced strong CDC, which approached the potency of veltuzumab, no activity was evident for 20-2b. Epratuzumab did not have CDC, while 22-(20)-(20) achieved modestly increased activity, and 22*-(20)-(20) induced even greater CDC. In vitro, epratuzumab induced minimal ADCC and 22-(20)-(20) did not show a statistically significant improvement. However, 22*-(20)-(20) exhibited potent ADCC, which was similar to that of veltuzumab. Finally, the Ck-based conjugates were more effective than the Fc-based counterparts for therapy of disseminated NHL (Daudi) xenografts. Using a single low dose of 0.25 mg, superiority (P=0.0351) was demonstrated for 20*-2b (median survival time [MST]>189 days, 87% cures), compared to 20-2b (MST=134.5 days, 37.5% cures). At a high (1 mg) dose, 22*-(20)-(20) (MST>98 days, 100% survival) was superior (P<0.0001) to 22-(20)-(20) (MST=71 days, 10% survival). With low-dose (10 μg) treatment, the MST was 91 days for 22*-(20)-(20), compared to 50.5 days for 22-(20-(20) (P=0.0014). Conclusions. These new constructs demonstrate the further enhancement of two different classes of fusion proteins with already potent anti-lymphoma efficacy. Due to extended Pk, improved stability and enhanced effector function, the Ck-based design is superior for in vivo applications. The strategy of designing antibody fusion proteins at the C-terminus of the light chain, instead of at the commonly used Fc, may improve the in vivo efficacy of most immunoconjugates in general, and the DNL conjugates in particular. Disclosures: Rossi: Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Cardillo:Immunomedics, Inc: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership. Chang:Immunomedics, Inc.: Employment.

Modification of renin isoelectric heterogeneity in Goldblatt hypertensive rat

1985 ◽  
Vol 248 (6) ◽  
pp. E694-E698 ◽  
Author(s):  
F. M. Sessler ◽  
R. L. Malvin
Keyword(s):  
Isoelectric Focusing ◽  
Rat Kidney ◽  
Venous Blood ◽  
Renin Activity ◽  
Hypertensive Rat ◽  
Isoelectric Points ◽  
The Difference ◽  
Cortical Slices

Six forms of renin have been described in rat kidney. Different stimuli resulted in secretion of unique profiles of those forms. We studied their storage and secretion in the two-kidney, one-clip Goldblatt hypertensive rat (GHR). Renal venous blood, kidney homogenates, and incubation media from cortical slices were subjected to isoelectric focusing. In all samples tested, six peaks of renin activity were found with isoelectric points at pH 5.90, 5.70, 5.40, 5.20, 5.00, and 4.80. The quantity of renin activity for each form was expressed as a percentage of the total recovered from the gel. In control kidneys the profile of renin stored and that released by in vitro slices were similar. However, in plasma, the percentage of renin focusing at the more basic pH was decreased. This is in agreement with other work showing that the liver removes the more basic forms more rapidly than the acidic forms. The clipped kidney of GHR secreted, both in vivo and in vitro, a profile of renin forms that was significantly different from the control kidney. The difference was expressed by an increase in the secretion of the more acidic forms by the clipped kidney. It is hypothesized that changes in the secretory profile of renin may reflect changes in storage and synthesis of those forms.

Human myosin-IXb is a mechanochemically active motor and a GAP for rho

1998 ◽  
Vol 111 (7) ◽  
pp. 941-950 ◽  
Author(s):  
P.L. Post ◽  
G.M. Bokoch ◽  
M.S. Mooseker
Keyword(s):  
Light Chain ◽  
Rho Gtpase ◽  
Actin Binding ◽  
Maximal Velocity ◽  
Heavy Chains ◽  
In Vitro Motility ◽  
Gtpase Activating Proteins ◽  
Active Motor ◽  
Gtpase Activation

The heavy chains of the class IX myosins, rat myr5 and human myosin-IXb, contain within their tail domains a region with sequence homology to GTPase activating proteins for the rho family of G proteins. Because low levels of myosin-IXb expression preclude purification by conventional means, we have employed an immunoadsorption strategy to purify myosin-IXb, enabling us to characterize the mechanochemical and rho-GTPase activation properties of the native protein. In this report we have examined the light chain content, actin binding properties, in vitro motility and rho-GTPase activity of human myosin-IXb purified from leukocytes. The results presented here indicate that myosin-IXb contains calmodulin as a light chain and that it binds to actin with high affinity in both the absence and presence of ATP. Myosin-IXb is an active motor which, like other calmodulin-containing myosins, exhibits maximal velocity of actin filaments (15 nm/second) in the absence of Ca2+. Native myosin-IXb exhibits GAP activity on rho. Class IX myosins may be an important link between rho and rho-dependent remodeling of the actin cytoskeleton.

In Vitro Interaction between Venous Blood Clots and Radiopharmaceuticals

1985 ◽  
Vol 24 (04) ◽  
pp. 173-179 ◽  
Author(s):  
B. R. R. Persson ◽  
V. Kempi
Keyword(s):  
Room Temperature ◽  
Venous Blood ◽  
Gel Chromatography ◽  
Time Intervals ◽  
Ph Values ◽  
Blood Clots ◽  
Glass Tubes ◽  
Ph Range ◽  
In Vitro Interaction

SummaryClots of 1 ml venous blood formed in glass tubes after 10 min at room temperature were incubated at 37° C with the radiopharmaceutical to be studied. Methods for quality control of the radiopharmaceuticals were compared. Gel chromatography scanning was found to give reliable information. The incorporation into the clot was studie’d at different pH values and after various time intervals. The highest incorporation was found for 125I-fibrinogen and for 99mTc-mac-roaggregates of albumin, followed by 99mTc-sulphur colloid and 99mTc-strep-tokinase at pH less than 2. The titrated initial dose of 99mTc-streptoki-nase was studied at various pH levels. The lysing effect was less in the pH range 1-2.5, where the best labeling yield was obtained. The inactivation of streptokinase by the labeling procedure was also studied with im-munoelectrophoresis and decomposition of casein. In vitro studies of the interaction of radiopharmaceuticals with clots add information for the clinical use of radiopharmaceuticals for thrombus localization.

scholarly journals BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly

1999 ◽  
Vol 10 (7) ◽  
pp. 2209-2219 ◽  
Author(s):  
Young-Kwang Lee ◽  
Joseph W. Brewer ◽  
Rachel Hellman ◽  
Linda M. Hendershot
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Protein Complexes ◽  
Critical Role ◽  
Light Chains ◽  
Heavy Chains ◽  
Chain Complexes ◽  
Chain Synthesis

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP–heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

scholarly journals Chlamydomonas Outer Arm Dynein Alters Conformation in Response to Ca2+

2007 ◽  
Vol 18 (9) ◽  
pp. 3620-3634 ◽  
Author(s):  
Miho Sakato ◽  
Hitoshi Sakakibara ◽  
Stephen M. King
Keyword(s):  
Light Chain ◽  
In Vitro Experiments ◽  
Iq Motif ◽  
Heavy Chains ◽  
Γ Subunit ◽  
Close Proximity ◽  
Entire Complex ◽  
Consensus Motifs ◽  
Dependent Interaction

We have previously shown that Ca2+ directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the β and γ heavy chains (HCs). The γ HC–associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca2+ with KCa = 3 × 10−5 M in vitro, suggesting it may act as a Ca2+ sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and γ HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the γ heavy chain. In vitro experiments indicate that LC4 undergoes a Ca2+-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca2+. The sedimentation profile of the γ HC subunit changed subtly upon Ca2+ addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated γ subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca2+ addition. We propose that Ca2+-dependent conformational change of LC4 has a direct effect on the stem domain of the γ HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm.

scholarly journals Multiplicity of clathrin light-chain-like polypeptides from developing pea (Pisum sativum L.) cotyledons

1992 ◽  
Vol 103 (4) ◽  
pp. 1127-1137 ◽  
Author(s):  
H.B. Lin ◽  
S.M. Harley ◽  
J.M. Butler ◽  
L. Beevers
Keyword(s):  
Pisum Sativum ◽  
Light Chain ◽  
Calcium Binding ◽  
Heat Stability ◽  
Coated Vesicles ◽  
Light Chains ◽  
Heavy Chains ◽  
Heat Stable ◽  
Pisum Sativum L

A comparative study has been made of clathrin-coated vesicles from developing pea (Pisum sativum L.) cotyledons and bovine brains in order to characterize the clathrin light chains from a plant system. Four polypeptides of 31 kDa, 40 kDa, 46 kDa and 50 kDa are considered as candidates for clathrin light chains in the developing pea cotyledons. The 31 kDa, 40 kDa, 46 kDa and 50 kDa polypeptides, together with the 190 kDa heavy chain, are dissociated as triskelions when coated vesicles of developing pea cotyledons are treated with 2 M urea. Partially purified 46 kDa and 50 kDa polypeptides have been demonstrated to bind to purified clathrin heavy chains. The 40 kDa, 46 kDa and 50 kDa polypeptides are sensitive to elastase. They are readily solubilized by neutralization of 10% trichloroacetic acid precipitates of clathrin. The 50 kDa polypeptide of plant clathrin-coated vesicles is heat-stable as are the light chains from bovine brains, while the heat stability of the 31 kDa, 40 kDa and 46 kDa polypeptides of plants is dependent on pH and ionic strength. The 40 kDa, 46 kDa and 50 kDa polypeptides bind calmodulin. The calcium binding properties of these polypeptides are ambiguous. The 40 kDa and 46 kDa polypeptides can be phosphorylated more extensively than the 31 kDa in vitro in the presence of polylysine, as can the smaller light chain of brains. The 50 kDa polypeptide can also be phosphorylated, even without the addition of polylysine. Unlike brain light chains, phosphorylation of the 31 kDa, 40 kDa, 46 kDa and 50 kDa polypeptides from peas is greatly reduced by N-ethylmaleimide (NEM). Our findings contrast with earlier reports of clathrin light chains of 30 and 38 kDa from zucchini and 57 and 60 kDa from carrots, respectively.

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