The ultrastructure of normal and pathological IgM immunoglobulins.
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Free IgM immunoglobulins were examined in the electron microscope using the negative contrast technique. Normal human and rabbit IgM and Waldenstrom macroglobulins were indistinguishable from one another and revealed flexible spider-like particles with five appendages joining a central ring. The average total span of the molecules was 300 A. The appendages were about 125 x 30 A; the central ring had an outer diameter of approximately 100 A and an inner diameter of 40 A. Some purified 19S IgM preparations tended to form massive aggregates (>/=50S) which, when examined in the electron microscope, revealed enormous clumps of IgM molecules whose appendages were entangled with one another. Electron microscopy of reduced-alkylated IgM revealed total absence of intact spider-like molecules. The predominating structure observed was a round electron-dense knob about 50 A in diameter which in some cases had a fine fiber-like extension with approximate dimensions 100 x 15 A. Rabbit and human IgM molecules with antibody activity to poliovirus dried in sodium tungstosilicate on a carbon film as in preparation for electron microscopy were shown to retain nearly 100% of their poliovirus neutralizing activity after redissolving in a physiological buffer. (+info)
In vitro synthesis of immunoglobulin by rheumatoid synovial membrane.
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A technique for the in vitro culture of rheumatoid synovial tissue with (14)C-amino acids and isolation and quantitation of the newly synthesized immunoglobulins has been developed. This technique has been used to compare immunoglobulin synthesis of 12 rheumatoid synovia with that of synovia from nonarthritic patients and with that of normal human lymph nodes and spleen. In addition, the spleen of a patient with Felty's syndrome has also been examined. Immunoglobulin synthesis in rheumatoid synovia has been shown to be quantitatively and qualitatively similar to that of normal human spleen and lymph nodes although somewhat less active than the Felty's syndrome spleen examined. 79% of the immunoglobulin produced in rheumatoid synovia was of the IgG type, whereas IgM comprised 10% and IgA, 11% of the total. Less than 10% of the IgM synthesized was found to be rheumatoid factor. A fraction containing approximately 90% of its radioactivity in the form of IgG has been obtained for further studies. (+info)
Idiotypy of rabbit antibodies. II. Comparison of idiotypy of various kinds of antibodies formed in the same rabbits against Salmonella typhi.
(43/100)
The idiotypic patterns detected in the anti-Salmonella typhi serum of one given bleeding have been sought in other bleedings of the same rabbit. Idiotypic patterns carried by antibodies of the second bleeding are not always found in those of the first bleeding. Bifurcated precipitation zones in gels (double diffusion in cells) have been observed in the reaction of several anti-idiotypic sera with the sera of the first and second bleedings of three rabbits, and apparently indicate that idiotypic patterns which were carried by the same molecule in the first bleeding were carried by two separate molecules in the second bleeding. In a comparison of idiotypy of two anti-S. typhi serum samples of one rabbit, the collection of which had been separated by a very long interruption of the immunization, all the idiotypes detected in the early bleeding were also detected in the late bleeding; several idiotypes were detected in the serum of the late bleeding and not in that of the early bleeding. Idiotypy has been observed and compared in IgM and in IgG antibodies. There are, in each of these two classes, antibodies that are not distinguished from antibodies of the other class by anti-idiotypic sera, and consequently that carry the same idiotypic patterns. A comparison has been made between the idiotypy of the two kinds of antibodies which, in anti-S. typhi sera, are precipitated and not precipitated by the polysaccharide. Certain idiotypic patterns are carried only by the antibodies of the first kind. There are also idiotypic patterns which are carried by antibodies of these two kinds, among which certain anti-idiotypic antibodies do not discriminate. Possible cellular implications of observations made on idiotypy at different stages of the immunization of one individual have been discussed. (+info)
An enzymic method for the trace iodination of immunoglobulins and other proteins.
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1. A method is described for the trace iodination of immunoglobulins and other serum proteins by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. gammaG immunoglobulin that had been labelled to a specific radioactivity of 5muc/mug. by use of carrier-free [(125)I]iodide gave no evidence of denaturation when analysed by electrophoresis and density-gradient ultracentrifugation. 3. Tryptic hydrolysis and peptide ;mapping' of a completely characterized peptide radioiodinated by this method showed that the [(125)I]iodide was bound to tyrosyl residues. 4. Proteins differ in their susceptibility to iodination by this method. Human gammaG immunoglobulin, for example, is iodinated more than ten times as readily as is human alpha(2)-macroglobulin under the same conditions. 5. Lactoperoxidase catalyses the iodination of proteins much more readily than does horseradish peroxidase. (+info)
Inhibition by alpha-macroglobulin and other serum proteins.
(45/100)
1. Normal human serum was found to inhibit human cathepsin B1. 2. The major inhibitor present in serum was purified and identified as alpha(2)-macroglobulin. 3. alpha(2)-Macroglobulin was found to bind cathepsin B1 in an approximately 1:1 molar ratio. When bound, the enzyme retained about 50% of its proteolytic activity, and up to 80% of its activity against alpha-N-benzoyl-dl-arginine 2-naphthylamide. 4. Pretreatment of alpha(2)-macroglobulin with cathepsin B1 inactivated by exposure to pH8.5 or iodoacetic acid, in large molar excess, did not prevent the subsequent binding of active enzyme. Active enzyme, once bound, was not protected from inhibition by 1-chloro-4-phenyl-3-tosylamido-l-butan-2-one. 5. Cathepsin B1 was also inhibited by human immunoglobulin G, at high concentration. 6. Because it had been suggested that haptoglobin is responsible for the inhibition of ;cathepsin B' by serum, a method was devised for the selective removal of haptoglobin from mixtures of serum proteins by adsorption on haemoglobin covalently linked to Sepharose. No evidence was obtained that haptoglobin has any inhibitory activity against the enzyme. (+info)
Monoclonal macroglobulinemia in NZB-NZW F1 mice.
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Serum monoclonal macroglobulins were detected in over 30% of NZB/NZW F(1) mice greater than 11 mo of age. The monoclonal nature of the IgM was shown by restricted electrophoretic mobility, characteristic appearance on immunoelectrophoresis, restriction to a single light chain type, and ability to induce anti-idiotypic antisera. The monoclonal macroglobulins were separated from antibodies to DNA and RNA that migrated in the 7S region of sucrose gradients. Enlarged lymph nodes were often present in mice with monoclonal IgM, and a transplantable IgM-producing lymphoid tumor was established from the spleen of one animal. (+info)
The interaction of alpha 2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism.
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1. alpha(2)-Macroglobulin is known to bind and inhibit a number of serine proteinases. We show that it binds thiol and carboxyl proteinases, and there is now reason to believe that alpha(2)-macroglobulin can bind essentially all proteinases. 2. Radiochemically labelled trypsin, chymotrypsin, cathepsin B1 and papain are bound by alpha(2)-macroglobulin in an approximately equimolar ratio. Equimolar binding was confirmed for trypsin by activesite titration. 3. Pretreatment of alpha(2)-macroglobulin with a saturating amount of one proteinase prevented the subsequent binding of another. We conclude that each molecule of alpha(2)-macroglobulin is able to react with one molecule of proteinase only. 4. alpha(2)-Macroglobulin did not react with exopeptidases, non-proteolytic hydrolases or inactive forms of endopeptidases. 5. The literature on binding and inhibition of proteinases by alpha(2)-macroglobulin is reviewed, and from consideration of this and our own work several general characteristics of the interaction can be discerned. 6. A model is proposed for the molecular mechanism of the interaction of alpha(2)-macroglobulin with proteinases. It is suggested that the enzyme cleaves a peptide bond in a sensitive region of the macroglobulin, and that this results in a conformational change in the alpha(2)-macroglobulin molecule that traps the enzyme irreversibly. Access of substrates to the active site of the enzyme becomes sterically hindered, causing inhibition that is most pronounced with large substrate molecules. 7. The possible physiological importance of the unique binding characteristics of alpha(2)-macroglobulin is discussed. (+info)
Degradation of human fibrinogen by plasms alpha2-macroglobulin-enzyme complexes.
(48/100)
This study demonstrates that human plasma alpha(2)-macroglobulin preparations possess an enzymic activity that degrades fibrinogen, resulting in the formation of products whose structure resembles that of circulating fibrinogen catabolites. The sequence of degradation is similar to that observed in plasmin-catalyzed digests, in that Aalpha-chain fragmentation precedes that of Bbeta-chain. The addition of plasminogen activators to plasma induced an increase in the N-alpha-tosyl-l-arginine methyl ester HCl esterase and fibrinogenolytic activity associated with alpha(2)-macroglobulin purified from this plasma, indicating that the enzymic activity of the complex was preserved and could be increased in the presence of other plasma enzyme inhibitors. Immunochemical studies demonstrated that an alpha(2)-macroglobulin-plasmin complex had formed in urokinase-treated plasma. This alpha(2)-macroglobulin preparation manifested an esterolytic profile like that of a complex prepared from plasmin and purified alpha(2)-macroglobulin. After complex formation with alpha(2)-macroglobulin in plasma, plasmin retained less than 0.1% of its fibrinogenolytic activity. That plasmin expressed its activity while bound to alpha(2)-macroglobulin was suggested by immunoprecipitation of this activity with alpha(2)-macroglobulin antibody and by the demonstration that pancreatic trypsin inhibitor did not effectively inhibit its fibrinogenolytic or esterolytic activity. These results raise the possibility that, in addition to its activity as a major plasma proteolytic enzyme inhibitor, alpha(2)-macroglobulin may modulate enzyme-substrate interactions, such as those resulting in the formation of circulating fibrinogen catabolites, by providing a mechanism for the preservation and protection of a portion of the enzymic activity in the presence of other circulating inhibitors. (+info)