Oliver A. Roholt

Antibody Combining Site: The B Polypeptide Chain

The antibody molecule consists of several polypeptide chains. Peptides, which appear to have been derived from the binding region of the rabbitantibody molecule directed against pazobenzenearsonate, have been isolated. The particular polypeptide chain from which these peptides are derived has now been identified as the B chain described by Fleischman, Pain, and Porter.

[35] Iodination—Isolation of peptides from the active site

Publisher Summary This chapter presents that for iodinating a protein, several reagents or combinations of reagents, including triiodide ion, iodine monochloride, iodine in alcohol, and iodide ion with an oxidizing agent such as nitrous acid, iodate ion, or chloramine T, are used. In each case, the reactive iodinating agent appears to be hypoiodous acid, HOI. The effect of iodination of an enzyme on its activity depends on the level of iodination of the enzyme and on whether this level disturbs or disrupts the tertiary structure of the protein or causes iodination of a specific residue in the active site of the enzyme. If iodination leads to a loss of activity and the activity can be preserved by carrying out the iodination in the presence of a competitive inhibitor of the enzyme, then the simplest conclusion is that there is an essential residue in the active site of the enzyme that is modified in the absence of the inhibitor, but is protected against this modification by the presence of the inhibitor. If the presence of the inhibitor during iodination does not protect the enzyme activity, then the inactivation may be due to tertiary structure alteration rather than attack at the site. When an iodinatable residue is found to be in the active site of an enzyme, iodopeptides containing this residue can be obtained by proteolysis and the residue can be identified.

Antibodies of limited heterogenety: L chains of a single mobility

Abstract The specifically purified anti-p-azobenzoate antibody from two rabbits (out of nine rabbits that had been injected with a heterogensous antigen (azo-p-benzoate-bovine-γ-globulin conjugate)) was found to be relatively homogensous on the basis of several criteria including homogeneity of the hapten-binding constant, and light chains giving only a single band upon disc electrophoresis, a relatively unique amino acid composition, and a relatively simple tryptic peptide map. Such homogeneity would not be expected on the basis of the concept that antibodies produced to a particular hapten by an individual animal represent a continous heterogeneous population composed of antibodies with binding constants following a probability distribution function. It appears rather, that only a few molecular species of antibody make up the bulk of the antihapten antibody produced in each individual animal. Thus, the observed limited antibody heterogeneity in an individual rabbit appears to depend on the stumulation of only a few of the many cells capable of producing antibody againts a given hapten, rather than on a structural identity of the environment around each individual group on the antigen molecule as postulated by others.

Polypeptide Chains of Antibody: Effective Binding Sites Require Specificity in Combination

Separated chains from antibody of different rabbits do not always give effective binding sites although combination of the chains does occur. There is a specificity of combination such that only chains from the same (or perhaps a related) rabbit form effective binding sites. There appears to be preferential combination between those chains which yield effective sites.

A differential method for determining the relative reactivity to iodination of different tyrosyl residues in a protein molecule

1. (1) A method has been developed for determining the relative rates of iodination of various tyrosyl residues in a protein. This is done by the use of a paired-label technique in which one portion of the protein is iodinated with iodine labeled with 125I and another portion is iodinated to incorporate a different number of iodine atoms labeled with 131I. The proteins are mixed and enzymatically digested. The resulting iodinated peptides are separated. Each iodinated peptide separated is a mixture of the 131I-labeled peptide from the first iodination and the 125I-labeled peptide from the second iodination. 2. (2) The relative reactivities of particular tyrosyl residues (identified by surrounding sequences) toward iodination can be determined from an analysis of the ratios of the amounts of iodine incorporated in the corresponding tyrosyl peptides derived from each of the two iodinated preparations. 3. (3) There is no need to recover quantitatively all the monoiodotyrosine and diiodotyrosine peptides derived from a particular tyrosine, a recovery which is difficult since for a single tyrosyl residue both of these forms can be distributed among several peptides.

ESTABLISHMENT OF TWO NONMETASTASIZING AND ONE METASTASIZING RAT MAMMARY CARCINOMA CELL LINES

SummaryTwo continuous rat mammary tumor cell lines have been established in culture from the lymphogenously metastasizing rat mammary carcinoma TMT-081 and one from the nonmetastasizing MT-100 and some of their in vitro and in vivo characteristics studied. Cell line TMT-081-MS was established as a free-floating cell suspension from the metastasis-free spleen of a rat bearing TMT-081 in the ascites form and is characterized by a high level of mammary tissue specific antigen (MTA), an antigen present on lactating or hormonally stimulated rat mammary tissues but not detected on normal mammary tissue. This line metastasizes in the syngeneic host but is rejected by the nude mouse without metastases. Cell line TMT-081-NM is a line derived from the ascites of a rat also bearing TMT-081 ascites. Cell line MT-100-TC is a line derived from the ascites of a rat bearing the ascites form of MT-100. Neither TMT-081-NM nor MT-100-TC has ever shown metastases in the syngeneic host but they are lethal; in the nude mice they grow rapidly, are lethal, and sometimes show hematogenous metastases. Both grow in small clusters and show a low level of MTA. These cell lines have been in continuous culture for a year and have proliferated and maintained their individual in vitro and in vivo growth characteristics during more than 100 consecutive subcultivations.

Limited heterogeneity of antibodies resolution of hapten binding curves into linear components

Abstract The hapten bindign curve for a heterogeneous antibody preparation was resolved by a graphical procedure into two linear binding curves. The antibody, obtained from the earlier bleedings of an individual rabbit, consisted principally of two homogeneous components on the basis of light chain disc electrophoresis and specific fractionation. Production of one of the two components subsequently ceased and the other component persisted. The measured binding constant for the persistant component, 104M−1, was used in the resolution as K for one of the components of the heterogeneous preparation; this component was found to amount to 40 per cent of the mixture. The binding constant of the other component was found to be 2·2 × 105M−1 and amounted to 60 per cent of the mixture. The resolution procedure should be useful in describing other antibody preparations.

Environments of individual tyrosines in protein: Differences in rates of mono- and dhodination

Abstract The relative rates of iodination of tyrosine to monoiodotyrosine and of monoiodotyrosine to diiodotyrosine in proteins: Tyrosine → k 1 monoiodotyrosine → k 2 diiodotyrosine have been a subject of controversy for some time. We have now shown that within a particular protein, a human Bence Jones protein, there are tyrosines for which k1 is greater than k2 and other tyrosines for which k2 is greater than k1. The value of the ratio, k1/k2, is dependent on the particular tyrosine involved.

Reactivity of tyrosine residues in the constant portion of type K Bence Jones protein

Abstract Tyrosine 173 of the constant portion of a human type K Bence Jones protein (Col) appears to be on the surface of the protein since it is the most reactive tyrosine toward iodination. This surface position for tyrosine 173 may be the case in general for type K Bence Jones proteins and L chains since it seems to be highly reactive in another type K Bence Jones protein (Koo) and the κ type L chain from a human myeloma IgG. Tyrosine 192, next to the valine characteristic of InV (b+) allotype, and tyrosine 186 and 140 appear to be less exposed. Tyrosine 140 is much less reactive than any of the other three of the constant portion and thus appears to be least exposed.

Structural differences between antibodies produced against the same hapten by individual rabbits

Abstract Structural differences have been observed in antibodies directed against the p-azobenzenearsonate group and obtained from individual rabbits of the allotype a1a1b4b4P+. Differences were observed in the iodination patterns of the antibodies as a whole, in the iodination patterns of the antibody site as shown by analysis of the peptides isolated from the sites and in the effect of iodination on the number of sites and on the binding constants. The binding contants of the original antibodies were also found to differ. The nature of the difference of antibody from rabbit to rabbit was such that it would be necessary for antibody from each rabbit to be composed of predominantly only a few different types of antibody molecules to give the results obtained.