Chaviva Isersky

Amyloid Fibril Proteins: Proof of Homology with Immunoglobulin Light Chains by Sequence Analyses

The sequences of the 35 and 36 amino-terminal amino acids of two purified amyloid fibril proteins have been determined. Results indicate that these two proteins are derived from homogeneous immunoglobulin light chains of variable region subgroup VκI. The relation between amyloidosis and immunoglobulins is thus more firmly established.

Soluble and Particulate Forms of Rat Catechol‐O‐Methyltransferase Distinguished by Gel Electrophoresis and Immune Fixation

Abstract: Catechol‐O‐methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDSPAGE) of all tissue homogenates examined revealed three major immune‐specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 × g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an “aggregate” of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two‐dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pl of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1–5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is “soluble” with an apparent Mr of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.

Immunglobulin origin of localized nodular pulmonary amyloidosis

SummaryThe major protein of an amyloid fibril concentrate from a patient with localized nodular pulmonary amyloidosis has been purified by sequential gel filtration on Sepharose 4 B and Sephadex G-100 columns using 5 M guanidine-HCl in 1 N acetic acid. The molecular weight of 15250, unreactive amino-terminal amino acid, peptide map pattern, and immunochemical cross-reactivity with some, but not all, lambda Bence Jones proteins identifies this amyloid fibril protein as being derived primarily from the aminoterminal segment of a homogeneous lambda light polypeptide chain of an immunoglobulin protein.Although the immunoglobulin origin of amyloid fibrils has been demonstrated in several pathologic settings, this is the first example of their immunoglobulin origin in a case of amyloidosis with restricted tissue involvement.

The Purification of Amyloid Fibril Proteins

Abstract Amyloid fibril concentrates have been fractionated and shown to have homogeneous fragments of the variable region of immunoglobulin proteins as their major protein constituent. Amyloid fibril protein purification was performed on ten amyloid preparations by sequential gel filtration on Sepharose 4 B and Sephadex G-100 columns equilibrated with 5 M guanidine-HCl in 1 N acetic acid.

Characterization of the receptors for IgE on membranes isolated from rat basophilic leukemia cells

Abstract Mast cells, basophils and a tumor analog, rat basophilic leukemia cells, have a surface receptor for immunoglobulin E (IgE). Membranes from such cells should be highly useful for the study of how the α- and β-subunits of the receptor interact with the plasma membrane and function therein. Membranes were prepared from rat basophilic leukemia cells. The cells were reacted with radioiodinated IgE to saturate a fraction of their receptors and the radiolabel was used as a surface marker. When isolated from sucrose gradients, 40 ± 10% yields of plasma membranes were obtained and these were typically purified 13-fold with regard to cellular proteins. Unoccupied receptors for IgE were almost all recovered (as assessed on membranes solubilized with detergent) as were surface-labeled proteins in general. The membrane-bound IgE appeared fully exposed as assessed with anti-IgE antibodies but surprisingly 70–80% of the unoccupied receptors present were unable to bind IgE when unsolubilized vesicles were tested. By electron microscopy the preparations appeared free of recognizable intracellular organelles. Though some of the membranes appeared multivesicular many showed large breaks in continuity. Assessment of permeability, carried out on dextran gradients, indicated that a significant number (⩾50%) of the IgE-carrying membranes are not sealed. It is therefore most likely that both external and cytoplasmic aspects of the plasma membrane were exposed in many of the vesicles. Treatment of such membrane vesicles with trypsin or papain did not alter the apparent mol. wt of the α-chain of the receptor or its binding activity for IgE. Additionally, when the vesicles were examined by enzyme-catalysed iodination, no labeling of the receptor was observed under conditions where it is not labeled on intact cells (i.e. in the presence of IgE). These results suggest that either the α-chain of the receptor does not penetrate the membrane or that it cannot be modified by the particular probes we used.

IgE-immunotoxins. II. IgE-ricin A-chain.

In order to develop a reagent capable of killing cells with high-affinity IgE Fc receptors, such as mast cells and basophils, ricin A-chain (the toxic portion of ricin) was conjugated to rat IgE myeloma protein, IR 162, via derivatization of the IgE by n-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) thus creating an IgE-immunotoxin. Monensin (10(-7)-10(-8)M), a carboxylic ionophore, facilitated IgE-ricin A-chain (3 X 10(-7)M) toxicity in a dose-related fashion ith significant reductions in [3H]leucine incorporation compared to cells exposed only to monensin. This enhanced toxicity could be inhibited by the addition of both anti-ricin A-chain or anti-IgE, suggesting that different routes of intracellular processing may play a role in determining the toxicity of the IgE-ricin A-chain conjugate. Ricin B-chain (5 X 10(-7) and 5 X 10(-8)M) added to free ricin A-chain (10(-6)-10(-8)M) reproducibly facilitated toxicity, and this toxicity could be inhibited (30-90%) by lactose (50 mM). Ricin B-chain also facilitated IgE-ricin A-chain (2.75 X 10(-8)M) toxicity; however, this toxicity was not affected by lactose. The data suggest that ricin B-chain potentiates the cytosolic access of internalized IgE-immunotoxin and that the binding and internalization of the toxin was mediated via the IgE Fc receptor. A second type of IgE-ricin A-chain conjugate was synthesized whereby both IgE and ricin A-chain were derivatized with SPDP. RBL cells were killed in a dose-dependent manner by this IgE-ricin A-chain conjugate (2.5 X 10(-6)-2.5 X 10(-9)M) without requiring the addition of monensin or ricin B-chain. These data indicate that the intracellular route and processing of internalized immunotoxin is critical to eliciting toxicity.