David Pressman

Production of free light chains of immunoglobulin by a hematopoietic cell line derived from a patient with multiple myeloma.

Summary A cell line derived from a myeloma patient was found to produce only λ−type light chains of immunoglobulin which were secreted into the culture medium. There appears to be no formation of γ−, α−, or μ−heavy chains or of intact immunoglobulins. The light chains produced gave a reaction of identity with the Bence-Jones protein of the original patients urine upon immunodiffusion and showed a similar molecular size upon gel filtration. The cells produced about 15 μg λ−chain per day per 106 cells (45 × 107 chain molecules per day per cell) under the culture conditions used.

Molecular identification of human Ia antigens coded for by a gene locus closely linked toHLA-DR locus

Human Ia(-like) specificities controlled by gene loci other thanHLA-DR were searched for at the molecular level in cells of human B-cell-type cell lines which carrytwo established DR specificities. Chevalier cells of DRw3 and 7 and U698M cells of DRw2 and 4 were used. Their Ia molecules were partially purified, radioiodinated and analyzed for Ia specificities by the direct binding and sequential binding assays with a selected panel of human Ia alloantisera. It was possible in both the cell lines to define a third subset of Ia molecules carrying a new specificity in addition to two Ia subsets carrying the established DR specificities. The new specificity was detected by putative anti-DRw4 and anti-DRw7 antisera and was closely associated with DRw4 and DRw7 at population level. It was thus designated provisionally as BR4X7. These results suggest that the BR4X7 specificity is coded for by a separateIa locus closely linked toHLA-DR locus. The determinant(s) responsible for BR4X7 was located on the small subunit of Ia molecules.

Plasma and Blood Volumes of Mouse Organs, As Determined with Radioactive Iodoproteins.

Summary The plasma and blood volumes of mice and of various mouse organs were determined by injecting mice with a protein iodinated with iodine containing tracer quantities of radioactive iodine, and determining the amount of radioactivity present in the various organs. The average plasma and blood volumes of the mice were found to be 6.7 ml and 12.7 ml per 100 g of body weight, respectively. The average plasma volume in ml per 100 g of wet tissue for the brain was 1.6; kidney, 19.1; liver, 20.2; lung, 23.9; small intestine, 5.0; spleen, 9.2; submaxillary gland, 5.9; testes, 3.4.

Characterization of soluble substances in plasma carrying HL-A alloantigenic activity and HL-A common antigenic activity.

SUMMARY The soluble HL-A-active substances found in plasma have been investigated using a radioimmunoassay to determine their HL-A alloantigenic activities, such as HL-A1 and HL-A2, and the HL-A common activities that are characteristic of a number of the HL-A antigens. Plasma, which accounts for about 10% of a particular HL-A alloantigenic activity that can be measured in the whole blood, contains a series of soluble molecular fragments related to HL-A antigens of different sizes which appear to be degradation products of cell membranes. Gel filtration of plasma yields three fractions carrying HL-A alloantigenic or HL-A common activities or both. The molecules with a molecular size of about 2–8 × 105 daltons appear to represent a definite portion of the cell membrane structure that carries the intact HL-A antigen. Molecules of 48,000 daltons appear to be identical to the molecular fragments that have been derived from cultured lymphoid cells by papain digestion of membrane fractions. Papain digestion of the first fraction yields fragments apparently identical to those in the second. The third fraction of 10,000 daltons contains a soluble component which carries no HL-A alloantigenic activity and only a part of the HL-A common activities present on the molecules of the other two fractions.

[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.

Amino acid composition and physicochemical properties of mouse β2-microglobulin

Summary Highly purified preparations of an 11,000-dalton substance with a pI value of 7.5 have been obtained from 3 M sodium thiocyanate extracts of spleen and liver cell membranes of A/J strain mouse. This substance appears to be the mouse homologue of human β 2 -microglobulin since a) it has a homology in amino acid composition, including the presence of twohalf cystine residues, to constant domains of mouse immunoglobulins as well as to human β 2 -microglobulin, b) it is not distinguishable antigenically from the 11,000-dalton component isolated from H-2 antigen molecules and c) it has a weak but significant cross-reactivity with human β 2 -microglobulin.

Partial amino acid sequence of mouse β2-microglobulin

Abstract A highly purified preparation of mouse β2-microglobulin has been obtained from sodium thiocyanate extracts of liver cell membranes of A J strain mice and the amino acid sequence has been partially determined. The first 40 residues have been assigned except for position 34. In the sequence, the mouse protein differs only at 9 positions from human β2-microglobulin at 8 positions from the dog homologue and at 11 positions from the rabbit homologue. The sequence has also a homology to the constant regions of mouse γG2a, most closely to the CH3 region. These data support the conclusion that the mouse protein is indeed the mouse homologue of human β2-microglobulin.

IgM and IgG anti-hapten antibody: hemolytic, hemagglutinating and precipitating activity.

Summary Hemolytic and hemagglutinating activities of IgM and IgG antibody against the p-azobenzenearsonate (Rp) group were compared using purified antibody preparations. IgM antibody showed a considerably higher activity in both hemolytic and hemagglutinating systems. The hemolytic and hem-agglutinating activities of IgM antibody were both 180 to 60 times higher on a molar basis than those of IgG antibody when measured with erythrocytes to which Rp-groups were attached. The reduced-alkylated product of IgM antibody showed little activity in both hemolytic and hemagglutinating systems, although the reduced-alkylated product had been shown previously to retain hapten-binding activity. IgM antibody also lost precipitating activity with a polyvalent hapten-protein conjugate upon reduction and alkylation. Loss of hemagglutinating and precipitating activities is probably due to univalency of 6S subunits produced by reduction.

Specific Localization of Anti-Rat-Lung Serum in the Lung.

Summary By labelling anti-rat-lung serum with radioiodine and injecting it into rats, it was found that the anti-rat-lung serum contained antibody which localized specifically in the lung. It also contained antibody which cross-reacted with and localized in the kidney. Anti-rat-brain, -liver, and -heart sera were also investigated and showed no specific localization, but these experiments were considered inconclusive. The data presented show that kidney tissue is not unique in being able to localize antibody from tissue homologous antiserum.

HLA-DR typing by radioimmunoassay

A radioimmunoassay procedure is described by which peripheral blood lymphocytes can be typed for HLA-DR specificities. The major advantages of this method are the following: simple and reproducible procedure, no need for B lymphocyte separation, no need for optimal viability, and no need for preabsorption of antisera with platelets. This method will find an application in the genetic and biochemical analysis of the HLA complex, and in the clinical tests of Ia antigens for diagnostic or prognostic purposes and in retrospective transplant studies.