Biomedical subjects

M L Organist

Publications and source records attributed to M L Organist.

6 recordsLinked to original sources

PubMed1988

Processing of the human IM-9 lymphoblast substance P receptor. Biosynthetic and degradation studies using a monoclonal anti-receptor antibody.

Cellular membrane receptors for the immunostimulatory neuropeptide substance P have been previously identified on the cultured lymphoblast cell line, IM-9. The regulation of this receptor by ligand and the contribution to its molecular weight by N-linked sugars was studied by incubating IM-9 cells for 14 hr in the presence of [35S]met with or without substance P and tunicamycin, respectively. Cells were lysed and the receptor proteins were immunoprecipitated with an anti-receptor monoclonal antibody. SDS-PAGE analysis of untreated cellular lysates revealed specifically precipitated proteins of 38 kD and 33 kD, which were down-regulated by substance P. In tunicamycin-treated cells, whose substance P binding was not affected, the major immunoprecipitated protein had an apparent Mr of 29 kD. The time course of receptor processing was studied by pulse chase analysis. Three proteins of molecular weights 38 kD (mature receptor), 36 kD and 33 kD (receptor precursors) were identified for time periods of 30 min to 4 hr. The half life of the mature receptor and its precursors was approximately 1 hr and 0.5 hr, respectively. Results from the present studies suggest that the lymphocyte substance P receptor is translated as a precursor protein that is glycosylated.

Antibodies, Monoclonal↗

PubMed1987

Characterization of a monoclonal antibody against the lymphoblast substance P receptor.

A murine monoclonal antibody to the IM-9 lymphoblast substance P (SP) receptor has been produced which recognizes the membrane-associated proteins of the SP receptor as demonstrated by immunoprecipitation of [125I]SP affinity-labeled and [35S]methionine biosynthetically labeled IM-9 soluble membranes. SP and anti-SP receptor binding to [35S]methionine-labeled IM-9 cell proteins were directly compared by attachment of each to affinity supports. Eluants from these affinity columns were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed an equivalent 33-kDa protein in both cases. This protein corresponds to one of the previously described [125I]SP specifically affinity-labeled membrane-associated proteins. In addition, two-color fluorescence-activated cell sorter analysis with human peripheral blood T lymphocytes with fluorescein-SP and rhodamine-labeled antireceptor antibody revealed a distinct population of cells (20 to 30%) that were equally labeled by both the fluorescent peptide and antibody. This result indicates that the anti-SP receptor antibody recognizes an epitope of the receptor that is common to both human peripheral blood T lymphocytes and IM-9 lymphoblast cells.

Animals↗

PubMed1987

Immunoaffinity purification of membrane protein constituents of the IM-9 lymphoblast receptor for substance P.

Substance P (SP) is an undecapeptide neuromediator that stimulates human T-lymphocyte function by binding to stereospecific membrane receptors. Human IM-9 cultured B-lymphoblasts express approximately 20,000 receptors per cell for [125I]SP with a Kd of 0.3 nM. [125I]SP was specifically crosslinked by disuccinimidyl suberate to IM-9 cell membrane proteins of 78, 58, and 33 kDa. An indirect immunoaffinity purification procedure has now been developed based on immunoabsorption of detergent-solubilized [125I]SP-labeled IM-9 cell membrane proteins to anti-SP antibody that was bound to an epoxide ultraffinity high-performance liquid chromatography column, followed by elution in acidic 8 M urea. The 58- and 33-kDa SP-receptor complexes were purified to apparent homogeneity by immunoaffinity chromatography and identified by autoradiography and silver staining of sodium dodecyl sulfate-polyacryl-amide gels.

Affinity Labels↗

PubMed1987

Purification of the 33,000-dalton ligand binding-protein constituent of the lymphoblast substance P receptor.

Substance P (SP) acts as an immunoregulator by binding to specific functional cell surface receptors on a subpopulation of human T-helper lymphocytes. Receptors with similar properties have also been characterized on the human IM-9 B-lymphoblast cell line. Four distinct proteins of molecular weight (MW) 33,000, 58,000, 78,000, and 116,000 can be specifically affinity labeled using [125I]-SP Bolton-Hunter reagent and disuccinymidyl suberate (DSS). Peptide-mapping studies of these individually purified affinity labeled proteins have shown that the 33,000 MW membrane protein is present in the higher molecular weight cross-linked proteins. In the present studies, the 33,000 MW affinity-labeled protein was purified using semipreparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by high-performance hydroxylapatite chromatography. Starting with 800 mg of affinity-labeled protein, the final yield was 39.0 micrograms of 33,000 MW affinity-labeled protein. Based on an estimate of 53 mg receptor per 800 mg membrane protein, this represents an overall yield of greater than 70%. Peptide mapping was done by digesting 20 micrograms of the receptor protein with bovine trypsin. The proteolytic fragments were separated by reverse-phase high-performance liquid chromatography, and the amino acid content of 13 distinct peptides was determined. With this procedure, sufficient receptor can now be purified so that partial amino acid sequences can be obtained for further structural studies.

Amino Acids↗

PubMed1987

Substance P and immunoregulation.

Peptides released from peripheral nerve endings in mammals, including the tachykinin substance P (SP) and vasoactive intestinal polypeptide (VIP), are potent mediators of smooth muscle and vascular functions. Significant neurophysiological activities of SP and VIP include the transmission of nociceptive and interneuron excitatory signals, respectively. SP has been shown to modulate distinct immediate hypersensitivity responses by stimulating the generation of arachidonic acid-derived mediators from mucosal mast cells but not basophils. Functions of mononuclear and polymorphonuclear leukocytes that characterize the inflammatory response and that are altered by SP include chemotaxis, lysosomal enzyme release, and phagocytic activities. The effects of SP on cell-mediated immunity are largely stimulatory, in that synthesis of DNA, protein, and immunoglobulin by mature T and B lymphocytes, respectively, is significantly enhanced at nanomolar concentrations of the neuropeptide. Functionally relevant receptors for SP on T lymphocytes have been demonstrated by cell sorter and radioligand-binding techniques, and the lymphocyte membrane proteins that comprise the SP receptor are currently being isolated and purified to homogeneity. The characterization of the structure of the SP lymphocyte receptor and identification of the receptor gene will permit detailed analyses of the molecular interactions between the immune and nervous systems.

Animals↗

PubMed1986

Binding characteristics and affinity labeling of protein constituents of the human IM-9 lymphoblast receptor for substance P.

The neuropeptide substance P (SP) stimulates human T-lymphocyte function in vitro. Human blood T-lymphocytes and cultured human IM-9 B-lymphoblasts express 7,000-10,000 and 25,000-30,000 substance P receptors per cell, respectively. The specific binding of 125I-SP is retained in IM-9 lymphoblast membranes solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) at a detergent-to-protein ratio of 1.0. In addition, specific and reversible SP binding to soluble IM-9 cell membrane proteins is demonstrated by gel filtration. The saturation of binding of 125I-SP to both intact and solubilized IM-9 cell membranes attained a steady state after 40-50 min at 4 degrees C. Scatchard analysis of the concentration dependence of 125I-SP binding to IM-9 cell membranes revealed a KD of 0.87 +/- 0.8 nM (mean +/- S.D., n = 4), which is similar to that observed in intact cells, and a density of receptors of 21 +/- 3 fmol/mg of membrane protein (mean +/- S.D.). Binding of 125I-SP to solubilized membranes demonstrated a KD of 0.75 +/- 0.33 nM (mean +/- S.D., n = 3) and a density of receptors of 3.7 +/- 1.5 fmol/mg of membrane protein (mean +/- S.D., n = 3). Affinity cross-linking of 125I-SP by disuccinimidyl suberate to intact IM-9 cells and membranes revealed specifically labeled proteins of Mr 58,000 and 33,000 in cells, and 58,000, 33,000, and 16,000 in membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Competitive effects of substituent peptides of SP on cross-linking and 125I-SP binding to membranes demonstrated that the SP receptor recognized the carboxyl-terminal domain of the peptide. Membranes from cells preincubated in vitro for 12 h at 37 degrees C with 10(-8) M SP demonstrated a decrease in SP receptor density to 13 +/- 2 fmol/mg (mean +/- S.D., n = 2), and a parallel diminution in the specific labeling of membrane proteins of Mr 58,000 and 33,000. These observations suggest that solubilization in CHAPS preserves the binding characteristics of the IM-9 lymphoblast receptor for SP, and that affinity cross-linking techniques identify by sodium dodecyl sulfate-polyacrylamide gel electrophoresis membrane proteins that are specifically labeled by SP.

Affinity Labels↗