PubMed Health⌕ Search

Biomedical subjects

M J Crumpton

Publications and source records attributed to M J Crumpton.

At least 91 records · Page 5Linked to original sources
PubMed1984

Cellular distribution of p68, a new calcium-binding protein from lymphocytes.

A Ca2+-binding protein of mol. wt. 68 000 ( p68 ) is a major component of a Nonidet P-40 insoluble fraction of human and pig lymphocyte plasma membrane. An affinity-purified rabbit antibody has been produced against p68 and used to study its cellular distribution. The antibody stained fixed and permeabilised human B lymphoblastoid cells, peripheral blood lymphocytes and sections of human tonsil. Whole cells, however, were not stained, indicating that the protein was not represented at the cell surface. This assignment was consistent with the detection of p68 in immunoprecipitates from biosynthetically- but not surface-labelled cells. It is concluded that p68 is located on the cytoplasmic face of the plasma membrane. Subcellular fractionation experiments confirmed that p68 was largely membrane-bound in lymphocytes, although a small soluble fraction (approximately 10% of the total) was detected. Sub-fractionation of lymphocyte membranes revealed that p68 was associated not only with the plasma membrane but also with other endomembrane systems. As judged by immunoprecipitation, p68 was present in a variety of cultured cell lines of both lymphoid and non-lymphoid origin. p68 demonstrated a diffuse distribution in fixed and permeabilised fibroblasts which did not correspond to the distribution of either microfilaments or intermediate filaments. However, in detergent-extracted cells the protein was localised in a lamina-like network. A similar immunofluorescent staining pattern has recently been observed for spectrin-related proteins in the detergent-resistant cytoskeleton of fibroblasts. It is suggested that p68 is part of a sub-membranous cytoskeletal complex not only in lymphocytes but also in other cell types.

Animals↗
PubMed1983

Human class I histocompatibility antigens (HLA-A,B,C). A small proportion only is phosphorylated.

Human Class I HLA antigens (HLA-A,B,C) were isolated by immune precipitation from cells labelled with 32P, [35S]methionine or 125I (by lactoperoxidase-catalysed cell-surface iodination) and were analysed using both one- and two-dimensional electrophoretic systems. In several B-lymphoblastoid cell lines and in human peripheral blood lymphocytes the electrophoretic mobility of the 32P-labelled HLA-A,B,C heavy chains consistently differed from that of molecules labelled by other means. Thus the 32P-labelled heavy chains appeared to be larger and to possess a more acidic pI than did heavy chains labelled with [35S]methionine or 125I, or identified by Coomassie Blue staining. Phosphatase treatment of immunoprecipitates, under conditions where 32P-labelled antigens were shown to be dephosphorylated, did not affect the mobilities of the [35S]methionine-labelled heavy chains. On glycosidase treatment, the positions of the 32P-labelled heavy chains were affected by neuraminidase but not by endo-beta-N-acetylglucosaminidase H. These results imply that phosphorylated HLA-A,B,C antigens comprise only a small proportion (relative to the total cellular HLA-A,B,C antigens) of the biosynthetically mature molecules. The possible significance of such heterogeneity is discussed.

B-Lymphocytes↗
PubMed1983

Biosynthesis and molecular nature of the T3 antigen of human T lymphocytes.

Immunoprecipitates of the T3 antigen prepared from HPB-ALL cells by using the monoclonal antibody UCH-T1 were analysed by SDS-polyacrylamide gel electrophoresis. Cells which had been biosynthetically labelled for up to 4 h gave a major polypeptide of mol. wt. 19 000 plus two weaker, more diffuse bands of mol. wts. 21 000 and 23 000, whereas surface labelled cells gave a prominent band of mol. wt. 19 000, a major band of 21 000 and a weaker diffuse band of approximately 26 000. As judged from their sensitivity to proteinase-K digestion, all the above polypeptides possess a transmembrane orientation. Digestion with endoglycosidases H and F (endo-H and endo-F), and tunicamycin treatment indicate that all the polypeptides, except that of 19 000 mol. wt. are N-glycosylated. The 21 000 and 23 000 mol. wt. chains possess both immature and mature oligosaccharide units, whereas the 26 000 mol. wt. band apparently has mature units only. Pulse chase experiments combined with digestion by endo-F and endo-H suggest that the N-glycosylated polypeptides are derived from two polypeptides of mol. wts. 14 000 and 16 000. It is concluded that the T3 antigen is derived from three different non-glycosylated polypeptides two of which are subsequently N-glycosylated to give the 21 000, 23 000 and 26 000 forms. The cell surface T3 antigen most probably comprises at least two distinct, non-covalently associated polypeptides, but the number and types of polypeptides giving rise to the whole molecule and whether different complexes exist is at present unclear.

Antibodies, Monoclonal↗
PubMed1983

The cell surface and its metabolism.

The cell surface structure is highly dynamic. In particular, binding of ligand induces the redistribution of receptors on the cell surface as well as the internalisation of ligand-receptor complexes. Internalisation in turn leads to a recycling of the receptor or to a decrease in the cell's responsiveness to the ligand. Modulation of the cell surface structure is apparently regulated intracellularly by components of the cell's cytoskeleton. A crucial component in this respect is likely to be a sub-membranous filamentous network that is linked directly to the cytoplasmic face of the surface membrane. In erythrocytes this network can be separated from purified preparations of the plasma membrane by virtue of its insolubility in nonionic detergents. Application of this procedure to the plasma membrane fraction of human B lymphoblastoid cells has yielded a detergent-insoluble residue comprising actin and a 68,000-Mr polypeptide as major components, together with polypeptides of 28,000-, 33,000- and 120,000-Mr as prominent but more minor components. The association of the 68,000-Mr protein with the detergent-insoluble residue and the original plasma membrane is Ca2+-dependent. Burkitt lymphoma cells differ noticeably from lymphoblastoid cells in that the 68,000-Mr protein is not associated with the inner face of the surface membrane. This difference may reflect the malignant phenotype of Burkitt lymphomas or the hypothetical sub-population of normal B lymphocytes from which the lymphomas are derived.

Burkitt Lymphoma↗
PubMed1983

Biochemical analysis of the polymorphism of three HLA-D region antigens.

Two-dimensional gel electrophoretic analyses of immunoprecipitates of the HLA-D region antigens revealed three beta (Mr 28 X 10(3) to 30 X 10(3) structurally polymorphic polypeptides. Analyses of the nonglycosylated beta polypeptides indicated that their polymorphism is most probably due to variations in amino acid structure. The polymorphism has been examined using lymphoblastoid cell lines that were derived from different geographical populations (North European and Italian) and that expressed various HLA-DR alloantigenic specificities. The results confirm that the positional variation of one beta polypeptide (namely beta-1) correlated with the HLA-DR allospecificity and was not influenced by the ethnic origin of the individual donating the cells. The positional variation of a second beta polypeptide (namely beta-2) was consistent with strong linkage dis-equilibrium between the corresponding genetic locus and the HLA-DR locus. Further, the association with a particular HLA-DR allospecificity was population-dependent; that is, different beta-2 polypeptides were associated with the same HLA-DR specificity in different geographical populations. In contrast, the position of the third beta polypeptide (namely, beta-3) which corresponds to the HLA-DC locus, did not necessarily vary with the HLA-DR specificity; that is, apparently the same beta-3 polypeptide was associated with different HLA-DR specificities provided the cells were of the same ethnic origin. The data indicate that lymphoblastoid cells express at least three sets of HLA-D region antigens that are encoded by different loci including HLA-DR and DC.

Antibodies, Monoclonal↗
PubMed1982

Demonstration of two distinct light chains in HLA-DR-associated antigens by two-dimensional gel electrophoresis.

The polypeptide composition of HLA-DR-associated antigens was analyzed by two-dimensional nonequilibrium pH gradient electrophoresis (NEPHGE)/sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Glycoprotein fractions from B lymphoblastoid cell lines homozygous for the DR antigen were used as antigen source. The HLA-DR-associated antigens were isolated by immunoprecipitation using a monoclonal anti-DR antibody, TDR31.1. Two polypeptides corresponding in molecular weight to the HLA-DR-associated antigen beta chain were detected. These polypeptides were shown to be different molecules on the basis of their separation on NEPHGE/SDS-PAGE analysis, their rates of turnover relative to the other polypeptides of the HLA-DR-associated antigens, and their noncoordinate alteration in position on NEPHGE/SDS-PAGE two-dimensional analysis of different cell lines. Of the two beta polypeptides, only one was invariant in position on NEPHGE/SDS-PAGE analyses of cell lines homozygous for the same DR specificity. It appears therefore that the position of one of these beta polypeptides correlates with DR specificity, while the other beta polypeptide exhibits positional variation on NEPHGE/SDS-PAGE analysis, indicative of polymorphism at a second HLA-DR-associated locus.

Antibodies, Monoclonal↗
PubMed1982

Two-dimensional gel analysis of the polypeptides precipitated by a polymorphic HLA-DR1,2,w6 monoclonal antibody: evidence for a third locus.

The SDR1 monoclonal antibody reacts only with cells which express the HLA-DR1, 2,w6 or w8 allogeneic specificities. Two-dimensional nonequilibrium pH gradient/sodium dodecyl sulfate polyacrylamide gel electrophoretic analyses of SDR1 immunoprecipitates from [35S]methionine biosynthetically labeled cells revealed the typical pattern of Ia antigens, namely two polypeptides of about 34kDa and 29kDa (designated epsilon and beta-3) as well as the "basic invariant spot" of about 31 kDa. The epsilon and beta-3 polypeptides were only weakly represented in similar analyses of immunoprecipitates performed using a monomorphic HLA-DR monoclonal antibody, TDR31.1. The epsilon and beta-3 polypeptides of B lymphoblastoid cell lines homozygous for HLA-DR2 and w6 were structurally polymorphic as judged by two-dimensional gel analyses. This polymorphism was independent of the HLA-DR specificity. It is concluded that the SDR1 antibody recognizes a polymorphic set of Ia antigens that are coded by a locus other than HLA-DR. These antigens probably also express the MT1 (DC1, LB12) alloantigenic specificity and are most likely the human equivalent of the murine I-A subregion antigens.

Animals↗
PubMed1982

Glycoproteins in relation to cell differentiation and malignancy.

Differentiation and malignancy have been correlated with particular cell surface glycoproteins and may, in fact, be mediated by these glycoproteins. Theoretically, two fundamentally distinct mechanisms exist by which changes in cell surface glycoproteins in differentiation and malignancy can be effected. Firstly, a particular surface protein can be glycosylated differently. This amounts to a change in carbohydrate structure only. Secondly, a new surface protein can be synthesised that either carries a pre-existing [i.e. also attached to another surface protein (s)] or a new carbohydrate moiety; synthesis of a pre-existing protein(s) may also be suppressed. This amounts to a change in polypeptide structure that may or may not be coupled with a change in carbohydrate structure. Current evidence indicates that both mechanisms probably operate in both differentiation and malignancy.

Animals↗
PubMed1981

Biosynthesis and maturation of HLA-DR antigens in vivo.

The biosynthesis of the HLA-DR antigens was studied in the B lymphoblastoid cell line BRI 8. Three chains, of molecular weights 33,000 (alpha), 31,000 (p31), and 26,000 (beta) were detected intracellularly in pulse-labeled cells by immunoprecipitation with anti-(HLA-DR) sera. The alpha and beta chains were inserted asymmetrically into the rough endoplasmic reticulum as transmembrane polypeptides with the majority of the polypeptide chains oriented in the lumen. At this stage, both chains carried "high mannose" oligosaccharide units which were processed to the complex form during subsequent intracellular transport to the cell surface. The Mr = 31,000 polypeptide was also glycosylated but was structurally distinct from the alpha chain and was probably oriented differently in the lipid bilayer, with a much greater proportion of its polypeptide chain exposed in the cytoplasm. It ws not, therefore, a precursor of the alpha chain. The mature HLA-DR antigens at the plasma membrane comprised polypeptides of Mr = 34,000 and 28,000. These chains corresponded to the processed alpha and beta chains. Although the Mr = 31,000 component was only detected intracellularly, it was not ruled out that some or all of it may have been processed and exposed on the cell surface with an apparent molecular weight indistinguishable from that of the alpha chain.

B-Lymphocytes↗
PubMed1981

Phosphorylated lymphocyte plasma-membrane proteins.

Lymphocytes were labelled by incubation with [32P]Pi and their plasma membranes isolated. Analysis by one-dimensional and two-dimensional gel electrophoresis revealed a small number of strongly phosphorylated polypeptides. Two of these were especially prominent; they had molecular weights of about 52000 and 90000, were acidic and were apparently not glycosylated. Similar patterns were obtained for quiescent T- and B-lymphocytes from different species and for cultured lymphoblastoid cells, although the relative amounts of the labelled polypeptides varied. Immunoprecipitation analyses of the detergent-solubilized 32P-labelled plasma membranes indicated that the glycosylated polypeptide of the human major transplantation (HLA-A and HLA-B) antigens and its mouse and pig counterparts are phosphorylated. In contrast, no phosphorylation of the membrane-associated immunoglobulin, the mouse Thy-1 antigen or the human HLA-DRw(Ia) antigen was detected. The phosphorylation patterns of human peripheral blood and nude-mouse spleen lymphocytes did not change during the period 5-30min after mitogen stimulation. Therefore a change in the phosphorylation of plasma-membrane protein(s) is probably not an early biochemical event in the initiation of T-lymphocyte and B-lymphocyte growth, although a rapid transient change cannot be ruled out. Similar plasma-membrane phosphorylation patterns were also obtained by incubating the purified plasma membrane with [gamma-32P]ATP. The phosphorylation of the 90000-mol.wt. polypeptide was particularly rapid and was stimulated by the addition of cyclic AMP.

Animals↗
PubMed1980

Albumin associated with purified pig lymphocyte plasma membrane.

Plasma-membrane preparations purified from pig lymphocytes contained a major polypeptide component of mol.wt. about 68 000. This component was identified as pig albumin by the following comparisons with authentic pig serum albumin: (a) co-migration when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions; (b) identical isoelectric points; (c) similar "fingerprints" of arginine-containing tryptic peptides; (d) reactivity with anti-(pig albumin) serum. The albumin was bound tightly to the plasma membrane. Biosynthetic labelling of pig lymphocytes under a variety of conditions failed to provide evidence that albumin was synthesized by lymphocytes, suggesting that the plasma-membrane-associated albumin was of extraneous origin. Radiolabelled pig serum albumin, however, failed to bind to the plasma-membrane fraction when added before cell disruption. Although lymphocyte plasma membrane preparations from other species possessed a polypeptide of about 68 000 mol.wt., this was judged not to be albumin on the basis of electrophoretic mobility under non-reducing conditions; also, no polypeptide was precipitated by anti-albumin sera. It is concluded that pig lymphocyte plasma-membrane preparations possess albumin which, although firmly attached, was probably of extraneous origin. This association appeared not to be common to lymphocytes from other species.

Animals↗
PubMed1980

Preparation of resealed pig lymphocyte plasma-membrane vesicles.

Pig lymphocyte plasma-membrane vesicles that are impermeable to large molecules were prepared by means of centrifugation on a continuous dextran gradient. They were isolated in a yield of 60--80% of the total plasma membrane recovered, and were characterized as being impermeable to macromolecules by their exclusion of inulin (mol.wt. 5200) and Dextran T10 (mol.wt. 10 000).

Animals↗
PubMed1980

Antisera recognising HLA-A, -B and DRW antigens raised in rhesus monkeys.

Rhesus monkeys were immunized with partially purified HLA-A, -B, -C and DR antigens. The resulting sera were shown to have activity against species-specific determinants on both HLA-A, -B, -C chains and beta 2 microglobulin by the use of somatic cell hybrids. When this was removed by absorption, the sera showed activity against three of the four HLA-A and -B antigens in the immunogen when tested on a panel of peripheral blood lymphocytes and T cells. Antibodies recognizing HLA-DR antigens were detected by testing platelet absorbed sera on a panel of typed lymphoblastoid cell lines. After absorption to remove activity against species-specific determinants on the HLA-DR antigens, two cross reacting specificities were defined. One consisted of a determinant in common between HLA-DRw1, 2 and 6 and the other a putative determinant in common between HLA-DRw4, and 5. The nature and significance of these cross-reacting groups of HLA-DR antigens is discussed in the light of current HLA-DR serology and the nature of HLA antigens in general.

Animals↗