D.L. Deane

Glycoproteins of the Chromaffin Granule Membrane: Separation by Two-Dimensional Electrophoresis and Identification by Lectin Binding

Abstract: The proteins of highly purified chromaffin granule membranes were separated by one or two‐dimensional electrophoresis, then transferred to nitrocellulose sheets; glycosylation was investigated by binding of several different radioiodinated lectins. Over 20 different glycosylated components were identified; comparison with mitochondrial and microsomal fractions suggested that most of the major glycoproteins are genuine components of the chromaffin granule membrane, rather than contaminants originating in other organdies. Two dimensional electrophoresis revealed heterogeneity within several of the glycoproteins, and this is ascribed to differences in the state of glycosylation, on the basis of shifts in electrophoretic mobility produced by treatment with neuraminidase. Neuraminidase treatment of chromaffin granule membranes also enhances the binding of many lectins. The identities of the lectin‐binding bands are discussed: neither cytochrome b561 nor the F1‐like ATPase appears to be glycosylated. Chromogranin A, although a glycoprotein, does not bind any of the lectins tested, but a number of concanavalin‐A binding proteins, as well as dopamine β‐hydroxylase, are present in the chromaffin granule lysate.

Association between pregnancy-associated alpha2-glycoprotein (alpha2-PAG) and mixed leucocyte reaction determinants on the leucocyte surface.

α2-PAG is present on the surface of mononuclear blood leucocytes and can be demonstrated predominantly on B-lymphocytes and monocytes. Pretreatment of cells with antibody to α2-PAG leads to a marked reduction in Fc-rosette formation. Competitive blocking experiments with specific antisera reveal a particularly close asociation between α2-PAG and MLR (mixed leucocyte reaction) determinants on the cell surface. These findings suggest one mechanism whereby α2-PAG may modify cell-mediated immune responses.

Subsets of Human D Locus Products Identified by a Series of Monoclonal Antibodies

Abstract A series of monoclonal antibodies, prepared against the Burkitts lymphoma line Daudi, define antigens predominantly expressed on cells of the B lymphoid series. Four antibodies which react with HLA-DR or DR-like antigens have enabled the serological definition of at least two and possibly three distinct subsets of molecules. Of these four antibodies, one (DA6.164) defines a DR allotype associated epitope common to all DR types tested except DR 7 homozygotes. A second (DA6.147) recognizes a DR-like antigen which is expressed in abundance on B lymphoblastoid cells, weakly on peripheral blood B cells and not at all on activated T cells. Two further antibodies (DA6.231 and DA6.121), with indistinguishable specificity, detect an epitope apparently common to more than one set of DR molecules.

Methods of detecting mature human Ia-like antigen and their metabolic precursors

Ia antigens were shown to be present in the cell almost exclusively as mature alpha beta dimers which split into separate alpha and beta chains after boiling in SDS. In contrast metabolically labelling the cells with [35S]methionine resulted in only free alpha and beta chains being labelled. It is concluded that this widely used type of labelling, although useful for studying intermediate synthesis, should not be used for labelling mature cell surface molecules.

Monoclonal Anti-B Cell Antibodies: their Effects on Human Mixed Lymphocyte Reactions

Abstract Three monoclonal antibodies which recognise determinants on the surface of human B cells have been studied for their effect on mixed lymphocyte reactions (MLR). MLR inhibition was observed under a variety of conditions. While it was not possible to ascribe lymphocyte activation to a single determinant recognised by any of the antibodies, further analysis of the mechanisms involved in lymphocyte activation should be possible through the use of monoclonal reagents.

TOWARD ANALYSIS OF THE HLA-DR SYSTEM WITH MONOCLONAL ANTIBODIES

Publisher Summary This chapter describes three monoclonal antibodies—DA6 231, 164, and 147— that precipitate glycoprotein dimers consisting of 34 K and 29 K subunits. 231 and 147 recognize DR-common epitopes, whereas 164 binds to all except DR7 homozygous cell lines. Protein transfer from sodium dodecyl sulfate (SDS) polyacrylamide gels to nitrocellulose paper, followed by probing with the monoclonal antibodies, has permitted assignment of the 231 and 147 epitopes to 29 K and 34 K subunits, respectively. Mutual binding inhibition between 231 and 164 is incomplete in one direction, suggesting that there is a major proportion of 231 + 164 + and a minority of 231 + 164 – 29K molecules expressed on all DR positive cells tested, except DR7 homozygous cells that have lost the 164 epitope. The serological expression of the 147 epitope at the cell surface is complex. The 231/147 ratio on peripheral and activated β lymphocytes is much higher than on β lymphoblastoid lines or on chronic lymphocytic leukemia cells. No significant binding of 147 can be detected on the strongly 231 + 164 + -activated cultured τ cells. Preliminary evidence suggests that molecules that can bind 147 are released on cell lysis even from 147 – activated τ cells.