Barry P. Peters

The use of fluorescein-conjugated Bandeiraea simplicifolia B4-isolectin as a histochemical reagent for the detection of α-d-galactopyranosyl groups. Their occurrence in basement membranes

The Bandeiraea simplicifolia B4-isolectin, which combines specifically with α-d-galactopyranosyl groups, has been conjugated with fluorescein isothiocyanate and demonstrated to be a reliable histochemical probe for the detection of these groups in normal tissues of mouse, rabbit, rat and man. Specificity of binding of the fluorescein-conjugated B. simplicifolia B4-isolectin to native cryostat tissue sections was demonstrated in two ways: n1. n1. The hapten inhibitor methyl α-d-galactopyranoside prevented the binding of the lectin to tissues whereas the non-hapten methyl α-d-glucopyranoside did not. n n2. n2. Pretreatment of tissue sections with coffee bean α-galactoside abolished lectin binding whereas pretreatment with A. niger or E. coli β-galactosidase did not. The fluorescein-conjugated isolectin visualized α-d-galactopyranosyl groups in basement membranes and on the surface of certain epithelial cells of mouse, rat, rabbit, and on the surface of the TA3 murine mammary carcinoma. These studies suggest that the B. simplicifolia B4-isolectin may be of great utility in studying the family of α-d-galactosyl-containing glycoconjugates of basement membranes in pathological states accompanied by basement membrane changes, such as diabetes mellitus, and in neoplasms that secrete basement membrane.

Isolation of laminin by affinity chromatography on immobilized Griffonia simplicifolia I lectin

Laminin is a non-collagenous glycoprotein which occurs as a major component of basement membrane [1]. It consists of two polypeptide chains (M r 200 000 andM r 400 000)joined to each other by disulfide bonds [ 1 ]. Various cultured cells including epithelial cells elaborate laminin and it is believed that this glycoprotein may mediate epithelial cell attachment [2]. The EHS sarcoma is a transplantable mouse tumor which produces an extracellular matrix of basement membrane and which provides a rich source of laminin [3]. Here, we describe a one-step purification of laminin extracted from the EHS sarcoma.

Fluorometric analysis of amino sugars and derivatized neutral sugars.

Abstract A rapid and sensitive procedure for the analysis of neutral and amino sugars is presented. Neutral sugars are separated after conversion to the corresponding glycamines, while the amino sugars are analyzed without modification, using an automatic amino acid analyzer and fluorometric detection. The method has been applied for the analysis of glycoproteins and oligosaccharides of the complex and high-mannose types.

Laminin production by murine melanoma cells: possible involvement in cell motility

Three lines of B16 melanoma cells (B16-F1, B16-F10 and B16-BL6) were examined for motility in the micropore filter assay and for synthesis in culture of the basal lamina glycoprotein laminin. All three lines synthesized laminin as judged by the incorporation of [35S]methionine into immunoreactive laminin and secreted (or shed) laminin into the culture medium as indicated by biosynthetic labeling studies and enzyme-linked immunosorbent assays. Immunoreactive laminin was also seen on the surface of the cells as indicated by immunofluorescence staining and by complement-mediated killing. Analysis of [35S]methionine-labeled laminin immunoprecipitates by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) both with and without reduction of intersubunit disulfide bonds revealed that all three cell lines produced a similar array of laminin forms, and that the Mr=950kD laminin molecule (but not the uncombined subunits) was secreted into the culture medium. Laminin biosynthesis appeared to be limited by the availability of the Mr=400kD A subunit as shown by the intracellular accumulation of excess B subunit in the form of uncombined B subunit (Mr =200kD) and as a disulfide-linked B dimer (Mr=400 kD). The motility of all three cell lines was stimulated four- to five-fold by the addition of either exogenous laminin from the EHS sarcoma or culture medium from the B16 cells containing the secreted laminin. The stimulated motility was inhibited by antilaminin serum. These observations suggest that the laminin synthesized by the B16 melanoma cells themselves may facilitate their motility.