Transplantation Antigens

114

There is increasing evidence that developmental associations among embryonic cells are mediated by specific components of the cell surface. Earlier work has indicated that such components are extruded into the medium of primary monolayer cultures of embryonic cells, and that they represent the active constituents of the tissue-specific cell-aggregating factors isolated from the supernatant medium of such cultures. We presently report that tissue-specific cell-aggregating factors can be obtained directly from embryonic tissues, and describe the isolation and partial purification of retina-specific factor from a cell-membrane preparation derived from retina tissue of the chick embryo. Extraction of the purified membrane preparation with 1-butanol yielded an activity in the aqueous phase which resides in a protein, probably a glycoprotein, with an estimated molecular weight of 50,000 in solution. This material could be obtained from retinas of embryos not older than 13 days, and only pre-13-day cells responded to its cell-aggregating activity. By these characteristics, this membrane-derived retina factor closely resembles the retina cell-aggregating glycoprotein previously purified from the supernatant medium of retina cell cultures. It is of special interest that the cell-aggregating protein is obtainable from cellular membranes during those stages of development when retina cells are most actively engaged in histological organization. Work in progress indicates that, by the procedures described herein, tissue-specific cellaggregating factors can also be obtained from membrane preparations of other embryonic tissues.

Section: Resultsmentioning

confidence: 99%

Isolation of retina-specific cell-aggregating factor from membranes of embryonic neural retina tissue.

Hausman¹,

Moscona²

1976

114

“…Various methods have been used to solubilize the cell surface-bound glycoproteins with serologic HL-A activity (1). The current methods used most successfully involve proteolytic treatment with papain, sonication, or extraction with high concentrations of salt (2)(3)(4), and apparently yield HL-A antigens differing in physical-chemical characteristics (4)(5)(6).…”

mentioning

confidence: 99%

Highly Purified Papain-Solubilized HL-A Antigens Contain β ₂ -Microglobulin

Peterson¹,

Rask²,

Lindblom³

1974

234

HL-A antigens comprising 11 different antigenic specificities were isolated after papain solubilization of spleen-cell membrane constituents. During the entire purification procedure, /32-microglolbulin appeared together with the HL-A antigens. The highly purified antigens were composed of two polypeptide chains. The large subunit carried the antigenic specificity whereas the small polypeptide chain was very similar, if not identical, to f32-nmicroglobulin. The two HL-A antigen polypeptide chains were held together by noncovalent interactions only, and 32-mnicroglobulin, isolated from urine, could replace the small subunit in forming a complex with the large polypeptide chain. The topographical relationship in the cell menibrane between 32-microglobulin and the large HL-A antigen polypeptide chain is unknown. The two polypeptide chains may be fortuitousdy bound as a result of the solubilization procedure.The major human histocompatibility determinants, HL-A antigens, are cell-surface markers present on lymphocytes and other nucleated cells. The great polymorphism exhibited by the histocompatibility antigens has severely impeded their isolation and chemical characterization. Various methods have been used to solubilize the cell surface-bound glycoproteins with serologic HL-A activity (1). The current methods used most successfully involve proteolytic treatment with papain, sonication, or extraction with high concentrations of salt (2-4), and apparently yield HL-A antigens differing in physical-chemical characteristics (4-6).Papain-solubilized HL-A antigens contain two polypeptide chains, one carrying the serologic activity and the other without serologically detectable HL-A antigenic determinants (6, 7). The latter of the two polypeptide chains has an estimated molecular weight of 10,000-12,000 (6, 7) and occurs in serum and urine (7,8). We earlier demonstrated that. the lowmolecular-weight plasma proteinB2-microglobulin, with molecular weight 11,800, is present on the surface of leukocytes (9). Amino-acid sequence analysis of this protein has revealed extensive homology with heavy and light chains of immunoglobulin G (9, 10). It was suggested that 02-microgiobulin is a free immunoglobulin domain that evolved before the gene duplication events giving rise to regular immunoglobulin polypeptide chains (9). Later work is compatible with this idea, since 02-microglobulin is manufactured by various mesenchymal and epithelial human cell lines (11). Furthermore, no correlation between immunoglobulin synthesis and 02-microglobulin production has been found (11-13). Isolation of HL-A Antigens. Four preparations of HL-A antigens were made. Briefly, a crude spleen-cell membrane fraction was suspended in 0.01 M Tris HCl buffer, pH 8.1, containing 5 mM cysteine and the protein concentration was adjusted to 10 mg/ml. To this membrane fraction, 2 mg of twice crystallized papain (20 units/mg, Sigma) were added per ml and proteolysis proceeded for 30 min at 370. The supernatant recovered after centrifugation of the papain...

“…They occur principally on the surface membrane (3,4) and can be removed by various procedures, including the use of proteolytic enzymes (5), autolytic procedures (6), detergents (7), ultrasonic radiation (8), and high salt concentrations (9). Among enzymic methods, papain treatment of membrane preparations, introduced by Nathenson and Shimada (10), reproducibly yields a soluble product of molecular weight 30,000-60,000 about which considerable structural information is now being accumulated.…”

mentioning

confidence: 99%

Enzymic Removal and Re-expression of a Histocompatibility Antigen, HL-A 2, at the Surface of Human Peripheral Lymphocytes

Turner

Strominger

Sanderson

1972

The removal by papain of a histocompatibility antigen, HL-A 2, from the surface of intact peripheral blood lymphocytes has been examined. Up to 80% of the antigenic determinant can be removed within 90 minutes. If the cells are reincubated in nutritive medium, re-expression of HL-A 2 is complete within 6 hours. Reexpression is reversibly inhibited by puromycin (50 ug/ml), but actinomycin D (6 ug/ml) only partially inhibited reexpression after 2 hours of prior incubation with the drug.