Sequential Lectin Research Articles

Galactose oxidase labeling of membrane proteins from human brain white matter

The use of galactose oxidase (EC 1.1.3.9) and tritiated sodium borohydride for labeling of membrane glycoproteins, described by Gahmberg and Hakomori 2, has previously been applied to the study of myelin glycoproteins of experimental animals 8. Rat brain myelin glycoproteins have been studied by sequential lectin affinity chromatography 12 and recently the lectin-binding capacity of rat central nervous system myelin glycoproteins has been characterized 7. Complex heterogeneity of the glycoprotein pattern of rat central nervous system myelin has been reported 7,8,12, and so a variety of glycoproteins can be expected to exist in human white matter membranes. Application of the galactose oxidase procedure to the study of human brain membranes could be useful in research concerning certain neurological diseases if the properties of autopsy brain material are taken into account. In this study, membrane proteins of human autopsy brain white matter were subjected to the galactose oxidase/NaB 3H 4 labeling procedure and the membranes labeled by this method or by the [ 3H]acetic anhydride techniques 6 were studied by lectin affinity chromatography using Lens culinaris phytohemagglutinin (lentil lectin) attached to Sepharose 4B beads.

Enhanced purification of Mullerian inhibiting substance by lectin affinity chromatography

Mullerian inhibiting substance (MIS), a secreted testicular product responsible for regression of the Mullerian ducts in the male mammalian embryo, was purified 7000 fold, exploiting the glycoprotein nature of this important fetal regressor to achieve enhanced purification. The present procedure employs media incubation of newborn calf testis, passage through DEAE Bio-Gel A and CM Bio-Gel A and sequential lectin affinity chromatography on wheat germ lectin (WGL)-Sepharose 6MB and concanavalin A (Con A)-Sepharose 4B. Strongly bioactive MIS was released from both lectin columns in the bound glycoprotein fraction only after elution with lectin-specific sugar. Carbohydrate analysis of the highly purified glycoprotein fraction eluted from Con A indicated the presence of both N-acetyl glucosamine and mannose, as would be expected from its sequential lectin affinity, as well as of galactose, galactosamine and N-acetyl neuraminic acid. Electrophoresis of this fraction on polyacrylamide-SDS gels showed an identical band pattern after staining with either Coomassie blue or periodic acid-Schiff reagent, further indicating that MIS is a glycoprotein.

Chromatographic behavior of pea lectin receptors from the 6C3HED murine ascites tumor

We have previously shown that pea lectin and concanavalin A bind to different receptors as well as to common receptors on the cell surface of 6C3HED murine ascites tumor cells (Allen, H.J. and Johnson, E.A.Z. (1976) Biochim. Biophys. Acta 436, 557–566). We have characterized the metabolically labeled pea lectin receptor from trypsinates of viable tumor cells by gel filtration and affinity chromatography. The pea lectin receptor eluted as an apparent high molecular weight glycopeptide ( M r 200 000) from Sephadex G-200 columns. Other glycopeptides were present in the trypsinates but they did not bind to either concanavalin A or to pea lectin in significant amounts. The high molecular weight glycopeptide was resistant to digestion by a high concentration of trypsin but it was absent in non-specific protease digests of viable cells. Sequential lectin affinity chromatography of Nonidet P-40 extracts of cells permitted the probable identification of the native glycoprotein corresponding to the high molecular weight glycopeptide which binds to pea lectin.

Isolation and immunologic characterization of the human platelet alloantigen, P1 A1

Platelets from patients with Glanzmann's thrombasthenia, acongential disorder of platelet function, are deficient in the membrane alloantigen. P1 a1 (Zw a). present on platelets of nearly all normal subjects. Because thrombasthenic platelets are also deficient in two membrane glycoproteins, designated IIb and IIIa. it has been suggested that one or both of these glycoproteins may carry the P1 a1 antigenic determinant. To address this question, the P1 a1 antigen was isolated by sequential lectin affinity chromatography of sodium deoxycholate extracts of platelet membranes, by indirect immunoprecipitation of Nonidet P40 extracts of lactoperoxidase-iodinated intact platelets, and by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane preparations. By each of the three separatory procedures, the P1 a1 antigenic marker was shown to be associated with glycoprotein IIIa. Studies of the effects of proteolytic enzymes, disulfide-reactive agents and temperature on P1 a1 activity and of the effect of various sugars on the P1 a1-anti-P1 a1 reaction indicate that the P1 a1 antigen, situated on a portion of the molecule susceptible to cleavage in situ by trypsin, but not by chymotrypsin. bromelain, or papain, requires intact disulfide bonds for its full expression, and is probably determined by a polypeptide sequence in GPIIIa. Virtually all of the P1 a1 activity of platelets appears to be located on the external plasma membrane. P1 a1 appears to be the first alloantigen to be assigned to a specific platelet membrane constituent and the third peptide-determined alloantigen to be assigned to a human cell membrane protein.