Leland M. Shannon

The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C.

The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence.

Concanavalin A is synthesized as a glycoprotein precursor.

Concanavalin A (Con A) is a tetrameric lectin which is synthesized in the cotyledons of developing jack-bean (Canavalia ensiformis (L.) D.C.) seeds and accumulates in the protein bodies of storage-parenchyma cells. The polypeptides of Con A have a molecular weight of 27000 and a relative molecular mass (Mr) of 30000 when analyzed by gel electrophoresis on denaturing polyacrylamide gels. In-vitro translation of RNA isolated from immature jack-bean cotyledons shows that Con A is synthesized as a polypeptide with Mr 34000. In-vivo pulse labeling of cotyledons with radioactive amino acids or glucosamine also resulted in the formation of a 34000-Mr polypeptide. In-vivo labeling with radioactive amino acids in the presence of tunicamycin yielded an additional polypeptide of 32000 Mr. Together these results indicate that Con A is cotranslationally processed by the removal of a signal sequence and the addition of an oligosaccharide side chain of corresponding size. Analysis of the structure of the oligogosaccharide side chain was accomplished through glycosidase digestion of glycopeptides isolated from [3H]glucosamine-labeled Con A. Incubation of the labeled glycopeptides with endoglycosidase H, α-mannosidase or β-N-acetylglucosaminidase, followed by gel filtration, allowed us to deduce that the oligosaccharide side chain of pro-Con A is a high-mannose oligosaccharide. Pulse-chase experiments with labeled amino acids are consistent with the interpretation that the glycosylated precursor of Con A is processed to mature Con A (Mr=30000). The 4000 decrease in Mr is interpreted to result from the removal of a small glycopeptide. The implications of the conversion of a glycoprotein pro-Con A to mature Con A are discussed in the context of the unique circular permutation of the primary structure of Con A.

Immunocytochemical localization of concanavalin A in developing jack-bean cotyledons.

The lectin, concanavalin A (Con A), was localized in the cotyledon of developing jack beans (Canavalia ensiformis (L.) DC) by electron-microscope immunocytochemistry. In mature seeds, Con A was present in protein-storage vacuoles (protein bodies) of storage-parenchyma cells. Although protein bodies could be seen in other cell types, only protein bodies in storage-parenchyma cells contained Con A. During seed development, Con A was also localized on the endoplasmic reticulum and Golgi apparatus, presumably en route toward deposition within the protein bodies. The intensity of labeling of the endoplasmic reticulum was much greater during the developmental stage of protein-body filling (66% final seed weight) than in mature seeds.

Purification of a cellulase from kidney bean abscission zones

Abstract The purification of a cellulase isoenzyme with a pI of 9.5 from kidney bean abscission zones is described. An important step in the purification involved the adsorption of the cellulase isoenzyme onto an affinity column of CF-11 cellulose and the subsequent elution with cellobiose. Native and SDS polyacrylamide gel electrophoresis established that there was only one component in the purified cellulase samples. Antibodies raised against the purified pI 9.5 cellulase precipitated this isoenzyme from crude or purified solutions but did not cross react with pI 4.5 cellulase from 2,4-D-treated abscission zones. The antibody was shown to be monospecific by immunoelectrophoresis and by the fact that it precipitated only a single 14C-labeled protein from an abscission zone extract heavily labeled with 14C amino acids.

A barley lectin that binds free amino sugars. I. Purification and characterization.

A lectin was isolated from barley seen which bound the coat glycoprotein of barley stripe mosaic virus (Type strain) and precipitated the virus from solution. Purification of the barley lectin was achieved by fractionation with ammonium sulfate and successive column chromatography on DEAE cellulose and cellulose phosphate. The barley lectin was homogeneous as ascertained by polyacrylamide gel electrophoresis, isoelectric focusing, and from immunochemical tests. No isolectins were detected. The lectin has a molecular weight of 31 000 daltons and is not a glycoprotein. Each virion can accomodate between 200 to 300 molecules of lectin. Barley lectin was shown to be specific for D-glucosamine, D-galactosamine and D-mannosamine with little distinction among the epimeric configurations at carbons 2 and 4. Free amino groups of D-glucosamine and D-galactosamine were detected on the coat glycoprotein of Type strain barley stripe mosaic virus and these sugars appear to serve as receptors for the barley lectin.

A preparative column electrophoresis apparatus using Sephadex G-25

Abstract A preparative column electrophoresis apparatus using Sephadex G-25 as a support medium is described. The apparatus has been successfully employed in the separation of closely related horesradish peroxidase isoenzymes. Rechromatography of the isoenzymes on carboxymethyl- or diethylaminoethyl-cellulose columns was time consuming and often resulted in the irreversible adsorption of some of the protein. The apparatus described here offers both a convenient and an inexpensive method of purification and one in which the entire sample can be recovered. Quantities as large as 120 mg of protein can be applied to each of several columns with excellent resolution. The column may be reused repeatedly and under a variety of conditions by merely equilibrating the column with a different buffer. The system has also been applied to the separation of “membrane-bound” enzymes in the presence of detergent. These results will be published elsewhere. The method also offers a potential means to measure interactions between proteins or other macromolecules by observing altered electrophoretic rates.

Conservation of antigenic determinants among different seed lectins

Abstract Immunochemical techniques were employed to examine seed lectins for structural similarities. Antisera raised against eight homogeneous lectins were used to test for cross-reacting material in crude seed extracts as well as highly purified lectins. The data provide immunochemical evidence that lectins isolated from different species may be structurally related proteins. As structurally related proteins, the cross-reacting lectins may also possess a similar function. In addition, antisera raised against Ulex agglutinin I or Bandeiraea agglutinin, appeared to recognize an identical set of determinants on those lectins showing cross-reactivity. This highly conserved region of the lectin molecule may be important for the proper functioning of these proteins.

Absence of carbohydrate in crystalline Fraction I protein isolated from tobacco leaves

Abstract By means of gas—liquid chromatography—mass spectra analyses, three-times recrystallized Fraction I protein from tobacco leaved contains less than 0.0005% carbohydrate and is therefore different from other species of protein which contain up to 5% carbohydrate.

A rapid, quantitative, and highly specific assay for carbohydrate-binding proteins

Abstract A rapid, quantitative, and highly specific assay capable of measuring 0.1 μg of soybean agglutinin is described. The method is applicable to determining soybean agglutinin in crude extracts as well as in purified preparations. The highly specific nature of the assay is the product of the chemical specificity between the Sepharose-GalNAc conjugate and soybean agglutinin and the antigenic specificity between soybean agglutinin and 125 I-labeled antibody raised against soybean agglutinin. The assay can, in principle, be used to measure any carbohydrate-binding protein. It is necessary only to link the appropriate hapten to the Sepharose beads and to raise antibody against the binding protein. While maximum specificity is attained using 125 I-labeled antibody raised against a highly purified protein, the inclusion of two distinct specificity requirements in the assay may allow use of antibodies raised against proteins only partially purified.

Interaction of Soybean Agglutinin with Soybean Callus Cells

Summary Depending on the stage of cell growth, small additions of soybean agglutinin to soybean cells in culture, can either inhibit or stimulate leucine incorporation into protein. Cell age also affects the amount of soybean agglutinin that can bind to the cells as well as the amount of endogenous soybean agglutinin-like material on the cell surface. It is speculated that the age dependent response of exogenous soybean agglutinin may be related to the ratio of unoccupied : occupied soybean agglutinin receptors on the cell surface.