Interaction of Soybean Agglutinin with Soybean Callus Cells

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  • Original Papers

    Interaction of Soybean Agglutinin with Soybean Callus Cells

    ") Department of Biochemistry, University of California, Riverside, California 92521 U.S.A. . ,') Stauffer Chemical Company, Richmond, California 94804 U.S.A.

    Received March 30,1981 . Accepted May 23,1981

    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 unoccu-pied: occupied soybean agglutinin receptors on the cell surface.

    Key words: Glycine max, lectins, soybean agglutinin, cell culture, receptors, development, protein synthesis, mitogenesis.

    Lectins are multivalent carbohydrate binding proteins that can be detected and measured by their ability to agglutinate cells. Cell agglutination is a consequence of the formation of numerous lectin bridges between surface oligosaccharide determinants on neighboring cells. By virtue of this characteristic, lectins have been widely used as a tool to analyze properties of the cell surface (Nicolson, 1974). The binding of lectins to the cell surface has been shown to induce a variety of biological events including cell division (Blomgren and Svedmyr, 1971), and cell differentiation (Southworth, 1975). These observations led investigators to inquire as to whether plant lectins might serve as selective agents to stimulate mitosis of plant cells. Particularly interesting in this regard was the demonstration of a mitogenic effect of soybean agglutinin (SBA) on soybean callus cells (Howard et al., 1977). These experiments were unique in that they examined the effect of a lectin on plant cells derived from the same species from which the lectin was isolated. The present report describes experiments which examine some of the features of the interaction of SBA with soybean cells at different times throughout the cell growth cycle.

    Materials and Methods A soybean callus culture was derived from root tissue (Murashige, 1974) and transferred to

    liquid Murashige and Skoog culture media as previously described (Uchimiya and Murashige,

    Z. Pjlanzenphysiol. Bd. 104. S. 97-102. 1981.

  • 98 ELENA DEL CAMPILLO, JOHN HOWARD and LELAND M. SHANNON

    1974). SBA was purified from crude seed extracts by affinity chromatography using N-acetyl-galactosamine covalently attached to Sepharose beads (Vector Laboratories, Burlingame, Ca). The adsorbed SBA was displaced with PBS containing 100 mM D-galactose. Following exhaustive dialysis against PBS, the SBA was lyophilized and stored at -20C. The purified SBA showed a single protein band following SDS gel electrophoresis (Webber and Osborn, 1969).

    SBA was filter sterilized and added to growth media to yield a final concentration of 1.5 I-Ig/ml. The initial cell density was 40 mg/ml. Cell growth was determined by measuring changes in the packed cell volume following centrifugation for five minutes at 2000 x g. Protein synthesis was estimated by measuring the incorporation of U-HC leucine into the methanol-chloroform-water (12: 5 : 3) insoluble fraction (Ferrari and Widholm, 1973). U-HC leucine was added to the culture medium, and after the addition of the cell inoculum, 0.4 ml of the cell suspension was dispensed into individual culture tubes. At 5 hr intervals the cells were collected onto filter pads, washed three times with methanol-chloroform-water, and the insoluble fraction counted. Quadruplicate assays were run at each time interval.

    Rabbit antisera to purified SBA was prepared by methods described previously (Howard et aI., 1979). Lactoperoxidase (501-11, 1 mg/ml) and 250 I-ICi of 1251 (specific activity: 17 Ci/mg; ICN, Irvine, CA) were added to 2 ml of antiSBA-IgG (5 mg/ml). The reaction was initiated by the addition of 25 1-11 of 0.1 M H 20 2. After 30 and 60 min an additional 25 1-11 aliquot of H 20 2 was added. After 90 min the mixture was applied to a Bio-Gel P-100 column (2.5 x 27 cm) and the effluent fractions monitored for IgG using Ouchterlony double diffusion assays. The fractions containing IgG were combined and 1251 measured in a gamma counter.

    A suspension of callus cells was centrifuged in a graduated tube at 2000 x g for five minutes. The packed cells were then suspended in phosphate buffered saline (PBS) to obtain a 0.25 % (v/v) suspension of cells. The cell suspension (100 1-11) was then added to a microfuge tube containing one milligram of bovine serum albumin, included to reduce non-specific binding. Cells were then incubated with SBA (100 I-Ig/ml) or PBS for 10 minutes and washed three times in PBS at room temperature. 125I-IgG (100 1-11, 5 mgl ml, specific activity, 5000 cpml mg) was added and incubated with the cells for 30 minutes. The cells were pelleted in a microfuge, washed three times with PBS, and the bound radioactivity was measured using a gamma counter.

    Results Previous studies showed that the addition of 1.5 ~g/ ml of SBA to soybean

    suspension cultures caused an increase in cell number, mitotic index and rate of 3H-thymidine incorporation into nucleic acid (Howard et aI., 1977). In the present study we examined the effect of SBA on the incorporation of He leucine into proteins. The results of a time course of u-He leucine incorporation into proteins in the absence and presence of 1.5 ~g/ ml SBA are shown in fig. 1. These data reveal that SBA caused a 2 fold increase in the rate of He leucine incorporation into protein. The cell inoculum used in this experiment was harvested during the logarithmic phase of growth.

    We next examined the effect of SBA on protein synthesis at different stages of the growth curve. A typical growth curve of soybean cells in suspension culture is seen in fig. 2 A, in which packed cell volume is used as an indicator of growth. For each group of cells harvested at different stages on the growth curve, a time course of U-He leucine incorporation was measured in the absence (control) and in the presence of 1.5 ~g/ ml SBA. The results are depicted in fig. 2 B as percentage of control. There

    Z. Pjlanzenphysiol. Bd. 104. S. 97-102. 1981.

  • Interaction of soybean agglutinin with callus cells 99

    9 SBA

    N

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    6 ~ a. ()

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    6 12 18 24 30 INCUBATION TIME (hrs)

    Fig. 1: Effect of SBA on protein synthesis. Soybean cells were harvested during logarithmic growth and transferred to fresh media containing U-HC leucine II! Ci/ml in the absence (0---0) and in the presence of 1.5 I!gl ml SBA (e---e). HC leucine incorporation into proteins was measured at 6 hr intervals. The error bars represent the range of values among the 4 replicates of each assay.

    A B 0 E 8 /~ \ UJ 150 ::; ~ ::J 6 6 0 > o~ 0 -' 100 z -' ....

    ~ 4 0\ 0

    " 0 0

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    5 10 15 20 5 10 15 20 DAYS IN CULTURE

    Fig. 2: (A). Growth curve for soybean cells. Suspension cultures of soybean cells were initiated by transferring 1 g of soybean callus tissue to 25 ml Murashige and Skoog media in 125 ml Erlenmeyer flasks. The flasks were continuously shaken at 100-150 rpm on a rotatory shaker at room temperature. At the indicated times the entire contents of the flask were centrifuged for five minutes at 2000 x g and the volume of the packed cells was determined. - (B). Effect of SBA on protein synthesis at different cell ages. Soybean cells were transferred at different stages of the growth cycle to fresh media containing U-HC leucine in the absence (control) and presence of 1.5 I!gl ml SBA. Data are shown as percent of control:

    rate of IC leu incorporation with SBA x 100 = % Control rate of HC leu incorporation without SBA

    was no effect of exogenous SBA during the lag phase of growth. Addition of SBA during the initial phase of log growth (day 7) caused an inhibition of the rate of amino acid incorporation over the control. When cells were in the middle of the logarithmic growth phase (day 10), addition of SBA caused a two fold increase in the rate of 14C

    Z. Pjlanzenphysiol. Bd. 104. S. 97-102. 1981.

  • 100 ELENA DEL CAMPILLO, JOHN HOWARD and LELAND M. SHANNON

    leucine incorporation. Finally, addition of SBA to cells approaching the stationary phase did not influence protein synthesis.

    The next experiment was undertaken to determine the amount of exogenous SBA capable of binding to cultured soybean cells harvested at various times during the growth cycle. In these studies iodinated (1251) IgG against SBA was added to soybean callus cells which had been previously incubated with or wihtout SBA. The amount of 1251_lgG bound to cells which had been incubated in the absence of exogenous SBA (control), represents a measure of endogenous SBA-like material on the cell surface. The increase in 1251-lgG bound to cells treated with SBA represents a measure of unoccupied SBA receptor sites. When 1251-labeled pre-immune IgG was substituted for 1251-SBA IgG, negligible radioactivity was bound. Maximal endogenous SBA like material was present on cells collected in the log phase of growth. As cells approached stationary growth phase, the SBA content of the media increased dramatically (data

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  • Interaction of soybean agglutinin with callus cells 101

    Discussion The mitogenic effect of SBA on soybean cells growing in suspension culture was

    followed by measuring the incorporation of U-HC leucine into protein. The rational