The phytohemagglutinin mitogenic proteins derived from Phaseolus vulgaris comprise a class of five glycoproteins that are isomeric tetramers composed of varying proportions of two different subunits (L and R). Within the native tetramer, the L subunit is a potent leukoagglutinin and mitogen that lacks hemagglutinating properties, whereas the R subunit is a potent hemagglutinin with little or no mitogenic activity. The subunits have been isolated in homogeneous form by isoelectric focusing in 8 M urea. Previous work has shown that they have equal molecular weights and differ in amino-acid sequence from residues 1-7, but are identical in positions 8-24 [(1973) J. Exp. Med. 138, 939-9511. We now report amino-acid composition studies which reveal striking similarities between the subunits. Both lack methionine and cysteine. The twelfth residue in each subunit is a glycosylated asparagine, with the identical carbohydrate composition in each. The last three residues of the subunits, as determined by carboxypeptidase A digestion, are identical. Tryptic peptide mapping of the succinylated phytohemagglutinin subunits reveals a high degree of similarity. We conclude that the substantial difference in biological properties among the tetrameric phytohemagglutinin mitogens is a result of relatively restricted differences in the primary structure of their constituent subunits.The phytohemagglutinin (PHAP) mitogenic proteins derived from the red kidney bean, Phaseolus vulgaris, have been shown to comprise a family of five heterogeneous proteins (1, 2). They consist of isomeric, noncovalently bound tetramers which are made up of two different subunits, designated as L and R ( Fig. 1) (2-4). One of the five proteins is a potent leukoagglutinin with low hemagglutinating activity (L-PHAP); it is homogeneous, consisting of four identical subunits (1). Three other, closely related proteins, which have modest leukoagglutinating but potent hemagglutinating properties (H-PHAP), have also been isolated. They consist of hybrid tetramers containing varying proportions of the two subunits (2-2R, 1L3R, and 4R), the increasing R subunit content of which is reflected by increasingly cathodal migration on polyacrylamide gel electrophoresis. A fifth PHAP mitogenic protein (3L-1R) has been identified, but detailed study of its properties has been hampered in the past by its contamination with other proteins. We have tentatively concluded that, within each native tetramer, the L subunit has strong mitogenic activity and a high affinity for receptors of lymphocyte membranes, but little or no affinity for those of erythrocytes.Conversely, the R subunit has a high affinity for erythrocyte membrane receptors, but little for those of lymphocytes. As a result, the 4R tetramer displays little or no lymphocyte mitogenic activity (6). The hybrid molecules (1L-3R, 2L-2R, and 3L-1R tetramers) have been found to be mitogenic, to cause mixed agglutination of erythrocytes and lymphocytes, and to have enhanced lymphocyte-transforming capability in the p...

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Alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkalime pH optimum), EC 3.1.3.11purified from a Burkitt lymphoma cell line (Daudi) and Molney-virus-induced murine leukemia (YAC) showed unique catalytic properties in substrate specificity and inhibition by cysteamine-S-phosphate. It migrated on polyacrylamide gel electrophoresis in a single activity band. Alkaline phosphatase with similar properties was found in several human lymphoblastoid cell lines, in chronic lymphatic leukemic cells, in organs of leukemic mice, and in sera of patients with certain lymphoproliferative disorders.The unique kinetic properties of this enzyme were established using two kinds of substrate, namely, the monoesters of orthophosphoric acid (Type I) and the S-substituted monoesters of thiophosphoric acid (Type II). The enzyme catalyzed the hydrolysis of Type

Human alpha-fetoprotein (HAFP) isolated by immunoadsorbent column was shown to suppress the mitogenic response of human lymphocytes to phytomitogens, antihuman thymocyte antiserum, and the mixed lymphocyte culture. HAFP isolated from the sera and ascitic fluid of five hepatoma patients, and from fetal liver, varied in biological potency over three orders of magnitude. Extended agarose gel electrophoresis and crossed immunoelectrophoresis demonstrated three molecular species of HAFP. Quantitation of the three species revealed a correlation between the relative amount of the most negatively charged species and biological potency. Treatment of HAFP with neuraminidase to remove completely sialic acid residues did not alter the biological potency, but converted the three species to two species having slower electrophoretic mobilities. We conclude that differences in sialic acid content are only partly responsible for the microheterogeneity demonstrated by HAFP, and that variability in another charged moiety is also present. Variation in the relative proportions of the different molecular species of HAFP may be important in the regulation of its immunosuppressive properties. While characterizing the immunosuppressive effects of human alpha-fetoprotein (HAFP) (1), we noted that HAFP isolates from the serum and ascitic fluid of five patients with hepatoma or from fetal sources varied over three orders of magnitude in their capacity to inhibit in vitro human lymphocyte transformation induced by phytomitogens, antihuman thymocyte antiserum (ATS), and the mixed lymphocyte culture (MLC) (2). The most potent HAFP preparation was that isolated from fetal liver homogenates of stillborn human abortuses of 10-20 weeks' gestation (2). Because HAFP isolates display a microheterogeneity with respect to charge (3, 4), presumably due to differences in sialic acid content, we undertook a study of the effects of such charge differences upon the biological potency of our various preparations. The results indicate that the most negatively charged species of HAFP are those with the greatest capability to suppress the mitogenic responses of human lymphocytes in vitro. We also observed that total desialylation of HAFP preparations does not affect their biological potency, nor does it abolish their microheterogeneity. MATERIALS AND METHODSHuman alpha-fetoprotein was isolated from sera and ascitic fluid of five hepatoma patients (Od., McF., Ho., Cr., and Lu.) by a modification of the method of Hirai et al. (1,2,5). After elution from the immunoadsorbent column, and prior to passage over Sephadex G-150, the HAFP eluate was passed over a second immunoadsorbent column to which the Na2SO4-precipitated globulin fraction of rabbit antihuman serum antibody was attached. HAFP was also isolated from the livers of still-born human abortuses of 10-20 weeks' gestation. The livers were homogenized in phosphate-buffered saline at pH 7.4, centrifuged at 20,000 X g for 1 hr (40), and the soluble liver extract was subjected to the procedures described...

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We have studied the effects of human alphafetoprotein (HAFP), isolated from the serum and ascitic fluid of a hepatoma-bearing patient, on the in vitro transformation of human peripheral blood lymphocytes by a variety of mitogenic stimuli. At a concentration of 2.5 mg/ml, HAFP inhibited the Iymphocyte response to phytohemagglutinin, concanavalin A, and rabbit anti-human thymocyte serum, but failed to inhibit the response to pokeweed mitogen. HAFP Until recently, little was known of the biological role of the alpha-fetoprotein (1), although it was regarded as an embryonic serum albumin (2, 3) and was shown to possess estrogen hormone-binding properties (4). Our report on the inhibition of human lymphocyte transformation in vitro by fetuin (5), a bovine alpha-fetoprotein, and the simultaneous observations by Tomasi and his coworkers that murine alpha-fetoprotein suppresses murine lymphocyte responses in vitro to both mitogenic and antigenic stimuli (6, 7), suggest an immunoregulatory role for alpha-fetoproteins during fetal development. This paper presents experimental data that demonstrate the inhibitory effects of human alpha-fetoprotein (HAFP) on lymphocyte responses in vitro to a variety of mitogenic stimuli which include the one-way mixed lymphocytes culture. The results suggest that this effect may be due primarily to an inhibition of thymus-derived lymphocytic (T-lymphocyte) function. MATERIALS AND METHODSThe techniques for isolating, culturing, and harvesting cultures of human lymphocytes were previously described (5,(8)(9)(10). A 1 ml culture system was employed throughout, and all experiments with cultures were performed in triplicate. The effect of preexposure with HAFP on the viability of human lymphocytes was determined by trypan blue exclusion (5), and their ability to undergo subsequent transformation when subjected to a mitogenic stimulus was assessed. Lymphocytes (25-35 X 106), in a total volume of 2.0 ml of RPMI medium and 12.5% of human type AB serum, were incubated for 18 hr in the presence of 2.2 mg/ml of HAFP or HSA, with conditions identical to those used for lymphocyte cultures. A portion (4 X 106) of the lymphocytes was removed from the cell suspensions, spun down, and used for the determination of viability by the trypan blue method. The remainder of the cell suspension was diluted with 5 ml of Amos A gelatin (9) buffer and centrifuged at 150 X g for 15 min. The cell buttons were washed once more with 7 ml of Amos buffer, resuspended in culture medium, and used for transformation studies.The ability of HAFP to inhibit E-rosette formation by Ficoll-Hypaque-isolated human lymphocytes was determined by means of the E-rosette formation procedure, carried out in the presence of 2.5 mg/ml of HAFP . This procedure was similar to one already described (13), except that heat-inactivated human serum (type AB), previously absorbed with sheep red blood cells, was used in place of fetal calf serum.In addition to the effect of HAFP in the mixed lymphocyte culture (MLC), the effect of HAFP on l...

The phytohemagglutinins (PHAP) 1 obtained from the red kidney bean, Phaseolus vulgaris, have been shown to be a mixture of five glycoproteins that are heterogenous by several physicochemical and biological criteria (1). One of these proteins is a potent leukoagglutinin with low hemagglutinin activity (L-PHAP); a mixture of three other closely related proteins with modest leukoagglutinating activity but potent hemagglutinating properties (H-PHAP) has also been isolated (2). Both L-PHAP and H-PHAP are excellent mitogens. Of particular interest is the observation that the mitogenic activity of H-PHAP is markedly potentiated by the presence of autologous red blood cells (RBC) during in vitro human lymphocyte transformation (3, 4). In contrast, the ability of L-PHAP to transform lymphocytes is not affected by the presence of RBC. Enhancement of mitogenicity by RBC correlates well with the ability of these mitogens to effect mixed lymphocyte-RBC agglutination, a property potently displayed by H-PHAP but lacking in L-PHAP. The fifth PHAP mitogenic protein has been clearly defined by means of polyacrylamide gel electrophoresis, but detailed study of its properties has been hampered by contamination with L-and H-PHAP during preparative isolation procedures.We have postulated previously that the five P H A P molecules consist of tetrameric isomitogens made up of varying proportions of two different subunits (2, 4). Our model hypothesizes that L -P H A P consists of four subunits,