No abstract

No abstract

No abstract

No abstract

No abstract

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...

Malic enzyme from pigeon liver is alkylated by the substrate analogue bromopyruvate, resulting in the concomitant loss of its oxidative decarboxylase and oxalacetate decarboxylase activities, but not its ability to reduce alpha-keto acids. The inactivation of oxidative decarboxylase activity follows saturation kinetics, indicating the formation of an enzyme-bromopyruvate complex (K congruent to 8 mM) prior to alkylation. The inactivation is inhibited by metal ions and pyridine nucleotide cofactors. Protection of malic enzyme by the substrates L-malate and pyruvate and the inhibitors tartronate and oxalate requires the presence of the above cofactors, which tighten the binding of these carboxylic acids in accord with the ordered kinetic scheme (Hsu, R. Y., Lardy, H. A., and Cleland, W. W. (1967), J. Biol. Chem. 242, 5315-5322). Bromopyruvate is reduced to L-bromolactate by malic enzyme and is an effective inhibitor of L-malate and pyruvate in the overall reaction. The apparent kinetic constants (90 muM-0.8 mM) are one to two orders of magnitude lower than the half-saturation constant (K) of inactivation, indicating a similar tightening of bromopyruvate binding in the E-NADP+ (NADPH)-Mn2+ (Mg2+)-BP complexes. During alkylation, bromopyruvate interacts initially at the carboxylic acid substrate pocket of the active site, as indicated by the protective effect of substrates and the ability of this compound to form kinetically viable complexes with malic enzyme, particularly as a competitive inhibitor of pyruvate carboxylation with a Ki (90 muM) in the same order as its apparent Michaelis constant of 98 muM. Subsequent alkylation of a cysteinyl residue blocks the C-C bond cleavage step. The incorporation of radioactivity from [14C]bromopyruvate gives a half-site stoichiometry of two carboxyketomethyl residues per tetramer, indicating strong negative cooperativity between the four subunits of equal size, or alternatively the presence of structurally dissimilar active sites.

No abstract