Interaction of phosphofructokinase with antibodies. Kinetic properties of phosphofructokinase in complexes with antibodies.

The allosteric properties of phosphofructokinase (EC 2.7.1.11) from rabbit muscle are influenced by enzyme concentration, most probably due to changes in the association state of the enzyme. In this study, the behaviour of dispersed pre-cipitates of phosphofructolinase as produced by treatment with antibodies has been investigated. The enzyme is not capable of rapid dissociation in the precipitated state as is confirmed by the lack of inactivation upon dilution and by the absence of shifts in substrate saturation curves as measured in the presence of different concentrations of the enzyme. The Hill coefficient of phosphofructokinase is decreased from 1.96 to 1.04 by antibody treatment. The V at neutral pH is increased 3-fold while the K0.5 for fructose 6-phosphate is reduced significantly. On the other hand, antibody-treated phosphofructokinase retains its sensitivity to allosteric activation by glucose 1,6-bisphosphate in the rpesence of high ATP concentrations.     

Murine polyspecific antibodies. I. Monoclonal and serum anti-DNA antibodies cross-reactive with 2,4,6-trinitrophenyl derivatives

Six anti-DNA hybridoma autoantibodies were prepared by fusing spleen cells from unimmunized MRL/MpJ/lpr/lpr female mice with BALB/c myeloma cells. The monoclonal antibodies were analyzed by solid-phase ELISA for antigen-binding specificities. Three antibodies (62A2, 85A5, and 43B2) bound ssDNA, TNP-KLH, and recognized an epitope(s) present on insolubilized proteins such as BSA, KLH, ferritin, and insulin. The antibodies bound, with a marked preference, TNP-KLH, either soluble or insoluble. The other three antibodies (35A1, 32C5, and 39D2) bound only ssDNA. However, this binding was inhibited by free flavinic acid. None of the six antibodies bound either cardiolipin or proteoglycans, indicating that they do not recognize the repeating negatively charge units common to cardiolipin, proteoglycans, and DNA. All six monoclonal antibodies were purified by affinity chromatography with TNP-Sepharose. Moreover, both anti-DNA and anti-TNP antibodies from sera of nonautoimmune and autoimmune mice were purified easily on TNP-Sepharose.     

Anti-thyroid antibodies in patients with hyperprolactinemia.

To clarify the possible role of prolactin in the regulation of immune responses in man, we measured circulating anti-thyroid antibodies in 172 normal subjects, 84 patients with prolactinoma and 63 patients with acromegaly with normal thyroid and adrenocortical functions. Frequencies of positive thyroidal microsome and thyroglobulin antibodies were significantly (p < 0.05 and p < 0.01, respectively) higher in women with prolactinoma (20.6% and 20.6%) than in normal women (7.5% and 4.7%). Men with prolactinoma had a significantly (p < 0.05) higher frequency of positive thyroglobulin antibody (18.8%) than normal men (1.5%). When the subjects were divided by decade, women with prolactinoma in the 4th decade had significantly (p < 0.05) higher frequencies of positive thyroidal microsome and thyroglobulin antibodies (30.8% and 30.8%) than normal women of corresponding age (3.7% and 3.7%). In contrast, there was no significant difference in the frequencies of positive anti-thyroid antibodies in patients with acromegaly and in normal subjects. Analysis of the peripheral lymphocyte population revealed that patients with prolactinoma had a higher percentage of B cells than normal subjects, while there was no significant difference in the percentages of total T lymphocytes or in the helper and suppressor T cell ratios in the two groups of subjects. These results suggest that prolactin regulates humoral immune responses in man directly by stimulating B lymphocytes or indirectly by inhibiting suppressor T lymphocyte activity.     

Reaction of antimannan antibodies with oligomannosides and glycopeptides.

A number of oligomannosides and glycopeptides prepared from various sources were tested for their potency to inhibit the binding of 3H-mannotetraitol (Manalpha1 leads to Manalpha1 leads to Manalpha1 leads to 2Mannitol) to antimannan antibodies. It was found that antimannan antibodies are highly specific to the Manalpha1 leads to 3Man structure, reacting very poorly with the Manalpha1 leads to 2Man and Manalpha1 leads to 6Man structures. A Manalpha1 leads to 3Man structure at the non-reducing end is far more reactive than one at an inner position. In this respect, antimannan antibodies differ from concanavalin A which reacts with mannose residues substituted at C-2 as well as those at non-reducing ends. Glycopeptides prepared from ovalbumin, Taka amylase A and from membrane glycoproteins of rat liver cross-reacted with antimannan antibodies to various extents reflecting the characteristic structures of the individual glycopeptides.     

Studies with anti-growth hormone receptor antibodies.

Antisera against a partially purified growth hormone receptor derived from rabbit liver were generated in guinea pigs. The antisera specifically inhibited the binding of 125I-ovine growth hormone (oGH) to liver membranes but had no effect on the binding of 125I-ovine prolactin to rabbit mammary gland receptors. These antisera did not bind or destroy 125I-oGH. Moreover, the binding of labeled growth hormone to membrane particles derived from liver of several species was also inhibited by the antisera, thus suggesting that immunological determinants of the growth hormone receptor of several species are similar. gamma-Globulin fractions derived from the antisera were responsible for the inhibition. In addition 125I-gamma-globulin derived from one antiserum bound to membrane pellets with a corresponding decline in 125I-oGH binding. Kinetic analysis of inhibition of 125I-oGH binding suggested a hyperbolic competitive inhibition, a point of view which is favored by the demonstration of a hormone receptor . antibody complex. The availability of the antireceptor sera confirmed previous data that differential affinity chromatography separated growth hormone and prolactin receptors in solubilized rabbit liver membrane preparations. The antireceptor sera will be useful probes in further characterization of the growth hormone receptor.     

Probes of beta-galactosidase structure with antibodies. Reaction of anti-peptide antibodies against native enzyme.

Antibodies were prepared against 18 tryptic and cyanogen bromide peptides from beta-galactosidase ranging in size from 15 to 96 amino acid residues representing more than 80% of the polypeptide chain. They were tested for binding capacity and affinity toward their homologous antigens and toward the whole native protein. Nine antisera bound to beta-galactosidase; these had been raised against certain peptides from the central and carboxyl-terminal regions of the poly-peptide chain. Based on these results a preliminary model of the three-dimensional structure of the folded protein is suggested.     

Specificity of monoclonal antibodies against human thyroglobulin; comparison with autoimmune antibodies.

Ten monoclonal antibodies (mAb) directed against human thyroglobulin (hTgb) were produced, purified and characterized. The mAb avidity for hTgb ranged from 10(-10) to 10(-6) M. The species specificity of the mAb was as follows: eight mAb reacted with monkey Tgb, three with dog Tgb and one with pig Tgb; none with bovine and ovine Tgb. The binding of mAb to hTgb was not significantly inhibited in the presence of Tgb carbohydrate moieties, tyrosine, iodotyrosines and iodothyronines. The topology of the antigenic determinants recognized by the 10 mAb on hTgb was explored by inhibition of Tgb binding of radiolabeled mAb by the other antibodies. Six distinct clusters of reactivity were described. Localization of the antigenic determinants recognized by mAb on hTgb was attempted using tryptic fragments of hTgb to inhibit the binding of mAb to hTgb. The inhibitory effect of hydrolysis products was different for each mAb but exhibited partial analogies between mAb of the same cluster of reactivity. Anti-hTgb autoimmune antibodies (aAb) purified from sera of Graves patients cross-reacted essentially with mAb of one out of the six clusters. These results demonstrate that the large number of antigenic determinants presented by the hTgb are not disseminated on the molecule but are clustered in antigenic regions. Furthermore, from the six antigenic regions evidenced in this paper, only one is involved in autoimmune antibody production in Grave's disease.     

Naturally occurring antibodies to liposomes. II. Specificity and electrophoretic pattern of rabbit antibodies reacting with sphingomyelin-containing liposomes

Sera obtained from rabbits after immunization with a variety of unrelated antigens contain antibodies that induce complement- (C) mediated lysis of sphingomyelin-containing liposomes in the absence of the relevant antigen from the membrane. Absorption or inhibition with dimyristoyl-phosphatidyl choline-containing liposomes were less effective than with sphingomyelin-containing liposomes in decreasing or abolishing C-dependent lysis of target-liposomes. Phosphoryl choline chloride inhibited the C-dependent lysis mediated by these antibodies, but only when used in high molar excess and in the presence of low antibody concentrations. Purified anti-liposome antibodies displayed an isoelectric focusing pattern consistent with a polyclonal response. The findings confirm the antibody nature of the anti-liposome activity of rabbit sera and indicate that their predominant specificity is directed against conformations of the phospholipid molecule in which the polar (phosphoryl choline) group does not have a major contribution.     

Antipituitary antibodies in patients with lymphocytic hypophysitis.

BACKGROUND: Lymphocytic hypophysitis is one of the causes of hypopituitarism, which is considered an autoimmune reaction in the anterior pituitary. METHOD: We examined antipituitary antibodies in patients with lymphocytic hypophysitis and related diseases by immunoblotting method. RESULTS: Autoantibodies to a 22-kDa human pituitary cytosolic protein were identified in significantly higher frequencies in sera from patients with lymphocytic hypophysitis (11 of 15, 73.3%) and isolated ACTH deficiency (7 of 9, 77.8%) compared with Hashimoto thyroiditis, Basedow's disease and normal control subjects. Also, reactivity against a 49-kDa human pituitary cytosolic protein was seen in 6 of 15 patients (40%) with lymphocytic hypophysitis. N-terminal amino acid sequences of 22-kDa human and rat pituitary cytosolic protein were FPTIPLSVL and FPAMPLSSLFAN, respectively, suggesting that they are human and rat growth hormone, respectively. The pituitary dysfunction (at least one hormone dysfunction) was observed in 11 of 14 patients. Nine of them (82%) showed 22 kDa antibody but 2 of them (18%) did not. CONCLUSION: The present study demonstrated that pituitary autoantibodies could be involved in the pathogenesis of lymphocytic hypophysitis and could be a positive marker for the disease.     

Naturally occurring antibodies to liposomes. I. Rabbit antibodies to sphingomyelin-containing liposomes before and after immunization with unrelated antigens

Liposomes were prepared from a mixture of sphingomyelin, cholesterol, and dicetylphosphate or L-alpha-dimyristoyl phosphatidylcholine, cholesterol, and dicetylphosphate, in the presence of glucose. The amount of trapped glucose released from these liposomes was monitored after incubation with a variety of normal and immune sera in the presence of guinea pig complement. All normal rabbit sera tested were found to release, in the presence of complement, detectable amounts of trapped glucose from sphingomyelin-containing liposomes. After immunization with a variety of unrelated antigens, the anti-sphingomyelin liposome activity increased signficantly and in direct proportion to the number of injections, despite the fact that the liposomes used in the assay did not contain the relevant antigen used for immunization. Liposomes prepared from dimyristoyl phosphatidylcholine showed only marginal release of their trapped marker when assayed with the same rabbit sera and complement. These liposomes, however, were fully reactive when the appropriate antigen was inserted in their bilayer structure. The antiliposome activity was associated mainly with the IgM antibody class. These results raise the interesting possibility that antigenic stimulation may trigger the activation of lymphocyte clones directed against autologous cell-membrane components that cross-react with artificial model membranes containing sphingomyelin.     

Identification and purification of collagen-synthesizing polysomes with anti-collagen antibodies.

Antibodies against embryonic chick bone collagen were prepared in rabbits and were purified by affinity and ion exchange chromatography until collagen-specific and RNase-free. 125I-anti-collagen antibodies were used to locate the collagen-synthesizing polysomes of 8-day chick embryo wings and legs on sucrose gradients by measuring the polysome associated radioactivity. The 125I-anti-collagen antibodies bound predominantly to polysomes in the heavy region of sucrose gradients. These binding sites could only be saturated with homologous anti-collagen antibodies. Further evidence for the specificity of this reaction was provided by a correlation of the amount of anti-collagen antibodies bound in the heavy regions of sucrose gradients with the amount of collagen being synthesized by a particular tissue. The validity of this immunochemical method was confirmed by localizing collagen-synthesizing polysomes by an independent method which utilizes their ability to incorporate [3H]proline into collagen peptides in a cell-free system. The collagen-synthesizing polysomes are found in a single, rather broad peak in these gradients. The results of shortening the centrifugation time indicate that larger species of collagen-synthesizing polysomes are not present in these tissues. Partial purification of the collagen-synthesizing polysomes may be achieved by specifically sedimenting them after treatment with anti-collagen antibodies followed by goat anti-rabbit antibodies.     

Catalytic antibodies and biomimetics.

Of the various approaches being studied to mimic the catalytic properties of enzymes, catalytic antibody research is advancing most rapidly and successfully; the discovery of new reactions and new catalytic antibody-producing haptenic structures continues unabated. One of the highlights of the past year was the design and synthesis of a catalytically active peptide. The overall area of catalytic antibodies and biomimetics will be prominent in future biotechnological applications, as further advances are made and the nature of the catalyzed reactions becomes better understood.     

Human acetylcholinesterase. Immunochemical studies with monoclonal antibodies

Monoclonal antibodies were used to investigate the immunochemistry of human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). A series of experiments on the sedimentation velocity and Stokes radius of acetylcholinesterase and its immune complexes indicated that each antibody recognized a single high-affinity binding site (epitope) on the monomeric enzyme. Further analysis suggested that the antibody-binding sites were replicated on multimeric enzyme forms but were subject to steric hindrance between nearby IgG molecules or adjacent enzyme subunits. The cellular localization of the epitopes was studied by measuring the binding of monoclonal antibodies to the cholinesterase of intact erythrocytes. The results implied that most of the epitopes are exposed to the external media. However, one antibody failed to bind to intact cells, despite a relatively high affinity for detergent-solubilized antigen, possibly because its epitope is buried in the lipid bilayer.     

Varicella-zoster viral glycoproteins analyzed with monoclonal antibodies.

Monoclonal antibodies to varicella-zoster virus were used to study viral glycoproteins by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on the viral glycoproteins immunoprecipitated, the five monoclonal antibodies fell into three groups. Two antibodies, 4B7 and 8G9 (group 1), immunoprecipitated a single glycoprotein of molecular weight (MW) 118,000 (118K glycoprotein) and had high neutralizing activity in the absence of complement. One antibody, 3C7 (group 2), which lacked neutralizing activity, immunoprecipitated two glycoproteins of MWs 120,000 and 118,000 and a glycoprotein giving a diffuse band in the region of 64,000 to 65,000. Pulse-chase experiments and experiments with monensin as an inhibitor of glycosylation suggested that the 120K polypeptide was derived by glycosylation of the 118K polypeptide and that a 43K antigen was processed into the 64 to 65K glycoprotein. Two antibodies, 3G8 and 4E6 (group 3), both had neutralizing activity only in the presence of complement, and both immunoprecipitated at least five polypeptides, with MWs ranging from 50,000 to 90,000. Antibody 3G8 was isotype immunoglobulin G2b (IgG2b), and its immunoprecipitating activity was stronger than that of 4E6, which was isotype IgG1. Pulse-chase experiments with antibody 3G8 showed that lower-MW glycopeptides chased into three polypeptides of MWs 90,000, 80,000, and 60,000 by 24 h. Immunoprecipitation experiments with antibody 3G8 on infected cells treated with glycosylation inhibitors 2-deoxyglucose, monensin, and tunicamycin, suggested that a prominent, early-appearing 70K polypeptide may have been processed into the glycoproteins of higher MWs and that the 60K polypeptide may have been derived by glycosylation of polypeptides of lower MWs.