Novel allosteric sites on human erythrocyte acetylcholinesterase identified by two monoclonal antibodies.

Monoclonal antibodies against human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) have been examined for inhibition of enzyme activity. Of sixteen antibodies analyzed, only one (C1B7) inhibited enzyme activity, indicating selection of an unusual susceptible site. The inhibitory activity of C1B7 was characterized and compared to another inhibitory antibody, AE-2, previously described by Fambrough et al. (Proc. Natl. Acad. Sci. USA 79, 1078, 1982). Maximal demonstrated inhibition was 84% for C1B7 and 72% for AE-2 and antibody inhibition of enzyme activity was equivalent for the reduced and alkylated acetylcholinesterase monomer and the intact dimer. The Ki (stoichiometry of the enzyme-antibody reaction estimated from enzyme kinetics) was 1.0 for C1B7 and 4.8 molecules of antibody per monomer of acetylcholinesterase for AE-2. The antibodies did not compete with one another for binding to acetylcholinesterase, indicating that they have different target epitopes on the enzyme. Antibody binding to the enzyme was not specifically affected by any of the anticholinesterase agents tested: (a) the irreversible esteratic site-directed inhibitor diisopropylfluorophosphate; (b) the reversible active site-directed inhibitors edrophonium, neostigmine, BW284c51, and carbachol; and (c) allosteric site-directed compounds propidium and gallamine. Kinetic analysis of their effects provide evidence that both antibodies decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.     

Antibodies and autoantibodies of glycogen phosphorylase b: inactivation of pig and rabbit enzymes.

Pig skeletal muscle glycogen phosphorylase b was purified using ammonium sulfate fractionation, DEAE-Sephadex A-50 and Sephadex G-200 column chromatography. The purified enzyme was used to immunize rabbits in the presence or in the absence of complete Freund adjuvant. Antibodies against pig phosphorylase in pure form were isolated from rabbit antisera using insoluble immunoadsorbents of pig phosphorylase. Autoantibodies against the rabbit enzyme were obtained from the same antisera using insoluble immunoadsorbents of rabbit phosphorylase. Complete inactivation of pig phosphorylase was accomplished by an antibody/enzyme molar ratio equal to 4 and autoantibody/enzyme molar ratio equal to 130. Complete inactivation of rabbit phosphorylase was accomplished by an antibody/enzyme molar ratio equal to 250 and autoantibody/enzyme molar ratio equal to 160. Passive haemagglutination technique gave positive results with minimum amounts of 0.02 microng/ml and 0.8 microng/ml for pig and rabbit phosphorylase respectively. Kinetic experiments have shown that antibodies and autoantibodies act as noncompetitive inhibitors of both enzymes with respect to AMP and glucose 1-phosphate but exhibit a mixed type of inhibition with respect to glycogen. When glycogen hydrolysates were used as substrate in place of intact glycogen molecules a pronounced decrease in the inhibitory capacity of antienzyme on the enzyme was demonstrated.     

Antiamylase-pullulanase enzyme monoclonals which specifically inhibit amylase or pullulanase activity

Monoclonal antibodies against amylase-pullulanase enzyme from Bacillus circulans F-2 have been produced to locate and characterize the catalytic sites of the enzyme. The antibodies have been examined for inhibition of both enzyme activities of amylase and pullulanase and then classified into four types: Type I which inhibited amylase activity, Type II which inhibited pullulanase activity, Type III which inhibited both enzyme activities, and Type IV which had no effect on either enzyme activity. Only two monoclonal antibodies (MAP-12 and MAP-17) as Type I and two antibodies (MAP-3 and MAP-5) as Type II were isolated. The inhibitory activities of the antibodies were characterized and compared. In Type II antibodies, the maximal demonstrated inhibition on the pullulanase activity was 88% for MAP-3 with 1 microg of antibody and 90% for MAP-5 with 2 microg of antibody, but did not inhibit the amylase activity. In Type I antibodies, in contrast, the maximal demonstrated inhibition on the amylase activity was 94% for MAP-12 and 97% for MAP-17 with 1 microg of antibody, respectively, but no inhibition of the pullulanase was noted. MAP-12 recognized sequential epitope, while MAP-17 recognized conformation-dependent epitope of amylase activity-related regions. However, both MAP-3 and MAP-5 recognized the conformation-dependent epitope of the pullulanase activity-related region. Furthermore, the antibodies of MAP-3, MAP-5, MAP-12, and MAP-17 did not compete with one another for binding to the enzyme, indicating that they have different target epitopes on the enzyme. Antibody binding of MAP-12 and MAP-17 to the enzyme was not specifically affected by any of the antiamylase compounds tested: (a) nojirimycin; and (b) 1-deoxynojirimycin. Kinetic analysis of their effects provides evidence that both antibodies of MAP-12 and MAP-17 decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.     

The effect of pH on the kinetics of beef-liver fructose bisphosphatase.

1. The kinetics of the reaction catalysed by fructose bisphosphatase have been studied at pH 7.2 and at pH 9.5. The activity of the enzyme was shown to respond sigmoidally to increasing concentrations of free Mg2+ or Mn2+ ions at pH 7.2, whereas the dependence was hyperbolic at pH 9.5. At both pH values the enzyme responded hyperbolically to increasing concentrations of fructose 1,6-bisphosphate, although inhibition was observed at higher concentrations of this substrate. This high substrate inhibition was shown to be partial in nature and the enzyme was found to be more sensitive at pH 7.2 than at pH 9.5. 2. The properties of the enzyme, are consistent with the enzyme obeying either a random-order equilibrium mechanism or a compulsory-order steady-state mechanism in which fructose bisphosphate binds to the enzyme before the cation. 3. Reaction of the enzyme with a four-fold molar excess of p-chloromercuribenzoate caused activation of the enzyme when its activity was assayed in the presence of MN2+ ions but inhibition when Mg2+ ions were used. Higher concentrations of p-chloromercuribenzoate caused inhibition. This activation at low p-chloromercuribenzoate concentrations, and the reaction of 5,5'-dithio-bis(2-nitrobenzoate) with the four thiol groups in the enzyme that reacted rapidly with this reagent, were prevented or slowed by the presence of inhibitory, but not non-inhibitory, concentrations of fructose bisphosphate. After reaction with a four-fold molar excess of p-chloromercuribenzoate the enzyme was no longer sensitive to high substrate inhibition by fructose bisphosphate.     

Hysteretic enzyme response induced by inhibitory antibodies against human leukocyte elastase

Polyclonal antibodies raised in rabbits against human leukocyte elastase contained two distinct populations of enzyme-inhibiting immunoglobulins. The enzyme-catalyzed reaction in the presence of antibodies (both IgG or monovalent Fab fragments) showed a transient state lasting up to several minutes depending on the inhibitor and substrate concentrations, which was followed by a linear steady-state. The transient was a concave upward or concave downward lag phase depending on whether the enzyme had been preincubated with the antibodies or not, respectively. The kinetic analysis of reaction progress curves showed that both antibody populations were slow inhibitors, which completely and reversibly excluded the substrate from binding to the enzyme. For both antibody populations, the formation of the enzyme-inhibitor complex was characterized by an initial rapid interaction followed by a slow isomerization to a catalytically inactive complex. The apparent pseudo first-order rate constant of the transient slow phase was a hyperbolic function of the inhibitor concentration for both antibodies, from which relevant kinetic constants and the half times for enzyme inactivation could be calculated. For instance, with a total antibody concentration of 1 mg/ml (as IgG), leukocyte elastase was inactivated with t1/2 = 0.31s and 24.8s by the faster and the slower of the two antibodies, respectively. It is suggested that the hysteretic response of the enzyme to the inhibitory action of its antibodies may be due to a kind of memory of the antibody molecule for a special inactive enzyme conformation resulting from inhibition by proteinase inhibitors during the immunization procedure. In turn, the purified antibodies would be able to reversibly induce a slow transition of the enzyme molecule from an active to a substrate-excluding conformation ("induced misfit").     

Anti-neuraminidase antibodies: Which substrate should be used to test their effect on influenza virus neuraminidase

The effect of two monoclonal anti-neuraminidase antibodies RANA 1 and WANA 1 on the enzymic activity of A/NWS_HA-Tokyo/3/67_NA HON2 influenza virus has been demonstrated to differ according to the substrate. Three substrates were tested, a low molecular weight substrate N-acetylneuraminosyl lactose, the glycoprotein fetuin, and cultured human ALL T cells. The enzyme's activity was enhanced when the virus was complexed with either antibody and exposed to the cultured human lymphocytes. Furthermore, no inhibition was observed at a 20:1 ratio of antibody to enzyme. The enzymic activity was inhibited by both antibodies when the two non-physiological substrates were used.     

Human cathepsin B1. Inhibition by α2-macroglobulin and other serum proteins

1. Normal human serum was found to inhibit human cathepsin B1. 2. The major inhibitor present in serum was purified and identified as alpha(2)-macroglobulin. 3. alpha(2)-Macroglobulin was found to bind cathepsin B1 in an approximately 1:1 molar ratio. When bound, the enzyme retained about 50% of its proteolytic activity, and up to 80% of its activity against alpha-N-benzoyl-dl-arginine 2-naphthylamide. 4. Pretreatment of alpha(2)-macroglobulin with cathepsin B1 inactivated by exposure to pH8.5 or iodoacetic acid, in large molar excess, did not prevent the subsequent binding of active enzyme. Active enzyme, once bound, was not protected from inhibition by 1-chloro-4-phenyl-3-tosylamido-l-butan-2-one. 5. Cathepsin B1 was also inhibited by human immunoglobulin G, at high concentration. 6. Because it had been suggested that haptoglobin is responsible for the inhibition of ;cathepsin B' by serum, a method was devised for the selective removal of haptoglobin from mixtures of serum proteins by adsorption on haemoglobin covalently linked to Sepharose. No evidence was obtained that haptoglobin has any inhibitory activity against the enzyme.     

Properties and application to immunoassay of monoclonal antibodies to neuron-specific γγ enolase

Two monoclonal antibodies to human and bovine neuron-specific γγ enolase have been produced in the isolated hybrid cell lines, which were obtained by fusion between γγ-immunized mouse spleen cells and mouse myeloma cells (P3-NS-1/1-Ag4-1), followed by a screening procedure with an enzyme immunoassay. The monoclonal antibody to human γγ enolase (E1-G3) and that to bovine γγ enolase (B1-D6) consisted of γ2a/κ and γl/κ immunoglobulin chains, respectively. Both antibodies could bind with the respective antigen with a molar ratio of about 1:1, and were found to be specific for the γ subunit of enolase, showing reactivities with human γγ and αγ, rat γγ and αγ, and bovine γγ enolases. However, the antibodies did not cross-react with the α or β subunit of human and rat enolase isozymes. Both antibodies could partially inhibit the activity of γγ and αγ enolases. E1-G3 antibody inhibited γγ and αγ enolase activity by 70 and 30%, respectively, and B1-D6 antibody, by 90 and 40%, respectively. Both antibodies had no effect on the activity of αα and ββ enolases of human and rat origins. The applicability of E1-G3 and B1-D6 antibodies to the sandwich-type enzyme immunoassay for neuron-specific enolase (enolase γ subunit) was examined, and it was found that the assay system using E1-G3 and B1-D6 as the labeled antibodies were sufficiently sensitive for the assay of serum neuron-specific enolase concentrations.     

Reaction of phosphofructokinase 2/fructose 2,6-bisphosphatase with monoclonal antibodies. A proof of the bifunctionality of the enzyme

Monoclonal antibodies were derived from mice immunized against homogeneous chicken liver phosphofructokinase 2/fructose 2,6-bisphosphatase. Of 112 clones, 30 were found to secrete antibodies that specifically reacted with the antigen in enzyme-linked immunoabsorbant assay (ELISA) while 17, which were ELISA-negative, produced antibodies that affected the enzymic activity of the antigen. Four clones were subcloned and used for an extensive investigation of the reaction of the corresponding antibodies with the supposedly bifunctional enzyme. A definite proof of the bifunctionality of the enzyme was obtained from the two following observations. First, the two activities were similarly retained by the four antibodies that had been coupled to Sepharose. Second, one of the antibodies inhibited both activities with the same efficiency. Furthermore, the antigen-antibody reaction led to the formation of aggregates with an apparent molecular mass of several megadaltons, showing that the two subunits of the antigen reacted with the same antibody and were therefore identical. The four monoclonal antibodies affected the activity of phosphofructokinase 2. This effect was seen as an up to 17-fold activation as well as an up to 85% inhibition. Only one of the four antibodies (antibody 10) had inhibitory effects on fructose 2,6-bisphosphatase, an effect which was in part explained by a decrease in the rate of formation of the intermediary phosphoenzyme. All the effects described above were obtained on both the chicken liver and the pigeon muscle enzymes but with lower doses of antibody in the case of the former enzyme. Antibody 10 was also shown to react with mouse liver phosphofructokinase 2/fructose 2,6-bisphosphatase, and with phosphofructokinase 2 from chicken brain, heart and testis and from frog skeletal muscle and liver. None of the four antibodies cross-reacted with phosphofructokinase 2 from Saccharomyces cerevisiae or from spinach leaves.     

Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody

A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.     

Preparation of antibodies to chick-embryo galactosylhydroxylysyl glucosyltransferase and their use for an immunological characterization of the enzyme of collagen synthesis

Antibodies were prepared against chick-embryo galactosylhydroxylysyl glucosyltransferase and further purified by immunoaffinity chromatography. The antibodies gave a single precipitation line of identity by double immunodiffusion against crude or pure chick-embryo glucosyltransferase. The ability of the antibody to precipitate the transferase was not altered by destroying the secondary structure of the enzyme. The antibody also inhibited the enzyme activity. The degree of inhibition was higher with denatured citrate-soluble rat skin collagen as the substrate than with gelatinized rat skin insoluble collagen or free galactosylhydroxylysine. The cross-reactivity of the glucosyltransferase between different species was low when studied by double immunodiffusion or inhibition kinetics. The antiserum showed no detectable cross-reactivity against other intracellular enzymes of collagen biosynthesis. A line of complete identity was found in double immunodiffusion between the transferases from whole chick embryos and chick embryo tendon, kidney and cartilage. Inhibition by the antiserum of the enzyme from chick embryo tissues synthesizing different collagen types was relatively similar. The data do not support the hypothesis that galactosylhydroxylysyl glucosyltransferase has isoenzymes with markedly different specific activities or immunological properties.     

Inhibition of microtubule assembly in vitro by anti-tubulin monoclonal antibodies

Five monoclonal antibodies against N-terminal domains of alpha- or beta-tubulin were tested for their ability to interfere with the in vitro formation of microtubules. Although all the antibodies exhibited similar association constants for immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. For the most potent antibody, TU-13, the antibody/tubulin molar ratio of about 1:320 was sufficient for a 50% inhibition. The data indicate that the surface regions of N-terminal domains of tubulin are involved in the formation of microtubules.     

ATP inhibits insulin-degrading enzyme activity

We studied the ability of ATP to inhibit in vitro the degrading activity of insulin-degrading enzyme. The enzyme was purified from rat skeletal muscle by successive chromatographic steps. The last purification step showed two bands at 110 and 60 kDa in polyacrylamide gel. The enzyme was characterized by its insulin degradation activity, the substrate competition of unlabeled to labeled insulin, the profile of enzyme inhibitors, and the recognition by a specific antibody. One to 5 mM ATP induced a dose-dependent inhibition of insulin degradation (determined by trichloroacetic acid precipitation and insulin antibody binding). Inhibition by 3 mM adenosine 5'-diphosphate, adenosine 5'-monophosphate, guanosine 5'-triphosphate, pyrophosphate, beta-gamma-methyleneadenosine 5'-triphosphate, adenosine 5'-O-(3 thiotriphosphate), and dibutiryl cyclic adenosine 5'-monophosphate was 74%, 4%, 38%, 46%, 65%, 36%, and 0%, respectively, of that produced by 3 mM ATP. Kinetic analysis of ATP inhibition suggested an allosteric effect as the plot of 1/v (insulin degradation) versus ATP concentration was not linear and the Hill coefficient was more than 1 (1.51 and 2.44). The binding constant for allosteric inhibition was KiT = 1.5 x 10(-7) M showing a decrease of enzyme affinity induced by ATP. We conclude that ATP has an inhibitory effect on the insulin degradation activity of the enzyme.     

Mechanism by which antibodies inhibit hapten-malate dehydrogenase conjugates. An enzyme immunoassay for morphine.

Antimorphine antibodies inhibit the activity of morphine conjugates of mitochondrial malate dehydrogenase. Conjugation of malate dehydrogenase through tyrosine and amino groups resulted in only moderate losses of enzyme activity. On conjugation through disulfide bonds the enzyme activity first increased but dropped sharply with increasing substitution. Only the former conjugates were inhibited by excess antibodies. The degree of inhibition (up to 86%) was directly related to the number of morphine residues bonded directly to amino groups. The maximum number of antibody binding sites that bind to enzyme was nearly equal to the number of haptens provided there were 16 or less haptens/enzyme. However up to 26 haptens/enzyme became completely bound by antibody on long incubation. Inhibition of enzyme activity was detectably reduced by 2 times 10 minus 9 M morphine or 2 times 10 minus 10 M codeine, thus providing a sensitive assay for these drugs. The data suggest that enzyme inhibition occurs by conformational freezing of the enzyme when antibody binds to a morphine residue attached to one specific amino group.     

Purification and properties of agmatine amidinohydrolase of Evernia prunastri

An agmatine amidinohydrolase (EC 3.5.3.11) has been purified from Evernia prunastri (L.) Ach. thallus incubated on 40 m M L-arginine at 26°C in the dark. The enzyme was purified 485-fold with an overall yield of 55%. It shows a pH optimum of 6.9, a temperature optimum at 35–40°C and a molecular mass (weight) of about 320 000. The Evernia hydrolase is significantly activated by L-arginine, L-ornithine and putrescine for agmatine concentrations lower than 14 m M and inhibited for agmatine concentrations producing inhibition by an excess of substrate. Urea was always a powerful inhibitor of the enzyme. The K_m for agmatine was estimated to be 6.4 m M .     

Immunochemical and enzymatic studies and glutamate dehydrogenase and a related mutant protein from Neurospora crassa

Roberts, D. B. (University of Cambridge, Cambridge, England). Immunochemical and enzymatic studies on glutamate dehydrogenase and a related mutant protein from Neurospora crassa. J. Bacteriol. 91:1888-1895. 1966.-When an investigation was made of the inhibition of Neurospora glutamate dehydrogenase by bivalent and univalent antibodies, it was shown that the enzyme inhibition is not complete even with excess antibodies. The residual activity was some three times greater with bivalent antibodies, in spite of the observation that the ratio of inhibiting antibodies to catalytic sites was 2:1 for both types of antibody. Substrates did not affect the inhibition of enzyme activity, nor did antibodies affect the K(m) for either substrate. An allosteric mechanism for the inhibition of glutamate dehydrogenase by antibodies is proposed. It was also demonstrated that the mutant protein am-3 can be activated, to show glutamate dehydrogenase activity, by a number of activators. The requirement for the activation was the presence of a carboxymethyl group. The data suggest that the nonactivated protein has two combining sites for l-glutamate: the catalytic and activating sites. The wild-type enzyme has only one of these sites. Because the activating site is distinct from the catalytic site, an allosteric mechanism was postulated for activation. Inhibition of am-3 activity by antibodies is achieved either by a mechanism similar to the inhibition of wild-type activity or by the antibodies preventing the activation of the mutant protein. The am-3 protein can be activated by antibodies. Consequently, there appeared to be a relation the phenomena of enzyme inhibition and am-3 activation by antibodies; i.e., they alter the configuration of the catalytic site. This alteration was necessary for the activation of am-3, but inhibited the activity of the wild-type enzyme.     

Ornithine decarboxylase activity is inhibited by the polyamine precursor amino acids at the protein stability level in Caco-2 cells

High concentrations of certain amino acids are known to affect hormonal secretion, immune function, electrolyte balance or metabolic functions. However, there is a lack of knowledge regarding the molecular mechanisms responsible for these effects. We showed that, as well as spermidine transport, the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, is decreased in human colon adenocarcinoma cells, Caco-2, following a 4-h supplementation with one of the two polyamine precursor amino acids, L-arginine or L-methionine. Dose-response assays indicated that the inhibitory effect of supplemental L-methionine was stronger than that of supplemental L-arginine. However, it was transient, being even replaced by ODC induction after 8 h, whereas the inhibitory effect of L-arginine lasted for at least 8 h. Unlike L-cysteine, neither L-methionine nor L-arginine could inhibit ODC activity in a crude acellular preparation of the enzyme. The inhibition of ODC activity in cells exposed to L-methionine or L-arginine was due to a decreased abundance of ODC protein without change at the mRNA level and each of these amino acids could counteract ODC induction by a glycine supplement. Contrary to the latter, supplemental L-methionine or L-arginine induced a marked decrease in ODC half-life, concomitantly with an increase in the activity of antizyme, an ODC inhibitory protein. Thus, depending on their nature, amino acids can up- or downregulate ODC activity at the protein stability level.     

Effect of specific antibodies on D-glyceraldehyde-3-phosphate dehydrogenase.

The inhibition of rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase by specific antibodies produced in rabbits has been studied. The results suggest that no influence on the enzyme active site is caused by the interaction with antibody, the inhibition being due entirely to the restricted accessibility for substrates of a part of dehydrogenase molecules included in the immune precipitate. Soluble complexes of the enzyme with monovalent Fab antibody fragments retain full catalytic activity. Modification of 8 -SH groups per mole of glyceraldehyde-3-phosphate dehydrogenase with p-chloromercuribenzoate results in no alterations in the quantitative precipitin curve, thus supporting the conclusion about the different localization of species-specific antigenic determinants of the enzyme and its active center. Interaction with monovalent Fab fragments of antibody stabilizes the structure of the dehydrogenase. Eight molar equivalents of Fab fragments almost completely protect the enzyme from cold inactivation in the presence of 0.15 M NaCl. Complex formation with Fab fragments does not prevent, however, the ADP-induced inactivation of the enzyme.