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.     

Ecdysterone induces acetylcholinesterase in mammalian brain

The effects of ecdysterone on brain acetylcholinesterase (AChE) in immature and adult rats of both sexes have been studied in in vitro conditions. Ecdysterone produced an increase of AChE in rat brain slices. The most remarkable effect was found in immature male rats. In vitro assay using a purified AChE from electric eel showed no effect. Pretreatment with cycloheximide or actinomycin D abolished the ecdysterone action on brain AChE. These results support the idea that induction of AChE may be involved in the heterophilic action of ecdysterone.     

Immunochemical differences among molecular forms of acetylcholinesterase in brain and blood

Molecular forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) differ in their solubility properties as well as in the number of their catalytic subunits. We used monoclonal antibodies to investigate the structure of acetylcholinesterase forms in brain, erythrocytes and serum of rats, rabbits and other mammals. Two antibodies were found to bind tetrameric acetylcholinesterase in preference to the monomeric enzyme. These antibodies also displayed lower affinity for certain forms of 'soluble' brain acetylcholinesterase than for the 'membrane-associated' counterparts. Furthermore, one of them was virtually lacking in affinity for the membrane-associated enzyme of erythrocytes. The basis for the antibody specificity was not fully determined. However, the immunochemical results were supported by measurements of enzyme thermolability, which showed that the catalytic activity of 'soluble' acetylcholinesterase was comparatively heat-resistant. These observations point toward structural differences among the solubility classes of acetylcholinesterase.     

Active-site determinations on forms of mammalian brain and eel acetylcholinesterase.

Three forms of brain acetylcholinesterase were purified from bovine caudate-nucleus tissue and determined by calibrated gel filtration to have mol.wts. of approx. 120 000 (C), 230 000 (B) and 330 000 (A). [3H]Di-isopropyl phosphorofluoridate (isopropyl moiety labelled) was purified from commercial preparations and its concentration estimated by an enzyme-titration procedure. Brain acetylcholinesterase preparations and enzyme from eel electric tissue were allowed to react with [3H]di-isopropyl phosphorofluridate in phosphate buffer until enzyme activity was inhibited by 98%. Excess of [3H]di-isopropyl phosphorofluoridate that had not reacted was separated from the labelled enzyme protein by gel filtration, or by vacuum filtration or by extensive dialysis. The specificity of active-site labelling was confirmed by use of the enzyme reactivator, pyridine 2-aldoxime. The forms of brain acetylcholinesterase were calculted to contain approximately two (C) four (B) and six (A) active sites per molecule respectively. Acetylcholinesterase (mol.wt. 250 000) from electric-eel tissue was estimated to contain two active sites per molecule. Gradient-gel electrophoresis was used to confirm the estimation of molecular weights of brain acetylcholinesterase forms made by gel filtration. Under the conditions of electrophoresis acetylcholinesterase form A was stable, but form B was converted into a species of approx. 120 000 mol. wt. Similarly, form C of the brain enzyme was converted into a 60 000-mol.wt. form during electrophoresis. These results are in general accord with the suggestion that the multiple forms of brain acetylcholinesterase may be related to the aggregation of a single low-molecular-weight species.     

Comparative studies on glucose phosphorylating isoenzymes from vertebrates--VIII. Immunochemical studies on mammalian hexokinases A

An immune serum elicited in a rabbit by injection of homogeneous brain hexokinase A was shown to be specific for the antigen. Other rat hexokinase isoenzymes (hexokinases B, C or D) did not present cross-reaction when tested by immunoinhibition of enzyme activity, double immunodiffusion and immunoadsorbent columns. The enzyme activity of hexokinase A from several mammals (rodents, lagomorphs, artiodactyls) was partially inhibited by the immune serum. In the case of mouse enzyme, the amount of serum required to inhibit 50% of the activity was five-fold higher than in the case of the rat enzyme. Enzymes from cow or sheep brain were only marginally affected. Hexokinases A isolated from various mammals, tested against the rat enzyme, showed faint lines of precipitation and marked spurs in double immunodiffusion plates even when enzymes from closely related rodents were analyzed. Immunoadsorbent columns, on the other hand, were able to retain most of the activity of hexokinases A from the mammals studied. Micro-complement fixation tests showed that hexokinases A from mammals outside the Order Rodentia were only partially recognized by the anti-hexokinase Arat serum. The results suggest that amino acid substitutions on the hexokinase A molecule have occurred at a rather fast rate.     

Immunochemical studies of isolated human brain ganglioside components

Abstract— Gangliosides G₁ to G₅ were isolated from human brain by means of TLC and tested with respect to their specificity to antisera against normal brain and Tay-Sachs brain gangliosides by agar double diffusion analysis. Gangliosides G₂ and G₄ gave precipitation reactions with antisera to normal human gangliosides (NHG) while only ganglioside G₆ reacted with antisera to Tay-Sachs gangliosides (TSG). Additional specificity information was also obtained by use of the enzyme neuraminidase for the removal of specific sialic acid (NANA) residues. It was concluded from these data that the specificity of the anti-NHG antibodies is determined by the presence of a galactose (β1, 3) N-acetyl galactos-amine–while that of anti-TSG antibodies is due to a N-acetyl galactosamine (β1, 4) galactose-end sequence. By means of natural compounds of known structure it was found that both the sequence of carbohydrate residues and position of NANA residues in the molecule played a critical role in the formation of precipitation bands with NHG-antisera. This information was utilized to distinguish one isomeric form of disialoganglioside from another, i.e. G₂ from G₃ and to confirm the structure of the trisialoganglioside, G₁. The immunochemical method appears to be a useful one for elucidating structural differences in ganglioside molecules.     

Immunochemical studies on alpha-amylase. 3. Immunochemical relationships among amylases from various microorganisms

Sirishinha, Stitaya (University of Rochester School of Medicine and Dentistry, Rochester, N.Y.), and Peter Z. Allen. Immunochemical studies on alpha-amylase. III. Immunochemical relationships among amylases from various microorganisms. J. Bacteriol. 90:1120-1128. 1965.-Immunochemical relationships among amylases obtained from a selected group of microorganisms were examined, and a cross-reaction was detected between the alpha-amylases of Bacillus stearothermophilus and B. subtilis. Immunodiffusion and quantitative precipitin studies, as well as cross-neutralization tests, indicate that B. stearothermophilus alpha-amylase reacts with a portion of antibody present in antisera to crystalline B. subtilis alpha-amylase. Amylases from these two species thus have some aspects of structure in common. Limited data obtained by immunodiffusion suggest that groupings which confer cross-reactivity to the B. stearothermophilus enzyme are lost after exposure to mercaptoethanol in the presence of ethylenediamine-tetraacetate, followed by treatment with iodoacetamide. With the antisera employed and within the concentration range examined, no immunochemical cross-reaction was observed among amylases from Aspergillus oryzae, B. subtilis, B. polymyxa, B. macerans, Pseudomonas saccharophila, and Euglena sanguinis. Immunoelectrophoresis of partially purified B. stearothermophilus alpha-amylase by use of antiserum to the crude enzyme, together with localization of amylase activity in immunoelectrophoretic plates, suggests that B. stearothermophilus alpha-amylase is antigenic in the rabbit.     

Gene structure of mammalian acetylcholinesterase. Alternative exons dictate tissue-specific expression.

The genes encoding mouse and human acetylcholinesterases have been cloned from genomic and cosmid libraries. Restriction analysis and a comparison of sequence with the cDNAs have defined the exon-intron boundaries. In mammals, three invariant exons encode the signal peptide and the amino-terminal 535 amino acids common to all forms of the enzyme whereas alternative exon usage of the next exon accounts for the structural divergence in the carboxyl termini of the catalytic subunits. mRNA protection studies show that the cDNA encoding the hydrophilic catalytic subunits represents the dominant mRNA species in mammalian brain and muscle whereas divergent mRNA species are evident in cells of hematopoietic origin (bone marrow cells and a erythroleukemia cell line). Analyses of mRNA species in these cells and the genomic sequence have enabled us to define two alternative exons in addition to the one found in the cDNAs; they encode unique carboxyl-terminal sequences. One mRNA consists of a direct extension through the intervening sequence between the common exon and the 3' exon deduced from the cDNA. This sequence encodes a subunit lacking the cysteine critical to oligomer formation. Another mRNA results from a splice that encodes a stretch of hydrophobic amino acids immediately upstream of a stop codon. This exon, when spliced to the upstream invariant exons, should encode glycophospholipid-linked species of the enzyme. Homologous sequence, identity of exon-intron junctions, and identity of position of the stop codon are seen for this region in mouse and human. Polymerase chain reactions carried out across the expected intron region and mRNA protection studies show that this splice occurs in mouse bone marrow and erythroleukemia cells yielding the appropriate cDNA.     

Immunochemical studies of estrogen receptors

Fusion of splenic lymphocytes from Lewis rats, immunized with affinity-purified estrogen receptor from the cytosol of MCF-7 human breast cancer cells, with two different mouse myeloma lines, has provided 13 monoclonal hybridoma lines secreting antiestrophilin antibodies, each of which (with one possible exception) recognizes a different antigenic determinant in the human receptor molecule. Of this library of monoclonal antibodies, some react with estrophilin from all sources tested, some react with mammalian but not avian receptors, whereas one preparation appears specific for estrophilin from primate sources. By proteolytic digestion under controlled conditions with mercury-deactivated papain, chymotrypsin, and trypsin, respectively, it is possible to remove sequentially the determinants recognized by one, two or three of the monoclonal antibodies, leaving the epitopes for the six remaining antibodies investigated on the steroid-binding portion of the receptor. The proteolytic fragment containing the epitope most readily removed (by mercuripapain) also contains the DNA-binding domain of the activated receptor molecule. Immunocytochemical staining, using the peroxidase procedure with various monoclonal antibody preparations, of frozen sections of human breast cancer tissue, fixed in ethanol or in picric acid-formaldehyde reagent, shows clearly that the majority of the native receptor, which appears in the cytosol after tissue homogenization, is actually localized within the nuclear compartment in the intact cell. The immunocytochemical technique also permits the identification of mixed populations of receptor-containing and non-containing cells in human breast cancers.