Multiple forms of acetylcholinesterase from pig brain

1. A number of methods of solubilization of pig brain acetylcholinesterase (EC 3.1.1.7) were studied. The multiple enzymic forms of the resultant preparations were examined by polyacrylamide-gel electrophoresis. 2. Butanol extraction, Nagarase treatment and ultrasonication proved unsuitable as preparatory methods, but detergent treatment (Triton X-100, Triton X-100-KCl and lysolecithin) gave good yields. 3. Separation of soluble enzyme in three systems of polyacrylamide-gel electrophoresis were compared and the relative advantages are discussed. 4. By using a 6% (w/v) gel and continuous buffer system two forms of acetylcholinesterase were detected in Triton X-100-solubilized enzyme, but the incorporation of a sample and spacer gel and a discontinuous buffer system resolved this into four components. The forms of the soluble enzyme extracted by different methods differed in mobility. 5. With gradient polyacrylamide-gel electrophoresis between two and six forms were detected, depending on the method used for extraction. The average molecular weights of the five forms most frequently found were 60000, 130000, 198000, 266000 and 350000. 6. Treatment of the Triton X-100-extracted enzyme with 2.5m-urea altered the pattern and evidence of dissociation was observed. 7. The results are discussed in the light of present theories on the molecular structure of acetylcholinesterase.     

Molecular forms of sucrose extractable and particulate acetylcholinesterase in the developing and adult rat brain

The activity of acetylcholinesterase in the rat striatum increased considerably during development, while activities in the cerebellum and midbrain increased only slightly. During maturation the activity of butyrylcholinesterase increased in all the brain regions examined except in cerebellum. The percentage of acetyl-cholinesterase extractable by isotonic sucrose solution from mature striatum was much smaller than those obtained for other regions of the rat brain. For the developing striatum, the percentage of isotonic sucrose extractable activity was almost three times that for adult striatum. Density gradient centrifugation showed that the membrane-bound particulate fraction of adult rat brain was mostly composed of the 10 S form of acetylcholinesterase with little activity of 4 S form of the enzyme. However, a much higher proportion of the 4 S form was found in the isotonic sucrose soluble fraction. In contrast to the particulate fraction from adult brain, that from 6-day old rats contained a much higher proportion of the 4 S form of the enzyme. The sucrose soluble fraction from 6-day old rat brains contained in general much smaller proportion of 4 S form as compared to those from adult rat brains.     

The multiple forms of brain acetylcholinesterase

Summary The pattern of the multiple forms of the acetylocholinesterase (AChE, E.C. 3.1.1.7) of the rat brain is investigated using polyacrylamide gradient micro-gel electrophoresis with regard to a possible functional importance of this individual forms. The patterns of the AChE-forms of selected regions of the CNS are compared and certain differences could be shown. After increased cholinergic input (into the hippocampus by electrical stimulation of the nc. septi medialis) an aggregation of AChE subunits is detectable. Subletal intoxication with an irreversible inhibitor of AChE is followed by a faster recovery of the smaller forms. A suggestion of a possible functional role of the multiple forms of AChE is discussed.The research reported in this paper was supported by the Ministerium für Wissenschaft und Technik der DDR     

The multiple forms of brain acetylcholinesterase

Summary Micro-polyacrylamide gradient electrophoresis followed by active staining is applied for the demonstration of the multiple forms of acetylcholinesterase. Among other advantages the very small samples that enable the analysis of well-defined brain material as well as the almost histochemical conditions of incubation enable its successful use in topochemical investigations of the multiple form pattern of brain acetylcholinesterase.The acetylcholinesterase of bovine nc. caudatus could be separated into 4 multiple forms and the pattern was analysed microdensitometrically. These forms differ in their molecular weight as well as well as in their degree of membrane binding. Increasing ionic strength (NaCl) is followed by changes in the pattern. This result is discussed as caused by aggregation of enzyme subunits.The research reported in this paper was supported by the Ministerium für Wissenschaft und Technik der DDR     

Molecular forms of acetylcholinesterase in mammalian smooth muscles

A comparative study of the molecular forms of acetylcholinesterase (AChE) was made in various smooth muscles (intestine, vas deferens, ciliary body, iris, nictitating membrane retractor, ureter, arteries, anococcygeus muscles) of some mammals (cat, guinea-pig, rat, rabbit, mouse), seeking for a correlation between the presence of 16 S (asymmetric, tailed) form of AChE in smooth muscles and their type of innervation defined by morphological criteria, as well as by the nature of the main neurotransmitters involved in their neuroeffector junctions. Contrary to previous assertions, many smooth muscles contain 16 S AChE, although all those examined here exhibited a proportion clearly less than that of striated muscles. There are large species-specific and individual variations in the percentage of 16 S AChE. The highest percentages of 16 S AChE were found in ciliary and iris muscles, which are provided with an individual (= multiunit) cholinergic innervation. The vas deferens muscles, which are also individually, but noradrenergically innervated contain practically no 16 S AChE. In the muscles having a fascicular (= unitary) innervation, the differences are striking: 16 S AChE is in rather high amount in intestine muscle layers, whereas it is very low or virtually absent in ureter or arterial muscles. Thus, the type of innervation is not clearly involved in the amount of 16 S AChE present in smooth muscles. As for the nature of neurotransmitter a clear correlation exists only in the case of individual innervation, in which only one neurotransmitter is involved or largely predominant.     

Regional grey and white matter changes in heavy male smokers

Cigarette smoking is highly prevalent in the general population but the effects of chronic smoking on brain structures are still unclear. Previous studies have found mixed results regarding regional grey matter abnormalities in smokers. To characterize both grey and white matter changes in heavy male smokers, we investigated 16 heavy smokers and 16 matched healthy controls, using both univariate voxel-based morphometry (VBM) and multivariate pattern classification analysis. Compared with controls, heavy smokers exhibited smaller grey matter volume in cerebellum, as well as larger white matter volume in putamen, anterior and middle cingulate cortex. Further, the spatial patterns of grey matter or white matter both discriminated smokers from controls in these regions as well as in other brain regions. Our findings demonstrated volume abnormalities not only in the grey matter but also in the white matter in heavy male smokers. The multivariate analysis suggests that chronic smoking may be associated with volume alternations in broader brain regions than those identified in VBM analysis. These results may better our understanding of the neurobiological consequence of smoking and inform smoking treatment.     

Distribution of acid and alkaline deoxyribonucleases in white and grey matter regions of growing and aging chick brain

The activities of acid and alkaline deoxyribonucleases in the white and grey matter areas of growing and old chick cerebrum were measured. Two marker enzymes for glial cells, butyrylcholinesterase and carbonic anhydrase were also measured in these regions. Higher specific activities of both butyrylcholinesterase and carbonic anhydrase were found in the white matter region at all the stages studied. Acid and alkaline deoxyribonuclease activities were observed in both white and grey matter. The decrease in the specific activity of acid deoxyribonuclease with advancement of age was more pronounced as compared to the alkaline deoxyribonuclease Marked reduction in total acid deoxyribonuclease activity in white matter, beyond the age of 130 days, was observed. On the other hand, total alkaline deoxyribonuclease activity in both white and grey matter continued to increase with age Further, the activity per mg of DNA also increased in white matter of the old brain. These results indirectly suggest a continued role for alkaline deoxyribonuclease in glial cells formed at a later age.     

Molecular forms of acetylcholinesterase in Xenopus muscle

Xenopus adult muscle, whole Xenopus embryos, and cultured embryonic myocytes together contain five acetylcholinesterase forms which can be resolved by sucrose density gradient centrifugation. These are identified as the collagenase-sensitive asymmetric forms A12 and A8, and the globular forms G4, G2, and G1. Asymmetric forms rise in whole embryos during the period of neuromuscular synapse formation, but their rise is not prevented by tricaine methanesulfonate, which abolishes motor activity. Aneural myocyte cultures synthesize primarily asymmetric acetylcholinesterase, much of which is extracellular. Prior nerve contact is not required for its expression. The proportion of asymmetric forms is neither decreased by tetrodotoxin, nor enhanced by veratridine and aconitine. We conclude that muscle activity does not modulate the expression of asymmetric acetylcholinesterase in Xenopus.     

Preferential inhibition of acetylcholinesterase molecular forms in rat brain

The effect of eight different acetylcholinesterase inhibitors (AChEIs) on the activity of acetylcholinesterase (AChE) molecular forms was investigated. Aqueous-soluble and detergent-soluble AChE molecular forms were separated from rat brain homogenate by sucrose density sedimentation. The bulk of soluble AChE corresponds to globular tetrameric (G₄), and monomeric (G₁) forms. Heptylphysostigmine (HEP) and diisopropylfluorophosphate were more selective for the G₁ than for the G₄ form in aqueous-soluble extract. Neostigmine showed slightly more selectivity for the G₁ form both in aqueous- and detergent-soluble extracts. Other drugs such as physostigmine, echothiophate, BW284C51, tetrahydroaminoacridine, and metrifonate inhibited both aqueous- and detergent-soluble AChE molecular forms with similar potency. Inhibition of aqueous-soluble AChE by HEP was highly competitive with Triton X-100 in a gradient, indicating that HEP may bind to a detergent-sensitive non-catalytic site of AChE. These results suggest a differential sensitivity among AChE molecular forms to inhibition by drugs through an allosteric mechanism. The application of these properties in developing AChEIs for treatment of Alzheimer disease is considered.Special issue dedicated to Dr. Morris H. Aprison.     

Lamprey brain contains globular and asymmetric forms of acetylcholinesterase

To obtain more information about the evolution of acetylcholinesterase in the vertebrates, we studied the cholinesterase activity from the brain of the lamprey Petromyzon marinus. We found that the enzyme is true acetylcholinesterase and that 98% of it is present in the G₄ globular form. Only 1% of the enzyme was found distributed among the asymmetric forms A₄, A₈ and A₁₂; an additional 1% of the activity could not be extracted from the brain. The identity of the asymmetric forms was confirmed by collagenase digestion. These data demonstrate that asymmetric acetylcholinesterase is present in the CNS of organisms representing all classes of vertebrates. However, our results are inconsistent with an evolutionary trend that has been observed for vertebrate brain acetylcholinesterase.     

Multiple forms of calpastatin in pig brain

Pig brain was found to contain two calpain-specific, heat-stable inhibitory fractions which could be separated by DEAE-cellulose chromatography. CS-0.1, which was eluted from the column at 0.1 M NaCl, was identified as an ordinary, well-known calpastatin. CS-0.2, eluted at 0.2 M NaCl, was different from CS-0.1 in that it inhibited calpain 1 more strongly than calpain II and that it did not cross-react with anti-calpastatin antibodies. Partial purification indicated that CS-0.2 contained inhibitor proteins smaller than ordinary calpastatin, but whether they are the products derived from CS-0.1 or entirely different genetic products has not yet been determined.     

Regional distribution of the muscarinic cholinoceptor and acetylcholinesterase in guinea pig brain

The muscarinic receptors in membranes prepared from guinea pig brain were studied using a radiolabeled antagonist, [³H]quinuclidinyl benzilate (QNB). The apparent dissociation constant of the QNB-receptor complex (K _d ) was similar in all regions, but the concentration of receptors was highest in the striatum, cerebral cortex, and hippocampus and lowest in the cerebellum. Similar distributions have been reported for other species, although the concentration of receptors in guinea pig brain is higher than in other species. Acetylcholine inhibited QNB binding with a Hill coefficient of 0.4–0.6. The concentration of acetylcholine required to inhibit binding by 50% (I₅₀) was lowest in the brain stem and more than 10 times higher in the hippocampus. Similar results have been reported for mouse brain. The activity of acetylcholinesterase was highest in the striatum, where the concentration of muscarinic receptors is highest, but did not vary greatly in other brain regions.RMD was seconded to the University of Melbourne to undertake this study.     

Molecular forms of acetylcholinesterase in developing Torpedo embryos

We have studied the evolution of acetylcholinesterase molecular forms during the embryonic development of Torpedo marmorata, in the electric organs and in the electric lobes of the central nervous system. In the early stages of development (35 mm embryos, ‘myogenic phase’ of electric organ development), globular forms of acetylcholinesterase (G₄ and G₂) are abundant in both tissues and the collagen-tailed form A₁₂ is already present. In the electric organs, this form accumulates rapidly after the 55–60 mm stage (‘electrogenic phase’), when synapse formation first commences. Although the molecular characteristics of the collagen-tailed forms, and particularly their aggregation properties, do not appear to change during development, their solubilization requires higher concentrations of MgCl₂, as the electrocytes mature, suggesting that they become more tightly integrated in a better organized basal lamina. The smaller collagen-tailed form A₈ shows a transient increase which coincides approximately with the maximal accumulation of A₁₂, suggesting that it is an intermediate in its synthesis. The accumulation of the hydrophobic G₂, which eventually becomes predominent in the adult electric organs, lags behind that of A₁₂. The functional significance of this important fraction of acetylcholinesterase is therefore not that of a pool of precursor for the synthesis of A₁₂. In the electric lobes, the tetrameric form (G₄) is abundant during development, as well as G₂ and G₁ at certain stages, but the A₁₂ form is predominant in the adult.     

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.     

Sexual dimorphism in the adolescent brain: Role of testosterone and androgen receptor in global and local volumes of grey and white matter

Here we examined sex differences in the volumes of grey and white matter, and in grey-matter “density,” in a group of typically developing adolescents participating in the Saguenay Youth Study (n = 419; 12-18 years). In male adolescents, we also investigated the role of a functional polymorphism in androgen-receptor gene (AR) in moderating the effect of testosterone on volumes of grey and white matter and grey-matter density. Overall, both absolute and relative volumes of white matter were larger in male vs. females adolescents. The relative grey-matter volumes were slightly larger in female than male adolescents and so was the grey-matter density in a large number of cortical regions. In male adolescents, functional polymorphism of AR moderated the effect of testosterone on relative white- and grey-matter volumes. Following a discussion of several methodological and interpretational issues, we outline future directions in investigating brain-behavior relationships vis-à-vis psychopathology.     

Two forms of GABA transaminase in pig brain

Two forms of GABA transaminase which could be distinguished by ion-exchange chromatography have been separated and purified in pig brain. The two forms have differentK _m values for -ketoglutarate and show different degrees of inhibition by various salts. Although the two forms are separable, they have identical antigenic properties, pH optima, and NH₂ terminal amino acid composition, and they appear to be of the same molecular size. The biological significance or the relationship between multiple forms of GABA transaminase is not yet understood.     

Multiple forms of adenylate kinase in pig brain

The rate of ATP formation from ADP by adenylate kinase is known to be easily followed by determination of the increase in fluorescence due to the NADPH that is formed by combined reactions of hexokinase and gluc-6-p dehydrogenase. We found that the rate of CTP formation from CDP also can be followed similarly by use of more units of hexokinase and extension of the reaction time. A crude enzyme sample containing most of the activity of adenylate kinase was prepared from pig brain and eluted from a CM-cellulose column. Enzymatic activity of ATP formation and that of CTP formation were compared for all the fractions. Two fractions were found in the eluate; one was rather specific for ADP as substrate, and the other was less specific for ADP and could form CTP at an appreciable rate.     

Different membrane-bound forms of acetylcholinesterase are present at the cell surface of hepatocytes

In the present study we have determinated the acetylcholinesterase molecular forms present in rat liver hepatocytes; we have also studied the association of acetylcholinesterase with the cell surface of the hepatocytes. Subcellular fractionation indicated that rough endoplasmic reticulum and plasma-membrane-enriched fractions contains G4 and G2 acetylcholinesterase forms bound to membranes. Hepatocytes incubated with phosphatidylinositol-specific phospholipase C released about 70% of the surface acetylcholinesterase. Sedimentation analysis showed that all the solubilized acetylcholinesterase activity comes exclusively from a G2 dimer. The G4 hydrophobic form of acetylcholinesterase accounts for the additional cell-surface activity. The existence of these two forms of acetylcholinesterase on the surface of hepatocytes was further established by analyzing the phosphatidylinositol-specific phospholipase C sensitivity of the acetylcholinesterase molecular forms present in isolated rat liver plasma membranes.