Molecular forms of acetylcholinesterase in frog skeletal muscle: effects of denervation

In the muscles of the frog, four main molecular forms of acetylcholinesterase are present, with sedimentation coefficients of 5.7, 10.4, 13 and 17.6 S. The heaviest forms, 13 S and 17.6 S are found in both nerve-free segments and endplates zones of sartorius muscle. They decrease in long-term denervation experiments. Consequently, these two forms are not specifically localized in endplates containing regions. However, they depend either on muscle activity or on neural influence or both.     

Acetylcholinesterase Molecular Forms in Chick Ciliary Ganglion: Pre- and Postsynaptic Distribution Derived from Denervation, Axotomy, and Double Section

Abstract: Four main molecular forms of acetylcholinesterase (AChE) characterized by their sedimentation coefficients (5S, 7.5S, 11.5S, and 20S), are found in chick ciliary ganglion. After transection of the preganglionic nerve (denervation), total AChE activity in the ganglion dropped by 35% in 2 days. By then, 11.5s and 20s forms had diminished by 60 and 75% respectively, where as 7.5s remained practically unchanged. Since presynaptic structures disappeared 2 days after denervation, we inferred that at most 35% of total ganglion AChE was presynaptic: 11.5s and 20s might be mainly presynaptic and 7.5S, postsynaptic. At later time intervals. total AChE continued to decline up to day 5, possibly as a result of orthograde transynaptic regulation of the enzyme activity. After transection of postganglionic nerves (axotomy), total ganglion activity showed little change; 11.5s and 20s decreased by 40 and 6076, respectively, in 5 days, but these drops were compensated for by an early increase in 7 5S, which started the day after axotomy. After simultaneous transection of both pre- and postganglionic nerves (double section), total ganglion AChE dropped rapidly by 35% in 1 day and remained at that level up to 21 days. The 11.5S diminished rapidly by 60% in 1 day. The early increase of the 7.5s form induced by axotomy alone did not occur. Since the effect resulting from double section was not the equivalent of the cumulative effects observed after denervation and axotomy, respectively, the level of AChE forms in the ganglion may be regulated by reciprocal interaction of pre- and postsynaptic elements. After denervation and double section but not after axotomy alone, the contralateral non-operated ganglion exhibited a fall in the 20s form. This suggests that a transynaptic effect is exerted on AChE by the contralateral preganglionic neuron. Taken together, these results indicate that the various AChE molecular forms in chick ciliary ganglion are preferentially but not exclusively distributed as follows: the pre- and postganglionic axons contain mainly the 11.5S form, whereas nerve endings and synaptic structures are enriched in 20S, and ganglion cell bodies, in 7.5s.     

CELLULAR DISTRIBUTION OF 16S ACETYLCHOLINESTERASE

Multiple molecular forms of acetylcholinesterase (AChE; EC 3.1.1.7), in crude extracts of various tissues from the rat, were distinguished by velocity sedimentation analysis on linear sucrose gradients. Skeletal muscle samples containing end-plate regions showed three different forms of AChE with apparent sedimentation coefficients of 16, 10 and 4s. The 16s form was not detected in non-innervated regions of skeletal muscle, large intestine smooth muscle, whole brain tissue, red blood cells or plasma. Spinal cord, a predominantly motor cranial nerve and mixed (sensory and motor) peripheral nerves contained 16, 10, 6.5 and 4S AChE. Ventral motor roots, supplying the sciatic nerve, contained these four forms of the enzyme, while corresponding dorsal sensory roots were devoid of the 16S form. The 16s-AChE confined to ventral roots can be attributed totally to motor neurons and not to Schwann cells composing these roots. Whether the 16s-AChE presently found in motor nerves has chemical identity with that found at motor end-plates is the basis of future experiments.     

Characterization of acetylcholinesterase molecular forms in slow and fast muscle of rat

Multiple molecular forms of acetylcholinesterase (AChE EC 3.1.1.7) from fast and slow muscle of rat were examined by velocity sedimentation. The fast extensor digitorum longus muscle (EDL) hydrolyzed acetylcholine at a rate of 110 mumol/g wet weight/hr and possessed three molecular forms with apparent sedimentation coefficients of 4S, 10S, and 16S which contribute about 50, 35, and 15% of the AChE activity. The slow soleus muscle hydrolyzed acetylcholine at a rate of 55 mumol/g wet weight/hr and has a 4S, 10S, 12S, and 16S form which contribute 22, 18, 34, and 26% of AChE activity, respectively. A single band of AChE activity was observed when a 1M NaCl extract with CsCl (0.38 g/ml) was centrifuged to equilibrium. Peak AChE activity from EDL and SOL extracts were found at 1.29 g/ml. Resedimentation of peak activity from CsCl gradients resulted in all molecular forms previously found in both muscles. Addition of a protease inhibitor phenylmethylsulfonyl chloride did not change the pattern of distribution. The 4S form of both muscles was extracted with low ionic strength buffer while the 10S, 12S, and 16S forms required high ionic strength and detergent for efficient solubilization. All molecular forms of both muscles have an apparent Km of 2 x 10(-4) M, showed substrate inhibition, and were inhibited by BW284C51, a specific inhibitor of AChE. The difference between these muscles in regards to their AChE activity, as well as in the proportional distribution of molecular forms, may be correlated with sites of localization and differences in the contractile activity of these muscles.     

Reciprocal regulation of acetylcholinesterase and butyrylcholinesterase in mammalian skeletal muscle

Developmental regulation, from the fetal period to 11 months of age, and the influence of denervation on the appearance and disappearance of the molecular forms of acetylcholinesterase (AchE) and butyrylcholinesterase (BuchE) in rat skeletal muscle were examined. The enzyme forms were extracted from anterior tibialis in 0.01 M sodium phosphate buffer, pH 7.0, containing 1 N NaCl, 0.01 M EGTA, 1% Triton X-100, and a cocktail of antiproteases, and analyzed by velocity sedimentation on 5-20% linear sucrose gradients. Three principal forms, denoted by sedimentation coefficients of 4, 10.8, and 16 S, were observed in muscle from all age groups. The amounts of each of the molecular forms of AchE and BuchE in skeletal muscle exhibited distinct and reciprocal patterns of appearance and disappearance during pre- and postnatal development. In tissue derived from animals less than 2 weeks of age, BuchE represented the predominant component of activity in the 4 S form, was present equally with AchE in the 10.8 S form, and was subordinate to AchE in the 16 S form. Between 1 and 2 weeks of age a progressive increase in AchE activities coincident with a reduction in BuchE activities resulted in inversion in the amounts of the two enzymes present in adult muscle. Denervation of muscle caused a dramatic reduction in the presence of AchE molecular forms with no discernable influence on the presence of BuchE molecular forms. These results indicate that biosynthesis of BuchE is strictly regulated in a reciprocal manner with that of AchE, and that BuchE metabolism is independent of the state of muscle innervation. Increased synthesis of AchE and either reduced synthesis or increased degradation of BuchE can account for the reciprocal regulation of these enzymes. These characteristics of mammalian muscle contrast sharply with characteristics deduced for avian tissue (Silman et al. (1979) Nature (London) 280, 160-162). The innervation-independent metabolism of BuchE and the diverse modes of its regulation in different tissue from different species signify that BuchE function may be unrelated to cholinergic neurotransmission.     

An evaluation of the hydrophobic interactions of chick muscle acetylcholinesterase by charge shift electrophoresis and gradient centrifugation

The hydrophobic interactions of globular forms of acetylcholinesterase from adult and embryonic chick muscles have been analyzed by sucrose gradient centrifugation and non denaturing polyacrylamide gel electrophoresis. The presence of positively- or negatively-charged detergents influences the electrophoretic migrations of hydrophobic globular forms, whereas the mobility of hydrophilic components is unchanged. We defined an hydrophobicity index (HI) which quantitatively reflects this interaction.Globular forms of acetylcholinesterase were isolated in preparative sucrose gradients of muscle extracts. The G₁ form (5 S) appeared as a single band in electrophoresis, the G₂ form (7 S) under two and the G₄ form (11 S) under three electromorphs. The G₁ and the G₂ forms interacted with detergents: this resulted in a shift in their sedimentation in sucrose gradients upon removal of detergents, and in a modification of their electrophoretic migrations in the presence of charged detergents (HI = 1.0 for G₁, HI = 1.7 for G₂). The G₄ form was heterogenous: one band (G_4f) did not interact with detergent (HI = 0.1). The other variants (G_4i and G_4s) were clearly hydrophobic (HI = 0.5 and HI = I respectively). The hydrophilic and hydrophobic variants of the G₄ form however, were not resolved by sedimentation analysis performed in the presence of Triton X100, but their separation was improved in the presence of 10-oleyl-ether. Therefore, the combination of electrophoretic and sedimentation methods, as described in this paper, can be used successfully for subdividing a single molecular form (size isomer defined by hydrodynamic parameters) into several constituents differing by their hydrophobic interactions.     

Solubilization of 20S acetylcholinesterase from chick retina

A 20S form of acetylcholinesterase has been solubilized from young chick retinas by means of a buffered salt-detergent solution containing EDTA. The release of this fast-sedimenting form of the enzyme is selectively blocked by the presence of even small amounts of Ca⁺⁺ in the homogenization medium. The collagen-tailed nature of this molecular species of acetylcholinesterase has been ascertained by collagenase digestion. This finding suggests that the avian central nervous system contains asymmetric, collagen-tailed quaternary structural forms of acetylcholinesterase as is the case in skeletal muscle and cholinergic ganglia.     

Acetylcholinesterase from Apis mellifera head. Evidence for amphiphilic and hydrophilic forms characterized by Triton X-114 phase separation.

The polymorphism of bee acetylcholinesterase was studied by sucrose-gradient-sedimentation analysis and non-denaturing electrophoretic analysis of fresh extracts. Lubrol-containing extracts exhibited only one form, which sedimented at 5 S when analysed on high-salt Lubrol-containing gradients and 6 S when analysed on low-salt Lubrol-containing gradients. The 5 S/6 S form aggregated upon removal of the detergent when sedimented on detergent-free gradients and was recovered in the detergent phase after Triton X-114 phase separation. Thus the 5 S/6 S enzyme corresponds to an amphiphilic acetylcholinesterase form. In detergent-free extracts three forms, whose apparent sedimentation coefficients are 14 S, 11 S and 7 S, were observed when sedimentations were performed on detergent-free gradients. Sedimentation analyses on detergent-containing gradients showed only a 5 S peak in high-salt detergent-free extracts and a 6 S peak, with a shoulder at about 7 S, in low-salt detergent-free extracts. Electrophoretic analysis in the presence of detergent demonstrated that the 14 S and 11 S peaks corresponded to aggregates of the 5 S/6 S form, whereas the 7 S peak corresponded to a hydrophilic acetylcholinesterase form which was recovered in the aqueous phase following Triton X-114 phase separation. The 5 S/6 S amphiphilic form could be converted into a 7.1 S hydrophilic form by phosphatidylinositol-specific phospholipase C digestion.     

Molecular forms of the cholinesterases inside and outside muscle endplates

Individual endplates were micro-dissected from chicken fast-twitch muscle, and the molecular forms of acetylcholinesterase and of pseudocholinesterase therein, identified by their sedimentation coefficients, were analysed directly. The forms actually present at the endplate, and those that are non-synaptic9 were established. This analysis was also extended to muscle of the chicken with inherited muscular dystrophy, showing altered distributions of these forms.     

Acetylcholinesterase activity and molecular forms during denervation and reinnervation in extensor digitorum longus muscle of the rat

The four principal molecular forms of acetylcholinesterase characteristic of the mammalian muscle (16.1 S., 12.5 S, 10.2 S, and 3.6. S) were identified by sucrose gradient sedimentation as the four activity peaks H, H₁, M and L.After denervation obtained by crushing the sciatic nerve five stages of the denervation-reinnervation process were examined. Days 7, 14, 22, 30, and 60 were chosen on the basis of previous electrophysiological and histochemical studies. The AChE activity showed an initial drop followed by recovery after nerve arrival at the muscle which was completed by day 60. Marked changes in the relative proportions of the four molecular forms were observed. The 16.1 S almost disappeared during the denervation period, reappeared after nerve arrival and was completely restored at day 60. Changes were also observed in the intermediate and lower forms and were tentatively related to processes of degradation, reaggregation and de novo synthesis.A comparison of the present data with those from parallel electrophysiological and histochemical studies suggests the presence and the functional role of molecular forms other than 16S in the neuromuscular junction.     

ACETYLCHOLINESTERASE OF RAT SYMPATHETIC GANGLION: MOLECULAR FORMS, LOCALIZATION AND EFFECTS OF DENERVATION

Abstract— In sucrose gradient centrifugation, acetylcholinesterase (AChE, EC 3.1.1.7.) from the rat superior cervical ganglion (SCG) has been found to contain four molecular forms, characterized by their sedimentation coefficients (4 S, 6.5 S, 10 S and 16 S). Homogenization of the ganglia in various media showed that the 4 S enzyme was readily solubilized in water whereas solubilization of the 6.5 S and 10 S forms was quantitative only in media containing Triton X-100. In order to solubilize the 16 S form, high concentrations of salt (NaCl 1 M) and detergent had to be present. AChE analysed by non-denaturing polyacrylamide gel electrophoresis separated into five bands. Although both distribution patterns were stable, i.e. each form or band preserved its characteristic sedimentation or electrophoretic migration when reanalysed, there was no 1:1 correlation between the forms isolated by sedimentation and the bands obtained by electrophoresis: one band might contain more than one form of enzyme, and conversely one form gave rise to several bands. It was therefore impossible to derive molecular weights from electrophoretic migration in non-denaturing gels. However, it could be shown that the results obtained by both methods of analysis were consistent. Acetylcholinesterase from other nervous structures was analysed: in pre- and postganglionic nerves, the main forms were 10 S and 6.5 S, with a small proportion of 4 S; the 16 S form was not detected. In other sympathetic ganglia, the distribution of forms was identical to that of the superior cervical ganglion. In rachidian ganglia, no 16 S form could be found. Following the section of the preganglionic nerve, the acetylcholinesterase activity of the superior cervical ganglion decreased by 50% in 3 days, and then rose again to about 80% of its original value after 2 weeks. These effects mainly reflected variations in the major 4 S and 10 S forms. The 16 S form, in contrast to its disappearance from denervated muscles, increased transiently during the first 2 weeks after denervation, reaching about twice its original activity. A concomitant cytochemical study of normal and denervated ganglia showed that after preganglionic denervation, AChE localized in the sympathetic neurones decreased markedly and remained low even during the recovery phase. During this period a cholinesterasic activity appeared in the perineuronal glia. Controls established that the enzyme synthetized in the glia is AChE.     

Changes in Acetylcholinesterase Molecular Forms During the Embryonic Development of Torpedo marmorata

Abstract: Multiple molecular forms of acetylcholinesterase from electric organ and electric lobe of Torpedo marmorata were examined at various developmental stages by sucrose density sedimentation. Four major forms were characterized by their apparent sedimentation coefficients of 6 S, 11 S, 13 S, and 17 S. Embryonic lobe possessed at early stages predominantly the 11 S form. With maturation the 17 S form became the most abundant. The early embryonic stages of the electric organ were characterized by predominating amounts of 6 S and 11 S forms. With differentiation of the postsynaptic membrane of the developing electrocytes, 13 S and 17 S forms replaced the slower-sedimenting forms. Concomitant with the formation of synaptic contacts, a transient increase in the 13 S form was followed by a dramatic accumulation of rapid-sedimenting 17 S form. The establishment of fully functional synapses was accompanied by an increase in the amount of the hydrophobic 6 S form. At birth, equal amounts of 6 S and 17 S form were found, with the other forms present in only trace amounts. The observed characteristic changes correlated with morphological and physiological events, indicating a close functional relationship between the accumulation of the 17 S form and synapse formation and the accumulation of the 6 S form and onset of function.     

Cobra venom acetylcholinesterase. Purification and molecular properties.

Acetylcholinesterase from cobra (Naja naja oxiana) venom has been purified by affinity chromatography to an homogeneous state, as ascertained by sodium dodecylsulfate/polyacrylamide gel electrophoresis and sedimentation analysis. The specific activity of the preparation was 5000 IU/mg with acetylcholine as substrate. Unlike acetylcholinesterases from insoluble cell structures, the native molecule of the cobra venom enzyme consists of a single polypeptide chain of molecular weight 67,000 +/- 2000. At high enzyme concentrations (greater than 0.2 mg/ml, greater than 1 microM) and ionic strength 0.1 M, it reversibly tends to form higher-molecular-weight 7.1-S aggregates. Despite the apparent structural simplicity of the venom acetylcholinesterase, the disc electrophoresis and isoelectric focusing experiments revealed that the enzyme exists in a number of forms with a common molecular weight but with different isoelectric points. Neuraminidase treatment did not reduce the number of the forms.     

Molecular Forms of Acetylcholinesterase in Pineal Cell and Sympathetic Neuronal Cultures

Abstract The activities of the various molecular forms of acetylcholinesterase (AChE) were measured in monolayer cultures of neonatal rat pineal cells grown alone and in co-culture with sympathetic neurons. AChE forms characterized by sedimentation coefficients of 4S, 6.5S, and 10S were found in the neuronal and pineal cultures, as well as in the co-cultures. The 16S AChE form was found only in the neuronal cultures. Total AChE activity increased with culture age in the co-cultures, but it decreased in pineal cells cultured alone. The low level of activity present in the neuronal cultures did not change markedly over the 27-day culture period. These results, which show bidirectional neuron-pineal cell effects, suggest that AChE molecular forms may be important markers to study the mechanisms underlying neuron-target cell interaction in the developing sympathetic nervous system.     

MOLECULAR PROPERTIES AND DIFFERENTIATION OF ACETYLCHOLINESTERASE IN SEA URCHIN EMBRYOS*

The two molecular forms of acethylcholinesterase (EC 3.1.1.7) in sea urchin embryos were characterized by several physical methods. The sedimentation coefficients determined by sucrose gradient centrifugation are 7.6S and 10.6S. The Stokes radii determined by gel filtration are 65 Å and 91 Å. From these parameters, molecular weights were estimated as 190,000 and 380,000; the one is twice as large as the other. Both forms have similar electric property and buoyant density in a CsCl gradient. When the enzyme solution was concentrated, the 10.6S form became predominant. These results suggest that the two forms are monomer and dimer. The sea urchin enzymes resemble globular forms of acetylcholinesterase of the electric organ of fishes. The activity of the enzyme abruptly increases in post-gastrulation embryos. Inhibition of concomitant protein synthesis by a specific inhibitor, emetine, does not affect the increase in enzyme activity. The result suggests that post-translational processes may be involved in the differentiation of this enzyme in sea urchin development. The following sea urchins were used in the study: Strongylocentrotus purpuratus, Strongylocentrotus franciscanus, and Dendraster excentricus.     

Presence and characterization of acetylcholinesterase in brush-border and basolateral membranes of rabbit enterocytes

Acetylcholinesterase is found in the brush-border and basolateral membranes purified from rabbit enterocytes. The sedimentation coefficients of the enzymes solubilized from two types of membrane are identical (5.5 +/- 0.2 S) and the apparent molecular weights are not significantly different (154 000 +/- 8000 for the brush-border and 145 000 +/- 8000 for the basolateral membrane enzyme). These results suggest a unique G2 molecular form for acetylcholinesterase from brush-border as well as from basolateral membranes.     

Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture,and recovery after inactivation

1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.     

Physical and chemical properties of the NH2-terminal glutamic acid and lysine forms of human plasminogen and their derived plasmins with an NH2-terminal lysine heavy (A) chain.

Comparative physical and chemical data are described for the human NH2-terminal Glu-plasminogen and Lys-plasminogen forms in order to determine the exact relationship between these two types of the zymogen. The molecular weights of Glu-plasminogen and Lys-plasminogen were similar and were determined to be 83, 800 plus or minus 4, 500 and 82, 400 plus or minus 3, 300, respectively, by sedimentation equilibrium methods. The molecular weights were identical in dodecyl sulfate solutions, approximately 83, 000, by sedimentation equilibrium methods. The sedimentation coefficients, s-020, w of Glu-plasminogen and Lys-plasminogen were determined to be 5.0 S, and 4.4 S, respectively. These two plasminogen forms had different partial specific volumes, and calculations of the frictional coefficients from sedimentation coefficients and molecular weights indicated conformation differences. Glu-plasminogen appeared to be larger in size than Lys-plasminogen in acrylamide gel-dodecyl sulfate electrophoresis. The amino acid compositions of Glu-plasminogen and Lys-plasminogen, and their major isolated isoelectric forms, were found to be similar, but several amino acid residues (glutamic acid, alanine, isoleucine, phenylalanine, and lysine) were found to be significantly higher in the Glu-plasminogen forms. The derived plasmins from both the Glu- and Lys-plasminogens with an nh2-terminal Lys- heavy (A) chain were found to have identical molecular weights of 76, 500 plus or minus 2, 500, and sedimentation coefficients, s-020, w of 4.3 S.     

Sedimentation velocity properties of catenated DNA molecules from HeLa cell mitochondria

Catenated molecules of closed circular DNA have been isolated from the mitochondrial DNA of HeLs cells. The sedimentation coefficients of several purified species have been investigated. The catenated dimer, made up of two interlocked duplex circles, sediments at 51 S in its superhelical (closed) form. Treatment with pancreatic DNase to relax the duplex circles converts the 51 S doubly closed dimer to a 42 S singly open species, then to a 36 S doubly open catenated dimer. The triply closed trimer sediments at 63 S and is converted to a 45 S triply open form by DNase. Electron microscopy of the DNA samples before and after DNase treatment shows that under the conditions used DNase does not change the catenated nature of the DNA. The measured sedimentation coefficients, have been compared with those estimated from previously proposed correlations of sedimentation coefficient and molecular weight, and with the sedimentation coefficients for catenated DNA presented by Wang. When all the interlocked circles in a catenane are relaxed, the DNA sediments about 5–10% faster than a relaxed multiple-length circular molecule of the same molecular weight. The sedimentation coefficient, 36 S, of the fully relaxed catenated dimer is 1.4 times that of the relaxed monomer.     

SUBCELLULAR ANALYSIS OF THE MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN RAT SKELETAL MUSCLE

Acetylcholinesterase (AChE, EC 3.1.1.7) activity of rat gastrocnemius muscle homogenized in 1 M-NaCl and 0.5% Triton X-100 was separated by velocity sedimentation in sucrose gradients into three molecular forms with sedimentation coefficients of about 4S, 10S and 16S. The distribution of homogenate AChE activity among the three peaks was 53, 34 and 13% respectively. The different molecular forms were found to be heterogeneously distributed in subcellular fractions prepared from sucrose homogenates of muscle, as follows: Subfractions of the crude sarcolemmal fraction were prepared by discontinuous sucrose gradient centrifugation. AChE was recovered in the greatest yield and with the highest specific activity in a light density subfraction (0.6/0.8 M-sucrose interface). The AChE activity in this light density subfraction was mainly (81-88%) the 10S form of the enzyme. The velocity sedimentation profiles of the AChE activity in the more dense subfractions were markedly different in that 16S AChE was a major component.