Acetyl- and butyrylcholinesterase in normal and diabetic rat retina

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinas. Tissues were sequentially extracted with saline (S₁) and saline-detergent buffers (S₂). 50% decrease in the amphiphilic G₄ and G₁ AChE molecular forms was observed in the diabetic retina compared to the controls. Less than 5% of the cholinesterase activity was due to BChE. 60% of the BChE activity in normal retina was brought into solution and evenly distributed between S₁ and S₂. In spite of the low BChE activity in the retina it was possible to detect globular forms (G^A ₁, G^A ₂, G^A ₄, G^H ₄) and a small proportion of an asymmetric form (A₁₂) in the S₁ extract. The G^A ₄ and G^A ₁ forms were found in the S₂ extract. In the diabetic retina the activity of G^A ₄ and G^A ₁ BChE molecular forms was reduced 60% and 40% respectively. Our results indicate that diabetes caused a remarkable decrease in the activity of cholinesterase molecular forms in the retina. These decrease might participate in the alterations observed in the diabetic retina.     

Developmental changes in the molecular forms of acetylcholinesterase during the life cycle of the lamprey Petromyzon marinus

• 1.1. We have determined the molecular forms of acetylcholinesterase (AChE) present in the skeletal muscle of the lamprey during the adult parasitic stage of its life cycle. AChE was found primarily in the globular G₄ form, as well as in the asymmetric forms A₄, A₈ and A₁₂.
• 2.2. We compare the complement of molecular forms present in skeletal muscle during the larval, parasitic, and spawning stages of the lamprey life cycle. The larval form, the ammocoete, contains elevated amounts of G₁ and G₂. However, the most striking change that we observed was in the proportion of asymmetric forms of AChE present: 5% in the ammocoete, 28% in the parasite and 9% in the spawner.
• 3.3. We speculate that these differences may be related to the physiological states of the lamprey during the various stages of its life cycle.

     

Collagen-tailed and globular forms of acetylcholinesterase in the developing chick visual system

We have carried out a comparative study of the developmental profiles of the enzyme acetylcholinesterase, and of its collagen-tailed and globular structural forms, solubilized in the presence of 1 M NaCl, 1% (w/v) sodium cholate and 2 mM EDTA, in the chick retina and optic lobes. The overall acetylcholinesterase activities, both per mg protein and per embryo or chick, are substantially higher in tectum than in retina, from embryonic day 16. The A₁₂ collagen-tailed form of the enzyme is present in similar amounts in the embryonic retina and optic tectum; however, while the A₁₂ activity increases significantly in retina after birth, both by percentage and in absolute terms, the tectal tailed enzyme follows a declining developmental profile, reaching a minimum after 6 months of life. On the other hand, the globular G₄ species shows developmental profiles, both in retina and tectum, rather similar to those obtained for the overall enzyme activity, while the G₂ and G₁ forms are present in comparable concentrations in both tissues. Besides, G₄ is the predominant globular form in the chick optic lobe after hatching, G₂ and G₁ being enriched in the embryonic tectum. In the case of retina, however, all the globular forms contribute more evenly to the total acetylcholinesterase activity, along the developmental period considered.The potential significance of some of the postnatal developmental profiles is discussed in terms of the progressive adjustment of retina and tectum to the requirements of visual function.     

Conformation similarities of the globular and tailed forms of acetylcholinesterase from Torpedo california

The conformation of the globular dimer (G₂), the tailed asymmetric dodecamer (A₁₂, also containing some tailed octamer A₈) and the globular tetramer (G₄, prepared by removing the collagen-like tail from A₁₂) of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) was studied by circular dichroism (CD) in the ultraviolet region. The G₂ and G₄ forms had similar conformation with about 40% α-helix, 35% β-sheets and 4% β-turns; the tailed form had a lower helicity (about 34%) and β-form (about 25%) content probably because of the presence of the tail whose CD spectrum resembles that of an unordered form, but it had about the same amount of β-turns as the other two forms. All three forms also had similar CD spectra in the near-ultraviolet region due to their non-peptide chromophores. The pH, thermal and urea denaturation of the three acetylcholinesterase forms was also similar to each other. The pH-dependency of both the enzymatic activity and CD intensity of the three forms showed bell-shaped curves with a plateau at pH 7–8. The activity was completely lost at pH below 5 or above 10, but the corresponding CD spectra retained 70–80% of the original magnitudes. Thermal denaturation of the three forms at pH 7.5 showed a conformational transition and loss of activity between 30 and 40°C, but the CD intensity of the helical band at 222 nm was reduced by only 20–30%. Urea denaturation of the three form began at 1 M urea; it was protein concentration- and time-dependent. Again, the activity disappeared faster than the decreasing CD intensity. Thus, the overall conformation of the three acetylcholinesterase forms appears to be relatively stable, but their active site is easily perturbed by changing the environment. The loss of activity correlated well with the disapperance of the CD band of tryptophan(s) in the near-ultraviolet region, suggesting that the Trp residue(s) might be at or near the active center of the enzyme.     

Intracellular distribution of molecular forms of acetylcholinesterase in rat brain and changes after diisopropylfluorophosphate treatment

The subcellular distribution of acetylcholinesterase activities was studied in the striatum and cerebellum of rat brain. The highest percentage of the enzyme activity was found in the crude synaptosomal (P₂) fraction, with striatum much higher than cerebellum. On sucrose density gradient centrifugation analyses all the particulate fractions (P₁, P₂, and P₃) showed a major peak of the 10 S form of acetylcholinesterase activity with very little activity of the 4 S form of the enzyme. The 10 S/4 S ratio was much higher in striatum than in cerebellum. In the soluble fraction (100,000g supernatant) the 10 S form was less than the 4 S form in the adult rat brain, but this was reversed in the 6-day-old rat brain. After diisopropylfluorophosphate administration the recovery of acetylcholinesterase molecular forms in various subcellular fractions differed at different recovery periods. These results indicate that the distribution of molecular forms of acetylcholinesterase in rat brain differs in various subcellular fractions, and also the pattern of distribution differs in different regions of the brain as well as in adult and developing brains.     

Differential effects of ethanol on membrane-bound and soluble acetylcholinesterase from sarcoplasmic reticulum membranes

The action of ethanol on the activity of membrane-bound and soluble acetylcholinesterase (AChE) in sarcoplasmic reticulum of skeletal muscle has been studied. Treatment of membranes with 2.5–12.5% v/v ethanol produced a slight stimulation of the AChE activity and inhibition at higher concentration. The enzyme remained associated with the membranes after these treatments. The enzyme solubilized with Triton X-100 was inhibited by ethanol in a time-independent manner. Isolated 16 S (A₁₂), 10.5 S (G₄) and 4.5 S (G₁) forms of AChE were inhibited by ethanol to a similar extent. Samples were reversibly inhibited by ethanol, up to 12.5% v/v, and irreversibly at higher concentrations. Kinetic studies performed with isolated forms in the presence of 5–12.5% v/v ethanol showed that the solvent behaved as a competitive inhibitor of the asymmetric form but as a mixed inhibitor of the tetrameric and monomeric forms. The results show that the solvent interacts with active and/or regulatory sites of AChE from muscle microsomes.     

The effect of temperature on the activity of acetylcholinesterase preparations from rat brain

Homogenization of rat brain with dilute buffer shows that about 15% of the acetylcholinesterase is soluble while the remaining 85% is present in a membrane-bound form which can be brought into solution by extraction with Triton X-100. The effect of temperature on the values of V_max and K_m of the buffer-soluble, the membrane-bound and the Triton-soluble forms of acetylcholinesterase have been compared and the results discussed in terms of possible changes in the conformation, dissociation or aggregation of the enzyme molecule.Gradient-gel electrophoresis of the soluble preparations carried out at 4°C or 37°C suggest that the normal tetrameric structure present at 4°C dissociates into monomers and forms some higher molecular weight species at 37°C.The effect of prior storage of the brains in toluene on these properties is also considered.     

Human intestine epithelial cell acetyl- and butyrylcholinesterase

The epithelial cells of the human intestine exhibit a cholinesterase activity which is restricted to the apex of the villi. This activity displays a maximum in the colon and a minimum in the jejunum. Contrary to most of the studied vertebrates, the human cells present both acetylcholinesterase and butyrylcholinesterase activities, acetylcholinesterase being predominant in all the intestinal segments: duodenum, jejunum, ileum and colon. Like in the other vertebrates, only globular forms are identified by sucrose gradient centrifugation. However, the simultaneous presence, on the one hand of three globular forms (G₁, G₂ and G₄) and, on the other hand of soluble as well as detergent-soluble molecular species seems to be a particular feature of the human cells.Abbreviations ChE Cholinesterases - AChE Acetylcholinesterase - BuChE Butyrylcholinesterase     

Multiple forms of phosphofructokinase in developing rabbit and guinea pig tissues.

Multiple forms of phosphofructokinase in striated muscle and cardiac muscle of developing rabbit (Oryctolagus cuniculus) undergo changes with development, but not in brain and liver. The cardiac muscle of the 1-day-old rabbit contains phosphofructokinase A₄ together with the four hybrid forms which were tentatively called A₃C, A₂C₂, AC₃, and C₄. In older animals, phosphofructokinase C₄ disappears first, followed by the hybrid forms, and only phosphofructokinase A₄ persists in the adult animal. Both phosphofructokinase A₄ and phosphofructokinase C₄, as well as their hybrid forms, are present in developing embryonic brain and also in the brains of adult animals. Developing rabbit liver contains a single form of phosphofructokinase, but two isoenzymes are consistently seen in guinea pig liver. In striated muscle from fetal and 1-day-old rabbit, two isoenzymes are found, tentatively identified as A₄ and the A₃C hybrid. The results suggest that fetal phosphofructokinase A₄ and phosphofructokinase C₄, and their hybrids, might be present in striated muscle. Guinea pig tissues show a pattern of phosphofructokinase isoenzymes different from that in rabbit tissues.     

Molecular Forms of Acetylcholinesterase in Bovine Caudate Nucleus and Superior Cervical Ganglion: Solubility Properties and Hydrophobic Character

Abstract: In the present paper, we report an analysis of acetylcholinesterase molecular forms in the bovine caudate nucleus and superior cervical ganglion. We show that: (1) The superior cervical ganglion contains a significant proportion (～ 15%) of collagen-tailed forms (mostly A₁₂ and A₈), but these molecules are found only as traces (ca. 0.002%) in the caudate nucleus, even in favorable extraction conditions (i.e., in the presence of 1 m -NaCl, 5 mm -EDTA, 1% Triton X-100). (2) The bulk of acetylcholinesterase corresponds to globular forms, mostly the tetrameric G₄ and the monomeric G₁ forms, with a smaller proportion of the dimeric G₂ form. (3) The tetrameric enzyme exists as a minor soluble component (G^S₄) that does not interact with Triton X-100, and a major hydrophobic component (G^H₄) that is partially solubilized in the absence of detergent in the caudate nucleus, but not in the superior cervical ganglion. (4) The monomeric G₁ form presents a marked hydrophobic character, as indicated by its interaction with Triton X-100, although it may be solubilized in large part in the absence of detergent in both tissues. (5) The detergentsolubilized forms aggregate upon removal of detergent. This property disappears after partial purification of G₄) that does not interact with Triton X-100, and a major hydrophobic component (G^H₄, but is restored upon addition of an inactivated crude extract, indicating that it is attributable to interactions with other hydrophobic components. (6) The proportions of molecular forms solubilized in detergent-free buffers vary with the ionic composition of the medium. Repeated extractions of caudate nucleus in Tris-HCl buffer produce a larger overall yield of G₁ form (e.g., 40%) than appears in a single quantitative detergent solubilization (<15%). This G₁ form apparently derives in part from a pool of G^H₄ form. (7) However, detergents that allow a quantitative solubilization of acetylcholinesterase yield the same proportions of forms (about 85% G₄) independently of the ionic conditions. (8) Modifications of the molecular forms occur spontaneously during purification, or storage of the crude aqueous ex-tracts, in a manner that depends on the ionic conditions. In Tris-HCl buffer, G₁ is converted into a well-defined 7.5S form. In Ringer, polydisperse components are formed. The effects observed in Ringer cannot be reproduced by addition of 5 mm -Ca_2- to the Tris buffer either during or after extraction. (9) Proteases, such as pronase, convert the hydrophobic forms into molecules that do not appear to interact with Triton X-100, and do not aggregate in its absence. These results raise fundamental questions regarding the status of acetylcholinesterase in situ, the structure and interactions of its molecular forms. They are discussed with reference to previous publications.     

Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide

To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChE(T) subunit, which should be able to produce all three globular forms of AChE: monomers (G(1)), dimers (G(2)), and tetramers (G(4)), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G(1)(a)) and non-amphiphilic tetramers (G(4)(na)) are found. Amphiphilic dimers (G(2)(a)) and non-amphiphilic tetramers (G(4)(na)) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A(12) form of the enzyme. Collagenase digestion of the A(12) AChE produces a lytic G(4) form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates.     

The functional role of molecular forms of acetylcholinesterase in neuromuscular transmission

The severity of poisoning following acetylcholinesterase (AChE) inhibition correlates weakly with total AChE activity. This may be partly due to the existence of functional and non-functional pools of AChE. AChE consists of several molecular forms. The aim of the present study was to investigate which of these forms will correlate best with neuromuscular transmission (NMT) remaining after partial inhibition of this enzyme. Following sublethal intoxication of rats with the irreversible AChE inhibitor soman, diaphragms were isolated after 0.5 or 3 h. It appeared that at 3 h after soman poisoning the percentage of G₁ increased, while those of G₄ and A₁₂ decreased. NMT was inhibited more strongly than in preparations obtained from the 0.5 h rats with the same level of AChE inhibition, but with a normal ratio of molecular forms. NMT correlated positively with G₄ as well as with A₁₂, but inversely with G₁. In vitro inhibition with the charged inhibitors DEMP and echothiophate resulted in higher levels of total AChE, relatively less G₁ and more G₄ and A₁₂ than after incubation with soman, but led to less NMT. Treatment of soman-intoxicated rats with the reactivating compound HI-6 resulted in preferential reactivation of A₁₂, persisting low levels of G₁ and concurrent recovery of NMT as compared with saline-treated soman controls with equal total AChE activity. Apparently, in rat diaphragm G₄ and A₁₂ are the functional AChE forms.     

Acetylcholinesterase in sea urchin spermatozoa

Flagella contain the bulk of spermatozoan acetylcholinesterase. Brief sonication of sea urchin sperm suspended in Tris-buffered (pH 8.0), Ca, Mg-free artificial sea water (F-ASW) containing 10 mM ethylene diaminetetracetic acid, (EDTA) doubled the specific activity over that of the intact spermatozoa. Lipids were removed from the solubilized supernatant of the tail membrane fraction by ether extraction. Hydrolysis of acetylthiocholine in the presence of dithiobisnitrobenzoic acid (DTNB) was monitored spectrophotometrically at 412 nm by the Ellman procedure. The enzyme was purified by affinity chromatography on a Sepharose cyanogen bromide gel to which the cholinesterase inhibitor trimethyl (para-aminophenyl) ammonium chloride was coupled. The enzyme was eluted from the column with a discontinous NaCl gradient (0.1–0.5 M). The active fraction recovered at 0.35 M NaCl contained 0.007% of the initial total sperm cell protein with a 500-fold increase in specific activity. Twenty-four hr centrifugation on a 5–20% sucrose density gradient at 50,000g in a Beckman L5-75 centrifuge yielded peaks at 14.7 S and 9.1 S. In the presence of 1% Triton X-100, three peaks appeared: 23.3 S, 13.7 S, and 9.1 S. These sedimentation coefficients resemble those of the electroplax acetylcholinesterase (AChE) forms A₈ and A₄. Eserine completely inhibited the activity of the purified enzyme, which exhibits a substrate optimum at 4 mM acetylcholine. The activity is depressed by 75% at 10 mM ACh and by 90% at 25 mM. The Km was 2.1 × 10^?4 M. In the sperm cell the enzyme that terminates the action of intracellularly synthesized ACh may be involved in controlling ionophoric channels that regulate transmembrane transport of calcium.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

Crystallography of Bacillus subtilis levansucrase

Levansucrase, an exocellular enzyme, has been isolated from a high producer mutant, the BS5C4 constitutive strain, of Bacillus subtilis. Three crystalline forms have been obtained, all three belonging to the orthorhombic space group P2₁2₁2₁. The most suitable form for a three-dimensional structure investigation has cell dimensions, $\text{a = 68}\text{A}\text{?}$, $\text{b = 125}\text{A}\text{?}$, $\text{c = 54}\text{A}\text{?}$. There is one molecule in the asymmetric unit.     

Manganese superoxide dismutases from Escherichia coli and from yeast mitochondria: Preliminary X-ray crystallographic studies

The Mn superoxide dismutase from Escherichia coli has been obtained in three crystal forms: (I) from 68% saturated (NH₄)₂SO₄, space group P222 or P222₁, $\text{a = 47}\text{A}\text{?}\text{, b = 103}\text{A}\text{?}\text{, c = 47.5}\text{A}\text{?}$, with one subunit per asymmetric unit; (II) from 50% polyethylene glycol 6000, space group C222₁ (with approx. P4₁2₁2 symmetry), $\text{a = 101}\text{A}\text{?}\text{, b = 108}\text{A}\text{?}\text{, c = 180}\text{A}\text{?}$, with four subunits (2 molecules) per asymmetric unit; (III) from 52% polyethylene glycol with a different method of preparing the enzyme solution, space group P2₁2₁2, $\text{a = 47}\text{A}\text{?}\text{, b = 51}\text{A}\text{?}\text{, c = 188}\text{A}\text{?}$, with two subunits per asymmetric unit.The yeast mitochondrial Mn superoxide dismutase has yielded the same crystal form both from 30% 2-methyl-2,4-pentane diol and from 23% polyethylene glycol 6000: space group P2₁2₁2₁, $\text{a = 63}\text{A}\text{?}\text{, b = 115}\text{A}\text{?}\text{, c = 125}\text{A}\text{?}$, with four subunits (one molecule) per asymmetric unit.A full X-ray crystallographic study of at least one of these enzymes is planned.     

Two-Site Immunoradiometric Assay of Chicken Acetylcholinesterase: Active and Inactive Molecular Forms in Brain and Muscle

Abstract: Several monoclonal antibodies were raised against chicken acetylcholinesterase (AChE; EC 3.1.1.7). Some of these antibodies react with quail AChE but not with AChEs from nonavian vertebrates or invertebrates and not with butyrylcholinesterase. They may be classified in several mutually compatible groups, i.e., that can bind simultaneously to the monomeric form of AChE. Most antibodies recognize a peptidic domain that does not exist in mammalian AChE and that may be digested by trypsin without loss of activity or dissociation of quaternary structure. The only exception is the antibody C-131, which is conformation dependent and preferentially recognizes active AChE. We have set up two-site immunoradiometric assays, using an immobilized capture antibody, C-6 or C-131, and a radiolabeled antibody, ¹²⁵I-C-54. The C-6/C-54 assay quantifies the totality of inactive and active AChE subunits: It detects 10^?3 Ellman unit (～40 pg of protein) and yields a linear response up to at least 25 10^?3 Ellman units. An analysis of gradient fractions, using C-6/C-54 and C-131/C-54 assays as well as activity determination, shows that the A₁₂ and G₄ forms are exclusively composed of active subunits, whereas inactive molecules cosediment with the active G₂ and G₁ forms. Both active and inactive G₂ and G₁ forms are amphiphilic, as indicated by the influence of detergents on their sedimentation coefficients and Stokes radii. In brain, the proportion of inactive forms decreases from 40% at embryonic day 11 (E11) to 20% at birth [day 1 (D1)]. In muscle, we observed no inactive AChE at E11 and a small proportion of inactive G₁ at D1. The proportion of inactive forms was much higher in cultured myotubes, obtained from E11 myoblasts. These results show that the proportion of inactive AChE depends on the tissue and varies during development. Thus, the cells seem to control actively the acquisition of AChE activity, as well as the formation of the various oligomeric forms.     

Effects of Aging and Amyloid-β Peptides on Choline Acetyltransferase Activity in Rat Brain

Choline acetyltransferase (ChAT, acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), involved in the learning and memory processes is responsible for the synthesis of acetylcholine. There are many discrepancies in literature concerning ChAT activity during brain aging and the role of amyloid beta peptides in modulation of this enzyme. The aim of the study was to investigate the mechanism of ChAT regulation and age-related alteration of ChAT activity in different parts of the brain. Moreover the effect of A peptides on ChAT activity in adult and aged brain was investigated. The enzyme activity was determined in the brain cortex, hippocampus and striatum in adult (4-months-old), adult-aged (14-months-old) and aged (24-months-old) animals. The highest ChAT activity was observed in the striatum. We found that inhibitors of protein kinase C, A, G and phosphatase A2 have no effect on ChAT activity and that this enzyme is not dependent on calcium ions. About 70% of the total ChAT activity is present in the cytosol. Arachidonic acid significantly inhibited cytosolic form of this enzyme. In the brain cortex and striatum from aged brain ChAT activity is inhibited by 50% and 37%, respectively. The aggregated form of A 25-35 decreased significantly ChAT activity only in the aged striatum and exerted inhibitory effect on this enzyme in adult, however, statistically insignificant. ChAT activity in the striatum was diminished after exposure to 1 mM H₂O₂. The results from our study indicate that aging processes play a major role in inhibition of ChAT activity and that this enzyme in striatum is selectively sensitive for amyloid beta peptides.     

Modulation in vitro of 3-hydroxy-3-methylglutaryl coenzyme A reductase in brain microsomes: Evidence for the phosphorylation and dephosphorylation associated with inactivation and activation of the enzyme

The activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in brain microsomes was modified in vitro. The inactivation of the enzyme required Mg²⁺ and ATP or ADP, and an inactivator present both in S₁₀₅ and microsomes. Inactivation was dependent on inactivator concentration and time of preincubation. The inactive reductase in brain microsomes could be completely reactivated by a factor present in brain S₁₀₅. Reactivation of the enzyme also depended on incubation time and the activator concentration. Activator activity was inhibited by NaF, a phosphatase inhibitor. Both the inactivator and the activator appear to be proteins. Our data thus suggest that the inactivation and the reactivation of the reductase in brain microsomes occurs via protein-mediated interconversion to phosphorylated and dephosphorylated forms of the enzyme with differing catalytic activity. The HMG-CoA reductase activity increases almost two-fold during isolation of the brain microsomes. This increase in activity is blocked when brain tissue is homogenized in the medium containing NaF. In rat brain about 50% of the reductase exists in an inactive form in both young and adult rats. The low reductase activity in brain of adult animals does not appear to be related to an increase in the proportion of an inactive phosphorylated form of the enzyme. This suggests that developmental change in the reductase activity is not associated with the change in the proportion of phosphorylated and dephosphorylated forms of the enzyme.     

Inactivation of acetylcholinesterase by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride

The neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to reversibly inhibit the activity of acetylcholinesterase. The inactivation of the enzyme was detected by monitoring the accumulation of yellow color produced from the reaction between thiocholine and dithiobisnitrobenzoate ion. The kinetic parameter, K _m for the substrate (acetylthiocholine), was found to be 0.216 mM and K _i for MPTP inactivation of acetylcholinesterase was found to be 2.14 mM. The inactivation of enzyme by MPTP was found to be dose-dependent. It was found that MPTP is neither a substrate of AChE nor the time-dependent inactivator. The studies of reaction kinetics indicate the inactivation of AChE to be a linear mixed-type inhibition. The dilution assays indicate that MPTP is a reversible inhibitor for AChE. These data suggest that once MPTP enters the basal ganglia of the brain, it can inactivate the acetylcholinesterase enzyme and thereby increase the acetylcholine level in the basal ganglia of brain, leading to potential cell dysfunction. It appears that the nigrostriatal toxicity by MPTP leading to Parkinson's disease-like syndrome may, in part, be mediated via the acetylcholinesterase inactivation.