Effect of Phospholipase C from <i>Bacillus cereus</i> on the Release of Membrane‐Bound Choline‐<i>O</i>‐Acetyltransferase from Rat Hippocampal Tissue

Carroll, Paul T.; Smith, L K

doi:10.1111/j.1471-4159.1990.tb02356.x

Cited by 12 publications

(5 citation statements)

References 30 publications

(38 reference statements)

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“…The soluble and detergent-solubilized ChAT forms appear to have similar affinities for choline and acetyl-CoA, similar molecular masses on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and similar epitope maps (Benishin and Carroll, 1983;Badamchian and Carroll, 1985;Bruce and Hersh, 1987). Insight into the possible nature of membrane-bound ChAT has been provided by Carroll and Smith (1990) and Smith and Carroll (1993), who reported that phospholipase C from Bucillus cereus and Bacillus thuringiensis, which are phosphatidylinositol-specific phospholipases, were able to release the membrane-bound form of ChAT from rat hippocampal tissue and to convert it from an amphiphilic to a hydrophilic form. This led to the proposal that at least some of the membrane-bound ChAT is anchored by an intracellular glycosylphosphatidylinositol linkage.…”

Section: Intracellular Localization Of Chatmentioning

confidence: 99%

Choline Acetyltransferase: Celebrating Its Fiftieth Year

Hersh

1994

Journal of Neurochemistry

154

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It is well known that the regulation of choline acetyltransferase (ChAT) activity under physiological and pathological conditions is important for the development and neuronal activities of cholinergic systems involved in many fundamental brain functions. This review focuses on recent progress in understanding the regulation of ChAT at the levels of both the protein and the mRNA. A deficiency in ChAT activity has been reported for neurodegenerative conditions such as Alzheimer's disease, amyotrophic lateral sclerosis, and schizophrenia. Although a major feature of ChAT regulation is likely to involve the spatial and temporal control of transcription, regulation of expression can also be at the level of RNA processing, transport/ translocation, turnover, or translation. In addition, there is increasing evidence that ChAT might be regulated at the posttranslational level by compartmentation and/or covalent modification, i.e., phosphorylation, as well as noncovalent modification (protein‐protein interaction, etc.). Synaptic activity and the state of neuronal transmission may also involve the regulation of ChAT at different levels via both positive and negative feedback loops, as was demonstrated in the characterization of two ChAT mutant Drosophila strains. Clearly, identification of cholinergic‐specific elements and the characterization of the trans‐acting factors that bind to them represent an important area of future research. Equally important is research on the mechanisms governing ChAT as an enzymatic entity. The future should be an exciting time during which we look forward to the elucidation of the cholinergic signal and its regulation as well as the determination of the three‐dimensional structure of the enzyme.

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Section: Intracellular Localization Of Chatmentioning

confidence: 99%

Choline Acetyltransferase: Celebrating Its Fiftieth Year

Hersh

1994

Journal of Neurochemistry

154

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“…The first information provided by the present work is that the sialidase contained in the pig forebrain total pellet is partly released in soluble form by treatment with a PLC specifically affecting phosphatidylinositol (B. cereus PLC) (PIPLC). In agreement with this conclusion are the observations that (a) addition of ZnClz, which inhibits PIPLC (Carrol and Smith, 1990), completely blocked sialidase liberation; (b) addition of EDTA, which inhibits the phospholipase activity on glycerophospholipids not containing inositol (Ikezawa et al, 1976), did not affect sialidase release; and (c) treatment with several proteolytic enzymes was unsuccessful in releasing sialidase. The released sialidase is distinct from the cytosolic sialidase, because this enzyme was completely washed off from the pellet (Venerando et al, 1982) before PIPLC treatment and has a pH optimum of 4.8 (Venerando et al, 1975), different from those of solubilized membrane-bound sialidase (4.2 and 6.6).…”

Section: Discussionmentioning

confidence: 62%

“…The amount of enzyme added was as follows: Pronase, 50 pg; chymotrypsin, phospholipase D, trypsin, pepsin, thermolysin, and sphingomyelinase, 100 pg; PLC (from B. thuringiensis), 0.3 unit; and PLC (from B. cereus), 1.5 units. Treatment with PLC (from B. cereus) was camed out also in the presence of ZnClz (5 mM), known to inhibit specifically the enzyme activity on phosphatidylinositol (Fox et al, 1986;Carrol and Smith, 1990), and of EDTA (1 mM), reported to inhibit selectively the enzyme activity on glycerophospholipids not containing inositol (Ikezawa et al, 1976). At the end of incubation, the mixtures were refrigerated by immersion into an ice-water bath and centrifuged at 150,000 g for 10 min.…”

Section: Sialidase Release By Treatment With Different Proteolytic Anmentioning

confidence: 99%

Solubilization of the Membrane‐Bound Sialidase from Pig Brain by Treatment with Bacterial Phosphatidylinositol Phospholipase C

Chiarini

Fiorilli

Siniscalco

et al. 1990

Journal of Neurochemistry

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The total pellet from pig forebrain (from which the cytosolic sialidase was completely washed out) was treated with phosphatidylinositol phospholipase C (PIPLC) and centrifuged at high speed. The supernatant contained sialidase and 5'-nucleotidase activities. The greatest liberation of sialidase was obtained after incubation for 20 min with PIPLC at 37 degrees C using pH 6.0 and a ratio between PIPLC (as units) and protein of 1.6. Under these conditions, the release of sialidase, 5'-nucleotidase, and protein was 22, 50, and 18.5%, respectively. On treatment with PIPLC, a purified preparation of pig brain neuronal (synaptosomal) membranes released 28% of its sialidase whereas a purified preparation of pig brain lysosomes did not liberate any sialidase activity. The pH optimum of sialidase present in the supernatant obtained after PIPLC treatment of the total pellet was 4.2, the same as that of the enzyme embedded in the membrane. When this supernatant was subjected to ammonium sulfate fractionation, 88% of its sialidase, having a pH optimum of 4.2, was recovered in the fraction precipitated between 20 and 45% of salt saturation and subsequently dialyzed. Ammonium sulfate treatment caused the appearance of a second sialidase activity, having a pH optimum of 6.6 and behaving on fractionation similarly to the pH 4.2 sialidase. The Km and Vmax values of pH 4.2 and pH 6.6 sialidase were similar (1.48 x 10(-4) and 0.98 x 10(-4) M for Km and 1.6 and 1.4 mU/mg of protein for Vmax, respectively), whereas the stability on standing at 4 degrees C or exposure to freezing and thawing cycles was greater for pH 4.2 sialidase.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Triton X-114 was previously used to reveal the existence of hydrophilic and arnphiphilic ChAT activities in synaptosomes isolated from the electric organ of the fish Torpedo, rat brain and human nucleus caudatus (Eder-Colli er ul., 1986Docherty andBradford, 1988: Carroll andSmith, 1990) and in the differentiated rat neuroblastoma cell line NS20Y (Barochovsky et ul., 1988). ChAT activity expressed in oocytes injected with pChAT4.2 was found to partition into the detergent-depleted and detergent-enriched phases.…”

Section: Discussionmentioning

confidence: 99%

Hydrophilic and Amphiphilic Forms of Drosophila Choline Acetyltransferase are Encoded by a Single mRNA

Salem¹,

Medilanski²,

Pellegrinelli³

et al. 1994

Eur J of Neuroscience

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We have previously shown that the enzyme choline-O-acetyltransferase (ChAT) exists in a hydrophilic and an amphiphilic form in Drosophila head. A complementary DNA clone of 4.2 kb containing the entire coding region of ChAT was isolated from a cDNA library of Drosophila heads. The cDNA was subcloned in an expression vector and injected into the nucleus of Xenopus oocytes. Injected oocytes expressed high levels of ChAT activity. This activity was inhibited by bromoacetylcholine, a specific inhibitor of the enzyme. In the present study the non-ionic detergent Triton X-114 was used to analyse whether the expression of hydrophilic and amphiphilic ChAT was or was not directed by a single cDNA. The two forms of ChAT were found to be synthesized in injected oocytes. Approximately 9% of the recombinant enzyme partitioned as amphiphilic activity. This value was similar to that found for native amphiphilic ChAT in Drosophila heads. Sedimentation in sucrose gradients of amphiphilic enzyme was found to be influenced by the type of detergent present in the gradient whereas this was not the case for hydrophilic ChAT. Hydrophilic and amphiphilic enzyme activities differed in some of their biochemical properties. Amphiphilic ChAT was less sensitive to inhibition by the product acetylcholine than was hydrophilic ChAT. Moreover, amphiphilic ChAT was found to be more resistant than hydrophilic ChAT to heat inactivation at 45 degrees C. These properties were observed for the native as well as for recombinant ChAT. These results demonstrate that the hydrophilic and amphiphilic forms of ChAT are derived from one mRNA.

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Effect of Phospholipase C from Bacillus cereus on the Release of Membrane‐Bound Choline‐O‐Acetyltransferase from Rat Hippocampal Tissue

Cited by 12 publications

References 30 publications

Choline Acetyltransferase: Celebrating Its Fiftieth Year

Choline Acetyltransferase: Celebrating Its Fiftieth Year

Solubilization of the Membrane‐Bound Sialidase from Pig Brain by Treatment with Bacterial Phosphatidylinositol Phospholipase C

Hydrophilic and Amphiphilic Forms of Drosophila Choline Acetyltransferase are Encoded by a Single mRNA

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