Studies on the characterization of human erythrocyte acetylcholinesterase and its interaction with antibodies

Human erythrocyte membranes were solubilized in 5% Triton X-100 and the acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was isolated by affinity chromatography utilizing a specific inhibitor, $\text{trimethyl}\text{-p-}\text{aminophenyl}$ ammonium chloride, bound to Sepharose 4B. After a repeated chromatography acetylcholinesterase was found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunization of rabbits with acetylcholinesterase elicited the formation of an antiserum which gave single precipitin lines with the enzyme on immunodiffusion and rocket, crossed and immuno-electrophoreses. The purified enzyme had a specific activity of 418 units/mg protein. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of acetylcholinesterase with acetylthiocholine as substrate was $\text{1.5 × 10}{}^{\mathrm{?4}}\text{M}$. Isoelectric focusing of acetylcholinesterase in the presence of Triton X-100 and within the pH ranges of 3–10 and 3–6 exhibited a single peak of enzyme activity with a $\text{P}\text{I}$ of 4.8. The results of amino acid and carbohydrate analyses showed that acetylcholinesterase is a glycoprotein with a carbohydrate/protein weight ratio of 0.16 and glucose, galactose, mannose, glucosamine, galactosamine and sialic acid as the sugar components. The N-terminal amino acid was blocked. Lipid, phosphorus and fatty acid analyses indicated phosphatidylserine and cholesterol as the major lipid components of acetylcholinesterase. The apparent subunit molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol was 160 000 and in its presence, 80 000. The kinetic studies showed a competitive inhibition of acetylcholinesterase by its antibodies. Agglutination of human red cells by monospecific antiserum to acetylcholinesterase confirmed that the antigenic site(s) of the enzyme is localized on the outer surface of the erythrocyte membrane.     

Prolonged membrane potential depolarization in cingulate pyramidal cells after digit amputation in adult rats

The anterior cingulate cortex (ACC) plays an important role in higher brain functions including learning, memory, and persistent pain. Long-term potentiation of excitatory synaptic transmission has been observed in the ACC after digit amputation, which might contribute to plastic changes associated with the phantom pain. Here we report a long-lasting membrane potential depolarization in ACC neurons of adult rats after digit amputation in vivo. Shortly after digit amputation of the hind paw, the membrane potential of intracellularly recorded ACC neurons quickly depolarized from ~-70 mV to ~-15 mV and then slowly repolarized. The duration of this amputation-induced depolarization was about 40 min. Intracellular staining revealed that these neurons were pyramidal neurons in the ACC. The depolarization is activity-dependent, since peripheral application of lidocaine significantly reduced it. Furthermore, the depolarization was significantly reduced by a NMDA receptor antagonist MK-801. Our results provide direct in vivo electrophysiological evidence that ACC pyramidal cells undergo rapid and prolonged depolarization after digit amputation, and the amputation-induced depolarization in ACC neurons might be associated with the synaptic mechanisms for phantom pain.     

18B1: An Axon-Specific Antigen Associated with Many Proteins

We describe an antigen, 18B1, defined by a monoclonal antibody. Immunoperoxidase staining of brain sections shows that 18B1 is selectively associated with neurofilament-rich axons. Antibody staining of sodium dodecyl sulphate-gel blots, on the other hand, shows that 18B1 is associated with a large number of proteins, none of which are structural components of brain neurofilaments. The antigen is sensitive to a variety of proteases but is not degraded by various glycosidases, suggesting that 18B1 is probably an amino acid sequence. Its association with neurofilament-rich axons together with its absence from neurofilaments themselves suggests that it may be involved in mediating interactions between neurofilaments and the proteins that bear it.     

A dot-immunobinding assay for monoclonal and other antibodies

A procedure for the assay of antibodies in sera based on the application of the antigen as a spot to nitrocellulose filters is described. The method has the merit of being simpler in operation and more sensitive than comparable existing procedures. Applications for screening supernatants of hybridomas making monoclonal antibodies, and the use of such antibodies in the determination of the tissue distribution of the corresponding antigens, are described. An application for the screening of human pathological sera for multiple antibodies in one operation is also described.     

Distinct cellular pools of perilipin 5 point to roles in lipid trafficking

The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~1.15 g/ml) and size (~575 kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~15 nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation.     

The Role of CaV2.1 Channel Facilitation in Synaptic Facilitation

1. Download high-res image (146KB)
2. Download full-size image

     

MIT-23: A mitochondrial marker for terminal neuronal differentiation defined by a monoclonal antibody

We describe a monoclonal antibody directed against a neuron-specific mitochondrial protein from rat brain. On protein blots the antibody recognizes a single polypeptide of apparent molecular weight 23,000. By solid-phase immunoassay the antigen was detected in all brain regions tested but was not detected in nonneural tissues. Within neurons, the antibody stains cytoplasmic granules that immunoelectron microscopy shows are mitochondria, hence the designation MIT-23. Immunocytochemical staining of the cerebellar cortex showed that MIT-23 occurs in all the neuronal types but is absent from glial and other nonneuronal cells. During neonatal development of the cerebellum, MIT-23 appears in neurons after their final cell division or migration is completed, suggesting that specific proteins associated with mitochondria participate in neuronal maturation.