Enzyme, cleft membrane

Biochemical reactions take place in the hydrophobic centers of membranes or enzyme clefts. Aqueous compartments of biological cells are usually taboo media for the formation of small molecular assemblies or covalent compounds. There is, however, one notable exception, namely the formation of inclusion... 

Acetylcholinesterase is a component of the postsynaptic membrane of cholinergic synapses of the nervous system in both vertebrates and invertebrates. Its structure and function has been described in Chapter 10, Section 10.2.4. Its essential role in the postsynaptic membrane is hydrolysis of the neurotransmitter acetylcholine in order to terminate the stimulation of nicotinic and muscarinic receptors (Figure 16.2). Thus, inhibitors of the enzyme cause a buildup of acetylcholine in the synaptic cleft and consequent overstimulation of the receptors, leading to depolarization of the postsynaptic membrane and synaptic block. 

Figure 3.1 Schematic representation of a generic excitatory synapse in the brain. The presynaptic terminal releases the transmitter glutamate by fusion of transmitter vesicles with the nerve terminal membrane. Glutamate diffuses rapidly across the synaptic cleft to bind to and activate AMPA and NMDA receptors. In addition, glutamate may bind to metabotropic G-protein-coupled glutamate receptors located perisynaptically to cause initiation of intracellular signalling via the G-protein, Gq, to activate the enzyme phospholipase and hence produce inositol triphosphate (IP3) which can release Ca from intracellular calcium stores...

Destruction of the neurotransmitter by enzymes located in the synaptic cleft or in the plasma membranes of presynaptic or postsynaptic... 

Many neurotransmitters are inactivated by a combination of enzymic and non-enzymic methods. The monoamines - dopamine, noradrenaline and serotonin (5-HT) - are actively transported back from the synaptic cleft into the cytoplasm of the presynaptic neuron. This process utilises specialised proteins called transporters, or carriers. The monoamine binds to the transporter and is then carried across the plasma membrane it is thus transported back into the cellular cytoplasm. A number of psychotropic drugs selectively or non-selectively inhibit this reuptake process. They compete with the monoamines for the available binding sites on the transporter, so slowing the removal of the neurotransmitter from the synaptic cleft. The overall result is prolonged stimulation of the receptor. The tricyclic antidepressant imipramine inhibits the transport of both noradrenaline and 5-HT. While the selective noradrenaline reuptake inhibitor reboxetine and the selective serotonin reuptake inhibitor fluoxetine block the noradrenaline transporter (NAT) and serotonin transporter (SERT), respectively. Cocaine non-selectively blocks both the NAT and dopamine transporter (DAT) whereas the smoking cessation facilitator and antidepressant bupropion is a more selective DAT inhibitor. 

The postsynaptic membrane opposite release sites is also highly specialized, consisting of folds of plasma membrane containing a high density of nicotinic ACh receptors (nAChRs). Basal lamina matrix proteins are important for the formation and maintenance of the NMJ and are concentrated in the cleft. Acetylcholinesterase (AChE), an enzyme that hydrolyzes ACh to acetate and choline to inactivate the neurotransmitter, is associated with the basal lamina (see Ch. 11). 

Mirex has considerable potential for chronic toxicity because it is only partly metabolized, is eliminated very slowly, and is accumulated in the fat, liver, and brain. The most common effects observed in small laboratory mammals fed mirex included weight loss, enlarged livers, altered liver enzyme metabolism, and reproductive failure. Mirex reportedly crossed placental membranes and accumulated in fetal tissues. Among the progeny of mirex-treated mammals, developmental abnormalities included cataracts, heart defects, scoliosis, and cleft palates (NAS 1978 Blus 1995). 

Acetylcholinesterase (AChE) This enzyme is found in the troughs of the junctional folds of the muscle membrane and is responsible for metabolizing ACh within the synaptic cleft. 

The first step is the synthesis of the enzymes that are involved in the formation of the neurotransmitters, which occurs on the rough endoplasmic reticulum in the cell body. They are then transferred to the terminus of the presynaptic neurone by axonal transport. Here the neurotransmitters are synthesized prior to packaging into vesicles. The contents of the vesicles are, upon stimulation of the presynaptic neurone, released into the synaptic cleft. After binding to the postsynaptic receptor they are inactivated either by uptake from the cleft back into the presynaptic neurone or by enzymic degradation (Figure 14.9). After exocytosis, the membrane of the vesicle can be recycled back into the presynaptic neurone for re-filling and further exocytosis (see Figure 14.8). 

The hepatocyte secretes biliary fluid into the bile canaliculi (dark green), tubular intercellular clefts that are sealed off from the blood spaces by tight junctions. Secretory activity in the hepatocytes results in movement of fluid towards the canalicular space (A). The hepatocyte has an abundance of enzymes carrying out metabolic functions. These are localized in part in mitochondria, in part on the membranes of the rough (rER) or smooth (sER) endoplasmic reticulum. 

The intracellular signaltransduction of ofi-adrenoceptors is effectuated by a G-protein-dependent activation of the phospholipase C. This enzyme cleaves phosphatidylinositol, a phospholipid present in cell membranes, into inositol-1,4-5-triphosphate (IP3) and diacylglycerol (DAG). IP3 is a strong inductor of intracellular calcium release which leads to an increase of smooth muscle tone or the liberation of hormones stored in vesicles. Noradrenaline which is released by exocytosis, spreads by diffusion only. Only a small fraction of the total amount of the transmitter released will actually reach the postsynaptic membrane and bind to its specific receptors. Another fraction escapes the synapic cleft by diffusion and is finally enzymatically degraded in the interstitial fluid. Another fraction is taken up postsynaptically and metabolized enzymatically by the target cells (uptake 2). By far most of the transmitter (90%) is actively taken up by the releasing neuron itself (uptake 1 or neuronal re-uptake). In the... 

The conformation of membrane-bound enzymes is undoubtedly restricted by the membrane. However, the mechanism of action of these enzymes appears to be similar to that of soluble enzymes, so that the presence of clefts and conformational flexibility is to be expected. The mitochondrial coupling factor apparently contains both the ATP synthesizing enzyme and a proton channel conformational changes undoubtedly play a role in the function of this system. A large movement of polypeptide chains has been proposed in the functioning of this system (and for other membrane-bound enzymes), but no convincing experimental evidence is available to support such a hypothesis. 

Schematic illustration of a generalized cholinergic junction (not to scale). Choline is transported into the presynaptic nerve terminal by a sodium-dependent choline transporter (CHT). This transporter can be inhibited by hemicholinium drugs. In the cytoplasm, acetylcholine is synthesized from choline and acetyl -A (AcCoA) by the enzyme choline acetyltransferase (ChAT). Acetylcholine is then transported into the storage vesicle by a second carrier, the vesicle-associated transporter (VAT), which can be inhibited by vesamicol. Peptides (P), adenosine triphosphate (ATP), and proteoglycan are also stored in the vesicle. Release of transmitter occurs when voltage-sensitive calcium channels in the terminal membrane are opened, allowing an influx of calcium. The resulting increase in intracellular calcium causes fusion of vesicles with the surface membrane and exocytotic expulsion of acetylcholine and cotransmitters into the junctional cleft (see text). This step can he blocked by botulinum toxin. Acetylcholine s action is terminated by metabolism by the enzyme acetylcholinesterase. Receptors on the presynaptic nerve ending modulate transmitter release. SNAPs, synaptosome-associated proteins VAMPs, vesicle-associated membrane proteins.

In the following we attempt to describe the acetylcholinesterase/choline acetyltransferase enzyme system inside the neural synaptic cleft in a simple fashion see Figure 4.49. The complete neurocycle of the acetylcholine as a neurotransmitter is simulated in our model as a simple two-enzymes/two-compartments model. Each compartment is described as a constant-flow, constant-volume, isothermal, continuous stirred tank reactor (CSTR). The two compartments (I) and (II) are separated by a nonselective permeable membrane as shown in Figure 4.50. 

The depolarization that accompanies the action potential induces an increase in membrane permeability to calcium ions. A large inward electrochemical gradient exists for calcium and it moves into the terminal. The calcium that enters the terminal activates enzymes that cause the attachment of some of the vesicles to releasing sites on the terminal membrane, membrane fusion, and the release of the vesicular contents into the synaptic cleft. Transmitter release is terminated by the removal of calcium from the terminal cytoplasm, either via a calcium pump, which pumps it out of the cell, or by uptake into the endoplasmic reticulum or into mitochondria. 

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