Transducing enzyme activation

All receptors are coupled to at least two transducing enzyme systems, phospholipase C and adenylate cyclase. The traditional model of transducing enzyme activation is based upon receptor activation of adenylate cyclase (see Birnbaumer, Chapter 1). In this model the dissociation of the G-protein from its receptor induces a lower-affinity receptor state and facilitates the interaction of the freed G-protein with the catalytic unit of adenylate cyclase, producing either a stimulation 

While the coupling functions of Gs and Gj are well characterized, additional pertussis toxin-sensitive G-proteins (for example G ) have been identified whose functions are still unknown [21]. Interestingly, in certain systems pertussis-toxin treatment prevents the activation of phospholipase C both by hormones and by nonhydrolysable guanine nucleotides [21], Thus, a pertussis-toxin-sensitive G-protein other than Gj (such as Gc) may couple receptors to this transducing enzyme. In other systems activation of phospholipase C is not sensitive to pertussis toxin, indicating that if a G-protein mediates the activation of PLC in these systems, it must be via yet another member of the G-protein family. 

In contrast to adrenal and hepatic tissue, adenylate cyclase inhibition may be physiologically relevant in renal vasculature, since pertussis toxin attenuates All-induced vasoconstriction [23]. The physiological role for adenylate cyclase inhibition may therefore depend upon the differential response of the three tissues to the second messenger, cAMP. In vascular smooth muscle, adenylate cyclase inhibition should enhance All-induced contraction by reducing cellular cAMP, the messenger of relaxation, while in the adrenal and the hepatocyte cAMP mediates a stimulatory response and adenylate cyclase inhibition would be expected to oppose All action. 

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FIGURE 5.6 Schematic representation of the immunosensor based on a Protein A-GEB biocomposite as a transducer, (a) Immobilization of RlgG on the surface via interaction with Protein A, (b) competitive immunoassay using anti-RIgG and biotinylated anti-RIgG, (c) enzyme labeling using HRP-streptavidin and (d) electrochemical enzyme activity determination. (Reprinted from [31] with permission from Elsevier.)... 

It is worth mentioning that membrane-bound forms of GC, which can be considered signal transducing enzymes , are structurally homologous to other signal transducing enzymes, such as certain protein tyrosine kinases and phosphatases, which also possess receptor moieties in their extracellular (amino terminus) domain and enzyme catalytic activity in their intracellular domain (see Ch. 24). Activation of many of these receptors occurs upon ligand-induced dimerization of the receptors, and a similar... 

Most electrochemical immunosensors use antibodies or antigens labelled with an enzyme that generates an electroactive product which can be detected at the electrochemical transducer surface. The combination of high enzyme activity and selectivity with the sensitive methods of electrochemical detection provides a basis for the development of immunosensors. Horse radish peroxidase (HRP) and alkaline phosphatase (AP) are popular enzyme labels and can be used with a variety of substrates. 

Fig. 1. Schematic diagram ot high-pressure apparatus tor enzyme activity tests. A, C02 cylinder B, syringe pump C, equilibrium cell D, sapphire windows E, magnetic stirrer F, white light source G, pressure transducer H, ball valve I, micrometering valve J, relief valve.

CNTs and other nano-sized carbon structures are promising materials for bioapplications, which was predicted even previous to their discovery. These nanoparticles have been applied in bioimaging and drag delivery, as implant materials and scaffolds for tissue growth, to modulate neuronal development and for lipid bilayer membranes. Considerable research has been done in the field of biosensors. Novel optical properties of CNTs have made them potential quantum dot sensors, as well as light emitters. Electrical conductance of CNTs has been exploited for field transistor based biosensors. CNTs and other nano-sized carbon structures are considered third generation amperometric biosensors, where direct electron transfer between the enzyme active center and the transducer takes place. Nanoparticle functionalization is required to achieve their full potential in many fields, including bio-applications. 

The application of chromatographic or electrophoretic systems leads to the separation of not only several substrates but also of sample constituents, which could infiuence the enzyme activity or lead to enz5rme-independent signals [75]. An alternative approach to eliminate these interferences is the restriction of the access of interfering compoimds to the enzyme and the transducer. This can be achieved by adapting the... 

On the other hand, the molecular recognition by enzymes, which are also applied in the form of organelles, microorganisms and tissue slices, is accompanied by chemical conversion of the analyte to the respective products. Therefore this type of sensor is termed a metabolism sensor2. The initial state is usually reached when the analyte conversion is complete. With metabolism sensors, under certain conditions cosubstrates, effectors, and enzyme activities can be measured via substrate determination. Amperometric and potentiometric electrodes and thermistors are the preferred transducers, but in some cases optoelectronic sensors have also been used. With biomimetic sensors physical signals such as sound, stress, or light are measured through their ability to... 

G-proteins are found on the inside of the membrane and act as transducers between receptors and cellular effectors. They are made up of three subunits (a, P, y) one of which (a) has GTPase (guanine triphosphatase, an enzyme) activity. 

Besides being used as adsorbent for gas molecules, both SWCNTs and MWC-NTs can be cast as a random network or a porous thin film on metal electrodes [57-59] or used as a three-dimensional scaffold [41,42] for biosensors. CNTs serve both as large immobilization matrices and as mediators to improve the electron transfer between the active enzyme site and the electrochemical transducer. Various enzymes, such as glucose oxidase and flavin adenine dinucleotide (FAD) can adsorb onto the CNT surface spontaneously and maintain their substrate-specific enzyme activity over prolonged times [57]. Recently, cells have been grown on CNT scaffolds which provide a three-dimensional permeable environment, simulating the natural extracellular matrix in a tissue [60-62]. 

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