Enzyme channeling systems

Triazines Flow-through amperometric immunosensor based on peroxidase chip and enzyme channeling system 1 ng L 1 8... 

Fig. 1. Free enzymes FIA system 1, enzymes and reagents solution 2, sample solution 3, buffer solution 4, peristaltic pump 5, three-channel valve 6, six-channel injection valve 7, colorimeter 8, computer 9, waste 10, coil.

The free enzymes FIA system permitted analysis in a linear range of 0.05-1.0 g of ethanol/L, a sampling frequency of 15 analyses/h, and a relative SD of 3.5%. The total volumetric flow was 5 mL/min, and the six-channel injection valve permitted the introduction of 0.185 mL (loop volume) of enzymes-reagents solution per analysis (4). Ethanol solutions of different concentrations were used to determine the linear working range and for the calibration curve construction (Fig. 4), presenting a linear relation up to 1 g of ethanol/L with a correlation factor of 0.9899 for six samples. 

Hundeck HG, Sauerbrei A, Hiibner U, Scheper T, Schiigerl K (1990) Four-channel enzyme thermistor system for process monitoring and control in biotechnology. Anal Chim Acta 238 211-222... 

Hundeck HG, Hiibner U, Liibbert A, Scheper T, Schmidt J, Weifl M, Schubert F (1992) Development and application of a four-channel enzyme thermistor system for bioprocess control. 

The implementation of natural ion channel systems for biosensor construction will suffer from the complexity of the structural and regeneration requirements imposed by the biological matrix. In a practical context, a more promising immediate solution seems to be the development of biosensors having an ion channel activity without implementation of ion channel proteins from natural sources, i.e. artificial ion channels. This involves modulation of ion conductivity through lipid membranes by means of alteration of phase structure by a wide variety of different selective binding interactions with, e.g. enzymes, antibodies, lectins, and others. 

Methylglutaryl-CoA hydratase (EC 4.2.1.18) catalyzes the reversible dehydration of HMG-CoA into 3-methylglutaconyl-CoA and is known from mammalian cells to be involved in leucine metabolism. This enzyme was detected in C. roseus suspension cultured cells and partially purified (87). Further enzymes channeling HMG-CoA away from isoprenoid biosynthesis in C. roseus cells were detected by a selective HPLC system showing that the CoA-esters may be rapidly dephosphorylated on the 3 -position by the action of 3 -nucleotidases (87). 

A review of enzyme analysers has described the automation of fixed-time and continuous-monitoring assays and the uses of partly or completely automated analysers and multi-channel systems. Automated analyses of polysaccharide hydrolases, in soluble or insoluble form, have been based on determination of the liberated reducing sugars with 3,5-dinitrosalicylic acid. A sensitive and specific method for locating glycoside hydrolases (e.g. oc-D-mannosidases) in polyacryl-... 

FIGURE 10.26 Glucose transport in E. coli is mediated by the PEP-dependent phosphotransferase system. Enzyme I is phosphorylated in the first step by PEP. Successive phosphoryl transfers to HPr and Enzyme III in Steps 2 and 3 are followed by transport and phosphorylation of glucose. Enzyme II is the sugar transport channel. 

Neutrophils represent an ideal system for studying osmotic effects on exocytosis. Stimulation of cytochalasin-B-treated neutrophils with the chemotactic peptide Jlf-formylmethionyl-leucyl-phenyl-alanine (FMLP) results in a rapid compound exocytosis up to 80% of lysosomal enzymes are released within 30 s (9-14). Secretion appears to be triggered by a rise in the level of cytosolic free calcium (15-18) promoted in part by entry of extracellular calcium through receptor-gated channels and in part by release of calcium that is sequestered or bound at some intracellular site (19-21). In this presentation, we augment our previously published data (22,23), which demonstrates that lysosomal enzyme release from neutrophils is inhibited under hyperosmotic conditions and that the rise in cytosolic calcium preceding secretion is inhibited as well. 

Many environmental toxins interact with specific cellular receptors, including enzymes, ion channels and ion pumps, and thus provide natural tools for the study of cellular signalling pathways. Palytoxin, a compound isolated from the coelen-terate of genus Palythoa, is one such useful and intriguing compound. The structure of palytoxin was first determined in 1981 independently by Hirata (7) and Moore (2). As one of the most potent marine toxins known, palytoxin has been studied in a variety of systems ranging from erythrocytes to neurons. As a tumor promoter of the non 12-O-tetradecanoylphorbol-13-acetate (TPA) type, palytoxin can also be studied in the context of a growth control system. 

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