Membrane-enzyme testing

Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

To test the above hypothesis, Reusch et al.30 analyzed the human erythrocyte Ca2+-ATPase pump for polyP and PHB. This protein is the first plasma membrane enzyme demonstrated to work as a Ca2+ pump, and it is the sole transporter of Ca2+ in the cell.121,122 Moreover, the Ca2+-ATPase is ubiquitous in plasma membranes of a variety of cell types in eukaryotic organisms.123... 

To provide a test of the effect of spacer length on enzyme activity, a third membrane-enzyme system was formed by reacting the product of the reaction between ethylenediamine and the cellulose ester membrane with suc-cinyl anhydride, then coupling this product to an amine on the enzyme using the carbodiimide route. The product may be represented as ... 

An enzymic test which allows the localization of yeast jS-D-fructofurano-sidase directly on sodium dodecyl sulphate gels is dependent upon the appearance of an activity band (mol. wt. 1.9 x 10 ) arising from membrane fractions and additional to those of the external and internal -D-fructo-furanosidases. The band showed kinetic characteristics of a precursor of the external enzyme, and when the increase in the external enzyme slowed down, this membrane-associated form almost disappeared completely. Tunicamycin-initiated inhibition studies indicated that the transient form contained already those carbohydrate parts which contain 2-acetamido-2-deoxy-D-glucose and are transferred via dolichyl phosphate-bound intermediates. 

A total protein content of 0.5% and less in a commercial product is considered safe regarding the product s immunogenic properties. Among the protein s impurities, the most antigenetic properties are attributed to ovalbumin, which has the ability to penetrate through skin and mucous membranes by bypassing the immune barrier. The ovalbumin content could be determined by sohd phase immune-enzyme test-system with sensitivity of -0.5 ng/ml. Its content must be lower than 0.2% from the overall protein content. 

The major difficulty in examining the effects of phospholipids on the properties of most membrane-bound enzymes is their firm attachment to the membrane, which precludes studies with highly purified systems. On the other hand, it is likely that if the technology were available for purification of these enzymes in homogeneous form, some enzymes would have properties completely different from those of the enzyme in intact membranes. It is impossible, therefore, to test conclusions and hypotheses derived from experiments with intact membranes in reconstitution experiments. For this reason, emphasis in the study of tightly-bound membrane enzymes should be directed to intact systems. In this regard, the basic design of experiments is to perturb the... 

Since in continuous degradation processes it is expected to reach a molecular weight distribution of the products, which is optimal for their further use, the investigation was devoted to test the effect of a key parameter such as the enzyme to substrate ratio (E/S). For a fixed mean retention time in the UF-membrane reactor, the following behaviour can be... 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

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