Soluble enzyme activity

The enzyme uptake varied significantly according to the enzyme source, type, loading, and incubation time. The typical uptake for amylase ranged between 20 and 60%, whereas for cellulase, only 7-10% uptake was obtained. Since these uptake measurements simply reflect losses in soluble enzyme activity from the immobilization solution, they set a maximum on the activity that may be expressed by the immobilized enzyme. The actual activity is expected to be less, once losses resulting from shear, conformational changes, and nonproductive binding are taken into account. Direct measurements of immobilized enzyme activity therefore provide the best measure of the effectiveness of a particular immobilization technique. 

Solubility, enzyme activity, chemical and conformational stability of pharmaceutically active proteins under non-aqueous conditions have been well characterized (Zaks and Klibanov, 1988a Zaks and Klibanov 1988b Houen, 1996). Many of the solvents utilized in the literature are not pharmaceutically acceptable, and much of this work has not been directly applied to non-aqueous pharmaceutical formulations. However, the fundamental science and elucidation of concepts important to successfully utilizing non-aqueous conditions are applicable from this literature base. Furthermore, prediction of activity, solubility, chemical and structural stability are not routine, and preformulation work must be done on a targeted basis. 

HERG channel inhibition Microsomal stability Metabolite identification Aqueous solubility Enzyme activity Permeability Cytotoxicity... 

The influence of colloidal forces on reactions involving Immobilized enzymes acting on Insoluble substrates has received less attention, yet it appears to offer some clear examples of fundamental phenomena important in enzyme kinetics. Datta examined lysis of Micrococcus Ivsodelktlcus by soluble and (polyacrylamide) immobilized lysozyme. He noted that the decrease in soluble enzyme activity with decreasing ionic strength (Table 1) paralleled the measured decrease in cell lysis measured in flow through a packed bed reactor of Immobilized enzyme. 

Ionic Strength [M] Soluble Enzyme Activity Immobilized Enzyme Relative Activity exp t (theory) Cell Surface Potential i ) (mV) P ... 

Immobilization was carried out using a homogeneous preparation of poly(ADP-ribose) synthetase obtained from calf thymus [6] and commercially available BrCN-activated Sepharose 4B. For successful immobilization of active enzyme, it was important to block a majority (% two-thirds) of the active groups of the gel by pretreatment with dithiothreitol. The treated gel was mixed with the enzyme solution in the presence of 0.35 M KCl, 0.2 mAf dithiothreitol, 20% glycerol, and 0.1 A/K phosphate buffer (pH 8.0). Figure 1 shows the time course of immobilization of the enzyme activity. The decrease in the soluble enzyme activity reflects mainly immobilization of the enzyme, whereas the decrease in the immobilized enzyme activity is indicative of inactivation of the enzyme. In order to avoid the increase of inactive... 

At the molecular level, little is known about the target(s) of the lethal photosensitized reactions of a-terthienyl in insects. The only report to date concerns the inactivation of the important enzyme acetylcholines-terease in larvae of the mosquito A. aegypti 156). After confirming that, in vitro, pure acetylcholinesterase rapidly decreased in activity upon irradiation (an oxygen-dependent process), the total soluble enzyme activity was tested in larvae irradiated for various lengths of time after initial incubation in the presence of a-terthienyl. A gradual decrease in enzyme activity was observed about 40% of the initial enzyme activity was lost at the point where all the larvae had died. Many other enzymes in the larvae are probably inactivated as a result of photosensitized treatments and the decrease of acetylcholinesterase activity may not be the sole direct cause of the death of the larvae. 

Squalene monooxygenase, an enzyme bound to the endoplasmic reticulum, converts squalene to squalene-2,3-epoxide (Figure 25.35). This reaction employs FAD and NADPH as coenzymes and requires Og as well as a cytosolic protein called soluble protein activator. A second ER membrane enzyme, 2,3-oxidosqualene lanosterol cyclase, catalyzes the second reaction, which involves a succession of 1,2 shifts of hydride ions and methyl groups. 

No enzymatic side effects are observed and substrate concentrations up to 20% by weight can be used without affecting the enzyme activity. The biocatalyst is used in soluble form in a batch wise process, thus poorly soluble amino adds can be resolved without technical difficulties. Re-use of the biocatalyst is in prindple possible. 

The rate of catalysis of membrane bound enzymes (Plot B, Figure 1) is more greatly affected than soluble enzymes by lowering the temperature. This is due to the effect of low temperatures on the solidification of the membranes. Thus, an Arrhenius plot of the rate of a membrane-bound enzyme as a function of temperature often shows a discontinuity with a sharp break point (transition temperature) and loss of activity at the temperature where the membrane becomes a gel or more solid phase. 

Hosono O, HommaT, Kobayashi H et al (1999) Decreased dipeptidyl peptidase IV enzyme activity of plasma soluble CD26 and its inverse correlation with HIV-1 RNA in HIV-1 infected individuals. Qin Immunol 91 283-295... 

Keane NM, Price P, Lee S et al (2001) An evaluation of serum soluble CD30 levels and serum CD26 (DPPIV) enzyme activity as markers of type 2 and type 1 cytokines in HIV patients receiving highly active antiretroviral therapy. Clin Exp Immunol 126 111-116 Khan MZ, Brandimarti R, Shimizu S et al (2008) The chemokine CXCL12 promotes survival of postmitotic neurons by regulating Rb protein. Cell Death Differ 15(10) 1663-1672... 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

This is the first example of a reaction for which the presence of a chelating ligand was observed to facilitate rather than retard metal-catalysed epoxidation (Gao et al., 1987). It was found that the use of molecular sieves greatly improves this process by removing minute amounts of water present in the reaction medium. Water was found to deactivate the catalyst. All these developments led to an improved catalytic version that allows a five-fold increased substrate concentration relative to the stoichiometric method. Sensitive water-soluble, optically active glycidols can be prepared in an efficient manner by an in situ derivatisation. This epoxidation method appears to be competitive with enzyme-catalysed processes and was applied in 1981 for the commercial production of the gypsy moth pheromone, (-1-) disparlure, used for insect control (Eqn. (25)). 

Hereditary methemoglobinemia is classified into three types a red blood cell type (type I), a generalized type (type II), and a blood cell type (type HI). Enzyme deficiency of type I is limited to red blood cells, and these patients show only the diffuse, persistent, slate-gray cyanosis not associated with cardiac or pulmonary disease. In type II, the enzyme deficiency occurs in all cells, and patients of this type have a severe neurological disorder with mental retardation that predisposes them to early death. Patients with type III show symptoms similar to those of patients with type I. The precise nature of type III is not clear, but decreased enzyme activity is observed in all cells (M9). It is considered that uncomplicated hereditary methemoglobinemia without neurological involvement arises from a defect limited to the soluble cytochrome b5 reductase and that a combined deficiency of both the cytosolic and the microsomal cytochrome b5 reductase occurs in subjects with mental retardation. Up to now, three missense mutations in type I and three missense mutations, two nonsense mutations, two in-frame 3-bp deletions, and one splicing mutation in type n have been identified (M3, M8, M31). 

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