Avances en el tratamiento de la retinopatía proliferativa mediante terapia génica / Advances in a gene therapy-based approach to treat proliferative vitreoretinopathy

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[Determination of the enzymatic activity of lysine amidase]. / Opredelenie fermentativnoi aktivnosti lizinamidazy.

A technique is proposed for determining lysinamidase and aminolactamase activities of lysinamidase (EC 3.5.1.n.). It is based on spectrophotometric measurement of the optical density decrease of the substrate solution at 227 nm. For cyclic lysinamide L-alpha-amino-epsilon-caprolactam epsilon 227 M = 151 M-1.cm-1, for linear lysinamide epsilon 227 M = 73 M-1.cm-1, and for lysine epsilon 227 M = 5 M-1.cm-1. The technique is simple and requires no additional reagents.

Determination of initial velocities of enzymic reactions from progress curves.

The present communication describes a novel method for estimating initial velocities (v) of enzyme-catalysed reactions. It is based on an approximation of experimental data obtained by the cubic spline function. The initial velocity of a reaction is calculated as a derivative of the approximating function at a time value equal to zero. The proposed method is usable on a computer with a FORTRAN IV program. The method can be successfully used in such cases as substantial extents of substrate conversion, the inactivation of an enzyme in the course of a reaction, the existence of large experimental error or when the reaction mechanism is unknown.

Lysine decarboxylase assay by the pH-stat method.

A simple assay of lysine decarboxylase is described. It is based on measuring the rate of titration of OH- ions, released during decarboxylation with the aid of a pH-stat apparatus. The continuous recording of the reaction progress may be easily performed. Using the pH-stat method the Km for lysine decarboxylase from E. coli is 2.0 mM.

[Choice of the initial enzyme correlation in immobilizing polyenzyme systems]. / Vybor iskhodnogo sootnosheniia fermentov pri immobilizatsii polifermentnykh sistem.

An optimal, in respect to productivity (activity X stability), enzyme ratio for immobilization of multienzyme systems was calculated by using the kinetic parameters (KM and Vmax), data on the stability and yield of each enzyme during immobilization. The experimental data, obtained during combined immobilization of invertase, mutarotase and glucose oxidase, illustrate the theoretical propositions.

[Isolation and characteristics of immobilized L-glutamate decarboxylase]. / Poluchenie i kharakteristika immobilizovannoi L-glutamatdekarboksilazy.

Glutamate decarboxylase from Escherichia coli was immobilized on inorganic macroporous carriers by the glutaraldehyde, carbodiimide and bromacetyl methods, on silicagel coated with a layer of a glutaraldehyde and m-phenylene diamine copolymer, and by polyacrylamide gel incorporation. The efficiency of the above methods of immobilization was evaluated. The bromacetyl method was found to be the most efficient. The dependence of activity of soluble and immobilized Glu-decarboxylase upon pH, temperature, substrate concentration, and stability was established. The differences in the properties of soluble and immobilized Glu-decarboxylase were due to the substrate diffusion in pores of the carrier. The immobilized Glu-decarboxylase obtained showed high activity and stability.