[Beta-galactosidase from E. coli as a marker in immunoenzyme analysis]. / Beta-galaktozidaza E. coli kak marker v immunofermentnom analize.

In the present review the authors analyze factors influencing sensitivity of the enzyme immunoassay (EIA). beta-Galactosidase from E. coli was chosen as a marker enzyme. Physico-chemical properties of the enzyme, detection methods and various ways of obtaining chemical conjugates with antigens and antibodies are discussed. Some examples of using beta-galactosidase as a label in homogeneous and heterogeneous EIA are given which enables different compounds to be analyzed with a high sensitivity. New approaches employing gene engineering for constructing "fusions" between beta-galactosidase and antigens (alternative to chemical conjugates) are discussed that can be used in the near future.

[Determination of beta-galactosidase activity on nitrocellulose plates using 5-bromo-3-indolyl-beta-D-galactopyranoside and tetrazolium salts]. / Opredelenie beta-galaktozidaznoi aktivnosti na nitrotselliuloznykh plastinakh s pomoshch'iu 5-brom-3-indolil-beta-D-galaktopiranozida v kombinatsii s soliami tetrazoliia.

A simple and convenient technique has been developed for detection of beta-galactosidase from E. coli on nitrocellulose sheets using a mixture of 5-bromoindol-3-yl-beta-D-galactopyranoside and nitro blue tetrazolium, which enables rapid detection of fmole (10(-15) mole) quantities of the enzyme at pH 9.5. The technique has the following advantages: the substrates are stable for a long period; reaction products give non-fading intense blue colour, resolution is extremely good with essentially no diffusion.

[Preparation of an insulin conjugate with beta-galactosidase from E. coli for immunoenzyme assay]. / Poluchenie kon''iugata insulina s beta-galaktozidazoi iz E. coli dlia tselei immunofermentnogo analiza.

A modified procedure has been worked out for preparing a conjugate of porcine insulin with E. coli beta-galactosidase employing a heterobifunctional reagent, N-hydroxysuccinimidyl m-maleimidobenzoate. Optimal conditions for insulin acylation and subsequent coupling with beta-galactosidase were selected that afforded the conjugate in a high yield. The ability of the modified antigen to react with antibody was evaluated in the reaction of conjugate binding with immobilized monoclonal antibody to insulin. The conjugate almost completely retained the enzymatic activity and reacted with high specificity with the antibody to insulin. The conjugate can be used in competitive ELISA of insulin.

[Specific features of intracellular serine proteinase of Bacillus amyloliquefaciens on native and denatured protein substrates]. / Osobennosti deistviia vnutrikletochnoi serinovoi proteinazy Bacillus amyloliquefaciens na nativnye i denaturirovannye belkovye substraty.

The effects of intracellular serine proteinase from B. amyloliquefaciens and of extracellular subtilisin BPN' on native and denaturated protein substrates were compared. The substrate hydrolysis by the enzymes was determined by a method initiating possible participation of intracellular serine protinease in intracellular protein degradation. This approach consists in a prolonged treatment of the products obtained after proteolysis with carboxypeptidase A with a subsequent amino acid assay. It was found that intracellular serine proteinase is capable of degrading denaturated protein substrates e. g. reduced, carboxymethylated ribonuclease A and bovine serum albumin, with an efficiency comparable to that achieved by subtilisin hydrolysis. On the other hand, the intracellular enzyme attacks the native proteins much slower than substilisin.

[Substrate specificity of Bacillus subtilis intracellular serine protease. Hydrolysis of insulin beta-chain, native ribonuclease A and p-nitroanilide peptide substrates]. / O substratnoi spetsifichnosti vnutrikletochnoi sepinovoi proteinazy v Bacillus subtlis. Proteoliz v-tsepi insulina i ribonukleazy A i kinetika gidroliza p-nitoranilidnykh substratov.

Intracellular serine protease, termed ISP-103, was isolated from Bacillus subtilis, strain 103. The substrate specificity of the enzyme was compared to that of secretory subtilisins. Similar to subtilisins, ISP-103 cleaves a single peptide bond Ala20-Ser21 within the native pancreatic ribonuclease A, which results in the accumulation of trypsin-sensitive ribonuclease S, consisting of a non-covalently bound S-peptide (20 amino acid residues) and S-protein (104 amino acid residues). The enzyme hydrolyzes a single peptide bond Leu15-Tyr16 of the B-chain of oxidized bovine insulin, in contrast to the subtilisins cleaving four additional bonds. ISP prefers Leu rather than Phe in the P1 binding site of the rho-nitroanilide peptide substrates and shows a more strict dependence of the activity on the presence of the hydrophobic residues in the P2 and P3 sites. The data obtained indicate that the substrate specificity of ISP, being within the borders of subtilisin specificity, is nevertheless much more restricted.