[Analytical biotechnology of recombinant peptides and proteins. I. Analysis of the purity, composition and structure of human, porcine and bovine insulins]. / Analiticheskaia biotekhnologiia rekombinantnykh peptidov i belkov. I.Analiz chastoty, sostava i struktury insulinov cheloveka, svin'i i krupnogo rogatogo skota.

A method for analysis of the type, purity, and possible structural modifications of insulins of bovine, porcine, and human origin was proposed. It is based on a combination of narrow-bore reversed-phase HPLC and mass spectrometry. The hydrolysis of insulins with highly specific Glu-protease V8 from Staphylococcus aureus followed by peptide mapping of the hydrolysis products and mass spectrometry of the isolated fragments helps rapidly and reliably localize and identify substitutions of amino acid residues in insulin structure by using insulin samples of less than 1 nmol.

Studies on the succinate dehydrogenating system. II. Reconstitution of succinate-ubiquinone reductase from the soluble components.

1. A protein fraction containing three polypeptides (the major one with Mr < 13 000) was isolated by means of Triton X-100 extraction of submitochondrial particles specifically treated to remove succinate dehydrogenase. 2. The mixing of the protein fraction with the soluble reconstitutively active succinate dehydrogenase results in formation of highly active succinate-DCIP reductase which is sensitive to thenoyltrifluoroacetone or carboxin. 3. The maximal turnover number of succinate dehydrogenase in the succinate-DCIP reductase reaction revealed in the presence of a saturating amount of the protein fraction is slightly higher than that measured with phenazine methosulfate as artificial electron acceptor. 4. The protein fraction greatly increases the stability of soluble succinate dehydrogenase under aerobic conditions. 5. The titration of soluble succinate dehydrogenase by the protein fraction shows that smaller amounts of the protein fraction are required to block the reduction of ferrycyanide by Hipip center than that required to reveal the maximal catalytic capacity of the enzyme. 6. The apparent Km of the reconstituted system for DCIP depends on the amount of protein fraction; the more protein fraction added to the enzyme, the lower the Km value obtained. 7. A comparison of different reconstituted succinate-ubiquinone reductases described in the literature is presented and the possible arrangement of the native and reconstituted succinate-ubiquinone region of the respiratory chain is discussed.

[Reconstitution of succinate-ubiquinone reductase of the respiratory chain of mitochondria]. / Rekonstruktsiia suktsinat-ubikhinon-reduktaznogo uchastka dykhatel'noi tsepi mitokhondrii.

A soluble protein fraction, which confers the reactivity of soluble succinate dehydrogenase towards ubiquinone, was isolated from beef heart mitochondria. This fraction contains three polypeptides as revealed by SDS-electrophoresis; the major peptide (about 80% of protein) has a molecular weight less than 13 000. Several properties of the reconstituted succinate-ubiquinone reductase, i. e. the turnover number of succinate dehydrogenase inhibitor sensitivity, stability and reactivity towards artificial electron acceptors were found to be identical to those of the native succinate-ubiquinone region of the respiratory chain. A model of the minimal functionally active structure capable of reduction of ubiquinone by succinate is proposed.