[Parameters of the succinate transport into Saccharomices cerevisiae cells after prolonged cold preincubation].

A nonconventional approach to the measurement of succinate transport through plasmalemma is proposed. It is based on the conditions in which the succinate oxidation rate is limited by transport through plasmalemma. Impermeable specific inhibitor of plasmalemma dicarboxylate transporter was employed as a tool to optimize conditions for the transport activity assay. For this purpose yeast culture was grown in synthetic medium under selected conditions. After aerobic preincubation of S. cerevisiae cells at 0 degree C (instead of incubation at 15 degrees C), the rate of endogenous respiration decreased substantially and was stabilized during measurements at a level that was five times lower than oxidation rates in the presence of exogenous substrate. This approach allowed for the reproducible determination of K(M) for the dicarboxylate transporter (7.3 +/- 2.1 mM) within a half-hour period. The advantages and drawbacks of this fast, but indirect, assay of slow substrates transport into the cell are compared with conventional methods.

Specific features of changes in levels of endogenous respiration substrates in Saccharomyces cerevisiae cells at low temperature.

The rate of endogenous respiration of Saccharomyces cerevisiae cells incubated at 0 degrees C under aerobic conditions in the absence of exogenous substrates decreased exponentially with a half-period of about 5 h when measured at 30 degrees C. This was associated with an indirectly shown decrease in the level of oxaloacetate in the mitochondria in situ. The initial concentration of oxaloacetate significantly decreased the activity of succinate dehydrogenase. The rate of cell respiration in the presence of acetate and other exogenous substrates producing acetyl-CoA in mitochondria also decreased, whereas the respiration rate on succinate increased. These changes were accompanied by an at least threefold increase in the L-malate concentration in the cells within 24 h. It is suggested that the increase in the L-malate level in the cells and the concurrent decrease in the oxaloacetate level in the mitochondria should be associated with a deceleration at 0 degrees C of the transport of endogenous respiration substrates from the cytosol into the mitochondria. This deceleration is likely to be caused by a high Arrhenius activation energy specific for transporters. The physiological significance of L-malate in regulation of the S. cerevisiae cell respiration is discussed.

[Isolation of cell membranes from Saccharomyces cerevisiae for evaluation of the protein composition]. / Poluchenie kletochnykh obolochek Saccharomyces cerevisiae s tsel'iu opredeleniia ikh belkovogo sostava.

We describe a simple method for the isolation of membrane fractions from Saccharomyces cerevisiae yeasts, containing the complex of plasma membranes and cell walls. The method is based on cell disruption on an INBI flow disintegrator. This device spares subcellular structures, which simplifies the isolation of cell membranes. The membrane fraction obtained by this method was suitable for studies of protein composition of these structures by means of two-dimensional electrophoresis.

[Turbidimetric method for quantitative determination of triton X-100 with silicotungstic acid]. / Turbidimetricheskii metod kolichestvennogo opredeleniia tritona X-100 s kremnevol'framovoi kislotoi.

The Formation of Triton X-100-silicotungstic acid complex was studied. Quantitative turbidimetric determination of the detergent based on this process was suggested. This method allows to determining the complex formation at any wavelength in the range from 350 (epsilon 350 = 15,600 cm-1 M-1) to 600 nm (epsilon 600 = = 9090 cm-1 M-1). The calibration curve for Triton X-100 recorded at 350 nm is linear in the concentration range of 0 to 30 micrograms/ml. A sigmoid calibration curve was observed at longer wavelengths. A linear fragment of the calibration curve recorded at 600 nm was found at a concentration of Triton X-100 of about 5 micrograms/ml. The complex nature of calibration curves can be explained by heterogeneity of the complex dispersion.

An equation for the reconstitution of membrane proteins and a method for determination of pore content in biological membranes.

The dependence between specific trapped volume of liposomes (W) and protein concentration (p) is proposed to be used for quantitative pore determination in biological membranes via pore reconstitution into liposomes. This dependence is described by the following equation: p = -p(e) x ln(W/W0), where W0 is initial trapped volume of liposomes and pe is an equivalent protein concentration at which molar concentrations of pores and liposomes become equal. Experimentally determined equivalent protein concentration pe is the basis of the method. This method also permits determination of molar mass of pore-forming complexes provided that preparations contain purified complex.

[A method of determining the specific volume of liposomes with Turnbull blue]. / Metod opredeleniia udel'nogo ob''ema liposom s pomoshch'iu Turnbulevoisini.

A method for measuring the specific volume of liposomes by using ferricyanide as a maker is proposed. Ferricyanide is determined by the formation of Turnbull's blue. Advantages of the use of this reaction are an eightfold increase in the sensitivity and the possibility to determine the marker compound in the spectral region (730 nm) free of any considerable interference of natural compounds. Results of the use of this method for measuring the specific volume of liposomes prepared by ultrasonic treatment of a lipid suspension are reported.

[Characteristics of the interaction between 2-alkylmalonates and the substrate-binding site of the dicarboxylate carrier of rat liver mitochondria]. / Osobennosti vzaimodeistviia 2-alkilmalonatov s tsentrom sviazyvaniia substratov dikarboksilatnogo perenoschika mitokhondrii pecheni krys.

The effect of thirteen synthetic 2-n-alkyl derivatives of malonic acid on the rate of the mitochondrial succinate oxidase reaction controlled by the dicarboxylate carrier, was studied. These compounds were shown to act as competitive inhibitors of succinate transport and could interact with the carrier according to the 1:1 stoichiometry. The affinity of the inhibitor for the carrier increased in parallel with the increase in the alkyl residue length. This dependence was impaired only in the case of pentyl-, hexyl- and heptylmalonates having close values of inhibition constants. The data obtained suggest that the polar site and two hydrophobic regions are located in the vicinity of the carrier active site. The possible organization of the carrier substrate-binding site is discussed.

[Trypsin-like proteinase from Actinomyces fradiae 119]. / Tripsinopodobnaia proteinaza Actinomyces fradiae 119.

Using electrophoresis and ultracentrifugation, a homogeneous proteinase was isolated from protofradine, a protease Act. fradiae 119 preparation. The purification procedure included filtration on DEAE-cellulose, gel filtration through Arcylex P-10, CM-chromatography and desalting on Sephadex G-15. The proteinase under study is an endopeptidase which hydrolyzes low molecular weight synthetic trypsin substrates as well as casein and denaturated collagen. Diisopropylfluorophosphate and soya bean trypsin inhibitor completely inhibit the proteinase activity, whereas pCMB and EDTA have no such effect. The stability maximum is observed at pH of 2.5-3.5, the action maximum at pH 8.7-9.5. The amino acid composition of the enzyme is similar to that of trypsin from Str. griseus. The molecular weights of the proteinase as determined by gel filtration and sedimentation equilibrium method are equal to 25400 and 26500, respectively. The isoelectric point lies at 5.3. The data obtained suggest that the proteinase can be attributed to the family of trypsin proteinases.

[Elastolytic enzymes from Actinomyces fradiae 119]. / Elastoliticheskie fermenty Actinomyces fradiae 119.

Using ion-exchange chromatography and gel-filtration, elastase II, the main elastolytic component of protofradin (preparation of proteases of Act. fradiae 119), was purified to an electrophoretically homogeneous state. The enzyme is a serine proteinase with mol. weight of 17800 +/- 1000 and a pI greater than 10. The enzyme is stable at pH greater than 4,0 and exhibits its maximal activity towards elastin at pH 9,2. An analysis of elastolytic products revealed that the enzyme hydrolyses natural elactin of bovine nuchal ligament to form large fragments with mol. weights ranging from 25 000 to 80 000 and small oligopeptides. The elastolysis is ceased at the stage of formation of short-chained peptides, predominantly tripeptides. Elastase I is a minor component of protofradin and in its molecular weight and some enzymatic properties is similar to elastase II.

[Isolation of individual proteases from protofradin]. / Vydelenie individual'nykh proteaz iz protofradina

An electrophoretically homogeneous trypsin-like proteinase, two homogeneous proteases (presumably metal-containing) and two elastases, possessing the ATEE-esterase activity, were isolated from protofradin, a protease preparation from Actinomyces fradiae 119, using fractionation on KM-cellulose K-32. The trypsin like proteinase of protofradin possesses the esterase activity, equal to the activity of pancreatic trypsin. Protofradin elastases differ in their pH optima, response to EDTA, stability upon storage and the degree of elastin hydrolysis. The specificity of elastase is probably the same, since in elastin both enzymes hydrolyze the peptide bonds, formed by the NH2-group of glycine and alanine residues, found in elastin in large amounts. The end products of elastin hydrolysis are tripeptides.