Periodic formation of the oriC complex of Escherichia coli.

We examined formation of an oriC-membrane complex through the chromosome replication cycle by dot-blot hybridization using an oriC plasmid as a probe. In a wild-type culture synchronized for chromosome replication, oriC complex formation was observed periodically and transiently corresponding to the replication initiation event. Prior to initiation of replication the oriC complex was recovered in the outer membrane fraction as well as at the time of initiation of replication. Moreover, periodic formation of the oriC complex was observed even when further initiation of replication was suppressed by culturing an initiation ts mutant at the restrictive temperature. Similar periodic formation of the oriC complex was also observed when DNA elongation was inhibited by addition of nalidixic acid to the culture. However, the second periodic peak did not appear when rifampicin or chloramphenicol was added. Cells which formed the oriC complex at the restrictive temperature could immediately initiate chromosome replication when the cells were transferred to the permissive temperature. We conclude that the oriC region of Escherichia coli forms a specific complex periodically just before and at the time of initiation of chromosome replication and that oriC complex formation is a prerequisite for initiation of chromosome replication.     

Dynamic light scattering studies on hydrodynamic properties of fibrinogen-fibronectin complex.

A high molecular weight 'cryogel' was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37 degrees C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20 degrees C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20 degrees C in 0.05 M TrisHCl(pII7.4) containing 0.5 M NaCl was estimated as 8.5 x 10(-8) cm2s-1. The complex did not dissociate over the temperature range from 20 to 37 degrees C. The diffusion coefficients of the complex decreased significantly at 12 degrees C and 40 degrees C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40 degrees C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15 degrees C, but not at near-freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.     

Characterization of a temperature-sensitive defect of enterovirus 70: effect of elevated temperature on in vitro transcription

A crude replication complex prepared from enterovirus 70-infected cells was used to study the temperature-sensitive characteristic of the virus. The complex showed a temperature sensitivity in the in vitro incorporation of radiolabeled ribonucleoside triphosphate. The endonuclease itself did not account for the restricted RNA synthesis at the nonpermissive temperature. Analyses of the in vitro products by both gel electrophoresis and sucrose density gradient centrifugation showed that the complex synthesized three types of viral RNA only when incubated for a short period of time at the nonpermissive temperature. When the replication complex was treated with a detergent (deoxycholic acid), incorporation of ribonucleoside triphosphate into RNA at the permissive temperature was reduced to the level of that at the nonpermissive temperature. In addition, the in vitro RNA synthesis by the enterovirus 70 replication complex at the permissive temperature required a higher concentration of ATP than of other ribonucleoside triphosphates, whereas such a preference for ATP was not found in the reaction at the nonpermissive temperature. The results indicate that the initiation step of RNA synthesis by the complex is blocked at the nonpermissive temperature. The possible implications of these findings are discussed.     

Dynamic Light Scattering Studies on Hydrodynamic Properties of Fibrinogen-Fibronectin Complex

Abstract

A high molecular weight ‘cryogel’ was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37°C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20°C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20°C in 0.05 M Tris- HCl(pH7.4) containing 0.5 M NaCl was estimated as 8.5× 10^?8 cm²s^?1. The complex did not dissociate over the temperature range from 20 to 37°C. The diffusion coefficients of the complex decreased significantly at 12°C and 40°C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40°C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15°C, but not at near- freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.     

Characterization and catalytic property of surfactant-laccase complex in organic media

The oxidation of o-phenylenediamine catalyzed in anhydrous organic solvents by surfactant-laccase complex was investigated. The complex was prepared by utilizing a novel preparation technique in water-in-oil (W/O) emulsions. The surfactant-laccase complex effectively catalyzed the oxidation reaction in various dry organic solvents, while laccase, lyophilized from an aqueous buffer solution in which its activity was optimized, exhibited no catalytic activity in nonaqueous media. To optimize the preparation and reaction conditions for the surfactant-enzyme complexes, we examined the effects of pH in the water pool of W/O emulsions, the concentration of enzyme and surfactant at the preparation stage, and the nature of organic solvents at the reaction stage on the laccase activity in organic media. Surfactant-laccase complex showed a strong pH-dependent catalytic activity in organic media. Its optimum activity was obtained when the complex was prepared at a pH of about 3. Interestingly, native laccase in an aqueous buffer solution exhibited an optimum activity at the same pH of 3. The optimum preparation conditions of surfactant-laccase complex were [laccase] = 0.8 mg/mL and [surfactant] = 10 mM, and the complex showed the highest catalytic activity in toluene among nine anhydrous organic solvents. The effect of a cosolubilized mediator (1-hydroxybenzotriazole (HBT)) on the reaction was also investigated. The addition of HBT at the preparation stage of the enzyme complex did not accelerate the catalytic reaction because HBT was converted to an inactive benzotriazole (BT) by laccase. However, the addition of HBT at the reaction stage enhanced the catalytic performance by a factor of five compared to that without HBT.     

Transformation of glucocorticoid-receptor complex from rat thymocytes and its accumulation in chromatin in a cell-free system.

The accumulation of glucocorticoid-receptor complex from rat thymocyte cytosol in a thymocyte chromatin preparation has been studied. A thymocyte 100 000 X g supernatant was prepared and the receptor and the receptor stabilized by the addition of glycerol until 40%. Tritiated glucocorticoid-receptor complex was formed by incubation of this solution with tritiated glucocorticoids at -5 degree C. The chromatin accumulated part of the complex at incubations at 4 degrees C. Receptor without hormone was not accumulated in the chromatin. The accumulation from cytosol diluted and preincubated at 4 degrees C prior to the addition of the chromatin occurred with a high rate, whereas a low rate was seen without preincubation. This indicated a transformation of the complex during the preincubation. This transformation was found to be obligatory for the accumulation and to be promoted by dilution of the supernatant and by high ionic strength. The transformed and the untransformed complexes differed with respect to partition coefficients in an aqueous dextran-polyethylene glycol two-phase system and in their behaviour during adsorptions with dextran-coated charcoal, where great loss of transformed complex was observed. The accumulation of complex in the chromatin was found to be unsaturable in the concentration interval studied (0.07--0.25 nM).     

The spectrophotometric determination of micromolar concentrations of Co2+ using o-phenanthroline

Co2+ and o-phenanthroline formed a 1:3 complex with absorption maxima at 346, 332, 313, and 301 nm. The complex obeyed Beer's Law at the first three maxima. Standard curves constructed by monitoring the E346 at different concentrations of Co2+ had a maximum sensitivity of about 1 microM Co2+. At this concentration of Co2+ the delta E346 was 0.003 absorption units. This assay was not affected greatly by Ca2+, Mg2+, K+, Na+, or Tris. Erbium ions (Er3+) produced a small, nonspecific increase in absorbance at all wavelengths. Zn2+ also formed a complex with o-phenanthroline with maxima at 343, 328, and 313 nm. The absorbance of the Zn2+-o-phenanthroline complex was about 10% that of the Co2+-o-phenanthroline complex at 346 nm, but was still sufficient to cause interference at Zn2+ concentrations above 10 microM.     

Transformation of glucocorticoid-receptor complex from rat thymocytes and its accumulation in chromatin in a cell-free system

The accumulation of glucocorticoid-receptor complex from rat thymocyte cytosol in a thymocyte chromatin preparation has been studied. A thymocyte 100 000 × g supernatant was prepared and the receptor stabilized by the addition of glycerol until 40%. Tritiated glucocorticoid-receptor complex was formed by incubation of this solution with tritiated glucocorticoids at −5°C. The chromatin accumulated part of the complex at incubations at 4°C. Receptor without hormone was not accumulated in the chromatin. The accumulation from cytosol diluted and preincubated at 4°C prior to the addition of the chromatin occurred with a high rate, whereas a low rate was seen without preincubation. This indicated a transformation of the complex during the preincubation. This transformation was found to be obligatory for the accumulation and to be promoted by dilution of the supernatant and by high ionic strength. The transformed and the untransformed complexes differed with respect to partition coefficients in an aqueous dextran-polyethylene glycol two-phase system and in their behaviour during adsorptions with dextran-coated charcoal, where great loss of transformed complex was observed. The accumulation of complex in the chromatin was found to be unsaturable in the concentration interval studied (0.07–0.25 nM).     

Properties of a DNA-adenylate complex formed in the reaction between mammalian DNA ligase I and DNA containing single-strand breaks.

The major DNA ligase from calf thymus (mammalian DNA ligase I) forms a covalent enzyme-AMP complex on incubation with ATP [S?derh?ll & Lindahl, J. Biol. Chem. 248, 672-675, (1973)]. The reaction of this complex with DNA has now been studied. When the ligase-adenylate complex is incubated at 0 degrees C for short time periods with DNA containing single-strand breaks, a DNA-AMP complex can be isolated from the reaction mixture by isopycnic centrifugation in CsCl. Incubation at pH 6.5 increased the amount of DNA-AMP complex that could be isolated 10-20-fold relative to that obtained at pH 7.4. Under the same conditions, incubation of the ligase-AMP complex with DNA free from single-strand breaks did not lead to detectable DNA-AMP formation. The DNA-AMP complex was resistant to treatment with dilute acid and alkali indicating the presence of a covalent linkage. Further, this complex was sensitive to DNase but resistant to pronase and RNase. Free AMP was released on further incubation of the isolated DNA-AMP complex with thymus DNA ligase I and Mg2+, suggesting that the complex is a reaction intermediate. Degradation of the DNA-AMP complex with several reagent enzymes indicated that the AMP residues were bound at the 5' ends of the single-strand breaks in DNA by pyrophosphate bonds.     

Conditions of formation of the heparin-fibronectin-collagen complex and the effect of plasmin

The formation and composition of the insoluble heparin-fibronectin-collagen complex and its degradation by proteolysis was investigated. At fixed concentrations of the other molecular components of the complex, the maximal rate of complex formation, measured turbidimetrically, was reached at a concentration of 4 microM heparin and 0.9 microM collagen, while the rate of complex formation was linearly related to concentrations of fibronectin as high as 3 microM. Heparin was incorporated into the complex in a saturable manner, and was released in active anticoagulant form by plasmin but not by urokinase. The complex formation was inhibited by 5 mM calcium or 250 mM NaCl as well as by polybrene or spermin. It is suggested that fibronectin binds both heparin and collagen cooperatively to form an insoluble ternary complex of the extracellular matrix.     

Characterization of the parameters affecting covalent binding stoichiometry in binary and ternary complexes of thymidylate synthase

Covalent binding stoichiometries for both the enzyme:5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) binary complex and the enzyme:FdUMP:5,10-methylenetetrahydrofolate (inhibitory ternary) complex at equilibrium were measured by the trichloroacetic acid precipitation assay and shown to be a function of temperature, time, pH, salt concentration, buffer composition and thiol concentration. Incubation at 37 degrees C yielded the maximum covalent binding ratio (mol FdUMP/mol enzyme) for the latter binary (0.7) and ternary (1.7) complexes. In most buffers studied, the maximum covalent binding ratio (1.5-1.7) for the inhibitory ternary complex occurred over a broad pH range (4.5-8.0), while the optimum covalent binding ratio for binary complex was observed at a much narrower region centered between pH 5.5-6.5. In the presence of increasing concentrations of phosphate buffer, the maximum binding ratio for the covalent binary complex decreased from 0.63 in the absence of phosphate to 0.1 in the presence of 225 mM phosphate, while that for the inhibitory ternary complex was unchanged. When a ternary complex was formed with enzyme, FdUMP and (+/-)-tetrahydrofolate in the absence of phosphate, the FdUMP:enzyme covalent binding ratio was 1.8, while in the presence of 75 mM phosphate, the binding ratio was only 1.0. When exogenous thiol was removed by centrifugal column chromatography, the maximum binding stoichiometry of the resulting inhibitory ternary complex was 1.7 and was independent of added thiol over a 2 h incubation period at 37 degrees C. When extensive dialysis at 5 degrees C was used to remove the thiol, the maximum binding stoichiometry of the resulting inhibitory ternary complex was found to be dependent on both the concentration of added thiol and the time of incubation at 37 degrees C and did not exceed a value of 1.0.     

Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex

Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.6 X 10(-4) min-1. The purified receptor sedimented at 8.3 S in high-salt and 9.4 S in low-salt sucrose gradients containing molybdate. A 7.0-nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high-salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90 000-Mr band. Three minor bands with Mr of 78 000, 72 000 and 48 000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high-performance ion-exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90 000-Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA-binding state after molybdate removal followed by warming 30 min at 25 degrees C in presence of 0.2% bovine serum albumin: 50-78% transformation yield could be demonstrated by DNA-cellulose chromatography. Partial transformation could also be obtained at 0 degrees C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.     

Inactivation of mitochondrial 2-oxoglutarate dehydrogenase complex as a result of phospholipid degradation induced by freeze-thawing

The inactivation of 2-oxoglutarate dehydrogenase complex by freeze-thawing was examined along with alterations of membrane phospholipids, in order to elucidate the mechanism of freezing injury in mitochondria.The dehydrogenase complex activity in slowly frozen and thawed mitochondria decreased to 70% as compared to intact mitochondria and further decreased during incubation. This inactivation during incubation was temperature dependent, i.e., at temperatures up to 25°C there was a slight decrease, while at higher temperatures there was a marked decrease in the dehydrogenase complex activity. Simultaneously, there was a significant accumulation of free fatty acids, generated from mitochondrial phospholipids, which inhibited 2-oxoglutarate dehydrogenase and subsequently enzyme complex activity. Oxoglutarate dehydrogenase activity in mitochondria was markedly inhibited by exogenous phospholipase A, and this inhibition was partially prevented with bovine serum albumin. Furthermore, when intrinsic phospholipase A was either inhibited or stimulated, there was a respective decrease or increase in the enzyme complex inactivation.The activity of the purified enzyme complex decreased slightly after slow freezing, but remained constant even when incubated at temperatures up to 32°C. However, the activity of this enzyme complex was markedly reduced when incubated either in the presence of venom phospholipase A or with exogenous fatty acid.The relationship between inactivation of the 2-oxoglutarate dehydrogenase complex, phospholipase A activation and production of free fatty acids in frozen and thawed mitochondria is discussed.     

Reactive oxygen and targeted antioxidant administration in endothelial cell mitochondria

We used fluorescent probes and EPR to study the mechanism(s) underlying reactive oxygen species (ROS) production by endothelial cell mitochondria and the action of mitoquinol, a mitochondria-targeted antioxidant. ROS measured by fluorescence resulted from complex I superoxide released to the matrix and converted to H(2)O(2). In contrast, EPR largely detected superoxide generated at complex III and effluxed outward. ROS fluorescence by mitochondria fueled by the complex II substrate, succinate, was substantial but markedly inhibited by rotenone. Superoxide, detected by EPR, in succinate-fueled mitochondria was not inhibited by rotenone and likely derived from semiquinone formation at complex III. Mitoquinol decreased H(2)O(2) fluorescence by succinate-fueled mitochondria but had little effect on the EPR signal for superoxide. This was not associated with a detectable decrease in membrane potential. Mitoquinol markedly enhanced ROS fluorescence in mitochondria fueled by the complex I substrates, glutamate and malate. Inhibitor studies suggested that this occurred in complex I, at one or more Q binding pockets. The above effects of mitoquinol were determined in mitochondria isolated and subsequently exposed to the targeted antioxidant. However, similar effects were observed in mitochondria after antecedent exposure to mitoquinol/mitoquinone in culture, suggesting that the agent is retained after isolation of the organelles. In conclusion, ROS production in bovine aortic endothelial cell mitochondria results largely from reverse transport to complex I and through the Q cycle in complex III. Mitoquinol blocks ROS from reverse electron transport but increases superoxide production derived from forward transport. These effects likely occur at one or more Q binding sites in complex I.     

Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects

Heparin has been shown to accelerate the inactivation of alpha-thrombin by antithrombin III (AT) by promoting the initial encounter of proteinase and inhibitor in a ternary thrombin-AT-heparin complex. The aim of the present work was to evaluate the relative contributions of an AT conformational change induced by heparin and of a thrombin-heparin interaction to the promotion by heparin of the thrombin-AT interaction in this ternary complex. This was achieved by comparing the ionic and nonionic contributions to the binary and ternary complex interactions involved in ternary complex assembly at pH 7.4, 25 degrees C, and 0.1-0.35 M NaCl. Equilibrium binding and kinetic studies of the binary complex interactions as a function of salt concentration indicated a similar large ionic component for thrombin-heparin and AT-heparin interactions, but a predominantly nonionic contribution to the thrombin-AT interaction. Stopped-flow kinetic studies of ternary complex formation under conditions where heparin was always saturated with AT demonstrated that the ternary complex was assembled primarily from free thrombin and AT-heparin binary complex at all salt concentrations. Moreover, the ternary complex interaction of thrombin with AT bound to heparin exhibited a substantial ionic component similar to that of the thrombin-heparin binary complex interaction. Comparison of the ionic and nonionic components of thrombin binary and ternary complex interactions indicated that: 1) additive contributions of ionic thrombin-heparin and nonionic thrombin-AT binary complex interactions completely accounted for the binding energy of the thrombin ternary complex interaction, and 2) the heparin-induced AT conformational change made a relatively insignificant contribution to this binding energy. The results thus suggest that heparin promotes the encounter of thrombin and AT primarily by approximating the proteinase and inhibitor on the polysaccharide surface. Evidence was further obtained for alternative modes of thrombin binding to the AT-heparin complex, either with or without the active site of the enzyme complexed with AT. This finding is consistent with the ternary complex encounter of thrombin and AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion along the heparin surface to a unique site adjacent to the bound inhibitor.     

Formation of a stable complex of thrombin and the secreted platelet protein glycoprotein G (thrombin-sensitive protein, thrombospondin) by thiol-disulfide exchange

When 125I-labeled thrombin was incubated with washed human platelets or with the supernatant solution of activated platelets, it formed a NaDodSO4-stable complex of apparent mass greater than 450 000 daltons. Formation of the complex was temperature dependent; with 20 nM thrombin incubated with the supernatant solution of ionophore-activated platelets, the initial rate of formation of the stable complex was 1 nM thrombin/min at 37 degrees C, 50 times the rate at 22 degrees C. Thrombin with all free amino groups methylated was still reactive. Active-site-blocked thrombin formed the complex only slowly. The complex that formed with active thrombin was not dissociated by hydroxylamine in urea. Reduction with 2-mercaptoethanol dissociated the complex, and its formation was blocked by the sulfhydryl-blocking agents iodoacetamide and 4,4'-dithiodipyridine. The complex was thus unlike those of thrombin and alpha 2-macroglobulin or antithrombin III, but it had characteristics of a disulfide-linked complex. Of the secreted proteins, albumin and glycoprotein G adhered to an activated thiol-Sepharose column, indicating that they contained free thiol groups. Purified glycoprotein G and thrombin formed a complex similar to the complex formed when thrombin was incubated with the supernatant solution of activated platelets. The purified glycoprotein bound 2.6 mol of radioactive N-ethylmaleimide/mol of protein, indicating three sulfhydryl groups per mole. After reacting with purified glycoprotein G, thrombin developed a new sulfhydryl group. It is concluded that glycoprotein G (thrombin-sensitive protein, thrombospondin) and thrombin form a dissociable complex that leads to a covalent complex by thiol-disulfide exchange of a thiol group on glycoprotein G and a disulfide on thrombin.     

Inactivation of mitochondrial 2-oxoglutarate dehydrogenase complex as a result of phospholipid degradation induced by freeze-thawing.

The inactivation of 2-oxoglutarate dehydrogenase complex by freeze-thawing was examined along with alterations of membrane phospholipids, in order to elucidate the mechanism of freezing injury in mitochondria. The dehydrogenase complex activity in slowly frozen and thawed mitochondria decreased to 70% as compared to intact mitochondria and further decreased during incubation. This inactivation during incubation was temperature dependent, i.e., at temperatures up to 25 degrees C there was a slight decrease, while at higher temperatures there was a marked decrease in the dehydrogenase complex activity. Simultaneously, there was a significant accumulation of free fatty acids, generated from mitochondrial phospholipids, which inhibited 2-oxoglutarate dehydrogenase and subsequently enzyme complex activity. Oxoglutarate dehydrogenase activity in mitochondria was markedly inhibited by exogenous phospholipase A, and this inhibition was partially prevented with bovine serum albumin. Furthermore, when intrinsic phospholipase A was either inhibited or stimulated, there was a respective decrease or increase in the enzyme complex inactivation. The activity of the purified enzyme complex decreased slightly after slow freezing, but remained constant even when incubated at temperatures up to 32 degrees C. However, the activity of this enzyme complex was markedly reduced when incubated either in the presence of venom phospholipase A or with exogenous fatty acid. The relationship between inactivation of the 2-oxoglutarate dehydrogenase complex, phospholipase A activation and production of free fatty acids in frozen and thawed mitochondria is discussed.     

The role of the cellulolytic high molecular mass (HMM) complex of the anaerobic fungus Piromyces sp. strain E2 in the hydrolysis of microcrystalline cellulose

The anaerobic fungus Piromyces sp. strain E2 produces extracellular cellulolytic enzymes present both in a high molecular mass (HMM) complex or as individual proteins. Although the HMM complex was present in the culture fluid during all growth stages, the highest amounts of complex were obtained when cultures were harvested at the end of fungal growth. The complex obtained after gel-filtration chromatography on Sephacryl S-300 HR was found to be the major factor in hydrolysis of cellulose to glucose (sole product, up to 250 mM). The complex was very stable as demonstrated by identical hydrolysis patterns with fresh preparations or preparations stored at 4° C for 2 months. From inhibition experiments with gluconic acid lactone and glucose, it was concluded that the HMM complex must contain at least one glucohydrolase. SDS-PAGE analysis revealed that a partially purified HMM complex was composed of at least ten polypeptides and contained numerous endoglucanases and one β-glucosidase. Received: 10 October 1996 / Accepted: 11 December 1996     

Mechanism of action of θ-amino acids on plasminogen activation and fibrinolysis induced by staphylokinase

Stimulation of Lys-plasminogen (Lys-Pg) and Glu-plasminogen (Glu-Pg) activation under the action of staphylokinase and Glu-Pg activation under the action of preformed plasmin-staphylokinase activator complex (Pm-STA) by low concentrations and inhibition by high concentrations of omega-amino acids (>90-140 mM) were found. Maximal stimulation of the activation was observed at concentrations of L-lysine, 6-aminohexanoic acid (6-AHA), and trans-(4-aminomethyl)cyclohexanecarboxylic acid 8.0, 2.0, and 0.8 mM, respectively. In contrast, the Lys-Pg activation rate by Pm-STA complex sharply decreased when concentrations of omega-amino acids exceeded the above-mentioned values. It was found that formation of Pm-STA complex from a mixture of equimolar concentrations of staphylokinase and Glu-Pg or Lys-Pg is stimulated by low concentrations (maximal at 10 mM) of 6-AHA. Negligible increase in the specific activities of plasmin and Pm-STA complex was detected at higher concentrations of 6-AHA (to maximal at 70 and 50 mM, respectively). Inhibitory effects of omega-amino acids on the rate of fibrinolysis induced by staphylokinase, Pm-STA complex, and plasmin were compared. It was found that inhibition of staphylokinase-induced fibrinolysis by omega-amino acids includes blocking of the reactions of Pm-STA complex formation, plasminogen activation by this complex, and lysis of fibrin by forming plasmin as a result of displacement of plasminogen and plasmin from the fibrin surface. Thus, the slow stage of Pm-STA complex formation plays an important role in the mechanism of action of omega-amino acids on Glu-Pg activation and fibrinolysis induced by staphylokinase. In addition to alpha-->beta change of Glu-Pg conformation, stimulation of Pm-STA complex formation leads to increase in Glu-Pg activation rate in the presence of low concentrations of omega-amino acids. Inhibition of Pm-STA complex formation on fibrin surface by omega-amino acids is responsible for appearance of long lag phases on curves of fibrinolysis induced by staphylokinase.     

Effects of temperature and energy inhibitors on complex formation between Escherichia coli male cells and filamentous phage fd

The effect of temperature and various energy inhibitors on the formation of a complex between Escherichia coli male cells and filamentous phage fd was studied by a novel filtration method. Centrifuged male cells were observed by electron microscopy to have lost the majority of pili and to produce complexes with fd only above 25 degrees C. After preincubation of the cells at 37 degrees C without addition of the phage, nearly half the level of complex formation observed at 37 degrees C was detected at 0 degrees C and fd was at a minimum at about 20 degrees C. Several energy inhibitors and uncouplers drastically reduced complex formation at 37 degrees C, and also at 0 degrees C if the cells were briefly exposed to the reagents at the end of preincubation. Alteration of the cellular ATP concentration, either by shift-down of temperature or by the addition of the reagents, accompanied alteration in the ability of cells to form a complex with fd as well as alteration of the number of pili on the cell surface. In contrast to earlier reports, these results indicate that the complex formation between male cells and filamentous phage does not proceed either when pili disappear from the cell surface because of a decrease in the cellular energy level or when pili are removed by mechanical forces. The results also show that phage fd adsorption itself is not energy-dependent.