Comparative study of the properties of preparations of native and modified collagenase

Collagenase was gradually modified by aldehyde dextran and hyaluronidase. The modification increased enzyme stability and widened pH-optimum of its activity against specific and biological substrates. The modified complex with collagenolytic and hyaluronidase activity accumulated in the lungs of mice after intravenous injection. These results demonstrate its possible use for the treatment of lung disorders.     

Artificial dimers of native actin: Preparation and properties in biological functions

With the aid of tartryl-bis--aminocaprylazide artificial dimers were produced from F actin from rabbit striated muscle. These derivatives will not polymerize by themselves but are able to copolymerize fully with native G actin. By modification of a single side chain per dimer, this copolymerization was completely inhibited. The dimers are able to activate subfragment I ATPase of myosin and bind to DNase I with inactivation of the enzyme in the same manner as native G actin. Within the dimer, one ADP is immobilized and will exchange against ATP extremely slowly. The dimers do not bind to the mushroom toxin phalloidin.     

Comparison of the properties of two kinds of preparations of human blood platelet actin with sarcomeric actin

A new procedure of purification of actin from human blood platelets was used. This method starting from acetone powder of whole platelets gives a much higher yield than the one previously described (actin I) (Landon et al. (1977) Eur. J. Biochem., 81, 571-577). This actin II preparation has the same reduced viscosity as skeletal muscle actin, while the reduced viscosity of actin I preparation is about 1/10 of this value. Moreover actin I has the form of very short filaments as shown by electron microscopy. After an extra step of purification actin I, when polymerized, acquired a high reduced viscosity. We confirmed that platelet and sarcomeric actins are similar in their polymerization properties and their ability to activate muscular myosin. A circular dichroism study showed that the overall conformation of both actins are similar, but the environment of their aromatic chromophores is different.     

Sedimentation properties of native and proteolysed preparations of ox glutamate dehydrogenase.

The concentration-dependent aggregation behaviour of purified ox liver and brain glutamate dehydrogenase preparations was compared with that of commercially-obtained preparations of the liver enzyme, which have recently been shown to have suffered proteolytic cleavage. Although there were no significant differences in these effects, the presence of 3 mM-GTP and 3 mM-NADH had markedly different effects on the two types of preparation. In this situation, at higher protein concentrations the commercially obtained preparations existed in a higher degree of aggregation than those which had not suffered proteolysis. Studies of the effects of GTP and NADH concentrations on the sedimentation coefficients at a fixed enzyme concentration suggested these effects to be largely due to differences in the affinities of the two preparations for nucleotides.     

Immunological properties of the preparations of the anti-anthrax antigen]

The results of examination of immunological properties of the preparations of anthrax protective antigen on laboratory animals (guinea pigs) confirmed the efficacy of using the lactic-peptone medium for obtaining the anthrax protective antigen. Incubation for 18 hours at 37 degrees C of the strain-producer (STI-1) and a double immunization scheme with the antigen obtained proved to be the most rational conditions for inducing the immunological response in the vaccinated laboratory animals. Three fractions of the anthrax protective antigen obtained possessed weaker immunobiological properties than the whole preparation of this antigen.     

Ox glutamate dehydrogenase. Comparison of the kinetic properties of native and proteolysed preparations.

Kinetic constants were determined for commercially available samples of ox liver glutamate dehydrogenase, which had previously been shown to have suffered limited proteolysis during preparation, with a range of substrates and effectors. These were compared with the values obtained with enzyme preparations purified in such a way as to prevent this proteolysis from occurring [McCarthy, Walker & Tipton (1980) Biochem. J. 191, 605-611]. The Km values and maximum velocities determined with different substrates revealed little difference between the two preparations although the proteolysed enzyme had lower Km values for NH4+ and glutamate when the activities were determined with NADPH and NADP+ respectively. This preparation was more sensitive to inhibition by Cl- ions but less sensitive to inhibition by high concentrations of the substrate NADH. The two preparations also differed in their sensitivities to allosteric effectors, with the proteolysed enzyme being the less sensitive to inhibition by GTP. At high concentrations of NADH, this preparation was also more sensitive to activation by ADP and ATP.     

Structural-dynamic and functional properties of native and modified streptokinase]

The process of streptokinase modification by a polymer and the dynamics of protein molecules and the polymer in a protein-polymer (dialdehyde dextran, DAD) conjugate were studied with the aid of luminescent methods. Conclusions were drawn on the structure of the protein-DAD conjugate and the selection of optimum conditions for the preparation of modified streptokinase. It is shown that modified streptokinase is more stable to the action of denaturating agents. The interaction between streptokinase and plasminogen activated by it and changes in the streptokinase-plasminogen complex for modified streptokinase were detected.     

Adjuvant and protective properties of native and recombinant Bordetella pertussis adenylate cyclase toxin preparations in mice

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin which is synthesised from the cyaA gene as an inactive protoxin that is post-translationally activated by the product of the cyaC gene. Purified active and inactive CyaA proteins were prepared from B. pertussis or from recombinant Escherichia coli expressing both cyaA and cyaC genes or the cyaA gene alone. respectively. In addition, a hybrid toxin (Hyb2) in which an internal region of CyaA had been replaced with the analogous region from the leukotoxin (LktA) of Pasteurella haemolytica, and which had low cell-invasive activity, was also prepared from E. coli expressing the cyaC gene. The CyaA preparations showed no evidence of toxicity in a mouse weight-gain test. Active toxin preparations were protective in mice against intranasal challenge with wild-type B. pertussis, as evidenced by lung:body weight ratios and bacterial numbers in the lungs, which were comparable to those in mice given whole-cell DPT vaccine. Hyb2 was not as protective as active CyaA and inactive CyaA preparations were not protective. Active CyaA, when co-administered with ovalbumin (OA), had a marked adjuvant effect on the anti-OA IgG antibody response which was not as apparent with inactive CyaA preparations. Similarly, active CyaA stimulated a greater anti-CyaA response than the inactive form.     

Protective properties of Vi-antigen preparations as dependent on their adhesin content

Adhesins contained in the preparation of Vi-antigen have been found to enhance its immunogenic and protective properties. In the preparations of Vi-antigen obtained from Salmonella typhi and Citrobacter freundii the presence of two antigenic determinants has been revealed. One of them is associated with the Vi-receptor and the other determinant, with adhesin. Both determinants take part in the protection of mice from Salmonella infection.     

Immunobiological properties and therapeutic effectiveness of preparations from Klebsiella bacteriophages]

The purified preparations of Klebsiella bacteriophages, viz. the monovalent preparation of K. pneumoniae bacteriophage and the polyvalent bacteriophage preparation for the treatment of infections caused by K. ozaenae, K. rhinoscleromatis scleromatis and K. pneumoniae sensu lato, have been obtained. The bacteriophage preparations have proved to be nontoxic and safe for laboratory animals after the intraperitoneal injection of these preparations followed by the pathomorphological study of the internal organs of the animals. The clinical study of the newly developed bacteriophage preparations in the course of the treatment of purulent inflammatory diseases in 109 patients has revealed that the preparations are not reactogenic and exhibit sufficient effectiveness in the therapy of ozena, rhinoscleroma and Klebsiella infections with different localization of the infectious process.     

Identification of a factor in conventional muscle actin preparations which inhibits actin filament self-association

Gel filtration of depolymerized conventionally purified muscle actin separates from the actin monomers a fraction of minor contaminants with a Stokes' radius of 4.7 nm which has the ability to block actin filament network formation. On the basis of heat and trypsin sensitivity, this inhibitory activity appears to be a protein. The inhibitory activity binds to actin filaments and reduces their low shear viscosity by up to 99% in a concentration dependent fashion while reducing polymerization to only a minor extent.     

Fluorescence of myosin and its subunits in concentrated salt solutions]

The parameters of fluorescence spectra of myosin and its subunits in Tris-HCl-buffer (pH 7.2) were studied. Analysis of the experimental results of myosin fluorescence quenching with I-ions and the quantum yield of the fluorescence at the excitation wavelength 296 nm shows that the greater part of the tryptophan residues (21 out of 28) is located in the hydrophylic environment. Concentrated solutions of NaCl and KCl do not affect myosin fluorescence, while LiCl, which changes the quaternary structure of the protein, brings about a change in the parameters of the myosin fluorescence spectra. This may be linked with structural changes accompanying the dissociation of the ligh subunits of myosin in the presence of LiCl.     

Fluorescence properties of native and photooxidised proteinase K: the X-ray model in the region of the two tryptophans.

The fluorescence properties of proteinase K are described and related to the X-ray model refined at 1.48 A resolution. Upon excitation of proteinase K at 295 nm the fluorescence is determined by the two tryptophan residues, Trp-8 and Trp-212. The tryptophans are partly buried just below the surface of the molecule. Neither Trp is in a highly hydrophobic environment, suggesting that this cannot be the explanation for the fluorescence at 330 nm: formation of exiplexes with adjacent peptide bonds would seem to be the more likely cause. Trp-8 is located in a 'cavity', close to an internal cluster of water molecules. The contribution of Trp-8 to the total indole emission is 60% and that of Trp-212 is 40%. The tryptophan fluorescence quantum yield is constant in the pH range 3-9. The fluorescence spectrum resulting from the simultaneous excitation of the tyrosyl and tryptophyl residues at 280 nm is dominated by the indole fluorophores: 61% of the light absorbed by the tyrosyl side chains is transferred to the two indole rings. Iodide and caesium are not efficient quenchers of the proteinase K tryptophan fluorescence, which is explained by restricted access of the ions to the somewhat buried Trp side chains and by electrostatic repulsion of caesium ions. Acrylamide quenching proceeds via both a dynamic and a static process and the data show homogeneity of the indole fluorescence arising from fluorophores in similar environments. The activation energy for the thermal deactivation of the excited tryptophans is 54 kJ mol-1. This value is substantially higher than those found for other proteinases from microorganisms and arises from the thermostability of proteinase K. Photooxidation of proteinase K in the presence of proflavine follows the kinetics of a first order reaction. The two tryptophans differ in their photoreactivity, Trp-212 being considerably more reactive.