Function of the activated fourth component of complement (C4b) in activation of C2.
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Complement Activating Enzymes↗
Biomedical subjects
B Hinsch
Publications and source records attributed to B Hinsch.
Reaction kinetics of some important site-specific endonucleases.
Reaction kinetics of the site-specific endonucleases BamHI, BgIII, C1aI, EcoRI, HpaII, PstI, SaII, SmaI, and XorII were investigated employing some frequently used substrates. Six of these enzymes could be analyzed under steady-state conditions. Kinetic data were obtained from progress curves applying an integrated Michaelis-Menten equation. KM ranged from 4 x 10(-9) M to 4 x 10(-11) M. Activities also spanned two orders of magnitude. In the case of C1aI the analysis of the pre-steady-state kinetics ("burst reaction") allowed the assessment of several rate constants. The rate-limiting step is the very slow dissociation of the enzyme-product complex (0.22 min(-1)). This complex is formed from the enzyme-bound nicked intermediate at a rate of 1.7 min(-1). The introduction of the first cut is again faster by a factor of about 6. SmaI and XorII resembled C1aI in their kinetics. The burst reaction can be used for the easy and unambiguous determination of molar concentrations of site-specific endonucleases in any preparation, which is free of non-specific DNases.
DNA Restriction Enzymes↗
Binding of non-substrate nucleotides to a restriction endonuclease: a model for the interaction of bam HI with its recognition sequence.
The kinetic constants of the site-specific endonuclease Bam HI for various substrates were determined and binding of non-substrate nucleotides to the enzyme was studied. Agarose gel assays in combination with an integrated Michaelis-Menten equation were used for the evaluation of data. The turnover number was 2.2 min-1 at 37 degrees C with pJC80 DNA as the substrate. It depends on the conformation and base composition of the substrate. Michaelis constants also depend on substrate conformation. Non-substrate polynucleotides were found to inhibit Bam competitively with KI ranging from 10(-6) to > 10(-3) M depending on base composition, base pairing, and helix conformation. Dinucleotides showed sequence-specific, competitive inhibition with KIs ranging from 10(-5) to > 10(-3) M. Mononucleotides and -nucleosides acted noncompetitively. Binding was influenced by the extent of phosphorylation, but not by the nature of the base. KIs varied between 10(-3) and 10(-2) M. The results are discussed with respect to the recognition requirements of Bam HI.
Bacteriophage lambda↗
Physical and kinetic properties of the site specific endonuclease Bam HI from Bacillus amylolique-faciens.
The site specific endonuclease Bam HI which is composed of subunits of a molecular weight of 22 000 [1] can aggregate to complexes of a molecular weight of 360 000. It is an acidic protein with an isoelectric point at pH 5.3. Optimal activity is reached at 13 mM MgCl2. A very simple method is presented to determine kinetic constants of restriction enzymes directly from agarose gel photographs without any further equipment applying the integrated Michaelis Menten equation. With pJC 80 DNA as a substrate KM was found to be 3.6 10(-10) M. The method can be used to redefine the unit activity of site specific endonucleases unambigously.
Bacillus↗
Comparison of Naja n. naja and Naja h. haje cobra-venom factors: correlation between binding affinity for the fifth component of complement and mediation of its cleavage.
Two cobra-venom factors, one from Naja n. naja (CVFn), the other from Naja h. haje venom (CVFh), have been purified and compared, functionally and structurally. Both factors interacted with human factors B and DS to form a potent C3 convertase, CVFBb. However, while the convertase formed with CVFn did also efficiently cleave C5, CVFhBb had very little C5-cleaving potency only, in particular when human C5 was used as substrate. Studies with agarose-linked CVF preparations indicated that CVFh has only low binding affinity for C5gp and C5hu whereas CVFn binds to both C5 species with much higher affinity. Since C5-binding (to CVF or to C3b) is a prerequisite for its cleavage by C3/C5 convertases, the difference in binding potency explains the different C5-cleaving activity of the two CVF preparations. When a ligand for C5, surface-fixed C3b, is present, CVFhBb is also capable of cleaving C5. The difference in activities of CVFn and CVFh is reflected in their different potency to interfere with immune haemolysis and in causing indirect lysis by their complexes with activated factor B. By gel chromatography of the CVF preparations in C5-containing medium, a stoichiometric complex CVFn-C5 (1 + 1) could be demonstrated. An analogous complex of C5 was neither found with CVFh, nor with C3hu or soluble C3bhu. Structural differences between CVFn and CVFh were revealed by immunodiffusion analysis and by polyacrylamide-gel electrophoresis in presence of SDS. The data available so far provide, however, no clear information about the structure of the C5 binding site.
Animals↗
Multiple effects of a diamidine (propamidine) on complement activation.
Propamidine, one of the diamidines used against infections with babesiae has inhibitory and enhancing effects on complement activation as assessed by immune haemolysis of sensitized sheep red cells. Utilization of C1 is powerfully, that of C3 weakly improved by propamidine while activation and/or fixation of C4, C5 and to a lesser degree of C8 and C9 are inhibited. At low concentrations of propamidine (less than 2 mM) the enhancing effects, at higher concentrations the inhibitory effects predominate. Inhibition is produced, in some cases certainly, in others likely, by interference of propamidine with binding properties of complement components. None of the complement enzymes, C1s, C42 or C3bBb was inhibited in its hydrolytic activity. The possible significance of propamidine actions is discussed.
Amidines↗
Interference of propamidine with binding of the fifth component of complement to surface-fixed C3b, and with C5 activation.
The effects of propamidine and their dose dependency, on utilization of the third and fifth complement components in immune haemolysis have been compared. While C3 utilization is not disturbed that of C5 is markedly inhibited by propamidine in concentrations as low as 0.5 mM. Both, binding of C5 to surface-fixed C3b and cleavage of C5 by convertases C42 and C3bBb, are also inhibited in the presence of propamidine. Since neither C3 cleavage by these enzymes nor even C5 cleavage by the cobra venom factor-supported convertase CFVBb is significantly reduced a general convertase-inhibiting effect of propamidine is ruled out. Rather the effect on utilization of C5 is the result of interference with binding of C5 is the result of interference with binding of C5 to C3b and hence impairment of its accessibility to the convertases. These findings thus further support the role of surface fixed C3b in C5 activation proposed earlier.
Amidines↗
Influence of viscosity of enzymatic reactions studied with glucoamylase of Aspergillus niger.
Viscosity can be interpreted in terms of transport of momentum and, therefore, it should influence the kinetics of enzyme reactions. A theory, developed by Somogyi and Damjanovich ((1975) J. Theor. Biol. 51, 393--401), is based on this idea. Transport of momentum must always be accompanied by the transport of mass and this second influence of viscosity is a limiting factor for fast reactions in the liquid phase. A third aspect is, that the chemical potentials of the components of viscous solutions are altered. This paper reports experiments concerning the influence of the viscosigens (compounds that increase the viscosity of solvents), alginate, sucrose, and maltose on the kinetic behaviour of glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3). The observed invariance of V and the decrease of Km are explained by the increase of chemical potentials and restriction of momentum transport.
Aspergillus niger↗