Csaba Bagyinka

Immune-complex-induced transglut aminase activation: Its role in the Fc-receptor-mediated transmembrane effect on peritoneal macrophages

Abstract The binding of soluble immune complexes (IC) or haemolysin-sensitized erythocytes (EA) to the Fc receptor of rat peritoneal macrophages was followed by a rapid increase of macrophage transglutaminase activity measured in cell homogenates and a time-dependent incorporation of 14 C-methylamine into proteins of the intact cells. Methylamine, a competitive substrate inhibitor of transglutaminase could inhibit EA rosette formation as well as the soluble IC- or EA-induced lipid reordering of the plasma membrane of macrophages. Cytochalasin B (CB) which prevents EA rosette formation as well as IC-induced lipid reordering did not affect the stimulation of transglutaminase activity by IC. The possible relation of transglutaminase activation, lipid reordering and the contractile system to each other in the IC (multivalent ligand)-induced Fc receptor redistribution is discussed.

Unconsidered factors affecting hydrogenase activity measurement

The effects of sample geometry and enzyme concentration on the H2-evolving activity of hydrogenase from Thiocapsa roseopersicina was measured. The specific activity increased linearly with increasing interface area between the liquid and the gas phase. Enzyme concentration was varied over four orders of magnitude and within this range the apparent specific activity depended on hydrogenase concentration. The experimental findings have been interpreted by a mathematical model involving competing H2 consumption reactions. The observed phenomena interfere with the widely used hydrogenase assay so that most of the previously published specific activity values are underestimated and should be corrected. The systematic error due to these hitherto unspecified parameters can easily exceed 10 000%; therefore, a thorough standardization of the assay procedure is necessary in order to make the data from various laboratories comparable.

Distribution and orientation of hydrogenase in various photosynthetic bacteria

All of the tested photosynthetic bacteria possessed hydrogenase activity. Cell membranes of representatives of the Chromatiaceae, Rhodospirillaceae, and Chlorobiaceae families were found to be impermeable to the oxidized redox dyes methyl viologen and benzyl viologen, whereas the reduced forms could easily penetrate the membranes. This permeability difference made possible the localization of the hydrogenase enzyme. Members of the Chromatiaceae and the Rhodospirillaceae contained a predominantly or exclusively membranebound enzyme. In contrast, the majority of hydrogenase activity was in the cytoplasm ofChlorobium limicola f. thiosulfatophilum. Hydrogenase, or at least its active center, was oriented toward the outer surface of the cell membrane in purple bacteria.

Location and quantification of metal ions in enzymes combining polyacrylamide gel electrophoresis and particle-induced X-ray emission.

A method is presented to identify and determine the relative amounts of protein-bound metal ionsin situ. Proteins or their subunits are directly scanned by a collimated proton beam of 3 MeV energy, and the characteristic X-rays produced are detected. The determination of Fe content of an iron-sulfur protein (HiPiP), as well as the Fe and Ni analysis of the hydrogenese fromThiocapsa roseopersina, have shown the feasibility of this technique.

Proteolytic resistance and its utilization in purification of hydrogenase from Thiocapsa roseopersicina

Abstract The effect of various proteinases on the hydrogenase from Thiocapsa roseopersicina has been studied by activity measurements accompanied with electrophoretic separation of the protein components. The native enzyme cannot be digested by the following proteinases: thermolysin, subtilisin, trypsin, papain, pepsin, proteinase-K, pronase E, Bacillus subtilis proteinase, Staphylococcus aureus strain V8 proteinase under standard conditions for optimal proteolytic cleavage. Pretreatments which commonly make resistant proteins accessible to proteolysis have been found ineffective in destabilizing the hydrogenase. The native hydrogenase is readily cleavable and inactivated by CNBr. Also under conditions which irreversibly inactivate the hydrogenase (boiling for 10 min) the enzyme loses its active conformation and becomes susceptible to proteolysis. The protein, as isolated, apparently contains no protective lipids or carbohydrates. The results suggest that a stable hydrogenase apoprotein structure is responsible for the proteolytic resistance. The remarkable stability of this enzyme offers new ways for its purification. A rapid and effective procedure, suitable for scale-up, has been established.

Protein structural changes associated with active and inactive states of hydrogenase from Thiocapsa roseopersicina

Electrophoretic and isoelectrofocusing behavior of the hydrogenase from Thiocapsa roseopersicina under various conditions revealed remarkable properties of this enzyme: there are two active forms which differ in their molecular masses as well as in oxygen sensitivity; the apparent molecular masses of the active hydrogenase forms (90 and 49 kDa) differ considerably from those in the inactive state (64, 34, and 15 kDa); the active forms and some of the inactivated ones can be transformed into each other reversibly; urea can unfold the 64 and 34 kDa proteins but not the 49 kDa form at room temperature; the pI of these proteins are different in the presence of urea. The results suggest large rearrangements in the hydrogenase protein structure which are associated with the enzymatically active and inactive states. It is concluded that reversible formation of disulfide bonds cannot be the major cause for maintaining the enzyme conformation. Strong hydrophobic interactions are suggested to be primarily responsible for the structural stability and for the rearrangements.

Concentration-Dependent Front Velocity of the Autocatalytic Hydrogenase Reaction

HynSL hydrogenase from Thiocapsa roseopersicina was applied to catalyze the oxidation of molecular hydrogen in a new, improved, thin-layer reaction chamber. Investigation of the nature of this catalysis via the development of reduced benzyl viologen showed clearly the typical characteristics of an autocatalytic reaction: propagation of a reaction front originating from a single point, with a constant velocity of front propagation. The dependence of the reaction velocity on enzyme concentration was a power function with a positive enzyme concentration threshold, with an exponent of 0.4 +/- 0.05. This indicates that the autocatalyst is an enzyme form. The front velocity decreased on increase of the electron acceptor concentration, as a sign that the autocatalyst interacts directly with the final electron acceptor. Overall, it may be concluded that the autocatalyst is an enzyme form in which [FeS]distal is reduced. Model calculations corroborate this. Because the reduction of all [FeS] clusters would be possible in a nonautocatalytic reaction, we hypothesize a small conformational change in the enzyme, catalyzed by the autocatalyst, which removes a block in the electron flow in either [NiFe] --> [FeS]proximal or the [FeS]proximal --> [FeS]distal reaction step, or removes a block of the penetration of gaseous hydrogen from the surface to the [NiFe] cluster.

Cross-crystallization method used for the crystallization and preliminary diffraction analysis of a novel di-haem cytochrome c4.

The newly discovered di-haem cytochrome c4 from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is the first cytochrome c4 to be crystallized from an anaerobic organism. It was crystallized using the addition of metal-ion salts to the standard vapour-diffusion method. Coloured well shaped three-dimensional crystals with dimensions of approximately 0.6 x 0.05 x 0.02 mm grew within 3-4 d at pH 5 and diffracted to 1.72 angstroms without radiation damage. Cytochrome c4 crystallized in space group P4(1)2(1)2 as a primitive tetragonal system with unit-cell parameters a = b = 75.29, c = 37.12 angstroms, alpha = beta = gamma = 90 degrees.

Effect of enzyme concentration on apparent specific activity of hydrogenase

The effect of enzyme concentration on the H2-uptake and H2-evolving activities of the reversible hydrogenase from Thiocapsa roseopersicina was examined. In the activity range assayed by a spectrophotometric technique the apparent H2-uptake specific activity varied greatly with hydrogenase concentration. Study of H2-evolving activity measured by the H2 electrode method and compared with a gas chromatographic assay also indicated that specific activity was highly dependent on enzyme concentration. The results indicate that the widely applied hydrogenase assays give systematically erroneous specific activity values. These assays should be used only for relative measurements and the hydrogenase concentration in the reaction mixture should be kept constant. To make the data from various laboratories comparable the assay parameters should be standardized.

The autocatalytic step is an integral part of the hydrogenase cycle

We earlier proved the involvement of an autocatalytic step in the oxidation of H(2) by HynSL hydrogenase from Thiocapsa roseopersicina, and demonstrated that two enzyme forms interact in this step. Using a modified thin-layer reaction chamber which permits quantitative analysis of the concentration of the reaction product (reduced benzyl viologen) in the reaction volume during the oxidation of H(2), we now show that the steady-state concentration of the product displays a strong enzyme concentration dependence. This experimental fact can be explained only if the previously detected autocatalytic step occurs inside the catalytic enzyme-cycle and not in the enzyme activation process. Consequently, both interacting enzyme forms should participate in the catalytic cycle of the enzyme. As far as we are aware, this is the first experimental observation of such a phenomenon resulting in an apparent inhibition of the enzyme. It is additionally concluded that the interaction of the two enzyme forms should result in a conformational change in the enzyme-substrate form. This scheme is very similar to that of prion reactions. Since merely a few molecules are involved at some point of the reaction, this process is entirely stochastic in nature. We have therefore developed a stochastic calculation method, calculations with which lent support to the conclusion drawn from the experiment.