Biomedical subjects

H Gruler

Publications and source records attributed to H Gruler.

43 records · Page 3Linked to original sources

PubMedDate not supplied

Automatic control and directed cell movement. Novel approach for understanding chemotaxis, galvanotaxis, galvanotropism.

It is shown that chemotaxis, galvanotaxis, galvanotropism, etc. are functions of cells having a goal-seeking system. Even when the involved physicochemical signals are unknown, the cellular system can be treated phenomenologically like an automatic controller having a closed-loop feedback system. The model is verified by means of galvanotaxis and chemotaxis data of human granulocytes. The galvanotaxis and chemotaxis coefficient quantifying the cellular sensibility can be predicted from the coefficient which characterizes the deterministic part of the signal transduction/response system of the cell divided by the coefficient which characterizes the noise strength in the cellular signal transduction/response system. The model is not restricted to directed movement of granulocytes. It is very general and can be applied to any cell type for directed phenomena like chemotaxis, galvanotaxis, phototaxis, magnetotaxis, directed growth, etc. The virus-disturbed directed migration of granulocytes is discussed and it is shown that the virus alters the deterministic part of the cellular controller.

Animals↗

PubMedDate not supplied

Light-induced activation and synchronization of the cytochrome P-450 dependent monooxygenase system.

The light-induced enhancement of the 7-ethoxycoumarin-O-deethylase activity was measured in a reconstituted system, consisting of the enzyme P-450PB-B and the NADPH-cytochrome P-450 reductase. The relative increase of the activity was about 15%. It is shown that the product release process is accelerated by light. The phases of the catalytic cycle of 2 x 10(12) protein complexes were locked by periodic application of light pulses (0.1 s duration, 1.32 s repetition time, and 390-470 nm, 0.27 joule/nmol P-450). More than 80% of the active reconstituted enzyme complexes (= "molecular machines") worked in phase after a few light pulses. The phase relation continued even after switching off the light pulses. The catalytic cycle time was 1.54 s, giving a turnover number of 39 min-1. The turnover number, as determined from the enzyme activity under optimum conditions, was 39 min-1. Due to the dissociation constant of the P-450PB-B:NADPH-P-450 reductase complexes [3] only 24% of the proteins were in the active (= working) state under the conditions used. The lifetime of this complexes is larger than 6 s since more than 4 cycles of the free running enzyme can be observed. This is the first report, that all catalytic active complexes in the test tube can be synchronized by an external light source, if the right repetition time of the pulses is chosen, so that all these "molecular machines" work in phase.

7-Alkoxycoumarin O-Dealkylase↗

PubMedDate not supplied

Directed cell movement with steric exclusion.

The space-dependent density of cells is evaluated for the following situation: (i) The cells are forced to make a directed movement (ii) the space for the cellular migration is restricted. The steady state distribution density is obtained when the drift current density equals the diffusion current density. The analogy to the Boltzmann statistics is shown. In a further step the cellular volume is introduced. For this case the density distribution is described in analogy to the Fermi statistics. The necrotactic response of granulocytes is used to verify the model.

Animals↗

PubMedDate not supplied

Complexation of membrane-bound enzyme systems.

The effect of changes in the N-terminal membrane-binding domain of cytochrome P450 forms and NADPH-cytochrome P450 reductase types on the cytochrome P450-dependent monooxygenase activities, has been examined. The nifedipine oxidase activity of two human P450 forms (CYP3A4, CYP3A4NF14) which differ only in their primary structure by ten amino acid residues in the N-terminal membrane-binding domain, yields nearly the same catalytic cycle time tau =2.65 +/- 0.15 s, due to their identical cytosolic catalytic protein structure. In contrast, the complex formation process ([P450]+[reductase] <--> [complex]) described by the dissociation constant KD, at high substrate concentration ([S]>>KS) and low product concentration ([P]<<KP) is determined to be KD/[P4501]o = 0.3 and 2.0, respectively. These values reflect large differences in the affinity of both P450 forms for the same type of reductase which is only due to their modified membrane-binding domains. In the present work, it has been shown for the first time, that the membrane-binding domain of cytochrome P450 enzymes determines the complexation process of the binary P450:reductase system. Furthermore, the nifedipine oxidase activity of the human CYP3A4 form reconstituted with two different types of reductase from human and rabbit also has the same catalytic cycle time tau = 2.65 +/- 0.15 s. This result is based on the similarity of the primary structure of the cytosolic catalytic domain of both reductase types. However, the complex was formed with different dissociation constants of KD/[P450]o = 0.3 and 4.7, respectively. This different affinity of both reductase types to the same P450 form is interpreted as a consequence of the substantial alteration of the amino acids in the N-terminal primary structure of their membrane-binding domains. 7-Ethoxycoumarin O-deethylase activity of two rat P450 forms (CYP2B1 and CYP1A1) were reconstituted with the same rat reductase. The catalytic cycle time for each P450 form is tau = 1.8 and 0.6 s, respectively. Correspondingly, the complex formation process controlled by the dissociation constant KD has changed from KD/[P450]o = 2.3 to 1.7, respectively. This is because both forms differ in their cytosolic as well as in their membrane-binding domains.

Animals↗

PubMedDate not supplied

Enzyme activity of the cytochrome P-450 monooxygenase system in the presence of single chain lipid molecules.

The influence of single chain lipids on the 7-ethoxycoumarin O-deethyase activity of the reconstituted binary protein complex of isolated cytochrome P450 and NADPH-cytochrome P450 reductase has been examined. The enzyme activity of this binary enzyme complex has been shown to be influenced by (i) altering the complexation process of both proteins, (ii) by altering the catalytic cycle time of the active binary protein complex and (iii) by altering the fraction of substrate molecules at the catalytic center of the enzyme. Competitive inhibition was measured for all single chain molecules. The following dissociation coefficients of substrate and lipids used for the catalytic center of the protein were obtained: 110 microM 7-ethoxycoumarin (substrate), 1.1 microM MOG (1-monooleoyl-rac-glycerol), 0.3 microM SPH (D-sphingosine), 1.5 microM OA (oleic acid), 3.0 microM LPC (L-alpha-lysophosphatidyl-choline), 15.5 microM MSG (1-monostearoyl-rac-glycerol), 9.5 microM AA (arachidonic acid), 9.0 microM PaCar (palmitoyl-L-carnitine), 3.5 microM MPG (2-monopalmitoyl-glycerol), 1.5 microM LPI (L-alpha-lysophosphatidyl-inositol), 50 microM LA (lauric acid), 60 microM MA (myristic acid), 85 microM PA (palmitic acid), >100 microM SA (stearic acid). Only competitive inhibition with the substrate molecule 7-ethoxycoumarin was observed for the single chain lipids LA, MA, PA, SPH, SA, and OA. Non-competitive effects were observed for MPG (-0.03 microM(-1)), PaCar (-0.02 microM(-1)), MSG (-0.023 microM(-1)), LPC (-0.03 microM(-1)), AA (-0.03 microM(-1)), and MOG (+0.04 microM(-1)). The negative sign indicates that the cycle time of the working binary complex is enlarged. The positive sign indicates that the formation of the binary complex is enhanced by MOG.

7-Alkoxycoumarin O-Dealkylase↗

PubMedDate not supplied

The activation of the cytochrome P-450 dependent monooxygenase system by light.

The cytochrome P-450 dependent monooxygenase system of rat liver microsomes is investigated by light dosimetry (action spectroscopy). The scoparone-O-demethylation activity is enhanced by light and depends on the wavelength of the irradiating light. The relative increase of the activity (about 15%) by the irradiating light (approximately 0.5 mW/cm2) is maximal at a wavelength of 400 nm. The light induced enhancement of the 7-ethoxycoumarin-O-deethylase activity was measured in reconstituted systems, consisting of one of two P-450 enzymes (P-450 beta NF-B and P-450 PB-B) with the NADPH-cytochrome P-450 reductase. The action spectrum of the reconstituted P-450 beta NF-B:NADPH-P-450 reductase complex shows a maximum between 420 and 440 nm. The relative increase of the activity induced by light of 420 nm was 7.3% and 9% for the reconstituted systems of P-450 beta NF-B and P-450 PB-B, respectively. The results are discussed in analogy to the classic experiments of Warburg with its blocked CO-enzyme-complex. The results can best be explained by the assumption that the light induced enhancement of the enzyme activity is due to an excitement of those intermediate states of the P-450 catalytic cycle (ferric and ferrous state of the heme iron) which are rate limiting.

Animals↗

PubMedDate not supplied

Directed cell movement in pulsed electric fields.

Human granulocytes exposed to pulsed electric guiding fields were investigated. The trajectories were determined from digitized pictures (phase contrast). The basic results are: (i) No directed response was induced by pulsed electric guiding fields having a zero averaged field. (ii) A directed response was induced by pulsed electric guiding fields having a non-zero averaged field. (iii) The directed response was enhanced for pulse sequences having a repetition time of 8 s. (iv) The lag-time between signal recognition and cellular response was 8-10 s. The results are discussed in the framework of a self-ignition model.

Capillary Action↗