Südamelihase rakkude struktuuri olulisus rakuhingamise regulatsioonis

Viimastel aastatel on järjest selgemaks saanud seos raku energeetilise ainevahetuse ja südamehaiguste vahel, mistõttu on oluline uurida seda mõjutavaid tegureid. Töös uuriti südamelihase rakkude mitokondriaalse hingamise regulatsiooni väga erineva rakustruktuuriga preparaatides: 1) permeabiliseeritud kardiomüotsüütides, kus mitokondrid on regulaarselt organiseeritud; 2) südamelihase fenotüübiga sarnastes kontraheeruvates HL-1 (B HL-1) rakkudes ja 3) HL-1 mittekontraheeruvates (NB HL-1) rakkudes. Nende preparaatide vahel esines suur erinevus mitokondriaalse hingamise regulatsioonis. Selline tulemus näitab raku struktuuri ja funktsiooni vaheliste seoste tähtsust südamelihase rakkudes ning võimaldab paremini mõista protsesse nii terves kui ka patoloogilises südamelihases. Eesti Arst 2008; 87(1):19−2

Chemical Modification of the Porcine Pancreatic Lipase

In this low-scope review we summarized the results which typically have not been publishedyet as full papers. To our opinion these data could be valuable for the discussion of the recently published [1] three-dimensional structure of human pancreatic lipase optained by the X-ray crystallography studies. The effect of the modification of several residues in porcine pancreatic lipase has been studied primarily in terms of changes in the steady-state kinetics binding and rate constants which could be determined in the assay system containing lipase (L), variable amounts ofcolipase (C), micellar NaTDC! (bs) and excess of tributyrylglycerol emulsion (S). The approach is based on the assumption that the enzyme does not have anyactivity in the presence of micellar NaTDC and absence ofcolipase due to the displacementof the enzyme from the substrate-water interphase [2, 3], and hasbeen first used by Rathelot etal. [4, 5]. This assumption is correct provided that traces of colipase have been removed from lipase preparations (on a Sephadex LH-60 column [6]). Formally, since the observed dissociation constant of the lipase/colipase interfacial complex, K,””, is typically less, or comparable, with the total enzyme concentration. In conditions K,*? >> [L], Eqn. 2 reduced to a common hyperbolic (Michaelis-type) relationship

Substrate Specifity Of Porcine Pancreatic Lipase Studied In Terms Of The Steady-State Kinetics Binding And Rate Constants

The steady-state kinetics binding constants have been rarely determined for emulsified lipase substrates since the apparent Michaelis constant has the dimension of the emulsion surface area in this case [1,2] which typically could not be determined precisely. On the other hand, only a combination of binding and rate parameters could be determined in experiments with substrate monolayers [3,4], and the range of suitable substratesis strictly limited in these experiments due to requirements on the stability of monolayers, productsolubility, etc. [4]. In this paper we review data on the substrate specificity of porcine pancreatic lipase on emulsified triacylglycerolsubstrates studied in terms of the steady-state binding and rate constants in the assay system lipase/colipase/micellar NaTDC'/-triacylglycerol emulsion’

In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography.

International audienceA method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest

Computational Study of Copper-Free Sonogashira Cross-Coupling Reaction

The copper-free Sonogashira cross-coupling reaction consisting of oxidative addition, <i>cis–trans</i> isomerization, deprotonation, and reductive elimination was computationally modeled using the DFT B97D/cc-pVDZ method for reaction between phenyl bromide and phenylacetylene. Tetrakis(triphenylphosphano)palladium was used as a catalyst and <i>sec</i>-butylamine as a base. The reaction mechanism was studied in dichloromethane solution. Oxidative addition proceeds through the biligated pathway, and the catalytically active palladium species is Pd(PPh₃)₃. Amines, present in the reaction mixture, can inhibit oxidative addition by coordinating to Pd(PPh₃)₃

Studies of mitochondrial respiration in muscle cells in situ: use and misuse of experimental evidence in mathematical modelling.

International audienceApplications of permeabilized cell and skinned fiber techniques in combination with methods of mathematical modelling for studies of mitochondrial function in the cell are critically evaluated. Mathematical models may be useful tools for explaining biological phenomena, but only if they are selected by fitting the computing results with real experimental data. Confocal microscopy has been used in experiments with permeabilized cardiomyocytes and myocardial fibers to determine the maximal diffusion distance from medium to the core of cells, which is shown not to exceed 8-10 microm. This is a principal index for correctly explaining high apparent Km for exogenous ADP (200-300 microM) in regulation of mitochondrial respiration in oxidative muscle cells in situ. The best fitting of the results of in silico studies may be achieved by using of the compartmentalized energy transfer model. From these results, it may be concluded that in cardiac muscle cells the mitochondria and ATPases are organized into intracellular energetic units (ICEUs) separated from the bulk phase of cytoplasm by some barriers which limit the diffusion of adenine nucleotides. In contrast, alternative models based on the concept of the cell as homogenous system do not explain the observed experimental phenomena and have led to misleading conclusions. The various sources of experimental and conceptual errors are analyzed

Regulation of respiration controlled by mitochondrial creatine kinase in permeabilized cardiac cells in situ Importance of system level properties.

International audienceThe main focus of this investigation is steady state kinetics of regulation of mitochondrial respiration in permeabilized cardiomyocytes in situ. Complete kinetic analysis of the regulation of respiration by mitochondrial creatine kinase was performed in the presence of pyruvate kinase and phosphoenolpyruvate to simulate interaction of mitochondria with glycolytic enzymes. Such a system analysis revealed striking differences in kinetic behaviour of the MtCK-activated mitochondrial respiration in situ and in vitro. Apparent dissociation constants of MgATP from its binary and ternary complexes with MtCK, K(ia) and K(a) (1.94+/-0.86 mM and 2.04+/-0.14 mM, correspondingly) were increased by several orders of magnitude in situ in comparison with same constants in vitro (0.44+/-0.08 mM and 0.016+/-0.01 mM, respectively). Apparent dissociation constants of creatine, K(ib) and K(b) (2.12+/-0.21 mM 2.17+/-0.40 Mm, correspondingly) were significantly decreased in situ in comparison with in vitro mitochondria (28+/-7 mM and 5+/-1.2 mM, respectively). Dissociation constant for phosphocreatine was not changed. These data may indicate selective restriction of metabolites' diffusion at the level of mitochondrial outer membrane. It is concluded that mechanisms of the regulation of respiration and energy fluxes in vivo are system level properties which depend on intracellular interactions of mitochondria with cytoskeleton, intracellular MgATPases and cytoplasmic glycolytic system

The creatine kinase phosphotransfer network: thermodynamic and kinetic considerations, the impact of the mitochondrial outer membrane and modelling approaches.

International audienceIn this review, we summarize the main structural and functional data on the role of the phosphocreatine (PCr)--creatine kinase (CK) pathway for compartmentalized energy transfer in cardiac cells. Mitochondrial creatine kinase, MtCK, fixed by cardiolipin molecules in the vicinity of the adenine nucleotide translocator, is a key enzyme in this pathway. Direct transfer of ATP and ADP between these proteins has been revealed both in experimental studies on the kinetics of the regulation of mitochondrial respiration and by mathematical modelling as a main mechanism of functional coupling of PCr production to oxidative phosphorylation. In cells in vivo or in permeabilized cells in situ, this coupling is reinforced by limited permeability of the outer membrane of the mitochondria for adenine nucleotides due to the contacts with cytoskeletal proteins. Due to these mechanisms, at least 80% of total energy is exported from mitochondria by PCr molecules. Mathematical modelling of intracellular diffusion and energy transfer shows that the main function of the PCr-CK pathway is to connect different pools (compartments) of ATP and, by this way, to overcome the local restrictions and diffusion limitation of adenine nucleotides due to the high degree of structural organization of cardiac cells