Estimation of Diffusion
Opponents: Jeroen Jeneson, PhD
BioModeling and Bioinformatics, Eindhoven University of Technology
Agu Laisk, DSc, Prof
Institute of Molecular and Cell Biology, University of Tartu
PhD Defense
fibers CM 0.3 - 0.5mM
mitochondria 0.015mM
Saks et al (Biochim Biophys Acta 1991)
Kümmel (Cardiovascular research 1988)
Restriction on MoM
Cardiomyocytes 0.3 - 0.5mM
Isolated mitochondria 0.015mM
Saks et al (Biochim Biophys Acta 1991)
Restriction on MoM
Seppet et al (Biochimica et Biophysica Acta 2001)
Saks et al (Biochem. J. 2001)
Saks 2001:
Skinned rat soleus fibers: "A powerful ADP-trapping system (PK+PEP) decreased the rate of respiration initiated by the addition of 2mM ATP not more than 20%. ...total activity of PK (20 units/ml) exceeded the combined rates of the ATPase and oxidative phosphorylation in oxygraphic cells (maximally 0.2umol/min per ml) by two orders of magnitude"
Skinned rat cardiac fibers:"...2mM MgATP, was inhibited not more than 40% by PK+PEP"
Boudina et al (Am J Physiol Heart Circ Physiol 2002)
PEP in solution activates
endogenous PK?
Diffusion: Fick's Law
Enzyme kinetics: Michaelis-Menten
Intracellular Na 10mM
Extracellular Na 135-145mM
14:1
Intracellular K 155mM
Extracellular K 3.5-5mM
1:30
Diffusion restrictions not intracellular but caused by clumping of cells and change in effective diffusion distances
Kongas et al (Am J Physiol Cell Physiol, 2002)
vs
Experiments performed by Natalja Jepihhina
Experiments performed by Natalja Jepihhina
Hypothesis: ADP accumulation in solution in population studies?
Dashed - fast diffusion
Dotted - ADP supplied by just diffusion
Black solid - flow + ADP diffusion
Red - DR Km=0.015mM, Green - 0.3mM, Blue - 0.45mM
WT vs GAMT-deficient mice
GAMT (Guanidinoacetate ethyltransferase) one of the enzymes on Cr synthesis
pathway
Experiments performed by Jelena Branovets
Analysis of kinetic data with mathematical models showed no differences in kinetic parameters WT vs GAMT-deficient mice
KmATPsyn=0.015mM
KmATPsyn=0.056mM Jacobus 1982
Sigmoidal relationship for VATPsyn Jeneson 1996
Wash solution (mM): NaCl 117, KCl 5.7, NaHCO3 4.4, MgCl2 1.7, KH2PO4 1.5, sucrose
120, BES 21, taurine 20, glucose 11.7 and creatine 10. pH was adjusted to 7.1 with NaOH at 25
Digestion solution (mM): NaCl 117, KCl 5.7, NaHCO3 4.4, MgCl2 1.7, KH2PO4 1.5, HEPES 21.1, taurine 20, glucose 11.7, creatine 11, phoshocreatine 10, pyruvate 2, 2 mg/ml BSA and 0.75-1 mg/ml
Mitomed solution (mM): KH2PO4 PO2 3, MgCl2 sucrose 110, K-lactobionate 60, taurine 20, HEPES 20, EGTA 0.5, DTT 0.5, malate 2, glutamate 5 and 5mg/ml BSA. pH adjusted to 7.1 with KOH at 25C
ATP 0 in vivo 5 - 10
ADP 0 in vivo measured 1-2mM, free10-50uM
Pi 3 in vivo < 1mM
pH 7.1
Na ca 4 (storage + Mitomed + pH adjustment)
K 3
Ca ~ 0
Mg 3
PEP 0
pyruvate 0
lactate 0
Jacobus et al, JBC 1982
Isolated mitochondria, VO2(ADP)
Jacobus et al, JBC 1982
Jeneson et al 1996 JBC