- Email: [email protected]
Non-muscle myoglobin expression and its respiratory consequences
Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S159–S164
dependence on haemoglobin(Hb) oxygenation state1. This coupling results, at least partly, from reactions between Hb and RBC membrane proteins, such as the polyanionic Nterminal cytoplasmic domain of Band 3 (cdB3) that binds either to the positively-charged phosphate binding sites of deoxygenated vertebrate Hbs2 at low O2 saturation, or to glycolytic enzymes (like aldolase, GAPDH and PFK) at high O2 saturation, whereby Hb may function as a transducer regulating RBC glycolysis in an O2-dependent manner. The effects of trout, chicken and human cdB3 peptides on O2 binding properties of fish, bird and mammalian Hbs are consistent with such a role in endothermic, but not in ectothermic, vertebrates3. Measurements of the interaction between Hbs and anionic domains of Band 3, other membrane proteins and intracellular proteins (band 4.1, glycophorin, aquaporin, sodiumproton exchanger, actin, tubulin) will be discussed as regards the oxygenation dependence of RBC ion-transporting functions and rheological properties. 1 Barvitenko, N.N., Adragna, N.C. and Weber, R.E. (2005). Cell. Physiol. Biochem. 15, 1–18. 2 Walder, J.A. et al. (1984). J. Biol. Chem. 259, 10238–10246. 3 Weber, R.E. et al. (2004). Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R454-R464. doi:10.1016/j.cbpa.2007.01.330
A10.7 Non-muscle myoglobin expression and its respiratory consequences A. Cossins, D. Williams, M. Berenbrink, A. Kipar, (Liverpool University) Myoglobin is famous for its specific expression in oxidative muscle in vertebrates, particularly including cardiac myocytes. We have recently conducted an extensive microarray screen of genes responding to hypoxia in the common carp, Cyprinus carpio. A prominent feature of this work was an up-regulated transcript expression of the myoglobin gene in the liver. We subsequently identified the myoglobin protein in liver homogenates using 2D gels and tandem mass spectrometry confirming that the protein is indeed expressed in this unusual location. Microarray analysis indicated that myoglobin was expressed in a range of other tissues, particularly including gill, intestine and kidney; in some tissues the transcript was substantially upregulated by chronic hypoxia. We also identified expression in brain, but this was linked mainly to a new myoglobin isoform discovered in the EST collection used to construct the cDNA microarray. To the best of our knowledge, this is the only vertebrate species known to possess two myoglobin isoforms. Most recently we have generated an anti-peptide antibody directed against the muscle isoform and have used this to explore the cellular location of the protein in different carp tissues. We will describe the location in brain, cardiac muscle, gill, liver and intestine, the results indicating small collections of expressing cells many of which are tissue-specific. Most
S161
tissues display expression within the vascular endothelial cells. The function of the myoglobin protein in these new locations is not clear but may involve functions other than oxygen storage. Similar tissue locations have been confirmed in zebrafish indicating that non-muscle location is not limited to the hypoxia-tolerant carp and its close relatives. doi:10.1016/j.cbpa.2007.01.331
A10.8 Oxygen dependence of membrane transport in normal and sickle human red blood cells J. Gibson, (University of Cambridge); C. Ellory, J. Browning, H. Robinson, (University of Oxford) Although the oxygen-dependence of transport systems in the membrane of red blood cells (RBCs) has been known about for some considerable time, its relevance to human RBCs has only recently become apparent. It has now been established that changes in oxygen tension have profound effects on a number of different transporters in human RBCs. In normal RBCs, the KCl cotransporter (KCC) is inactivated at low oxygen tensions, becoming quiescent to stimuli like low pH or cell swelling; the anion exchanger is also more active at high oxygen tensions; whilst the Na+/K+ pump is insensitive to oxygen. In RBCs from sickle cell patients, which contain HbS rather than the normal HbA, the oxygen-dependence of some of these systems is altered. The behaviour of KCC is markedly abnormal, with activity at its lowest at moderate oxygen tensions, then increasing again at lower values, until in anoxia, activity is similar or slightly elevated to that in normoxia. Sickle cells also exhibit an unique pathway, termed Psickle, which manifests apparently as a conductive channel, selective for cations, activated upon deoxygenation. Recently, we have used the patch clamp technique to investigate further the conductance of normal and sickle RBCs in response to changes in oxygen tension. In addition, we have also shown that sickle cells exhibit a non-electrolyte permeability, also activated upon deoxygenation, whose properties share many of those of Psickle, suggesting that this pathway is more promiscuous than hitherto realised. In this presentation, we highlight some of our more recent findings and discuss their significance. doi:10.1016/j.cbpa.2007.01.332
A10.9 Oxygen-dependent ion transporters in erythrocytes A. Bogdanova, (Zürich); M. Nikinmaa, (Turku) Although none of the erythrocytic ion transport systems are known to be directly coupled to an oxygen sensor, several transporters in erythrocytes respond to changes in oxygen tension. Most studies have concentrated on the oxygen-
dependence on haemoglobin(Hb) oxygenation state1. This coupling results, at least partly, from reactions between Hb and RBC membrane proteins, such as the polyanionic Nterminal cytoplasmic domain of Band 3 (cdB3) that binds either to the positively-charged phosphate binding sites of deoxygenated vertebrate Hbs2 at low O2 saturation, or to glycolytic enzymes (like aldolase, GAPDH and PFK) at high O2 saturation, whereby Hb may function as a transducer regulating RBC glycolysis in an O2-dependent manner. The effects of trout, chicken and human cdB3 peptides on O2 binding properties of fish, bird and mammalian Hbs are consistent with such a role in endothermic, but not in ectothermic, vertebrates3. Measurements of the interaction between Hbs and anionic domains of Band 3, other membrane proteins and intracellular proteins (band 4.1, glycophorin, aquaporin, sodiumproton exchanger, actin, tubulin) will be discussed as regards the oxygenation dependence of RBC ion-transporting functions and rheological properties. 1 Barvitenko, N.N., Adragna, N.C. and Weber, R.E. (2005). Cell. Physiol. Biochem. 15, 1–18. 2 Walder, J.A. et al. (1984). J. Biol. Chem. 259, 10238–10246. 3 Weber, R.E. et al. (2004). Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R454-R464. doi:10.1016/j.cbpa.2007.01.330
A10.7 Non-muscle myoglobin expression and its respiratory consequences A. Cossins, D. Williams, M. Berenbrink, A. Kipar, (Liverpool University) Myoglobin is famous for its specific expression in oxidative muscle in vertebrates, particularly including cardiac myocytes. We have recently conducted an extensive microarray screen of genes responding to hypoxia in the common carp, Cyprinus carpio. A prominent feature of this work was an up-regulated transcript expression of the myoglobin gene in the liver. We subsequently identified the myoglobin protein in liver homogenates using 2D gels and tandem mass spectrometry confirming that the protein is indeed expressed in this unusual location. Microarray analysis indicated that myoglobin was expressed in a range of other tissues, particularly including gill, intestine and kidney; in some tissues the transcript was substantially upregulated by chronic hypoxia. We also identified expression in brain, but this was linked mainly to a new myoglobin isoform discovered in the EST collection used to construct the cDNA microarray. To the best of our knowledge, this is the only vertebrate species known to possess two myoglobin isoforms. Most recently we have generated an anti-peptide antibody directed against the muscle isoform and have used this to explore the cellular location of the protein in different carp tissues. We will describe the location in brain, cardiac muscle, gill, liver and intestine, the results indicating small collections of expressing cells many of which are tissue-specific. Most
S161
tissues display expression within the vascular endothelial cells. The function of the myoglobin protein in these new locations is not clear but may involve functions other than oxygen storage. Similar tissue locations have been confirmed in zebrafish indicating that non-muscle location is not limited to the hypoxia-tolerant carp and its close relatives. doi:10.1016/j.cbpa.2007.01.331
A10.8 Oxygen dependence of membrane transport in normal and sickle human red blood cells J. Gibson, (University of Cambridge); C. Ellory, J. Browning, H. Robinson, (University of Oxford) Although the oxygen-dependence of transport systems in the membrane of red blood cells (RBCs) has been known about for some considerable time, its relevance to human RBCs has only recently become apparent. It has now been established that changes in oxygen tension have profound effects on a number of different transporters in human RBCs. In normal RBCs, the KCl cotransporter (KCC) is inactivated at low oxygen tensions, becoming quiescent to stimuli like low pH or cell swelling; the anion exchanger is also more active at high oxygen tensions; whilst the Na+/K+ pump is insensitive to oxygen. In RBCs from sickle cell patients, which contain HbS rather than the normal HbA, the oxygen-dependence of some of these systems is altered. The behaviour of KCC is markedly abnormal, with activity at its lowest at moderate oxygen tensions, then increasing again at lower values, until in anoxia, activity is similar or slightly elevated to that in normoxia. Sickle cells also exhibit an unique pathway, termed Psickle, which manifests apparently as a conductive channel, selective for cations, activated upon deoxygenation. Recently, we have used the patch clamp technique to investigate further the conductance of normal and sickle RBCs in response to changes in oxygen tension. In addition, we have also shown that sickle cells exhibit a non-electrolyte permeability, also activated upon deoxygenation, whose properties share many of those of Psickle, suggesting that this pathway is more promiscuous than hitherto realised. In this presentation, we highlight some of our more recent findings and discuss their significance. doi:10.1016/j.cbpa.2007.01.332
A10.9 Oxygen-dependent ion transporters in erythrocytes A. Bogdanova, (Zürich); M. Nikinmaa, (Turku) Although none of the erythrocytic ion transport systems are known to be directly coupled to an oxygen sensor, several transporters in erythrocytes respond to changes in oxygen tension. Most studies have concentrated on the oxygen-