Morphological and biochemical characterization of mitochondria in Torpedo red blood cells

1. Morphological and biochemical characterization ofmitochondria in Torpedo red blood cells 電鱝紅血球細胞中粒線體的型態學 與生物化學特性 Alessandra Picaa, Salvatore Scaccob, Francesco Papab, Emanuele De Nittob, Sergio Papab Comparative Biochemistry and Physiology Part B 128 (2001) 213-219 指導教授：莊守正 老師 黃寶貴 老師 學生：林嘉彥 學號：M97310022

2. Torpedo marmorata - Classification Kingdom Animalia Phylum Chrodata Subphylum Vertebrata Class Chondrichthyes Subclass Elasmobranchii Order Torpediniformes Family Torpedinidae Genus Torpedo http://fishbase.sinica.edu.tw/images/species/Tomar_u1.jpg

3. Distribution http://www.aquamaps.org/receive.php

4. Biology http://fishbase.sinica.edu.tw/images/species/Tomar_u0.jpg

5. Mitochondria

6. Respiratory chain • Complex Ⅰ ： • FMN & Fe-S • Complex Ⅱ ： • FAD & Fe-S • Complex Ⅲ ： • Fe-S & Cytochrome b • Cytochromec1 • Complex Ⅳ ： • Cytochrome a • Cytochrome a3

7. NADH：NADH →[complexⅠ] → CoQ → [complex Ⅲ] → Cyt C → [complex Ⅳ] → O2 • FADH2：FADH2 →[complexⅡ]→ CoQ→ [complexⅢ] → Cyt C → [complex Ⅳ] → O2

8. Introduction • In mammals maturation of red blood cells in bone marrow leads to erythrocytes devoid of nucleus and cytoplasmic organelles. • In non-mammalian vertebrates mature erythrocytes retain the nucleus and cytoplasmic organelles. • In poikilotherms, like fishes, the nucleated erythrocytes have a relatively long life span, therefore, represent a convenient material to study erythrocyte organelles.

9. Torpedo is a sluggish fish adapted to conditions of lower oxygen consumption than other elasmobranchs of comparable size (Hughes, 1978). • Little is known on the occurrence and functions of mitochondria in fish erythrocytes (Sekhon and Beams, 1969; Kreutzmann, 1976; Lane et al., 1982). • Torpedo red blood cells appeared to us a material of choice for our studies since these cells are larger (35 X 15 μm) than those from other non-mammalian vertebrates(Pica et al., 1983).

10. Materials and methods • Red blood cell preparation 200 ml Total 48 (Each determination 4-5) Ｘ (1-2 kg body weight) 800g 15min pellet

11. Isolation of red blood cell mitochondria 800 mM sucrose 2 mM K-EDTA 10 mM Tris pH 7.4 200 μg/ml digitonin 4℃ + incubated 400 μg/ml lisozyme 200 μg/ml nagarse 20 min 4 ℃ 8000 g 4 ℃ 10 min resuspended

12. supernatant 0 ℃ 1500 g Potter Elvehjem homogenizer 22000 g 10 min 7000 g resuspended pellet pellet

13. Obserations of mitochondria in living red blood cells Microscope slides with Janus green B A small drop of blood Covered with acoverglass Sealed with nail polish

14. Transmission electron microscopy Fixed in 4% paraformaldehyde, 2.5% glutaraldehyde in cacodilate buffer pH 7.2 containing 0.8 M sucrose Redblood cell (Mitochondria) + Postfixed in osmium tetroxide 2% for 2 h and then dehydrated Ultrathin sections TEM

15. Mitochondrial respiration Freshlyisolated mitochondria Suspended in 800 mMsucrose, 2 mM K-EDTA, 10 mM Tris, pH 7.4, 80M cytochrome c. Polarography

16. Determination of cytochrome contents 100μg mitochondrial proteins 100 μl of 100 mM Na-phosphatebuffer, pH 7.4, 1.5% Na-cholate + Spectrophotometer

17. Measurements of enzymatic activities 500 μl of 40 mMK-phosphate buffer, pH 7.4, 5 mM MgCl2 , 3 mMKCN Purified mitochondriaproteins + Complex I Oxidation of NADH Complex III reduction of 40 μM duroquinol Complex IV Aerobicoxidation of 10 M cytochrome c Decylubiquinone

18. Results and discussion • Morphological obserations Fig. 1. Light and electron microscopy images of red blood cells and mitochondria from Torpedo marmorata Risso. Panels 13. Light microscopy. Vital stained red blood cells. Scale bar, 10 m. (1a) Basophilic erythroblast with approximately 20 small round mitochondria. (1b) Acidophilic erythroblast. Note the 15 mitochondria around the nucleus. (2) Mature erythrocyte with 18 elongated mitochondria. (3) Mature erythrocyte with 12 mitochondria at the poles of the nucleus.

19. Fig. 1. Panels 4-5. TEM. Ultrathin sections. Scale bar, 3 m.(4) Basophilic erythroblast with approximately 30 mitochondria. (5) Acidophilic erythroblast with approximately 16 mitochondria around the nucleus. Panels 6-7. TEM. Ultrathin sections of isolated mitochondria. Note that most of mitochondria display condensed form. Scale bar, 1.5 m. Panels 8 and 9, magnification of single mitochondria in the orthodox and condensed configuration, respectively, scale bar 0.5 m.

20. Respiratory chain contents and activities Cytochrome c & c1 Cytochrome b Cytochrome a & a3 Fig. 2. Reduced minus oxidized difference absorbance spectrum of isolated mitochondria from Torpedo marmorata Risso red blood cells. Freshly isolated 100 g protein mitochondria were suspended in 100 l of 100 mM Na-phosphate buffer, pH 7.4, 1.5% Na-cholate. The oxidized spectrum was first recorded then the difference reducedoxidized spectrum after reduction with a few grains of Na-dithionite at room temperature was recorded.

21. Table 1 Cytochrome contents, enzymatic and respiratory activities of mitochondria from Torpedo marmorata red blood cells

22. Fig. 3. Polarographic trace of respiratory activity of isolated mitochondria from Torpedo Marmorata supported by various respiratory substrates. Mitochondria 0.65 mg protein were suspended in 0.65 ml of the reaction mixture described under Materials and methods. The following additions were made in the sequence shown in the figure: 10 mM glutamate plus 10 mM malate, 1 M CCP (carbonylcyanide m-chlorophenylhydrazone), 2 g rotenone, 10 mM succinate, 2 g antimycin A, 2 mM ascorbate plus 200 M TMPD (N,N,N,N,-tetramethyl-p-phenylene diamine), 80 M cytochrome c, 2 mM KCN. For other details see section Materials and methods.

23. Discussion • It is, therefore, apparent that although mature Torpedo erythrocytes maintain at difference of mammalian erythrocytes, a significant number of mitochondria, not much lower than that of mammalian tissues, the mitochondrial content of active respiratory enzymes is definitely lower than in mammalian tissues. • This can be related to the reported low respiratory activity of elasmobranches (Hughes, 1978). • In conclusion the biochemical data characterise a functional, canonical respiratory chain in the Torpedo mitochondria.

24. These functional parameters with the morphological data discussed above, qualify the red blood cells of Torpedo marmorata as a convenient material to study the bioenergetic activities, biogenesis and disappearance of mitochondria in these cells as well as in red blood cells in general. • The short life of red blood cell mitochondria, in addition to its relevance for the change from aerobic to anaerobic energy metabolism, which can favor the oxygen transport function of haemoglobin (Ogo et al., 1993), might be related to the role of these organelles in cell death (Bernardi et al., 1999).

25. Thanks for your attention !