- Email: [email protected]
On the nature and function of yellow aging pigment lipofuscin
Preliminary notes 419 elude that our micrographs present the transcribing rDNA of the nucleoli contained in the polytene chromosomes of the Chironomus salivary glands. We have to emphasize, however, that the relative length of free (‘spacer’) axis is much below the corresponding values found in amphibian oocytes [4, 5, 131and in the HeLa cells [I, 31. We thank Dr H. D. Berendes, University of Nijmegen, and Dr H. Falk, University of Freiburg, for various support and Miss Marianne Winter for skilful technical assistance. The study received financial support from the EMBO (short-term fellowship awarded to J. D.) and the Deutsche Forschungsgemeinschaft, SFB 46 (grant awarded to W. W. F.).
References 1. Bakken, A H & Miller, 0 L, J cell biol 55 (1972) 12a. 2. Dalgarno, L, Hosking, D M & Shen, C H, Eur j biochem 24 (1972) 498. 3. Miller, 0 L & Bakken, A H, Acta endocrinol, suppl. 168 (1972) 155. 4. Miller, 0 L & Beatty, B R, Science 164 (1969) 955. 5. - Genetics, suppl. 61 (1969) 134. 6. Miller, 0 L & Hamkalo B A, Int rev cytol 33
(1972) 1. I. Perry, R P, Cheng, T-Y, Freed, J J, Greenberg,
J R. Kellev. D E & Tartof. K D. Proc natl acad sci US 65 ii970) 609. 8. Reeder. R H & Brown./ D D. J mol biol 51 (1970)
361. ' 9. Ringborg, U, Daneholt, B, Edstrom, J-E, Egyhazi,
E & Lambert, B, J mol biol 51 (1970) 327. 10. Ringborg, U & Ryd!ander, L, J cell biol 5 I (1971) 355.
Il. Robert, M, Chromosoma 36 (1971) 1. 12. Rubinstein, L & Clever, U, Biochim biophys acta 246 (1971) 517.
13. Scheer, U; Trendelenburg, M F & Franke, W W, Exptl cell res 80 (1973) 175. 14. Wensink, P C & Brown, D D, J mol biol60 (1971) 235.
Received March 5, 1973 Revised version received May 14, 1973
On the nature and function of yellow aging pigment lipofuscin V. N. KARNAUKHOV, Institute of Biological Physits of the Academy of Sciences of USSR, Pushchino, Moscow Region, USSR, 142292 Summavy. A comparison of lipofuscin granules from
warm-blooded animals and carotenoid-containing granules (cytosomes) from molluscoid neurons was carried out. It was confirmed that the carotenoids are a component of the lipofuscin granules. Data in literature and the experimental data obtained showed that the liuofuscin granules contained carotenoids, myoglobin and some-respiratory enzymes. On the basis of identification of the properties of carotenoid-containing granules (cytosomes) of molluscoid neurones and the lipofuscin granules, it is proposed that the functions of the lipofuscin are those of forming the intracellular oxygen stock and providing the energy requirements of the cells under the conditions of low rate oxygen penetration into tissues.
It has been shown that carotenoids, in common with myoglobin, take part in the oxidative metabolism of molluscoid neurons [l-3]. In the cells the carotenoids and myoglobin are localized in specific granules [4], named cytosomes [5, 61. The cytosomes contain some respiratory enzymes [5, 61 and have the ability of energy-dependent accumulation of SPf under hypoxic conditions [7, 81. Based on the experimental data obtained, it was suggested that in common with myoglobin the carotenoids, having a large number of unsaturated double bonds in their molecules, can function as intracellular oxygen stock [2, 31 and the cytosomes can provide the energy required by the cell under the conditions of low-rate oxygen penetration into tissues [3, 41. When comparing the ultrastructural organization of the molluscoid cytosomes (fig. 1) and lipofuscin granules of warm-blooded animals, we were led to assume that there was a close similarity between the chemical composition and physiological function of these intracellular organoids [9, 10, 111. A considerable oxygen uptake was found [13] in the lipofuscin granule fractions [12]. Exptl Cell Res 80 (1973)
480
Preliminary notes
Fig. 1. Ultrastructural
organization of carotenoid-containing granules in molluscoid neurons.
The oxygen uptake of the lipofuscin granule fractions was stimulated by the addition of NADH [14]. Bjorkerud has demonstrated the presence of haemoproteins and carotenoids in lipofuscin obtained from bovine cardiac muscle [15]. However, in subsequent studies on the chemical composition of lipofuscin from human cardiac muscle carried out by Bjorkerud [16] and Hendley et al. [17], carotenoids were not found. Our experiments were carried out in order to test our concept of the role of carotenoids in the intracellular stock of oxygen and of the presence of carotenoids in the composition of lipofuscin granules [9-l 11.
An imuroved 1181high-sensitive microsoectrofluorimeter [Id] was used. The excitation of cell fluorescence was induced bv a mercurv arc lamp L[PIII-250 (250 watts). The em>ssion of the lamp at wavelengths of 365 nm was separated by glass filter YWZ-6. The dimensions of the measured part of the preparation were 10 x 50 pm. The sections were incubated in Tyrode solution for 30 min following isolation at 37°C and then placed into a thermostated (37°C) perfusion chamber for measuring. For chemical extraction of carotenoids, heart, brain, and liver tissues from bulls aged 2 and 12 were used. Ten gram of each type of tissue from 3 young and 3 old bulls were taken for the analyses. The tissue samples from each type of the same age category were mixed. Then, from this mixture (the total weight 30 g) after lyophilization the total lipid fraction was extracted by light petroleum (b.p. SOC). This fraction was then saponified and sterols were removed from it by a conventional technique [19, 201. The absorption spectra of one-tenth of the unsaponifiable fractions in light petroleum were measured with a UNICAM SP-800 spectrophotometer.
Materials and Methods
Results The fluorescent spectra of the brain cortex sections of young rats (fig. 2, curve l), show
Microspectrofluorimetric investigations were performed on fresh sections of brain cortex tissue from young (5 months) and old (14-15 months) white rats. Exptl
Cell Res 80 (1973)
Preliminary notes 48 1 two emission maxima. One of them (A = 480 nm) is due to the reduced pyridine nucleotides, whereas the other one (I =520 nm) is caused by the oxidized flavoproteins [21, 221. The fluorescence colour of the sections is blue-white without any inclusions. In contrast, the lipofuscin granules of various dimensions and forms are observed with bright yellow fluorescence on the brain cortex sections of old rats on the blue-white background. Accordingly, in fluorescent spectra of the tissues from old animals a new emission band with the maximum of I.560-565 nm (fig. 2, curve 2) appears in addition to the bands of reduced pyridine nucleotides (I =480 nm) and oxidized flavorproteins (21520 nm). If lipofuscin takes up a considerable portion of the viewing field, this maximum increases (fig. 2, curve 3) and becomes predominant in the spectrum (fig, 2, curve 4). The characteristics of the fluorescent spectra of rat brain lipofuscin coincide with the data obtained by Hydtn & Lindstriim [23]. It is of interest that the position of lipofuscin fluorescent bands in the spectral
OD.
I
454
Fig. 3. Absorption spectra of the unsaponifiable frac-
tion of the lipid extract from tissues of old (I, brain; 2, liver; 3, tieart) and young (4, brain; 5, liver; 6, heart) bulls.
range (1=560-565 nm) coincides with the fluorescent band of carotenoid-containing granules in molluscoid neurons [3] as well as with fluorescent bands of carotenoids in carrot root cells (x- and p-carotene) [24]. The results of the spectrophotometric analysis of the unsaponifiable fraction of the tissues from heart, brain and liver of young and old bulls are given in fig. 3. A characteristic three-band absorption spectrum with maxima at 422, 451 and 476 nm, indicates a considerable carotenoid concentration in tissues of old animals which is at least one order higher than the concentration of these compounds in tissues of young animals [24, 251. Discussion
I . 500
.
I
.
I 600-
660 Iin
Fluorescentspectraof the brain sectionsfrom young (1) and old (24) rats. Fig. 2.
The literature data and the data obtained enable us to suggest that the yellow aging pigment lipofuscin is identical with the carotenoid-containing granules (cytosomes) in molluscoid neurons [9-l 1, 24, 331. The lipofuscin granules contain carotenoids, myoglobin [26] and some flavoprotein [27] and haemoprotein respiratory enzymes [28-301. The carotenoids, together with the myoglobin function as intracellular oxygen stocks, while the above mentioned respiratory enzymes seem to be components Exptl Cell Res 80 (1973)
482
Preliminary notes The nitochondrial
of terminal
NADIR-6
system oxidation
42 d-d-q-c
-a+a3
The lipofuscin system of terminal oxidation
Carotenolds
. The
system
of oxygen stock
of the mitochondrial and cytosomal systems of terminal oxidation.
lipofuscin with age may be considered a result of adaptation of the cells to a decreased rate of oxygen diffusion into tissues due to the decrease of the blood vessels oxygen penetration ability which progresses with age. For the same reason the lipofuscin accumulation is observed in tissues of young animals living under the hypoxic conditions [341. Based upon these suggestions, the interrelation between carotenoids and reproduction processes, repeatedly mentioned [35], may be considered as an adaptive reaction of the organism directed to the maximal survival of posterity under the conditions of possible oxygen deficiency [24, 36-391.
Fig. 4. Interrelation
of a specific system for terminal oxidaton (fig. 4). It should be stressed that the system differs from the well known mitochondrial system for terminal oxidation in the fact that the terminal electron acceptor for the system is either oxygenated carotenoid or oxygen from the intracellular stock. Good electron-acceptor and electron-donor properties of carotenoid molecules [31, 321 provide this possibility. It may well be that the lipofuscin granules can provide energy requirements for the cells under hypoxic conditions. Support for this assumption may be found in observing energy-dependent accumulation of Sr2+ in mitochondria and cytosomes of molluscoid neurones. Tt was shown that under normal conditions the mitochondrial energy production was predominant in the neurons while the cytosomes indicated only little activity. In anaerobiosis the situation is reversed: the Sr2+ accumulation of mitochondria significantly decreases, while the cytosomal activity strongly increases [8]. It thus appears that the accumulation of Exptl Cell Res 80 (1973)
References 1. Karnaukhov, V N, Structure and functions of macromolecules and macromolecular systems, p. 200. Moscow (1969). 2. - Biophysics of living cell, vol. I (1970) 25. Puschino. 3. - Exptl cell res 64 (1971) 301. 4. Karnaukhov, V N & Varton, S S, Cytologij 13 (1971) 1088. 5. Nolte, A, Breucker, H & Kuhlmann, D, Z Zellforsch 68 (1965) 1. 6. Zs-Nagy, I, Anna1 biol Tihany 34 (1967) 25. 7. Zs-Nagy, I & Kerpel-Fronius, S, Abstr 17 int ;mt-g;) electron microscopy, p. 155. Grenoble 8. Zs-Nagy, I, Ann biol Tihany 38 (1971) 117. 9. Karnaukhov, V N, Rozanov, S 1 & Svoren,
VA, Biofysika 11 (1966) 1085. V N, Abstr of 2nd biochem meeting, sect 8, p. 30. Tashkent (1969). USSR 196 (1971) i221. 11. -DAN 12. Bjorkerud, S, J ultrastruct res, suppl. 5 (1963). 13. Bjorkerud, S & Cummins, J T, Exptl cell res 32 (1963) 510. 14. Bjorkerud, S, Advan gerontol res, vol. 1, pl 275. Academic Press, New York and London (1964). 15. - Exotl mol narhol 3 (1964) 377. 16. Hendley, D 6, Mildvan, A S, Reporter, M C & Strehler, B L, J gerontol 18 (1963) 250. 17. Karnaukhov, V N & Zintchenko, V P, Cytologij 13 (1971) 1243. 18. Karnaukhov, V N, Kulakov, V I, Melnikova, E V & Yashin, V A, Cytologij 10 (1968) 654. 19. Davies. B H. Chemistry and biochemistry of plant pigments (ed T Goodwin) p. 489. Academic Press, New York (1965). 20. Kamaukhov. V N. Medvedev. A I. Abdurakhmanov, A & Fin, d T, Biophysics of living cell 1 (1970) 13.
10. Karnaukhov,
Preliminary notes 21. Karnaukhov, V N, Khaspekov, L G, Zintchenko, 22. 23. 24. 25. 26. 21. 28. 29. 30. 31. 32. 33. 34. 35. :;: 38. 39.
V P, Brailovskaya, V A & Lukyanova, L D, DAN USSR 201 (1971) 227. Karnaukhov, V N & Pavlenko, V K, Studia biophysica 23 (1970) 69. HydCn, H & Lindstrcm, B, Disc Farad sot 9 (1950) 436. Karnaukhov, V N, Biophysics of living cell, vol. 2 (1971) 68. Puschino. Karnaukhov, V N & Tataryunas, T B, DAN USSR 203 (1972) 1197. Miyawaki, H, J natl cancer inst 34 (1965) 601. Hack, M H, Colcolough, H L & Helmy, F M, Acta histochem 35 (1970) 357. Fride, R L, Acta neuropathol 2 (1962) 113. Kumamoto, T & Bourne, G H, Acta histochem 16 (1963) 87. Nandy, K, J gerontol 23 (1968) 82. Pullman, B & Pullman, A, Quantum biochemistry. Interscience, New York and London (1963). Dingle, J T & Lucy, J A, Biol rev 40 (1965) 422. Karnaukhov, V N, Cytologij 15 (1973) 532. Sulkin, N M & Srivanij, P, J gerontol 15 (1960) 2. Goodwin, T V, The comparative biochemistry of carotenoids (1952). Smirnov, A I, DAN USSR 73 (1950) 609. Soin, S G, Zoologichesky zhurnal 35 (1956) 1362. - The adaptive peculiarities of fish evolution. MGU-press, Moscow (1968). Karnaukhov, V N, Tataryunas, T B & Petrunyaka. V V, Mech age dev 2 (1973) 201.
Received April 2, 1973
Detection of DNA polymerase activity in fixed cells Z. DARZYNKIEWICZ,
Department of Connectioe Tissue Research, Boston Biomedical Research Institute, Boston, Mass. 02114, USA
It was shown recently that RNA polymerase activity may be successfully localized and studied on cytological preparations. The enzyme retains its activity in situ and catalyses ribonucleotide synthesis in the presence of exogenous ribonucleoside triphosphates after a mild fixation of cells [l-4]. In the present studies, a similar approach was used to localize DNA polymerase activity in eukaryotic cells. After brief fixation, the cells become permeable to deoxyribonucleoside triphosphates (dNTPs) and incorporate them into DNAse hydrolysable macromolecules in the nuclei.
483
Materials and Methods Cell preparations. 3T3 mouse fibroblasts and their simian virus 40 transformed line (SV 3T3) were a gift from Dr P. H. Black, Massachusetts General Hospital, Boston, Mass. HeLa cell line was purchased from Microbiological Associates (Bethesda, Md Cat. No. 71-134). The cells were grown on glasscoverslips in basal Eagle’s medium supplemented with 10% fetal calf se&m and with antibiotics (penicillin 50 PI/ml; streptomycin 50 pg/ml). Coverslips bearing cells to be assayed for DNA polymerase were rinsed in Hanks salt solution, fixed in absolute ethanol : acetone (1 : 1 v/v) for 5 min at o”C, air dried at 4°C and stored at 4°C for not longer than 20 min. To determine the number of cells synthesizing DNA parallel cultures were treated with 3H-TdR (SH-thymidine-methyl, spec. act. 6.7 Ci/mmole; New England Nuclear Corporation, Boston, Mass. 0.5 ,&i/ml) for 1 h. The coverslips were then washed in saline, fixed in ethanol :acetic acid (3 : I v/v) and processed for autoradiography as described elsewhere [5]. autoradiography. One ml of standard incubation medium contained: 100 pmoles of TrisHCI buffer (pH 7.8), lOpmoles of 2 mercaptoethanol; 150,umoles of sucrose; 8 pmoles of MgCl,; 500 nmoles each of dTTP, dCTP and dGTP (Schwarz/Mann, Orangeburg, N.Y.) and 12.5 nmoles of 3H-dATP (12.9 Ci/mmole, New England Nuclear). The coverslips were incubated in this medium at 37°C for up to 20 min. Incubations were terminated by rinsing the coverslips in ice-cold 0.1 mM solution of unlabelled dATP in 0.15 M sucrose buffered with Tris-HCl (pH 7.8) followed by post-fixation in ethanol:acetic acid mixture (3:l v/v) for 2 h. The coverslips were then washed in 70% ethanol, air-dried and mounted on gelatinized slides. Before autoradiography, the slides were treated with 5 % trichloroacetic acid for 10 min at 0°C and rinsed in water for 30 min. Some coverslips were incubated with RNAse (boiled RNAse A, Bovine pancreas, Worthington Biochemical Corp., 1 mg/ml in acetate buffer, pH 5.0, 37”C, for 60 min) or with DNAse (DNAse I, Worthington, 1 mg/ml in 0.01 M MgS04, 37”C, for 2 h. The slides were covered with Kodak AR 10 stripping film and exposed for 6 days. Grains were counted over at least 100 cell nuclei from each individual slide; the mean values and S.E. of counts from the representative slides are presented in the tables and diagrams. All expt were run in triplicate; in all cases the mean values of duplicate cultures did not differ significantly (p < 0.05) from those presented in Results. The percentage of 3H-TdR-labelled cells (S phase cells) was calculated on the basis of counts of 500 cells from autoradiographs of cultures treated with 3HTdR.
Incubations,
Results Cells incubated in standard medium incorporate the label almost exclusively in the nuclei. Interphase cells exhibit the highest labelling; mitotic cells are distinctly less Exptl Cell Res 80 (1973)
References 1. Bakken, A H & Miller, 0 L, J cell biol 55 (1972) 12a. 2. Dalgarno, L, Hosking, D M & Shen, C H, Eur j biochem 24 (1972) 498. 3. Miller, 0 L & Bakken, A H, Acta endocrinol, suppl. 168 (1972) 155. 4. Miller, 0 L & Beatty, B R, Science 164 (1969) 955. 5. - Genetics, suppl. 61 (1969) 134. 6. Miller, 0 L & Hamkalo B A, Int rev cytol 33
(1972) 1. I. Perry, R P, Cheng, T-Y, Freed, J J, Greenberg,
J R. Kellev. D E & Tartof. K D. Proc natl acad sci US 65 ii970) 609. 8. Reeder. R H & Brown./ D D. J mol biol 51 (1970)
361. ' 9. Ringborg, U, Daneholt, B, Edstrom, J-E, Egyhazi,
E & Lambert, B, J mol biol 51 (1970) 327. 10. Ringborg, U & Ryd!ander, L, J cell biol 5 I (1971) 355.
Il. Robert, M, Chromosoma 36 (1971) 1. 12. Rubinstein, L & Clever, U, Biochim biophys acta 246 (1971) 517.
13. Scheer, U; Trendelenburg, M F & Franke, W W, Exptl cell res 80 (1973) 175. 14. Wensink, P C & Brown, D D, J mol biol60 (1971) 235.
Received March 5, 1973 Revised version received May 14, 1973
On the nature and function of yellow aging pigment lipofuscin V. N. KARNAUKHOV, Institute of Biological Physits of the Academy of Sciences of USSR, Pushchino, Moscow Region, USSR, 142292 Summavy. A comparison of lipofuscin granules from
warm-blooded animals and carotenoid-containing granules (cytosomes) from molluscoid neurons was carried out. It was confirmed that the carotenoids are a component of the lipofuscin granules. Data in literature and the experimental data obtained showed that the liuofuscin granules contained carotenoids, myoglobin and some-respiratory enzymes. On the basis of identification of the properties of carotenoid-containing granules (cytosomes) of molluscoid neurones and the lipofuscin granules, it is proposed that the functions of the lipofuscin are those of forming the intracellular oxygen stock and providing the energy requirements of the cells under the conditions of low rate oxygen penetration into tissues.
It has been shown that carotenoids, in common with myoglobin, take part in the oxidative metabolism of molluscoid neurons [l-3]. In the cells the carotenoids and myoglobin are localized in specific granules [4], named cytosomes [5, 61. The cytosomes contain some respiratory enzymes [5, 61 and have the ability of energy-dependent accumulation of SPf under hypoxic conditions [7, 81. Based on the experimental data obtained, it was suggested that in common with myoglobin the carotenoids, having a large number of unsaturated double bonds in their molecules, can function as intracellular oxygen stock [2, 31 and the cytosomes can provide the energy required by the cell under the conditions of low-rate oxygen penetration into tissues [3, 41. When comparing the ultrastructural organization of the molluscoid cytosomes (fig. 1) and lipofuscin granules of warm-blooded animals, we were led to assume that there was a close similarity between the chemical composition and physiological function of these intracellular organoids [9, 10, 111. A considerable oxygen uptake was found [13] in the lipofuscin granule fractions [12]. Exptl Cell Res 80 (1973)
480
Preliminary notes
Fig. 1. Ultrastructural
organization of carotenoid-containing granules in molluscoid neurons.
The oxygen uptake of the lipofuscin granule fractions was stimulated by the addition of NADH [14]. Bjorkerud has demonstrated the presence of haemoproteins and carotenoids in lipofuscin obtained from bovine cardiac muscle [15]. However, in subsequent studies on the chemical composition of lipofuscin from human cardiac muscle carried out by Bjorkerud [16] and Hendley et al. [17], carotenoids were not found. Our experiments were carried out in order to test our concept of the role of carotenoids in the intracellular stock of oxygen and of the presence of carotenoids in the composition of lipofuscin granules [9-l 11.
An imuroved 1181high-sensitive microsoectrofluorimeter [Id] was used. The excitation of cell fluorescence was induced bv a mercurv arc lamp L[PIII-250 (250 watts). The em>ssion of the lamp at wavelengths of 365 nm was separated by glass filter YWZ-6. The dimensions of the measured part of the preparation were 10 x 50 pm. The sections were incubated in Tyrode solution for 30 min following isolation at 37°C and then placed into a thermostated (37°C) perfusion chamber for measuring. For chemical extraction of carotenoids, heart, brain, and liver tissues from bulls aged 2 and 12 were used. Ten gram of each type of tissue from 3 young and 3 old bulls were taken for the analyses. The tissue samples from each type of the same age category were mixed. Then, from this mixture (the total weight 30 g) after lyophilization the total lipid fraction was extracted by light petroleum (b.p. SOC). This fraction was then saponified and sterols were removed from it by a conventional technique [19, 201. The absorption spectra of one-tenth of the unsaponifiable fractions in light petroleum were measured with a UNICAM SP-800 spectrophotometer.
Materials and Methods
Results The fluorescent spectra of the brain cortex sections of young rats (fig. 2, curve l), show
Microspectrofluorimetric investigations were performed on fresh sections of brain cortex tissue from young (5 months) and old (14-15 months) white rats. Exptl
Cell Res 80 (1973)
Preliminary notes 48 1 two emission maxima. One of them (A = 480 nm) is due to the reduced pyridine nucleotides, whereas the other one (I =520 nm) is caused by the oxidized flavoproteins [21, 221. The fluorescence colour of the sections is blue-white without any inclusions. In contrast, the lipofuscin granules of various dimensions and forms are observed with bright yellow fluorescence on the brain cortex sections of old rats on the blue-white background. Accordingly, in fluorescent spectra of the tissues from old animals a new emission band with the maximum of I.560-565 nm (fig. 2, curve 2) appears in addition to the bands of reduced pyridine nucleotides (I =480 nm) and oxidized flavorproteins (21520 nm). If lipofuscin takes up a considerable portion of the viewing field, this maximum increases (fig. 2, curve 3) and becomes predominant in the spectrum (fig, 2, curve 4). The characteristics of the fluorescent spectra of rat brain lipofuscin coincide with the data obtained by Hydtn & Lindstriim [23]. It is of interest that the position of lipofuscin fluorescent bands in the spectral
OD.
I
454
Fig. 3. Absorption spectra of the unsaponifiable frac-
tion of the lipid extract from tissues of old (I, brain; 2, liver; 3, tieart) and young (4, brain; 5, liver; 6, heart) bulls.
range (1=560-565 nm) coincides with the fluorescent band of carotenoid-containing granules in molluscoid neurons [3] as well as with fluorescent bands of carotenoids in carrot root cells (x- and p-carotene) [24]. The results of the spectrophotometric analysis of the unsaponifiable fraction of the tissues from heart, brain and liver of young and old bulls are given in fig. 3. A characteristic three-band absorption spectrum with maxima at 422, 451 and 476 nm, indicates a considerable carotenoid concentration in tissues of old animals which is at least one order higher than the concentration of these compounds in tissues of young animals [24, 251. Discussion
I . 500
.
I
.
I 600-
660 Iin
Fluorescentspectraof the brain sectionsfrom young (1) and old (24) rats. Fig. 2.
The literature data and the data obtained enable us to suggest that the yellow aging pigment lipofuscin is identical with the carotenoid-containing granules (cytosomes) in molluscoid neurons [9-l 1, 24, 331. The lipofuscin granules contain carotenoids, myoglobin [26] and some flavoprotein [27] and haemoprotein respiratory enzymes [28-301. The carotenoids, together with the myoglobin function as intracellular oxygen stocks, while the above mentioned respiratory enzymes seem to be components Exptl Cell Res 80 (1973)
482
Preliminary notes The nitochondrial
of terminal
NADIR-6
system oxidation
42 d-d-q-c
-a+a3
The lipofuscin system of terminal oxidation
Carotenolds
. The
system
of oxygen stock
of the mitochondrial and cytosomal systems of terminal oxidation.
lipofuscin with age may be considered a result of adaptation of the cells to a decreased rate of oxygen diffusion into tissues due to the decrease of the blood vessels oxygen penetration ability which progresses with age. For the same reason the lipofuscin accumulation is observed in tissues of young animals living under the hypoxic conditions [341. Based upon these suggestions, the interrelation between carotenoids and reproduction processes, repeatedly mentioned [35], may be considered as an adaptive reaction of the organism directed to the maximal survival of posterity under the conditions of possible oxygen deficiency [24, 36-391.
Fig. 4. Interrelation
of a specific system for terminal oxidaton (fig. 4). It should be stressed that the system differs from the well known mitochondrial system for terminal oxidation in the fact that the terminal electron acceptor for the system is either oxygenated carotenoid or oxygen from the intracellular stock. Good electron-acceptor and electron-donor properties of carotenoid molecules [31, 321 provide this possibility. It may well be that the lipofuscin granules can provide energy requirements for the cells under hypoxic conditions. Support for this assumption may be found in observing energy-dependent accumulation of Sr2+ in mitochondria and cytosomes of molluscoid neurones. Tt was shown that under normal conditions the mitochondrial energy production was predominant in the neurons while the cytosomes indicated only little activity. In anaerobiosis the situation is reversed: the Sr2+ accumulation of mitochondria significantly decreases, while the cytosomal activity strongly increases [8]. It thus appears that the accumulation of Exptl Cell Res 80 (1973)
References 1. Karnaukhov, V N, Structure and functions of macromolecules and macromolecular systems, p. 200. Moscow (1969). 2. - Biophysics of living cell, vol. I (1970) 25. Puschino. 3. - Exptl cell res 64 (1971) 301. 4. Karnaukhov, V N & Varton, S S, Cytologij 13 (1971) 1088. 5. Nolte, A, Breucker, H & Kuhlmann, D, Z Zellforsch 68 (1965) 1. 6. Zs-Nagy, I, Anna1 biol Tihany 34 (1967) 25. 7. Zs-Nagy, I & Kerpel-Fronius, S, Abstr 17 int ;mt-g;) electron microscopy, p. 155. Grenoble 8. Zs-Nagy, I, Ann biol Tihany 38 (1971) 117. 9. Karnaukhov, V N, Rozanov, S 1 & Svoren,
VA, Biofysika 11 (1966) 1085. V N, Abstr of 2nd biochem meeting, sect 8, p. 30. Tashkent (1969). USSR 196 (1971) i221. 11. -DAN 12. Bjorkerud, S, J ultrastruct res, suppl. 5 (1963). 13. Bjorkerud, S & Cummins, J T, Exptl cell res 32 (1963) 510. 14. Bjorkerud, S, Advan gerontol res, vol. 1, pl 275. Academic Press, New York and London (1964). 15. - Exotl mol narhol 3 (1964) 377. 16. Hendley, D 6, Mildvan, A S, Reporter, M C & Strehler, B L, J gerontol 18 (1963) 250. 17. Karnaukhov, V N & Zintchenko, V P, Cytologij 13 (1971) 1243. 18. Karnaukhov, V N, Kulakov, V I, Melnikova, E V & Yashin, V A, Cytologij 10 (1968) 654. 19. Davies. B H. Chemistry and biochemistry of plant pigments (ed T Goodwin) p. 489. Academic Press, New York (1965). 20. Kamaukhov. V N. Medvedev. A I. Abdurakhmanov, A & Fin, d T, Biophysics of living cell 1 (1970) 13.
10. Karnaukhov,
Preliminary notes 21. Karnaukhov, V N, Khaspekov, L G, Zintchenko, 22. 23. 24. 25. 26. 21. 28. 29. 30. 31. 32. 33. 34. 35. :;: 38. 39.
V P, Brailovskaya, V A & Lukyanova, L D, DAN USSR 201 (1971) 227. Karnaukhov, V N & Pavlenko, V K, Studia biophysica 23 (1970) 69. HydCn, H & Lindstrcm, B, Disc Farad sot 9 (1950) 436. Karnaukhov, V N, Biophysics of living cell, vol. 2 (1971) 68. Puschino. Karnaukhov, V N & Tataryunas, T B, DAN USSR 203 (1972) 1197. Miyawaki, H, J natl cancer inst 34 (1965) 601. Hack, M H, Colcolough, H L & Helmy, F M, Acta histochem 35 (1970) 357. Fride, R L, Acta neuropathol 2 (1962) 113. Kumamoto, T & Bourne, G H, Acta histochem 16 (1963) 87. Nandy, K, J gerontol 23 (1968) 82. Pullman, B & Pullman, A, Quantum biochemistry. Interscience, New York and London (1963). Dingle, J T & Lucy, J A, Biol rev 40 (1965) 422. Karnaukhov, V N, Cytologij 15 (1973) 532. Sulkin, N M & Srivanij, P, J gerontol 15 (1960) 2. Goodwin, T V, The comparative biochemistry of carotenoids (1952). Smirnov, A I, DAN USSR 73 (1950) 609. Soin, S G, Zoologichesky zhurnal 35 (1956) 1362. - The adaptive peculiarities of fish evolution. MGU-press, Moscow (1968). Karnaukhov, V N, Tataryunas, T B & Petrunyaka. V V, Mech age dev 2 (1973) 201.
Received April 2, 1973
Detection of DNA polymerase activity in fixed cells Z. DARZYNKIEWICZ,
Department of Connectioe Tissue Research, Boston Biomedical Research Institute, Boston, Mass. 02114, USA
It was shown recently that RNA polymerase activity may be successfully localized and studied on cytological preparations. The enzyme retains its activity in situ and catalyses ribonucleotide synthesis in the presence of exogenous ribonucleoside triphosphates after a mild fixation of cells [l-4]. In the present studies, a similar approach was used to localize DNA polymerase activity in eukaryotic cells. After brief fixation, the cells become permeable to deoxyribonucleoside triphosphates (dNTPs) and incorporate them into DNAse hydrolysable macromolecules in the nuclei.
483
Materials and Methods Cell preparations. 3T3 mouse fibroblasts and their simian virus 40 transformed line (SV 3T3) were a gift from Dr P. H. Black, Massachusetts General Hospital, Boston, Mass. HeLa cell line was purchased from Microbiological Associates (Bethesda, Md Cat. No. 71-134). The cells were grown on glasscoverslips in basal Eagle’s medium supplemented with 10% fetal calf se&m and with antibiotics (penicillin 50 PI/ml; streptomycin 50 pg/ml). Coverslips bearing cells to be assayed for DNA polymerase were rinsed in Hanks salt solution, fixed in absolute ethanol : acetone (1 : 1 v/v) for 5 min at o”C, air dried at 4°C and stored at 4°C for not longer than 20 min. To determine the number of cells synthesizing DNA parallel cultures were treated with 3H-TdR (SH-thymidine-methyl, spec. act. 6.7 Ci/mmole; New England Nuclear Corporation, Boston, Mass. 0.5 ,&i/ml) for 1 h. The coverslips were then washed in saline, fixed in ethanol :acetic acid (3 : I v/v) and processed for autoradiography as described elsewhere [5]. autoradiography. One ml of standard incubation medium contained: 100 pmoles of TrisHCI buffer (pH 7.8), lOpmoles of 2 mercaptoethanol; 150,umoles of sucrose; 8 pmoles of MgCl,; 500 nmoles each of dTTP, dCTP and dGTP (Schwarz/Mann, Orangeburg, N.Y.) and 12.5 nmoles of 3H-dATP (12.9 Ci/mmole, New England Nuclear). The coverslips were incubated in this medium at 37°C for up to 20 min. Incubations were terminated by rinsing the coverslips in ice-cold 0.1 mM solution of unlabelled dATP in 0.15 M sucrose buffered with Tris-HCl (pH 7.8) followed by post-fixation in ethanol:acetic acid mixture (3:l v/v) for 2 h. The coverslips were then washed in 70% ethanol, air-dried and mounted on gelatinized slides. Before autoradiography, the slides were treated with 5 % trichloroacetic acid for 10 min at 0°C and rinsed in water for 30 min. Some coverslips were incubated with RNAse (boiled RNAse A, Bovine pancreas, Worthington Biochemical Corp., 1 mg/ml in acetate buffer, pH 5.0, 37”C, for 60 min) or with DNAse (DNAse I, Worthington, 1 mg/ml in 0.01 M MgS04, 37”C, for 2 h. The slides were covered with Kodak AR 10 stripping film and exposed for 6 days. Grains were counted over at least 100 cell nuclei from each individual slide; the mean values and S.E. of counts from the representative slides are presented in the tables and diagrams. All expt were run in triplicate; in all cases the mean values of duplicate cultures did not differ significantly (p < 0.05) from those presented in Results. The percentage of 3H-TdR-labelled cells (S phase cells) was calculated on the basis of counts of 500 cells from autoradiographs of cultures treated with 3HTdR.
Incubations,
Results Cells incubated in standard medium incorporate the label almost exclusively in the nuclei. Interphase cells exhibit the highest labelling; mitotic cells are distinctly less Exptl Cell Res 80 (1973)