Mitochondrial enzyme activity during in vitro ageing of human diploid

0 downloads 0 Views 245KB Size Report
Mean specijic activities of mitochondrial enzymes during in vitro ageing. 0-S ATPase .... yeasts, insects and man (Baum & Palmer, 1985) points to a degree of ...
I I76

BIOCHEMICAL SOCIETY TRANSACTIONS

Table 1. The concentration of some metabolites in jilarial helminths The figures are the mean ~ s . E . M .of either n = 6 controls or n = 3 experimental. Worms were incubated in 50ml of MEM at 37°C for 2 h in the presence of 1 O p drug ~ (except KCN, 1 0 0 ~ ~Nucleotides ). were measured by h.p.1.c. using neutralized perchloric acid extracts. [Lactate] and [succinate] were measured in the medium by standard spectrophotometric methods. AEC, adenylate energy charge; ND, not determined; * P < 0.05; **P < 0.02. AMP

AEC

D. viteae B. pahangi (nmol/mg protein) Controls Rotenone Antimycin A KCN CCCP Rafoxanide Oligomycin BWA466C BWA728C

2.6 4.5 5.2 6.7 4.4 5.1 5.4 8.3 5.3

f 0.4 f 0.8* f OX** f 0.2** f 0.1** f 1.0** f 0.9** f 0.4** f 2.1

0.7 f 0.1 ND 1.2 f 0.2** 1.8 f 0.4** 1.9 & 0.2** 3.7 f 0.2** 0.8 0.2 5.6 f 0.3** 3.4 f 0.4**

+

D. viteae 0.900 0.853 0.852 0.822 0.862 0.832 0.854 0.707 0.821

f 0.004 f 0.016* f 0.015** f 0.011** f 0.005** f 0.022** f 0.013** f 0.016** 0.041

+

Lactate B. pahangi

D. vifeae

0.919 & 0.009 ND 0.833 f 0.010** 0.850 f 0.013** 0.845 f 0.009** 0.713 f 0.012** 0.909 f 0.012 0.661 f 0.034** 0.704 f 0.018**

828 f 28 920 & 67 1053 f 34** 1162 f 31** 1354 f 31** 734 f 58 1275 f 47** 607 f 53* 656 f 24**

suggest that the observed oxygen uptake was mitochondrial (O’Dowd et af., 1988) and that inhibition of oxidative phosphorylation caused significant metabolic effects (Table 1). The increased rate of lactate production caused by some agents might be explained as a mechanism to maintain ATP levels. However, in the case of B . pahangi, ATP was depleted and in D. viteae the concentration of AMP was increased with a concomitant decline in the AEC (Table 1) and ATP/ADP ratio (data not shown). Thus, the presumed increased rate of anaerobic ATP provision was unable to restore the energy status of the worms to control values; although motility was unaffected by the respiratory chain inhibitors (O’Dowd et al., 1987). Only three compounds affected motility, namely rafoxanide, BWA466C and BWA728C (O’Dowd et al., 1987) and the loss of movement was associated with a severe ATP depletion (data not shown). The effect of oligomycin was interesting because only D. viteae was affected (O’Dowd et al., 1987; Table I); this might reflect a difference in permeability of the compounds across the filarial cuticle or perhaps a difference in the ATPase enzyme. In summary, filarial helminths consumed oxygen which may be coupled to ATP synthesis. However since the P/O

Succinate

B. pahangi

D. viteae (nmol/2 h per mg wet wt.) 1266 f 33 ND 1636 f 85** 1679 f 30** 2097 f 41** 1020 f 42* 1411 f 57 819 f 24** 863 +_ 9**

0 10 21 0 19 21 9 18 11

B. pahungi 0

f3 f3 f3 f9 + 2 f 1 f9

ND 3 f 0.3 0 4 f I 13 f 3 0 ND ND

ratio is unknown (see Saz, 1981) experiments utilizing isolated mitochondria will have to be performed before making any detailed energy balance calculations. This work was supported by the World Health Organization, Onchocerciasis Chemotherapy Project, OCT contract No. 82005.

Bergmeyer, U . (1974) Methods ofEnzyma/ic Analysis, Academic Press, London and New York Chapman, A. G . & Atkinson, D. E. (1973) J . Biol. Chem. 248, 83098312 Mendis, A. H. W. & Townson, S. (1985) Mol. Biochem. Parasitol. 14, 337 -3 54 Murgatroyd, R. C., Rees, M. J., Hayes, D. J. & Comley, J . C. W. (1987) Trop. Med. Parasitol. 38, 69 ODowd, A. B., Hayes, D. J., Selwood, D. L. & Stables, J. N. (1987) Biochem. Soc. Trans. 15, 11741 175 Rew, R. S. & Saz, H. J. (1977) J . Parasitol. 63, 123-129 Saz, H. J. (1981) Annu. Rev. Physiol. 43, 323-341 Wang, E. J. & Saz, H. J. (1974) J . Parasirol. 60,316-321 Received 18 June 1987

Mitochondria1 enzyme activity during in vitvo ageing of human diploid fibroblasts NICOLA HARPER, IRAJ GHADIMINEJAD*, HAROLD BAUM* and ALAN H. BITTLES Departments of’ Anatomy & Human Biology and *Biochemistry, King’s College London, London W C 2 R 2 L S , U . K . At early CPD levels, the energy requirements of cultured human diploid fibroblasts (HDF) are met almost exclusively by glutamine oxidation and anaerobic glycolysis, the relative rates of glutamine and glucose utilization being subject to reciprocal regulation (Zielke et al., 1978; Sumbilla et al., 1981). With ageing in vitro, there is a highly significant shift to glycolysis (Bittles & Harper, 1984), and it has been suggested that this may indicate a decrease in oxidative phosphorylation caused by reduced structural and/or functional integrity of the mitochondria (Bittles & Sambuy, Abbreviations used: HDF, human diploid fibroblast(s); CPD, cell population doubling(s); PBS, phosphate-buffered saline; 0 - S ATPase, oligomycin-sensitive ATPase; GDH, glutamate dehyrogenase; MDH, malate dehydrogenase.

1986). To assess the possible effects of ageing in vitro on H D F mitochondrial enzyme activity, the specific activities of three representative enzymes were determined at four stages during the cellular life-span in culture. Human diploid embryonic lung fibroblasts, strain 2002 (Flow Labs), were roller cultured as previously described (Bittles & Harper, 1984). The life-span in vitro of this cell strain has been defined as 60 f 3 CPD. Cells were harvested at CPD 22, 33, 43 and 55 using trypsin/EDTA, washed three times with PBS ‘A + B + C’, resuspended in 0.03 Mphosphate buffer pH 6.8, and disrupted by maximum amplitude sonication at 0°C for 3 x 10s. Particulate material was removed by centrifugation, 12000g for 15 min at 4°C. 0 - S ATPase activity was measured by the method of Pullman & Penefsky (1 963); G D H by that recommended by the D.G.K.C. (1974) and M D H activity according to Bergmeyer & Bernt (1974). Total protein was determined by the Hartree (1972) method. All enzyme and protein assays were carried out in triplicate or quadruplicate. An analysis of variance was run on the means of each set of results to compare their statistical significance. 1987

1177

623rd MEETING, CANTERBURY Table 1. Mean specijic activities of mitochondrial enzymes during in vitro ageing CPD 22 33 43 55

0 - S ATPase (unit/mg protein)

Malate dehydrogenase (unit/mg protein)

Glutamate dehydrogenase (m unit/mg protein)

63. I 61.6 57.9 64.7

29 1 270 240 298

3.8 3.7 6.6 10.6

Between C P D 22 and 55 no significant changes were observed in the specific activities of either 0 - S ATPase, a mitochondrial inner membrane enzyme, or MDH, located in the matrix (Table 1). However, there was a highly significant, age-related increase in the activity of the matrix enzyme G D H (P < 0.0001). In an earlier study, H D F intracellular glutamine concentration was shown to increase concomitantly with advancing C P D in culture, ascribed to the general reduction in metabolic activity characteristic of ‘older cells’ (Sambuy & Bittles, 1982). The G D H results obtained in the present investigation suggest that this interpretation requires revision: the increased intracellular glutamine concentration more probably reflected a primary, age-related decline in glutamine oxidation. Although the precise nature of the putative defect(s) associated with this change remains to be elucidated, structural and compositional changes in H D F mitochondrial membranes have been demonstrated by electron microscopy (Johnson, 1984) and immunoblotting (Ghadiminejad et al., 1987). As glutamine provides 30-50% of the energy requirements of H D F at low C P D (Zielke et al., 1984), any perturbation in the availability of the amino acid for energy provision must have profound effects at the cellular level. In particular, the switch to glycolysis

previously observed may result in reduced availability of glucose as a source of ribose moieties for nucleic acid biosynthesis. Bergmeyer, H. U. & Bernt, E. (1974) in Meth0d.s of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd edn., pp. 613, Academic Press, New York Bittles, A. H. & Harper, N. (1984) Biosci. Rep. 4, 751-756 Bittles, A. H. & Sambuy, Y. (1986) in The Biology of Human Ageing (Bittles, A. H. & Collins, K. J., eds.), pp. 49, Cambridge University Press Deutsche Gesellschaft fur Klinische Chemie (1974) Z . Klin. Chem. Klin. Biochem. 12, 391-395 Ghadiminejad, I., Harper, N., Bittles, A. H. & Baum, H. (1987) Biochem. Soc. Trans. 15, I 1 77- I 1 78 Hartree, E. F. (1972) Anal. Biochem. 48, 422-427 Johnson, J. E. (1984) in Aging and Cell Structure (Johnson, J. E., ed.), vol. 2, pp. 37, Plenum Press, New York Pullman, M. F. & Penefsky, H. S. (1963) Methods Enzymol. 6,277-284 Sambuy, Y. & Bittles, A. H. (1982) Mech. Ageing Dev. 20, 279-287 Sumbilla, C. M., Zielke, C. L., Reed, W. E., Ozand, P. T. & Zielke, H. R. (1981) Biochim. Biophys. Acta 675, 301-304 Zielke, H. R., Ozand, P. T., Tildon, J. T., Sevdalian, D. A. & Cornblath, M. (1978) J . Cell Physiol. 95, 41-48 Zielke, H. R., Zielke, C. L. & Ozand, P. T. (1984) Fed. Proc. 43, 121-125

Age-dependent loss of a mitochondrial antigen in cultured human diploid fibroblasts IRAJ GHADIMINEJAD, NICOLA HARPER*, ALAN H. BITTLES* and HAROLD BAUM Departments of Biochemistry and *Anatomy and Human Biology, King’s College, London, W8 7AH, U.K.

The precise identity of these various antigens is still a mystery. The major (‘M2’) antigens are normally, but not exclusively, associated with the inner mitochondrial membrane (Ghadiminejad & Baum, 19876). The 52 kDa peptide (‘M4’) has been less extensively studied, but may Introduction be associated with the outer mitochondrial membrane The presence of antimitochondrial antibodies (AMA) in (Ghadiminejad & Baum, 1986). Whatever their identity, the the sera of patients with primary biliary cirrhosis (PBC) is cross-reactivity of these antigens from species as diverse as characteristic of this disease (Munoz et al., 1981). However, yeasts, insects and man (Baum & Palmer, 1985) points to a there is some diversity between patients as to the mito- degree of conservation compatible with some key cellular or chondrial antigens against which these AMA react (Baum & developmental role(s). Because of this, and since a change in Palmer, 1985). The majority of patients show reactivity on mitochondrial activity has been implicated in the process of immunoblots against a major antigenic peptide (‘M2’) of ageing (Harper et al., 1987), we have examined the reactivity Mr74 kDa (for bovine heart mitochondria) together with of homogenates of cultured human diploid fibroblasts of a number of less prominent bands, frequently including different cell population doubling levels (CPD) against relatively strong ones of Mr54 and 43 kDa (Frazer et al., the AMA of marker PBC sera, of the common (74kDa 1985; Lindenborn-Fotinos et al., 1985). The molecular mass reactive-‘M2’) and less common (52 kDa reactive-‘M4’) of the major band is species-dependent but, within a given type, to determine if any age-related difference in reactivity species, organ-independent (Ghadiminejad & Baum, 1987~). could be detected. However, a minority of patients, although clinically indistinguishable from the others, show reactivity predominantly Materials and methods against an antigen (‘M4’) of Mr52 kDa, (Lindenborn-Fotinos The ‘M2’ and ‘M4’ sera used were from patients with e t a l . , 1985; Ghadiminejad & Baum, 1986). In this case clinically defined, stage-three PBC, and exhibited reactivities, the size of the antigen is apparently species-independent in all immunological tests, fully characteristic of the ‘M2’ (Ghadiminejad & Baum, 1986). and ‘M4‘ classifications respectively. Human diploid fibroblasts were roller cultured, harvested Abbreviations used: AMA, antimitochondrial antibodies; PBC, and sonicated at CPD 22, 33, 43 and 55 (Harper e t a l . , primary biliary cirrhosis; ELISA, enzyme-linked immunosorbent 1987). Quantitative ELISA, cellular immunofluorescence assay. Vol. 15