IN 'VITRO Vol. 7, No. 1, 1971

TRANSHYDROGENASE ACTIVITY IN MAMMALIAN CELLS IN VITRO: ITS POSSIBLE PHYSIOLOGICAL

SIGNIFICANCE*

C. DE LUCA A~D R. P. GIOELI

Department o/Oral Biology, School o/Dentistry, State University o/New York, Buffalo, New York 14226

SUM'MARY

Transhydrogenase (NADH:NADP oxidoreductase, EC 1.6.1.1) activity has been demonstrated in mammalian cells cultured in vitro. Levels of activity of this enzyme were 10- to 20-fold higher in H4-II-E-C3 cells derived from the minimal deviation Reuber hepatoma than in three other cell lines tested. H4 cells lack the ability to reduce nicotinamide adenine dinucleotide phosphate by the glucose 6-phosphate dehydrogenase reaction. This raised the question of the physiological significance of transhydrogenase in these cells.

Cultured cells of an established line, H4-II-E- C3, derived (1) from a minimal deviation hepa- toma in the rat apparently lack glucose 6-phos- phate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) activity. This enzyme generally has a major role in the production of reduced nicotinamide adenine di- nucleotide phosphate (NADPI-I). I t was of in- terest, therefore, to investigate other possible sources of NADPH in this cell. Oldham, Bell, and Harding (2) have recently emphasized the fact that through the pyridine nucleotide trans- hydrogenase (NADH :NADP oxidoreductase, EC 1.6.1.1) described by Colowick et al. (3) the potential sources for NADP reduction are greatly expanded beyond those available from NADP-linked substrate dehydrogenases. Accord- ingly, studies have been initiated to determine whether this enzyme was present in the cultured hepatoma cells and to ascertain its physiological role in these cells. Transhydrogenase (TH) ac- tivity, apparently like that first described for animal tissues by Kaplan, Colowick, and Neu- feld (4), was indeed shown to be present in H4 cells (De Luca and Gioeli, manuscript in prepa- ration). We wish to report here preliminary ob- servations from studies concerned primarily with the possible physiological significance of this en- zyme in this cell line.

* This work was supported by Grant P513 from the American Cancer Society.

MATERIALS AND METHODS

Cell culture and methodology. The H4-II-E- C3 cell line used in these studies was derived from a minimal deviation hepatoma (1) and was obtained through the generosity of Dr. V. R. Potter. Details of the methods used for growth and maintenance of these cells have been pub- lished elsewhere (5). For these studies, mono- layer cultures were maintained in stationary flasks rather than in roller bottles. The growth medium was medium S-20 supplemented with 20% (v/v) heat-inactivated fetal calf serum. Penicillin and streptomycin (100 units and 50 ~g, respectively, per ml) were routine additions to the medium. Periodic checks for contamina- tion by Mycoplasma, using the culture technique of Hayflick (6), showed the H4 line to be free of this organism. Medium S-20 is now available through Associated Biomedic Systems, Inc. (Buffalo, N. Y.) ; fetal calf serum was purchased from Grand Island Biological Company (Buf- falo, N. Y.).

Comparative studies on the variation of TH throughout a culture growth cycle were done with three other cell lines all grown and treated in an identical manner. These were the NCTC- L929 line, derived from mouse subcutaneous connective tissue; the HTC line, derived from the Morris minimal deviation hepatoma 7288e (7) in the rat; and a fibroblast-like variant, F/rh, from an original H4 sample culture (De Luca, unpublished observations).

13

14 DE LUCA AND GIOELI

General methods for growth cycle studies used in this laboratory have been described elsewhere (8). Replicate 16-ounce prescription bottles each containing 10 ml of medium equilibrated at room temperature were inoculated with 1 to 2 • 100 cells depending oil the cell line used. The cultures were gassed with a mixture of 5% CO~- 95% air to adjust the pH to 7.2 and then incu- bated at 37~ Thereafter, the medium was not changed and the pH was not readjusted.

Enzyme preparation and transhydrogenase assay. Details for harvesting cells have been de- scribed (5, 8). Cells were collected in Potter-Elve- hiem homogenizer tubes and stored frozen at - 1 5 ~ in 5 m~ phosphate buffer. At the time of assay, the harvest was thawed and cells were homogenized. Neutralized digitonin was added (0.3 ml of 1% digitonin per 0.9 ml of cell ho- mogenate), and the mixture was allowed to stand on ice for 15 rain, This preparation was then centrifuged at 800 • g for 10 rain at 5~ The clear, colorless supernatant as the TH preparation. Enzyme cell lines to be compared were same time. The results expressed of two determinations.

fluid was used preparations of assayed at the are the average

The method used to measure TH activity was essentially that of Stein, Kaplan, and Ciotti (9) with modification. I t was based on the reduction of analogues of nicotinamide adenine dinucleo- tide (NAD) in the presence of reduced NAD (NADH) and the TH preparation. The assay mixture consisted of 200 ~moles of Tris buffer (ptI 8.6), water, about 1.00 ~mole of analogue, 0.15 ~mole of dithiothreitol, 0.10 ml of TH prep- aration (containing about 100 ~g of protein), and 0.26 /~mole of NADtt in a total volume of 1.00 ml. The mixture was equilibrated in the spectrophotometer for about 1 rain without NADH; the reaction was then started with the addition of NADH and followed at the wave- length appropriate for the analogue in use. All reaction rates were compared to a water blank and were measured between the interval of 20 to 80 sec after starting the reaction. Enzyme activ- ity was expressed as the change in absorbance (AA) per minute and was calculated per millili- ter of enzyme preparation, i.e., AA per minute per milliliter. All assays were performed at 30~ in 1-ml cuvettes with 1-cm light path. All meas- urements were made with the Gilford model 2400 spectrophotometer set to its highest sensi- tivity. Specific activity was based on protein

content as measured by the method of Lowry et al. (10) and was expressed as AA per minute per milligram of protein.

Analogues. The analogues used as acceptors in these studies were the 3-acetylpyridine analogue of NAD (3AP-NAD) and the thionicotinamide analogue (TN-NAD). Reduction of 3AP-NAD was followed at 380 nm; that of TN-NAD was followed at 400 nm, where the contribution to total absorbance by the NADt t present was minimal. NADH and its analogues were highest purity products of the Sigma Chemical Com- pany.

RESULTS

TH activity, like that of many other enzymes (8, 11), has been shown to undergo quantitative variation or modulation throughout the growth cycle of populations of cultured cells. In order to ascertain the maximum TH activity of which the cell was capable, it was necessary, therefore, to determine the activity each day throughout the entire growth cycle. Two- to 4-fold fluctua- tions between maximum and minimum values over a single growth cycle are commonly ob- served for mammalian cells. Shown in Table 1 are illustrative data for TH activity in It4 cells. These are compared with respective daily activi- ties observed in other mammalian cells cultured under identical conditions. I t can be seen that the TH activity of H4 cells could at a given time be at least 10- and up to 20-fold higher than that of the other cells examined.

Discuss ion

The first demonstration of transhydrogenase activity by Colowick et al. (3) constituted a classical contribution to the concept and mecha- nisms of regulatory processes in the living cell. Although the exact physiological significance of this enzyme has not been completely clarified, a role in regulating relative levels of the oxidized and reduced forms of both NAD and NADP would seem to be most obvious. An activity of this type logically becomes more significant for cells like those of the H4 line which cannot be shown to contain glucose 6-phosphate dehydro- genase (G6PD) either by spectrophotometric (1) or histoehemical methods (De Luea and Mason, manuscript in preparation).

Transhydrogenase was indeed found to be present in these cells. An over-all range of activ- ity could be measured equivalent to 52 to 86

TRANSHYDROGENASE IN CELL CULTURE 15

TABLE 1

C O M P A R A T I V E T R A N S n Y D R O G E N A S E A C T I V I T Y IN M A M M A L I A N C E L L S

Acceptor

3AP-NAI) 3AP-NAD 3AP-NAD TN-NAD TN-NAD

Cell Line

H4-II-E-C3 HTC - L-929 b H4-II-E-C3 F/ rh

0.486 0.054

__r

2

0.378 0.017

__v

Specific Act ivi ty on Day

3 4

AA ~8o/mln/mg ,rotdn

0.298 0.327 0.022 0.041 0.039 0.026

0.381 __c

0.662 __c

0.817 0.270

0.350

0. 463 0.163

All specific activities were corrected for endogenous activity. b TH activity was observed only after a 2.5-rain lag. c No TH activity detected.

m~moles of analogue reduced per min per mg of protein when 3AP-NAD was used as aceeptor. This activity is comparable to the 59 m/zmoles reduced per rain per mg reported by Stein et al. (9) for digitonized mitochondria from the Novi- koff hepatoma grown in vivo. On the other hand, it is only of the order of one-tenth to one-sixth that observed by these same authors for rat heart preparations (490 m/~moles per min per mg). Evans and Kaplan (12) reported a range of 4 to 18 m/xmoles per min per nag for particulate frsetions from sonicates of leuko- cytes from normal controls and leukemia pa- tients, respectively. Further, significantly higher levels of TH were found in H4 cells in compara- tive studies with three other cell lines all eul- tured simultaneously and under identical condi- tions. These results might suggest an inverse re- lationship between TH activity and cellular ca- pacity for substrate level reduction of pyridine nueleotides. A more complete survey of sub- strate-dependent potential and TH levels would be necessary to validate this suggestion. This is in progress.

The possible relationship between the TH found in H4 eells, presumed to be intramito- ehondrial, and the lack of G6PD, most often described as soluble, may not be readily appar- ent. However, the presence of shuttle systems for transporting reducing equivalents across the mitoehondrial membrane (13, 14) and considera- tion of the general capabilities of complex regu- latory circuits operative in cells make an indi- rect relation between these not at all an unrea- sonable possibility. Assignment of physiological significance on the basis of quantitative meas- urements must take into consideration the fact

that cellular metabolism is influenced by envi- ronmental conditions. This is a most important consideration in the celt culture system where direct contact, biochemical as well as physical, between cells and environment exists. I t is, how- ever, precisely this metabolic sensitivity, coupled with the feasibility of stringent control, that makes the cell culture system an ideal experi- mental model for elucidating questions of the type posed here. Fur ther studies on the physio- logical significance of TH in the H4 line are in progress.

REFERENCES

1. Pitot, H. C., C. Peraino, P. H. Morse, Jr., and V. R. Potter. 1964. Hepatomas in tissue culture compared with adapting liver in vivo. Nat. Cancer Inst. Monogr. 13: 229-245.

2. Oldham, S. B., J. J. Bell, and B. W. Harding. 1968. Role of the bovine adrenal cortical pyridine nucleotide transhydrogenase in ste- roid llfl-hydroxylation. Arch. Bioehem. Bio- phys. 123: 496-506.

3. Colowiek, S. P., N. O. Kaplan, E. F. Neufeld, and M. M. Ciotti. 1952. Pyridine nueleotide transhydrogenase. I. Indirect evidenee for the reaction and purification in the enzyme. J. Biol. Chem. 195 : 95-105.

4. Kaplan, N. O., S. P. Colowick, and E. F. Neu- feld. 1953. Pyridine nueleotide transhydroge- nase. III. Animal tissue transhydrogenases. J. Biol. Chem. 205: 1-15.

5. De Luca, C., G. B. Valls, and J. A. Mason. 1970. Drug effects on catalase activity in the mam- malian cell: the role of the cell in drug ac- tion. Bioehem. Pharmacol. 19: 2211-2220.

6. Hayflick, L. 1969. The Mycoplasmatales and the L Phase o] Bacteria. Appleton-Century- Crofts, New York.

7. Thompson, E. B., G. M. Tomkius, and J. F. Curran. 1966. Induction of tyrosine ~-keto- glutarate transaminase by steroid hormones

16 DE LUCA AND GIOELI

in a newly established tissue culture cell line. Proc. Nat. Acad. Sci. U. S. A. 56: 296-303.

8. De Luca, C. 1966. Effects of mode of culture and nutrient medium on cyclic variations in enzyme activities of mammalian cells cul- tured in vitro. Exp. Cell Res. 43: 39-50.

9. Stein, A. I-I., N. O. Kaplan, and M. M. Ciotti. 1959. Pyridine nucleotide transhydrogenase. VII. Determination of the reactions with co- enzyme analogues in mammalian tissues. J. Biol. Chem. 234 : 979-986.

10. Lowry, O. It., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

11. Pan, Y.-L., and R. S. Krooth. 1968. The influ-

ence of progressive growth on the specific catalase activity of human diploid cell strains. I. Effect of cellular genotype: homo~ zygous strains. J. Cell. Physiol. 71: 151-160.

12. Evans, A. E., and N. O. Kaplan. 1966. Pyridine nucleotide transhydrogenase in normal hu- man and leukemic leukocytes. J. Clin. Invest. 45 : 1268-1272.

13. Boxer, G. E., and T. M. Devlin. 1961. Pathways of intracellular hydrogen transport. Science 134 : 1495-1501.

14. Ball, E. G. 1966. Regulation of fatty acid syn- thesis in adipose tissue. In: G. Weber (Ed.), Advances in Enzyme Regulation. Vol. 4. Pergamon Press, New York, p. 11.

We wish to thank Dr. Joseph H. Kite, Jr., for checking our cultures for the presence of Mycoplasma.