Human cell dehydroascorbate reductase. Kinetic and functional properties

15

Bzochlmwa et Blophyswa Acta, 659 (1981) 15--22 Elsewer/North-Holland Bmmedmal Press

BBA 69249

HUMAN CELL DEHYDROASCORBATE REDUCTASE KINETIC AND FUNCTIONAL PROPERTIES

ROBERT BIGLEY, MA2~rHEW RIDDLE, DONALD LAYMAN and LIBUSE STANKOVA

Department of Medwme and Dlwszon of Medlcal Genetws, Unwers~ty of Oregon Health Sciences Center, Portland, OR 97201 (US A ) (Received November 5th, 1980)

Key words Dehydroascorbate reductase, Dehydroascorbate uptake, (Human)

Summary Dehydroascorbate reductase (glutathmne dehydroascorbate oxldoreductase, EC 1.8.5 1) actlwty was examined m crude cytosol extracts of human cells. In blood neutrophfl and lymphocyte extracts, the Km at pH 6.85 for dehydroascorbate was 1.3 mM, and for reduced glutathmne 3.8 mM Rates of dehydroascorbate uptake by mtact human neutrophfls, monocytes, lymphocytes and cultured flbroblasts were proportmnal to cytosol dehydroascorbate reductase actlwtms. Dehydroascorbate reductmn durmg dehydroascorbate uptake by these cells may be entirely enzymatm.

Introduction Dehydroascorbate, but not reduced ascorbate, is taken up from suspending media by human blood neutrophds, virtually all of the dehydroascorbate taken up is promptly reduced [1]. Dehydroascorbate reductase (glutathlone: dehydroascorbate oxldoreductase, EC 1.8.5.1) catalyzes the reduction of dehydroascorbate to reduced ascorbate m plant [2] and animal [3--5] tissues. Since dehydroascorbate is readily reduced nonenzymatmally by GSH, the function of dehydroascorbate reductase is problematm. Studms m glutathlone reductasedefmmnt cells suggest that dehydroascorbate reduction is a necessary condition for dehydroascorbate transport across neutrophfl membranes [6] To explore the relationship between enzymatm and nonenzymatm dehydroascorbate reduction m cells, and between cellular dehydroascorbate uptake and reduction, we have determmed some kmetm propertms of crude cytosol dehy0005--2744/81/0000--0000/$02 50 © Elsewer/North-Holland Blomedmal Press

16 droascorbate reductase, and measured this activity along with dehydroascorbate uptake In human neutrophfls, lymphocytes, m o n o c y t e s and cultured fIbroblasts Experimental procedures Venous blood and skin biopsies were obtmned from healthy, Informed and consenting human donors Neutrophfls, 90-+ 5% pure, were separated from heparmlzed blood by Dextran (Mr 117 000, Sigma Chem. Co , St. Louis, MO) sedimentation followed by lysls of contaminating erythrocytes with me-cold NH4C1, 0.87 g/100 ml. L y m p h o c y t e s and m o n o c y t e s were separated by centrlfugatlon over Fmoll-Hypaque (Flcoll-Paque, Pharmacla, Plscataway, NJ). After the m o n o c y t e s had adhered to the surface of plastic p e t n dishes [7], the lymphocyte-contmnmg supernatant was poured off. Adherent m o n o c y t e s were washed three times, then removed from the plastm by mcubatlon for 15 mln at 26°C with 30 mM hdocalne-HC1 (crystalline Xylocalne, kindly donated b y Astra Pharmaceutical Prod., Inc., Worcester, MA) [8]. Skin biopsies were minced and explanted into 25 cm 2 tissue culture flasks contmnlng 2 ml McCoy's 5a medium (Flow Laboratories, Rockvllle, MD) supplemented with heat-reactivated 20% (v/v) pooled human serum, 200 umts/ml pemcfllm and 200 pg/ml streptomycin. The cells that grew from the explanted tissue were dissociated by mcubatlon for 5 mln with 0.05% trypsm-EDTA and subcultured m 75 cm 2 Falcon plastic tissue flasks containing growth medium with 10% (v/v) human serum. Culture medium was changed twice weekly. One-to-two week confluent cultures contammg 4--6 • 106 flbroblasts per flask were used for assays. The procedure for measuring dehydroascorbate uptake by suspended, intact cells was as described previously [9]. Briefly, 5 • 106 cells were incubated for 20 mm at 37°C in 1 ml Ca2÷-free Krebs-Ringer phosphate buffer, pH 7.4, contammg 5.5 mM glucose and 100 pg [14C]dehydroascorbate, 0.02 /~Ci/~g, freshly prepared from L-[14C]ascorbIc acid (New England Nuclear, Boston, MA) and L-ascorbm acid (Sigma Chem. Co ). Dehydroascorbate was prepared by bubbling Br2 vapor through a freshly prepared solution of reduced ascorbate m 0.154 M NaC1, Br2 was washed o u t with water-saturated N2. Dehydroascorbate concentrations were measured colonmetncally, usmg the dmitrophenylhydrazme method of Roe et al. [10]. Uptake was stopped by placing reaction tubes m Ice water. Cells were unmedlately washed three times with 4°C 0.154 M NaC1, then counted with a h e m o c y t o m e t e r . Aquasol (New England Nuclear) was added to centrifuged cells and radioactivity was measured in a Packard TrlCarb scmtlllatlon counter. For those assays made on adherent fIbroblasts, culture medium was replaced with 9 ml Krebs-Rmger phosphate buffer contammg 1.1 mg/ml glucose. After premcubation for 25 m m at 37°C, 1 ml [14C]dehydroascorbate, 1 mg/ml, was added, and the incubation was continued for 20 mm. The adherent cells were then washed three times with 20 ml 4°C 0.154 M NaC1, dissociated with trypsm-EDTA, washed once more, and counted as above. Measured colommetrically by the m e t h o d of Roe et al. [10], m e d m m dehydroascorbate is quantitatively recovered as dehydroascorbate after 20 mm mcubatmn with cells, except for that fraction recovered as reduced ascorbate m cells, total recovery is 98.5 -+ 1.1% [1]. Using reagent concentrations as m

17 the present study, colorlmetncally-measured dehydroascorbate uptake and reduction are lmear over the range 1 - - 6 . 1 0 6 cells/ml reaction mixture [1]. Dlketogulonate is detected only as a minor fraction of cellular total ascorbate, it does n o t change in a m o u n t during incubation [1]. Measured radlochromatographlcally after 20 mln premcubatlon, 86--96% of the dehydroascorbate taken up by cells is in the form of reduced ascorbate [6]. This discrepancy between radlochromatographlc and colorlmetrm measurements may reflect a fraction of dehydroascrobate which was reduced upon uptake by cells and subsequently oxidized. Radlochemlcal measurement of cellular dehydroascorbate uptake is 98.5 + 3.6% effmlent as compared to colonmetrlc measurement m duplicate samples (n = 6) The present study includes radlochemmal measurements of cellular dehydroascorbate uptake. Reduction of dehydroascorbate upon uptake was assumed on the basis of previous measurements, it was n o t quant~tated m this study. Reduced glutathlone and glutathlone reductase actlwty were assayed accordmg to Beutler [11]. Protem was measured by the m e t h o d of Lowry et al. [12] usmg bovine serum albumm as a standard. Stimulated hexosemonophosphate shunt activity was measured contmuously usmg the gas flow-lomzatlon chamber m e t h o d [1]. Cell extracts for enzyme activity measurements were prepared by homogenizing suspensions of 6--10 • 107 cells/ml in 0.154 M NaC1, for 5 mln at 3500 rev./mln m a glass-Teflon homogemzer held m Ice, and centrffugmg for 90 m m at 4°C and 12 000 × g . The supernatants used for assaymg dehydroascorbate reductase were dialyzed at 4°C for 20--24 h agmnst 200 vol. 50 mM phosphate buffer, pH 6.85, in order to remove GSH. The resulting precipitate was separated by centrlfugatmn at 12 000 × g, 4°C for 10 mm, and discarded. The standard reactmn mLxture for the coupled dehydroascorbate reductase assay contamed 50 mM phosphate buffer/0.27 mM EDTA/0.2 mM NADPH/2.0 mM GSH/ 2 I.U./ml glutathlone reductase/2.0 mM dehydroascorbate/cell extract equivalent to 1 0 - - 2 0 . 1 0 6 cells/ml; the fmal pH was 6.85. The reactmn mLxture w i t h o u t dehydroascorbate and cell extract was mcubated for 5 mln at 37°C. After addition of dehydroascorbate, A340 was recorded for approx. 1.5 m m , as a control for reagent concentratmns. Cell extract was then added, and A340 was recorded for another 1.5 mln. The reaction mixture for direct assay contamed the same components as the coupled assay except for NADPH and glutathlone reductase; reduced ascorbate concentratmn was measured using the 2,6~hchlorolndophenol technique of Wolf et al. [13] after 0, 3 and 6 m m incubation at 37°C. In both assays, n o n e n z y m a t m reduction of dehydroascorbate by GSH was measured under ldentmal conditions except t h a t an equal volume of 0.154 M NaC1 was substituted for cell extract. This measurement was subtracted from that obtained with cell extract to d e t e r m m e the net enzymatic rate of dehydroascorbate reduction.

Results Dehydroascorbate reductase was measured both directly, as the rate of reduced ascorbate production (Eqn. 1), and m the coupled assay, as the rate of NADPH oxidation (Eqns. 1 and 2)

18 dehydroascorbate + 2 GSH dehydroa~corbatered~cta~ reduced ascorbate + GSSG (1) GSSG + NADPH + H ÷ GSSGr e d u c ~ s e 2 GSH + NADP ÷

(2)

The direct and coupled assays measure dehydroascorbate reductase activity with equal efflcmncy, with a measurement error of -+7.5%. This confirms that the oxidation of 1 mol NADPH in the coupled assay is eqmvalent to reductmn of 1 mol dehydroascorbate (algebraic sum of Eqns. 1 + 2). Since the coupled reaction can be measured more quickly, is less subject to error and reqmres less enzyme, it was used for the other expernnents described here. The pH dependence of n o n e n z y m a t m dehydroascorbate reductmn and of neutrophfl extract activity are illustrated m Fig. 1. The pH o p t i m u m of the enzymatic reaction is close to 7.4. Since the rapidity of the n o n e n z y m a t m reaction increases measurement error in the higher pH range the standard assay was performed at pH 6.85. Figs. 2 and 3 illustrate the effects of dehydroascorbate and GSH concentrations on neutrophfl dehydroascorbate reductase actlwty At pH 6.85, the Km for dehydroascorbate is 1.33 mM, for GSH It is 3.8 mM. Affinity of the enzyme for dehydroascorbate increases with lncreasmg pH. At pH 7.25, the Km for dehydroascorbate IS 0.54 mM. Substrate inhibition IS observed at dehydroascorbate concentrations above 2 mM. L y m p h o c y t e dehydroascorbate reductase exhibits the same kinetic properties. Table I shows rates of dehydroascorbate uptake by equal volumes of normal h u m a n cultured skin flbroblasts and blood neutrophlls, l y m p h o c y t e s and monocytes, along with cytosol GSH concentratsons and glutathlone reductase, stimulated hexosemonophosphate shunt and dehydroascorbate reductase actlv-

40

3C

~ 20 Q.

C

0

65

70

75

pW

Fig 1

Enzymatac and nonenzymatlc

dehydroascorbate reduction

pH dependence

Dehyd~oascorbate

reduet*on w a s m e a s u r e d b y t h e s t a n d a r d c o u p l e d a s s a y d e s c r i b e d m E x p e r i m e n t a l procedures, except t h a t t h e compos*t*on of t h e p h o s p h a t e b u f f e r w a s v a n e d t o a c h , e v e f i n a l p H as i n d i c a t e d E n z y m a t * c reduct*on represents act*wty m the presence of neut~ophfl extract equivalent to 6 t u r e , less m e a s u r e d n o n e n z y m a t l c a c t i v i t y • e, n e t e n z y m a t i c , o

10 5 cells m 1 m l o f r e a c t * o n rnLxo, n o n e n z y m a t * c

GSSG reductase nmol/mtn per mg protein

* M e d i u m d e h y d r o a s c r o t a t e , 0 55 m M , g l u c o s e , 5 5 m M ** M e t h y l e n e b l u e , 2 1 m M .

Dehydroascortate reductase nmollmm per mg protein 55

26

0 72

0 05

015

±

± 23

5

+ 159

4 28 ± 358

H M S actavzty, m e t h y l e n e b l u e stem ** n m o l N A D P H o x l d / n u n p e r 0 1 m l cells

G S H c o n t e n t n m o l / 0 1 m l cells

0 57 ±

HMS actawty during dehydroascortate uptake * n m o l N A D P H o x l d / r a m p e r 0 1 m l cells

+

048

Dehydroascortate uptake n m o l / m m p e r 0 1 m l cells

Ftbroblasts 107 c e I L s = O l m l

UPTAKE AND ITS POTENTIAL DETERMINANTS

HMS, hexosemonophosphate shunt

DEHYDROASCORBATE

TABLE I

±

170

64

678

412

042

090

±

±

22

24

± 220

44 42 ±

220±

300

Lymphocytes 3 108 = 0 1 m l

±

448

39

249

255

049

±

±

63

14

±

±

1190

51

172

277

069

200

+

96

± 18

± 16

28 9 0 ±

920

2100

Neutrophxls 108 = 0 1 m l

FIBROBLASTS

178

± 128

15 30 ±

405±

805

Monocytes 108 = 0 1 m l

IN HUMAN BLOOD CELLS AND CULTURED

¢0

20 20

20

15

15

0

IO

P

/

/

I0

0

i--,

•

Km05

0

/

~[A]

33m

I

I

[

I

I

2 [A], m M

3

4

~o~o~.... ~ I

0

I

I

I

2 3 [GSH],mM

"° I

4

Fig 2 N e u t r o p h d c y t o s o l d e h y d r o a s c o r b a t e r e d u c t a s e a c t i v i t y vs d e h y d r o a s c o r b a t e c o n c e n t r a t i o n Fig 3 N e u t r o p h d c y t o s o l d e h y d r o a s c o r b a t e r e d u c t a s e a c t i v i t y vs r e d u c e d g,l u t a t h t o n e c o n c e n t r a t i o n

1ties In each cell type, both glutathlone reductase activity and methylene bluestimulated hexosemonophosphate shunt activity were greater than the measured rates of dehydroascorbate uptake Thus, it is unhkely that dehydroascorbate uptake as measured here could have been hmlted by GSH avallablhty Among cell types, dehydroascorbate uptake rates had no consistent relationship with GSH content, glutathlone reductase activity or stimulated hexosemonophosphate shunt activity Fig 4, a plot of data from Table I, shows that rates of dehydroascorbate uptake for each cell type were d~rectly proportional to measured cytosol dehydroascorbate reductase activities To determine whether cell surface area or the dlssoclatmn procedure might affect the rate of dehydroascorbate transport across membranes, uptake was measured m four prated samples of cultured flbroblasts, one of each pair incubated with [14C]dehydroascorbate while stall adherent to the flask surface and the other incubated after harvesting with trypsm-EDTA. Both kinds of preparatmn t o o k up dehydroascorbate at the same rate.

~ 200 I00

'~

o

:°°cles

'

50 I00 A Reductose,nmol /mm/rngprotem

Fig 4. C y t o s o l d e h y d r o a s c o r b a t e r e d u c t a s e (A r e d u c t a s e ) activities vs r a t e s o f d e h y d r o a s c o r b a t e u p t a k e b y i n t a c t h u m a n cells

21 Dlscuss~on

Plants contam both dehydroascorbate reductase and reduced ascorbate oxldase [ 14] activities. In the absence of evidence that mammahan hssues possess reduced ascorbate oxldase activity, it is reasonable to question the function of mammahan dehydroascorbate reductase. Dehydroascorbate conshtutes 5--20% of total ascorbate m normal human plasma and leukocytes [15,16]. The plasma dehydroascorbate fraction may be considerably higher in certain disease states [15]. Although enzymatm dehydroascorbate productmn has not been demonstrated m intact mammalian tissues, it may occur, for example m those situations where reduced ascorbate appears to protect p-hydroxyphenylpyruwc acid oxldase from substrate mhlbltmn [17,18] and dunng norepmephrme bmsynthesls catalyzed by dopamme ~-hydroxylase [19]. Reduced ascorbate autoxldatmn catalyzed by transltmn metal ions and reactmns of reduced ascorbate with oxidants and free radmals are other potential sources of tissue dehydroascorbate. Cellular total and reduced ascorbate are depleted during the enhanced productmn of oxidants and free radmals whmh accompames phagocytosm, at the same time, cell dehydroascorbate increases [20]. One function of dehydroascorbate reductase may be to regenerate tissue reduced ascorbate whmh has been enzymatmally or nonenzymatmally oxidized. Its efflcmncy may determine the capacity of cellular reduced ascorbate to serve as an enzyme protector and substrate, and as a part of mechamsms whmh mactlvate potenhally injurious oxidants and free radmals. Dehydroascorbate taken up by mtact neutrophfls from suspending solutmn is wrtually completely reduced when measured lmmedmtely after uptake [1]. Dehydroascorbate uptake is restricted by the rate of GSH regeneratmn m glutathrone reductase-defmmnt human neutrophfls [6] Thus, at least m neutrophfls, dehydroascorbate reductmn is reqmred for dehydroascorbate uptake. The present study shows that the rate of dehydroascorbate uptake among four normal human cell types correlates with cytosol dehydroascorbate reductase actlwty, it does not correlate with GSH content or with the actlvltms of GSHregenerating enzymes. These observatmns suggest that the dehydroascorbate reductmn whmh occurs during dehydroascorbate uptake by intact cells depends on dehydroascorbate reductase actlwty. Acknowledgements This work was supported by grants from the Oregon Affiliate, Amerman Diabetes Association, the American Cancer Socmty, grant CH-30A; Nahonal Institutes of Health, grants HL 24216-01 and HD 27483-01, and Job's Daughters. References 1 2 3 4 5 6

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7 8 9 10 11 12 13 14 15 16 17 18 19 20

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