Thyroxine stimulation of rat liver microsomal NADH-cytochrome c reductase in vitro

Pergamon Preae

Life Sciences Vol . 15, pp . 2059-2068 Printed in the D .S .A .

THYROiXINE STIMULATION OF RAT LIVER MICR06OMAL NADH-CYTOCHROMB C RSDUCTASE IN VITRO Fred H . Faas, ililliam J . Carter, and James 0 . Wysn Department of Medicine, IIniveraity of Arkansas Medical Center Little Rock, Arkansas

72201

(Received in final form 12 November 1974) Rat liver microsomai NADH- cytochrome, reductase activity is stimulated by 20 yM thyroxine is vitro . Thyroxine does not influence microsamal NADH-dich~rophenolindophenol reductase, NADPH-cytochrome ç reductase, or NADPH-dichlorophenolindophenol reductase activity . Stimulation of NADHcytochrome ç reductase activity is not mediated by euperoxide and is likely due to enhanced reduction or oxidation of cytochrome ~ . Thyroxine stimulates rat liver microsomal fatty acid deaaturation in vitro (1) .

These reactions are known to involve the microeomal electron transport

chain, and the evidence to date indicates that cytochrome ~ but not cytochrome P450 is involved in microsomal deaaturation (2,3) .

In this study, the in vitro

effect of thyroxine on NADH-cytochrome c reductase and NADPH-cytochrome ç reductale activities in rat liver microsomee was investigated .

Localization of the

effect of thyroxine to this portion of the complex of deaaturation reactions may provide an understanding of the mechanism of the observed in vitro thyroxine stimulation of fatty acid deaaturation .

Thyroxine was found to stimulate the

NADH-cytochrome ç reductase activity but not NADH-dichlorophenolindophenol re ductase or NADPH-cytochrome ç reductase activity .

Thin stimulation ie not in-

hibited by superoxide diemutaae . Materials and Methods Male, white rata were purchased from Charlee Rivera' Laboratories .

Aorse

heart, cytochrome ç type III, NADH, NADPH, 2,6-dichlorophenolindophenol, auperoxide dismutase and sodium L-thyroxine pentahydrate were purchased from Sigma 2059

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NADH-Cyto C Reductase, Thyroxine Stimulation

Chemical .

Vol. 15, No . 12

Xanthine oxidase was purchased from Worthington Hiochemic~ils .

Microsomes were prepared from fed rats killed by a blow on the head followed by fracture of the neck .

The liver was minced in 5 ml 0.25 M

of liver .end homogenized with a teflon glass hoctogenizer . carried out at 0-4°C.

sucrose per g

Preparations were

The homogenate was centrifuged for 20 minutes at 16,000 X

g and the 16,000 g supernatant centrifuged at 105,000 X g for 30 minutes .

The

105,000 x g supernatant w .is discarded, the vicrosomal pellet was washed with 0.25 M sucrose, and the pellet was resuspended in 0.25 M sucrose to an appropriate protein concentration .

Replicate experiments were done on different days with

newly prepared microsomes . Microsomal enzyme activities were assayed at room temperature in a Cary model 14 recording spectrophotometer.

In e:ich experiment, non-thyroxine and

thyroxine reactions were carried out sequentially in duplicate over a 1 to 2 hour period of time .

Microsomal enzyme activity was stable during this time .

The duplicates were generally within lOx of each other, and the mean is reported eacept is Table 2 where individual values are shown.

Initial reaction rates were

calculated from the first 30 seconds of the reaction ; however, most reactions were linear with time for at 3 .0 ml .

least three minutes .

Reaction mixture volumes were

L-thyroxine was added in 0.1 ml of 0.01 N NaOH, which was also added to

non-thyroxine containing reactions . ing no microsomes .

Reactions were read against a blank contain-

Reactions were initiated by addition of NADH or NADPH to both

the blank and experimental cuvettes . a modification of the Lowry method

Protein determination

was carried out by

(4) .

NADH-cytochrome ç reductase and NADPH-cytochrome c reductase activities were determined epectrophotometrically by following the rate of reduction of cytochrome ç as measured by an increase in optical density at 550 nm (5) .

NADH

cytochrome c reductase reactions contained 80 mM Tris-acetate buffer (pH 8 .15), 5 to 20 Wg microsomal protein per ml, 0.05 mM cytochrome ç, 0.1 a!I NADH and varying L-thyroxine concentrations .

In superoxide dismutase containing reac-

tions, superoxide dismutase was added to the usual NADH-cytochrome ç reductase

NADH-Cyto C Reductaae, Thyroxine Stimulation

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reaction mixture shortly before initiating the reaction with NADH .

NADPH-cyto-

Vol. 15, No . 12

chrome ç reductase reactions contained 50 mM potassium phosphate buffer (pH 7.6), 12 to 75 4~g microsomal protein per ml, 0.05 mM cytochrome ç, 0.1 mM NADPH and 0 or 50 WM L-thyroxine .

The addition of 1 mM RCN to the reaction mixture did not

influence either NADH or NADPH-cytochrome ç reductase activity .

Reaction rates

were calculated using a millimolar ebsorbancy index for reduced minus oxidized cytochrome ç at 550 nm of 21 mM-lca-1 (6) . NADH and NADPH-dichlorophenolindophenol reductase activities were determined spectrophotometrically by following the rate of reduction of 2,6-dichlorophenolindophenol as measured by the decrease in optical density at 600 nm (5) . NADH-dichlorophenolindophenol reductase reaction mixtures contained 80 mM Trisacetate buffer (pH 8.15), 7 to 15 Wg microsomal protein par ml, 0.2 aM 2,6-dichlorophenolindophenol, 0.4 mM NADH, and 0 or 50 hM L-thyroxine .

NADPH-dichloro-

phenolindôphenol reductase reactions contained 50 mM potassium phosphate buffer (pH 7 .6), 5 to 12 Wg microsomal protein per ml, 0.05 mM 2,6-dlchlorophenolindophenol, 0.1 mM NADPH aad 0 or 50 ~M L-thyroxine .

The millimolar absorbancy index

used for calculating reaction rates was 21 .8 mM -lcm-1(7) " Results Thyroxine 8tinnlation of NADH -C9tochrome c Aeductase Activity ,

Control

NADH-cytochrome ç reductase activity varied from 165 to 643 nmoles cytochrome ç reduced/min/mB protein in different microsomal preparations, but was consistent No change in enzyme activity occurred between 0.02

in a given preparation (8) . mM and 0.2 mM NADH .

8azyme activity was directly proportional to protein con-

centration between 7.5 Fig and 22 .5 I+g protein/ml .

Lower protein concentrations

were associated with a alight decrease in enzyme specific activity . In Table 1, stimulation of microsomal NADH-cytochrome ç reductase activity by 50 ~M thyroxine is shown.

Thyroxine causes a statistically significant mean

stimulation of 58x over the mean control reaction rate of 328 nmoles cytochrome ç reduced/min/mg protein.

The percent thyroxine effect was independent of enzyme

specific activity and independent of protein concentration within the range

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NADH-Cyto C Reductase, Thyroxine Stimulation

tested .

Vol. 15, No . 12

There ie a stimulation by thyroxine in every case, regardless of the

control ensyme activity .

In figure 1, the effect of varying thyroxine concen-

tration on NADH-cytochrome ç reductase activity is shown.

An increasing stimu-

lation with increasing thyroxine concentration is seen with a maximum mean stimulation of 47X at 50 yM thyroxine.

A significant stimulation occurs at 20 yM

thyroxine, TABLE 1 Influence of Thyroxine on Microsomal NADH-cytochrome c Reductaae and NADH-dichlorophenolindophenol Reductase Activities NADH-cytochrome ç reductase (nmoles cytochrome ç reduced/ min/mg protein)

Experiment

Control

NADH-dichlorophenolindophenol reductase (nmoles dichloro phenolindophenol reduced/min/ mg protein)

50 yM Thyroxine

XT4 Effect

Control

50 y M Thyroxine

XT4 Effect

1

165

261

+58

129

129

0

2

351

635

+81

291

303

+4

3

347

535

+54

292

296

+1

4

198

344

+74

234

224

-4

5

277

429

+55

-

-

6

452

667

+48

-

-

7

503

701

+39

-

-

Mean

328

510

+58 ± 6

236

238

0

Mean protein concentration for the NADH-cytochrome ç reductase and NADH-dichlorophenolindophenol reductase reactions ie 13 yg/ml and 11 yg/ml respectively . The mean percent thyroxine stimulation is given as the mean ± S,E, The level of significance was determined by the paired t-teat . Influence of Thyroxine on NADH-dichlorophenolindophenol Reductase Activity , To obtain basic kinetic information on tha NADH-dichlorophenolindophenol reduttaee activity, initial rates of dichlorophenolindophenol reduction were obtained for various concentrations of dichlorophenolindophenol with saturating concen-

NADH-Cyto C Reductase, Thyroxine Stimulation

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tratione of NADH (0 .1 mM).

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The apparent Rm for dichloropheaolindophenol in

several experiments waa found to be between 0,02 ~ and 0,04 ~I,

A concen-

tration of 0 .2 mH dichlorophenolindophenol was snlected for studies of thyroxine influence on NADH-dichloropheaoliadophenol reductase activity .

At this concen-

tration and using 0.4 mM NADH, NADH-dichlorophenolindophenol reductase activity waa similar to NADH-cytochrome ç reductase activity in those microsomal preparations in which both activities were assayed (Experiments 1-4, Table 1) .

Con-

trol NADH-dichlorophenolindophenol reductase activity varied from 129 to 292 nmoles dichloropheaaliadophenol reduced/min/mB protein,

Enzyme activity was

directly proportional to microsomal protein concentration,

c4

5~ 0

rt~t

TIiYROXWE CONCENTRATION (M) FIG . 1

8ffect of varying thyroxine concentration on NADH-cytochrome ç reductase activity . Results shown are the mean of 3 experiments . Mean microsomal protein concentration is 16 .3 11g/ml . The data ie plotted ae the mean + S .E . of (T4-control), Significant differences (p C.05) are indicated by an asterisk and The curve waa drawn freewere determined by the paired t-teat . hand ae a beat fit . In Table 1, the influence of thyroxine on microsomsl NADA-dichlorophenolIndophenol reductase activity is shown .

50 F~!! thyroxine causes no aignificsnt

change in NADA-dichlorophenolindophenol reductase activity from the mean control

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NADH-Cyto C Reductase, Thyroxine Stimulation

Vol. 15,

No .

12

reaction rate of 236 amolea dichlorophenolindophenol reduced/min/mg protein.

Aa

seen in Table 1, parallel NADH-cytochrome ç reductase reactions using the same microsone preparation on the same day as the corresponding dichlorophenolindophenol reductase reactions revealed a mean 68X stimulation of NADH-cytochrome ç reductase activity by 50 EtI! thyroxine .

Mean control rate of cytochrome ç re-

duction in these experiments on these days was 265 nmoles cytochrome ç reduced/ min/mg protein ; thus, NADH-dichlorophenolindopheaol reductase and NADH-cytochrome ç reductase activities are comparable in these experiments . TABLE 2 Absence of Influence of Superoxide Diemutase on Thyroxine Stimulated NADH-cytochrome ç Seductase Activity nmoles cytochrome ç reduced/min/mg protein Ezperimant

Control

50 {LM XT4 Thyroxine effect

No superoxide dis~taee

Control

50 WM XT4 Thyroxine effect

10 kg/ml Superoxide dismutase

1

328 369

496 542

38X

337 364

419 501

31X

2

379 419

575 563

41X

430 441

631 597

41X

MicrosomSl protein concentration is 10 .5 4+g/ml and 8.5 ug/ml respectively . Absence of Influence of Superoxide Dismutase on Thyroxine Stimulated NADH cytochrome c Reductase Activity .

To determine if the thyroxine effect is medi-

ated by the superoxide radical, OQ, the influence of superoxide dismutase on thyroxine stimulated NADH-cytochrome ç reductase activity was tested,

Superoxide

dismutase activity was initially assessed by its inhibition of cytochrome c reduction in the presence of 80 mM Trie-acetate buffer (pH 8 .15), O.OS mM cytochrome ç, 0 .05

Td~t

xanthine and xanthine oxidase (9) .

Sufficient xanthine oxidase

was added to reduce 11 nmoles cytochrome c/min (.08 O.D ./min), a typical rate used in assaying NADH-cytochrome c reductase activity .

Under these conditions,

0 .174+g/ml superoxide dismutase inhibited the rate of cytochrome ç reduction 38X,

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NADH-C~rto C Reductase, Thyroxine Stimulation

0 .67 Ng/ml inhibited 60X and 1 .67 Ng/ml inhibited 74X . In Table 2, it can be seen that 10 Fag/ml superoxide diamutase does not influence the thyroxine stimulation of NADH- cytochrome ç reductase activity .

In

both experiments, the per cent thyroxine stimulation in the presence of auperoxide diamutase, 31X and 41X respectively, was similar to that seen in the abaence of auperoxide diamutase . enzyme activity .

Superoxide diamutase had no effect on control

These results indicate that the thyroxine stimulation of NADH-

cytochrome ç reductase activity is not mediated by auperoxide . Influence_ of Thyroxine on NADPH-~tochrome c Reduc tase and NADPH- dichlor opheaolindophenol Reductase Activities .

Control NADPH- cytochrome ç reductase

activity varied from 51 to 91 nmoles cytochrome ç reduced/min/mg protein . change in enzyme activity occurred between 0 .02 mM and 0 .2 mM NADPFi,

No

ßnzyme

activity was proportional to protein concentration between 12 and 75 vg protein/ ml in most experiments, although is nome experiments a decrease in enzyme epecific activity of up to 25X occurred at the higher protein concentrations . TABLE 3 Influence of Thyroxine on Microsomal NADPH- cytochrome ç Reductase and NADPfi-dichlorophenolindophenol Reductase Activities ßnzyme Activity Assayed

Control

50 4!i Thyroxine

XT4 ßffect

NADPH- cytochrome ç reductaae (nmolea cytochrome ç reduced/ min/mg protein)

64

63

NADPFi-dichlorophenolindophenol reductase (nmoles dichloropheaolindophenol reduced/min/ mg protein)

-2 ± 4 ~ .5 .)

121

116

-4 ± 7 (N .S .)

Results are the mean of four experiments . Mean protein concentrabona for the NADPH-cytochrome ç reductase and NADPH-dichloropheaolindophenol reductase reactions are 32 Ng/ml and 7 Fg/ml respectively . The percent T4 effect ie given as the mean ± S .ß . Table 3

shows that 50 E!i thyroxine causes no significant change is micro-

somal NADPH-cytochrome ç reductase activity from the control reaction rate of 64 nmoles cytochrome ç reduced/min/mg protein .

No thyroxine effect was seen at

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NADH-Gjrto C Reduotase, Thyroxine Stimulation

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varying protein concentrations between 12 and 72 Lig/ml . Control NADPH-dichlorophenolindophenol reductase activity varied from 74 to 153 nmoles dichlorophenolindophenol reduced/min/mB protein .

Table 3 also

ahoas that NADPH-dichlorophenolindophenol reductase activity is not influenced by 50 WM thyroxine with a mean control activity of 121 nmoles dichlorophenolindophenol reduced/min/s1B protein . Discussion As thyroxine has been shown to stimulate rat liver microsomal fatty acid desaturation reactions in vitro (1), these studies were undertaken to assess the possibility that thyroxine may stimulate the early reactions which eventuate in in vitro fatty acid desaturation .

The microsomal electron transport complex

effecting fatty açid desaturation is thought to involve the transfer of reducing equivalents fran NADH to flavoprotein to cytochrome ~ to the desaturase (2,3) .

Cytochrome ç reduction by NADH is thought to involve transfer of reduc-

ing equivalents from NADH to flavoprotein to cytochrome b5 to cytochrome ç (3) . Thus, the observed thyroxine stimulation of microsomal NADH-cytochrome ç reductase activity involves the first three members of the complex effecting fatty acid desaturation .

Dichlorophenolindophenol accepts electrons from the flavopro-

tein directly and reduction does not involve cytochrome ~ reduction (10) . Failure of thyroxine to stimulate microsomal NADH-dichlorophenolindophenol reductase activity implies that the stimulation occurs beyond flavoprotein oxidation .

Thus, thyroxine stimulation of microsomal NADH-cytochrome ç reductase

activity appears to indicate either enhanced cytochrome bS reduction or oxide-. tion .

This is consistent with the observation that the rate limiting step in

cytochrome ~ reduction by NADH is thought to be the transfer of electrons from the flavin of the reductase to cytochrome b5 (3,11) .

In a single report of the

effect of thyroxine in vitro on cytochrome ~ reduction kinetics, thyroxine was reported to enhance aerobic re-oxidation of cytochrome b5 in the absence of a terminal electron acceptor (12) . In recent years, it has been demonstrated that microsomal oxidatione may

Vol . 15, No . 12

NADH-Cyto C Reductase, Thyroxine Stimulation

generate the euperoxide radical

02 (13) .

2067

This work has been enhanced greatly by

the use of euperoxide dismutaee to inhibit euperoxide mediated reactions (14) . Since euperoxide is able to reduce cytochrome ç (9), the influence of euperoxide dismutase on the thyroxine sti~lated NADH-cytochrome ç reductase activity was studied.

The absence of influence of euperoxide diemutase on control or thyrox-

in stimulated NADH-cytochrome ç reductase activity indicates that euperoxide is sot involved is the thyroxine effect . The lack of stimulation of microsomal NADPH-cytochrome ç reductase activity is consistent with the thesis that the in vitro thyroxine stimulation of fatty acid desaturation may be the result of stimulation of the microsomal electron transport reactions which mediate fatty acid desaturation .

The NADPH-cytochrome

ç reductase is not thought to participate in the electron transport chain which results in microsomal fatty acid desaturation (15) . NADPH does not require the presence of cytochrome

S

Cytochrome c reduction by and is thought to involve

transfer of reducing equivalents from HADPH to flavoprotein to cytochrome ç (16) . The lack of thyroxine stimulation of NADPH-dichlorophenolindophenol reductase activity further indicates that enhanced flavoprotein reduction or oxidation does not occur.

In addition, failure of stimulation of microsomal NADPH-cytochrome c

reductase activity by thyroxine indicates that the thyroxine stimulation of NADH-cytochrome c reductase activity is not due to a thyroxine induced enhancement of cytochrome ç reduction itself . In these reactions relatively high coacentrationa of thyroxine are required to see the stimulation of NADA-cytochrome ç reductase activity .

Whether this re-

presents a pharmacologic or physiologic effect is not known at,thia time .

In

vivo studies have demonstrated that thyroxine treatment reeulte in an increase in microsomal NADPH-cytochrome c reductase activity but no increase in microsomal NADH-cytochrome ç reductase activity (17) . The basic observation developed is that thyroxine stimulates microeomal NADH-cytochrome ç reductase activity in vitro . bq euperoxide .

This stimulation ie not mediated

The observations support the interpretation that cytochrome

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NADH-Cyto C Reductase, Thyroxine Stimulation

is either more readily reduced or oxidized .

Vol. 15, No . 12

These possibilities will be further

studied using dual beam spectroscopy to study the extent and kinetics of reduction of cytochrome

3

using fatty acids as the terminal acceptor . A cknowledgments

This work was supported by Grants AM 12452 and AM 05635 from the National Institutes of Health to the Oniversity of Arkansas . References 1. .

F, H . FAAS, W . J. CARTER, AND J . WYNN, ENDOCRINOLOGY 91 1481-1492 (1972) .

2.

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V. MASSEY, BIOCHIM. BIOPHYS . ACTA 34 255-256 (1959) .

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J . M. McCCItD AND I, FRIDOVICH, J. HIOL . CiIEM, 244 6049-6055 (1969) .

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P . STRIITMATTER AND S, F. VE LICK, J . BIOL . CREM . 221 277-286 (1956) .

11 .

M. J. ROGERS AND P, STRITIMATTER, J . BIOL . CHEM . 248 800-806 (1973) .

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J . MODIRZADSH AID H. KAMIN, BIOCHIM. BIOPHYS, ACTA 99 205-226 (1965) .

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0. AVGIISTO, 8 . J . H, BECHARA, D . L . SANIOTO, AND G . CIIENTO, ARCH . BIOCHEM. BIOPHYS . 158 359-364 (1973) .

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I . FRIDOVICH, ADV . ENZYMÖL . 41 36-97 (1974) .

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T, OMURA, R . SATO, D, Y. COOPER, 0. ROSENTHAL AND R. W . ESTABROOK, FSDSRATION PROC . 24 1181-1189 (1965) .

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C . H, WILLLAMS AND H, KAMIN, J . BIOL . CREM . 237 587-595 (1962) .

17 .

A, S. FAIRHURST, J . C, ROBERTS AND R, E, SMITH, Al~R . J . PHYS . 197 370376 (1959) .