Intracellular distribution and properties of steroid 11β-hydroxylase and steroid 18-hydroxylase in rat adrenal

35 °

PRELIMINARY NOTES T A B L E I1[ EFFECT

OF INHIBITORS

ON NUCLEAR

RESPIRATION

AND

ATI'

S ' V N T H t,;Slt%

R e s p i r a t i o n w a s m e a s u r e d as d e s c r i b e d in T a b l e 1. A T t ' s y n t h e s i s was d e t e r m i n e d ai?er a I,o-nlin aerobic i n c u b a t i o n of "in vilro aged nuclei"". M o n o i o d o a c e t a t e was a d d e d before ageing.

Inhibition (%) A ddition Respiration

A n t i m y c i n ( 0 . 2 / , g per mg protein) R o t e n o n e ( 0 . 0 4 / , m o l e per m g protein) A m y t a l (2.5 raM) L e w i s i t e (O.l mlVl) M o n o i o d o a c e t a t e (1.5 raM) 9 5 % C O - 5 % O~

~>3 53 7° 7° 5° o

.4 7"I' synthesis

too i oo Loo 40 50 ,~

The substrates for nuclear oxidative phosphorylation are derived at least in part from glycolysis and the citric acid cycle. O~ uptake and ATP synthesis were partly inhibited by 1.5 mM monoiodoacetic acid and o. I mM lewisite (dichloro-2-chlorovinylarsine) (Table I I l ) . The substrates must be generated inside the nucleus, since we are unable to enhance respiration or phosphorylation by addition of glycolytic or citric acid cycle intermediates. From the results presented, the conclusion is justified that the isolated rat-thynms nucleus can perform oxidative phosphorylation coupled to a respiratory chain.

Radiobiological Institute T.N.O., Rijswijk, Z.H. (The Netherlands)

I. B E T E l .

H. M. KL()UWEN

1 S. OSAWA, V. G. ALLFREY AND A, E. MIRSKY, J. Gen. Physiol., 4 ° (I957) a B. C. Mc. EWEN, V. G, ALLFREY AND A, E. MIRSKY, ,]. Biol. Chem., 238 a 13. C. I~{c. EWEN, V. O. ALLFREY AND A. E. MIRSKY, .]. Biol. Chem., 238 4 B. C. Me. EWEN, V. G. ALLFREY AND A. E. MIRSKY, J. Biol. Chem., 238 H. M. KLOtlWEN AND 1. BETEL, Intern. J. Radiation Biol., 6 (I963) 441. " 1. BETEL AND H. M. KLOUWEN, Biochim. Biophys. dcla, 76 (I963) 327.

49 j. (1963) 758. (1963) 257[. (1903) 2579.

Received February I5th, 1964 Biovhim. Biophys..4cta, 85 (1964) 348 -35 o

PN

IOO99

Intracellular distribution and properties of steroid 1tfl- hydroxylase and steroid |8-hydroxylase in rat adrenal* The presence of i8-hydroxylase in rat adrenal has been reported z,~, but there has been not precise study on its intracellular distribution. On the other hand, it has been reported that ilfl-hydroxylase (EC 1.99.1.7) was present only in mitochondrial fractions 3& * P a r t of t h i s w o r k was p r e s e n t e d a t t h e 36th Gene ra l M e e t i n g of t h e J a p a n e s e B i o c h e m i c a l S o c i e t y on O c t o b e r 31, x963.

Biochim. Biophys. Acta, 85 (1964) 350-352

351

PRELIMINARY NOTES

In this paper, the intracellular distribution of the i8-hydroxylase and its enzymic character are reported in relation to the ii/5-hydroxylase. Rats were killed by a blow on the head, adrenal glands were removed, separated from fat, pooled and homogenized in five volumes of ice-cold o.33 M sucrose solution with a loosely fitting glass-Teflon homogenizer. The homogenate was differentially centrifuged and divided into nuclear fraction (6oo × g precipitate), mitochondrial fraction (6oo × g-5ooo × g precipitate), microsomal fraction (15 ooo × g-Io5 ooo × g precipitate) and soluble fraction (lO50ooXg supernatant). The nuclear and mitochondrial fractions were washed twice with small volumes of ice-cold o.33 M sucrose solution. After incubation of these subcellular fractions with substrate steroids, the media were extracted with dichloromethane. After isolation by silica-gel thin-layer chromatography in a dichloromethane:methanol (94:6, v/v) system, quantitative determinations of the products were performed by measurement of ultraviolet absorbancy at 24o m#. Reaction products were further identified by infrared and sulfuric acid chromogen spectra and melting point. TABLE I I

I~- A N D I 8 - H Y D R O X x-rLASE A C T I V I T I E S IN R A T - A D R E N A L S U B C E L L U L A R FRACTIONS

Reaction m e d i u m contained i mg I i-deoxycorticosterone (dissolved in o.i ml propylene glycol), ioo m g adrenal tissue preparation, o.8 mg TPN, 3 m g p o t a s s i u m glucose 6-phosphate, I Kornberg u n i t of glucose-6-phosphate dehydrogenase, 8 mM MgCI~, 4 ° mM Tris buffer (pH 7.4) in a volume of 5 ml. I n c u b a t i o n w a s carried o u t for one h o u r at 37 ° in a D u b n o f f t y p e i n c u b a t o r with c o n s t a n t shaking. Gas phase: O,,-CO2 (95:5) mixture. Products Subcellular fraction

Whole homogenate Nuclear fraction Mitochondrial fraction Microsomal fraction Soluble fraction Nuclear fraction plus soluble fraction Mitochondrial fraction plus soluble fraction Microsomal fraction plus soluble fraction Mitochondrial fraction plus heated soluble fraction*"

I8"OH'DOCt

Corticosterone (~ t-hydroxylated)

( ug)

(t~g)

323 42 85 2 2 14o

383 4° 85 2 2 16o

285 85

33 ° 95

158

16o

* i 8 - H y d r o x y - i i - d e o x y c o r t i c o s t e r o n e ( I 8 -+ 20 hemiketal). "" H e a t e d in boiling w a t e r b a t h for 6 min.

As shown in Table I, lift- and i8-hydroxylase activities were found in the microsomal fraction, though less abundantly than in the mitochondrial fraction. The microsomal 11/5- and iS-hydroxylase activities were clearly demonstrated when recombined with the soluble fraction, but the soluble or the microsomal fractions alone were inactive. The hydroxylase activities in the microsomal fraction activated by the soluble fraction were one-third to one-fifth of those found in the mitochondrial fraction. On Biochim. Biophys. Acta, 85 (1964) 350-352

352

I'RELIMINAI(Y N O T E S

the other hand, the succinate dehydrogenase (EC 1.3.99.1 ) activity in tit(' mic+-.~,rain in a boiling water bath, the activating eflbct was reduced about 5o?},. (2) Not dialyzable+ (3) Precipitable l>y (NH~)2S()a. (4) Effectively substituted by l)(>rcine-adrenal soluble fraction t)repared by a similar method. (5) Specificity. The soluble fraction was effective only ,m II13-all(l IS+hydroxylases, but not effective on microsomal 2i-hy(troxylase (E(" 1 .<)0. I. i i ) (Table I l ).

TAI31.E 1 [ EFFECT

Incubation

OF SOLUI~LE

conditions

I~'RACTION

ON MICROSOMAL

I [[]-,

1,~;-, A N D

2 I - I [ ' V I ) I ~ O X ' ~ ' L \~i£~',

w e r e a s in T a b l ( ' 1, e x c e p t t h a t ~ m g o f p r o g e s t e r o n e o f d e o x v c o r t i c o s t e r o n e (I)OC).

was used instead

Reaction products (2 r-hydroxlated steyroids) (pg) Enzyme preparalion

Microsome alone Microsome plus solublu traction Soluble fraction alone

I)OC

_,+~,> l '~3 o

z8-hydro~3, DOC

Corticosteronc

7"oral g z-OH steroids

t}

o

-,(,.

to o

47 ,>

27 ° ,~

(6) Not replaceable by even excess of glucose-0-phosphate dehydrogenase (EC 1.I.1.49). The results presented here suggest that the factor is one of the enzyme components of II{~- and iS-hydroxylases. A more detailed s t u d y will be reported in the near future. The authors are very much indebted to Dr. R. PAPPO of Searle Co. fl)r his gift of i 8 - h y d r o x y - i i - d e o x y c o r t i c o s t e r o n e (i8 ~. 2o heiniketal).

Deparlment of Biochemislry, Shionogi Research Laboratory, Shiotzogi and Co. Lld., Osaka Physiological Chemistry Division, Nalional Institute of Radiologieal Science, Chiba (Japan) 1 2 a 4

YUTAKA NAKAMURA I { U N - I ( ; H I "I'AMA()I(I

15".G. PI~RON, Endocri~olog)', 69 (1961) 39. M. t(. BIRMINGHAM AND I'. J . WARD, J . Biol. Chem., 2 3 6 (1961) I 6 0 1 . M. I.. SWEAT, J. A m . Chem. Soc., 73 ( I 9 5 I ) 4 0 5 6 . M. L. SWEAT, M. J . BRYSON AND 1~. B. YOUNG, 15"0C. 5lh [~zlern. Cong~. 13iochem., ~/Ioscoa~,, x96x , Vol. 9, P e r g a m o n P r e s s , N e w Y o r k , 1963, p. 323.

Received F e b r u a r y zoth, r904 Biochim. Biophys. Acla, 85 ( [ 9 6 4 ) 3 5 o - 3 5 2