Studies on biotransformation of elastase IV. Tissue distribution of 131I-labeled elastase and intracellular distribution in liver after intravenous administration in rats

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S T U D I E S ON B I O T R A N S F O R M A T I O N

OF ELASTASE

IV. T I S S U E D I S T R I B U T I O N O F 1~tI-'.ABELED E L A S T A S E A N D I N T R A C E L L U L A R D I S T R I B U T I O N IN L I V E R A F T E R I N T R A V E N O U S A D M I N I S T R A T I O N IN R A T S

KOUICH! KATAYAMA and TAKESHI FUJITA

Departnwnt of Drupe Metabolism, Section oJ Experimental Therapeutics Research (Director: Dr S. Ohtake). Research and Dereloprne~:t Divisiot~, Eisai Co.. Lid, Koishikawa, Buttkyo-ku. Tokyo (Japan) (Received August 13th, 1973)

SUMMARY

The tissue distribution o f trichloroacetic acid-precipitable radioactivity following intravenous administration o f 0.3 m g o f ~311-laUeled elastase (EC 3.4.4.7) per kg in rats has been investigated. T h e time courses o f the tissue levels were divided into two patterns. One of the patterns was observed in liver, spleen, bone marrow, kidney and adrenal gland: the levels o f trichloroacetic acid-precipitable radioactivity in these tissues reached the m a x i m u m 15 rain after administration and thereafter declined biexponentially. In the other pattern, which was observed in heart, lung, testis, stomach and intestinal wail, the tissue levels reached the peak 30 rain o r more after injection and then declined monoexponentially. Sephadex G-200 gel filtration profiles o f radioactivity solubilized by Triton X-100 from liver d e m o n s t r a t e d that the radioactivity in liver was mainly in the form o f ~3~I-labeled elastase b o u n d to a2-macroglobulin within 45 rain following administration of 13tI-lal?eled elastase, and thereafter i n / h e f o r m of 1311.labeled elastase b o u n d to ,t-antitrypsin. This finding suggests that the u p t a k e o f the f o r m e r complex by liver is more rapid than that of the latter. The inlracellular distribution of trichloroacetic acid-precipitable radioactivity in liver has been studied by the differential centrifugation o f liver homo:~enates d u r i n g the first I h after intravenous a d m i n i s t r a t i o n o f J3tl-labeled elastas¢ in rats. The tim~ course of the specific radioactivity in the 39 000 :~: g (60 min) fraction showed a m a x i m u m Io.vel 5 min after administration. In contrast, the specific radioactivity in the 27 000 / g ( 10 rain) fraction which was the highest level a m o n g a n o t h e r fractions within I h reached a m a x i m u m at 15 rain. The fact that the radioactivity in the 27 0C~0 g fraction was f o u n d in lysosomes, was confirmed by such procedures as sucrose density gradient centrifugation, and solubilization by Triton X-100 and by freezingthawing,

179 INTRODUCTION It has been suggested that a small a m o u n t o f elastase (EC 3.4.4.7) is absorbed f r o m the intestinal tract, and tl,at the lymphatic system plays a significant role in the a b s o r p t i o n o f this enzyme after intraintestinal administration o f latl-labeled elastase in rats [1, 2]. T h e preceding paper [3] has shown that 13,I-labeled elastase administered intravenously circulates as elastase b o u n d to two specific proteins, namely ttz-macroglobulin and t~-antitrypsin in serum, and the enzyme b o u n d to az-macroglobu;in is m o r e rapidly cleared from the blood t h a n that b o u n d to tq-antitrypsin. In o r d e r to obtain more information about the biotransformation of 1311labeled elastase in rat, the present study deals with the tissue and intracellular distribution o f trichloroacetic acid-precipitable radioactivities after intravenous administration of 13tl-labeled elastase. MATERIALS AND METHODS 1311-labeled elastase (abbreviated as [~l]elastase), of which the specific radioactivity was 40-501~Ci/mg, was prepared by the m e t h o d described pre,lously [4]. Sephadex G-25 and G-200 (Pharmacia Fine Chemicals}, Triton X-IC0 (Wako Pure Chemical Industries), bovine albumin ( A r m o u r Pharmaceutical Co.), glucose-6p h o s p h o r i c acid (B.D.H. Chemicals),/~-glycerophosphate (Merck), and cytochrome c (Type VI, Sigma Chemical Co.) were used.

Animal experiments Male rats o f Wistar strain (2413-280 g) were allowed to drink I m M KI aqueous solution in place of water for 4 days ad libitum and fasted for 24 h prior to use. A 0.15 M NaCI solution of ['Z~l]elastase was injected into the femoral vein at a dose o f 0.3 m g / k g . F o r tissue distribution experiments, rats were sacrificed by decapitation 15 rain, 30 min. I h, 6 h, ! 2 h and 24 h after administration o f ['z~ I]elastase. As the reference experiments, rats administered Kt311 at a dose of 0.2 mg as iodine (15/~Ci/kg) were sacrificed at !, 6 and 24 h. Tissues were removed, rinsed with 0.15 M NaCI and blotted with filter paper. After the tissue was homogenized in 3 vol. (v/w) of 0.25 M sucrose using a P o t t e r - E l v e h j e m type homogenizer with a Teflon pestle at 2000-25C0 rev./min with 3 strokes, the radioactivity in each h o m o g e n a t e was assayed by trichloroacetic acid precipitation (see below). F o r c h r o m a t o g r a p h i c analysis o f the cadioactivity in liver and the intracetlular distribution o f trichloroacetic acid-precipitable radioactivity in liver, rats were sacrificed by decapitation at predetermined intervals d u r i n g the first I h following intravenous administration o f [~l]elastase at 0.3 mg/kg. After in situ perfusion o f the liver with cold 0.15 M NaCI, the liver ,was removed, homogenized, solubilized by l ',~,, (w/v) Triton X-100 and then subjected to Sephadex G-200 column c h r o m a t o g r a p h y .

Subcellular fractionation of liver T h e liver h o m o g e n a t e was fractionated at 4 °C by differential centrifugation based on the m e t h o d o f Mego and McQueen [5]. The h o m o g e r a t e was centrifuged at 900 × g for 10 rain a n d the resulting pellet was washed twice with 0.25 M sucrose by

180

resuspension and reeentrifugation u n d e r the same conditions. After the washings had been added to the 900 × g s u p e r n a t a n t , the c o m b i n e d s u p e r n a t a n t was centrifuged at 27 000 × g for ! 0 rain and this pellet was also washed twice. The final s u p e r n a t a n t was cer, trifuged at 39 000 × g for ! h. In other experiments, the 900 × g s u p e r n a t a n t was centrifuged at 3300 × g for l 0 rain ( D e D u v e et al. [6]) and the resulting pellet was washed twice, a n d then the 3300 x g supernatant w a s centrifuged at 2 7 0 0 0 × g for 10 min, followed by 39 000 × g for I h, as described above. After all pellets had been h o m o g e n i z e d in 3 vol. o f 0.25 M sucrose at 800 rev./min with ! stroke, the radioactivity in each fraction was analyzed by trichloroacetic acid precipitation a n d the protein was d e t e r m i n e d . T h e light m i t o c h o n d r i a l fr~ction sedimented at 2 7 0 0 0 >: g was further centrifuged with sucrose density gradient and was treated with T r i t o n X-100 or repeated freezing-thawing.

Su~'r~ re density gradient centrifugation Linear sucrose density gradients (density 1.10-1.32) were prepared with a Hitachi gradienter D G K - U . 0.5 ml o f 0.25 M sucrose c o n t a i n i n g the light m i t o c h o n drial fraction isolated from 0.5 g o f original liver was layered on 4.5 ml o f the gradient, and centrifuged at 39 000 rev./min for 3 h at 4 °C in the swinging bucket rotor RPS-40A of the Hitachi Model 55P-2 centrifuge. The c o n t e n t of the tube were divided into 8 fractions by a tube slicer.

Solubilization of the light mitochondrial fraction Treatment with Triion X-IO0. The mixture o f 0.5 ml o f the light m i t o c h o n d r i a l fraction {equivalent to I g of original liver/ml) a n d 0.5 ml o f various concentrations o f Triton X-100 was allowed to stand for 30 rain in ice-cold water. T h e n the mixture was centrifuged at 27 000 "." g for 10 rain and the s u p e r n a t a n t was assayed. Treatment byfree:#1g and thawing. I ml o f light m i t o c h o n d r i a l fraction (0.5 g of original liver/ml) was frozen with solid CO2-acetone and thawed by incubation for 1 rain at 37 ~'C. After centrifugation at 27 000 ;~ g for 10 rain, the s u p e r n a t a n t was assayed.

Radioactivitr determination All counting was d o n e in a well-type scintillation c o u n t e r (Aloka JDC-207).

Trichloroacetic acid precipitation An equal volume o f 10% (w~ v) trichloroacetic acid solution was a d d e d to the aliquot o f h o m o g e n a t e . After centrifugation, the radioactivities o f supernatant and precipitate were counted.

St,~hade.v G-2(X) gel.filtrathm of radioactiviO" soluhili:ed from liver To 2 ml o f 253,/i (w/v) liver h o m o g e n a t e , 1 ml of 5 % Triton X-100 a n d 2 ml o f 0.25 M sucrose were added in an ice-cold water bath. After centrifugation at 105 000 / g fGr ! h, the supernatant was c o n c e n t r a t e d by addition o f the appropriate a m o u n t of dry Sephadex G-25. The c o n c e n t r a t e d s u p e r n a t a n t was charged on a Sephadex G-200 c o l u m n (I.5 cm ~'-~ 3 0 c m ) and then eluted with 0.15 M NaCI.

181

Determination o f protein and enzyme activio" Protein was d e t e r m i n e d with bovine albumin as a s t a n d a r d by the m e t h o d o f L o w r y et al. [7], Acid phospi-~atase was u e t e r m i n e d w i t h / ~ - g l y c e r o p h o s p h a t e in the presence o f 0 . 1 % T r i t o n X-100 by the m e t h o d o f W a t t i a u x and De Duve [8], cytoc h r o m e o x i d a s e was based o n the m e t h o d reported by Cooperstein a n d L a z a r o w [9], a n d g l u c o s e - 6 - p h o s p h a t a s e was by the m e t h o d o f H a r p e r [10]. RESULTS

Tissue distribution of trichloroacetic acM-precipitabh, radiouctivio'.follo wing intravenous administration o f [ 131ljelastase i n o r d e r to e v a l u a t e the analytical m e t h o d for tissue distribution o f injected [~J~llelastase, the c o n t e n t s o f radioactivity in tissues a n d the percentages o f trichloroacetic acid-precipitable radioactivity to the total radioactivity in each tissue after i n t r a v e n o u s a d m i n i s t r a t i o n o f [J3~l]elastase were c o m p a r e d with those follov, ing i n t r a v e n o u s a d m i n i s t r a t i o n o f K~3~I. The results are shown in Table I. TABLE 1 TISSUE CONTENTS OF RADIOACTIVITY FOLLOWING INTRAVENOUS ADMINISTRATION OF [13qiELASTASE OR Kl~lt Radioactivity contents in tissues were represented as the mean percentages of the dose of 2 experiments. The values in 0arentheses indicated the mean percentages of Irichloroacetic acid-precipitable radioactivity to the total radioactivity in each tissue. Tissue

Liver Lung Kidney Heart Spleen Adre~tal gland Thyroid gland Testis Brain Stomach Intestine Stomach contents Intestinal contents

[~JII]elastase, 0.3 mg ( 15/,Ci kg]

K TM I, 0.2 rng as I ( 15 pCi): kg

Ih

6h

24h

Ih

3,84 170.2) 0.66 173.41 1.17 (71 01 0.31 (80.5) 0.,14(69.5) 0.02 173.2) 0.04 (61.7) 0.39 (69.21 0,10 169.5) 1.11 (31.2) 2.20 (42.3) i .82 2.19

1.96 (62.4} 0.77 (68.3t 0.82 (55.31 0.23 (72.0) 0.20153.8) 0.01 (66.8) 0.09 (76.3) 0.79 (51.6) 0.06 (56.8) i.42 (10.4) 1.45 (45.2) 1.52 1.25

0.85 (29.3) 0.35 (41.3) 0.34 (24.61 0.07 (37.0) 0.10(27.9! 0.00 0.31 (95.1) 0.34 (18.8) 0.03 (15.2) 0.53 (14.7) 0.90 (24.3) 3,87 2.59

1181 ,5.6) 1.38 (6.1) 0.51 (9.6) 0~1 (9.9/ 0.79 4.8;' 0.45 14.81 0.19 (8.5) 0.10 (4.7) 0.22 I0.9) 0.18110.0) 0.01 ,0.0) 0.00 0.05 53.1) 0.07 (78.8/ 0.43,0.5) 0.53 (2.5) 0.04 ,0.0) 0.03 (2.7) 1.83. ~|.9) 2.78 (6.41 !.61 (4.5) 0.98 (8.3) 3.20 I 1,06 1.14 1.12

6h

24h 0.32 (4.7t 0.16 (4.9) 0.19 (5.4) 0.05 ~7.7) 0.07(12.6/ 0.03 (0.01 2.17 (93.7) 0.20 (0.0) 0.01 (0.0) 0.64 (4,31 0.21 (5.2t (I.51 0.44

As s h o w n in Table I, the c o n t e n t o f radioactivity in each tissue with [t3~l]elastase was not greatly different f r o m that in the same tissue with K TM l, but the percentages o f t r i c h l o r o a c e t i c acid-precipitable radioactivity with [13Zi]elastase were clearly greater t h a n t h o s e with K TM I. The results indicate that trichloroacetic acid-precipitable r a d i o a c t i v i t y in the tissues, apart f r o m the t h y r o i d gland, can be a s s u m e d to be due to the [t3~l|elastase, T h e trichloroacetic acid-precipitable r a d i o a c t i v i t y in tissue after intravenotJs

182 injection o f 0.3 m g o f P~ll]elastase per kg was represented as ["q]elastase equiv. (pg/g) o f tissue a n d is s h o w n in Fig. 1. The serum concentrations were approximately ten times as high as t h e concentration in all the tissues except for the thyroid gland, The levels in the thyroid gland increased with time, The trichloroacetic acid-precipitable radioactivity in the thyroid gland seems n o t to be in the f o r m of [~3tl]elastase b u t to c o m e f r o m the degraded p r o d u c t s o f injected [tatl]elastase; the radioactivity contents a n d t h e a

b u

~,.//

o.!

am

-

-

Time,{h)

Time{h)

Fig. I. i'issue distribution o f trichloroacetic a¢id-precipitable radioactivity after intravenous administration of 0.3 mg [~;~l}elastase per kg in rats. Mean concentration o f 2 experiments was represented

as elastase equiv. PII/g of tissue, (a) C, serum: 0, liver: :~, bone marrow; &, pancreas; V. kidney: V, lung: ~, stomach. (b) ;.,:, spleen: O, heart; /~, adrenal gland: &, brain~ ~ , testis: V, thyroid gland: ,~i~i.intestine. percentages of trichloroacetic acid-precipitable radioactivity to the total in the thyroid gland after administration o f [~Jll]elastase were similar to those following administration o f K~3~I. As ~hown in Fig. !, the time course o f tissue levels was divided into two patterns, io one of the patterns observed in liver, spleen, bone marrow, kidney and adrenal gland, the tissue levels attained the m a x i m a 15 rain after administration and then decli~,ed biexponentially. In the other pattern observed in heart, lung, testis, stomach and intestinal wall, the levels reached the peaks 0.5 h or more after injection and decreased monoexponentially. T h e elimination rates from tissues in the former pattern were similar to those in the latter pattern I h af,'er administration,

Uptake and nature of radioactivity in liver Since the time course o f the hepatic concentration o f trichloroacetic acidprecipitable radioactivity in Fig. I showed a characteristic pattern, further studies were performed in the liver perfused with cold 0.15 M NaCi. Fig. 2 shows the contents and concentrations o f radioactivity in the perfused liver d u r i n g the first 1 h after intravenous administration of 0.3 mg [13~1]elastase per kg in rats. As shown in Fig. 2, the radioactivity in liver attained a m a x i m u m level o f abo~t 4 0 % o f the dose 15 min after administration and thereafter declined. T h e concentration o f trichloroacetic acid-precipitable radioactivity at the peak time in the perfused

183 §e Ib 0

"6

10

8 1

~

(b)

s

!' "~0.5

0 5 10 15

30 Time

45

60

(mi, n )

Fig. 2. Time course of radioactivity in perfused liver after intravenous administration of 0.3 mg [t~l]elastase per kg in rats. (a) Amount of radioactivity in the perfosed liver ~as represented as the mean percentage of the dose with the S.E. of 3 experiments. (b) Concentration of radioactivity ( ) and of trichloro~cetic acid-precipitable radioactivity (0) in the perfused liver were repre~nted as the mean concentration of the elastase equiv, of 3 experiments. liver (Fig. 2a) was a p p r o x i m a t e l y 3 p g as [t311]elastase equiv, per g and was the same as that in the n o n - p e r f u s e d liver (Fig. I). F o r the c h r o m a t o g r a p h i c analysis o f the radioactivity in liver, Triton X - I t 0 was a d d e d to liver h o m o g e n a t e at the c o n c e n t r a t i o n o f I~°~, and the mixture was c e n t r i f u g e d at 105 0(10 :~. g for I h. The supernatant, in which 97~)~, o f the total radioactivity in h o m o g e n a t e were recovered, was c o n c e n t r a t e d with dry Sephadex G-25 a n d subjected to Sephadex G-200 c o l u m n c h r o m a t o g r a p h y . T h e elution patterns o f the radioactivity in liver or s e r u m obtained during the first I h after injection o f [ ~ i]elastase a r e s h o w n in Fig. 3. As s h o w n in Fig. 3, a gel filtration pattern o f s e r u m 2 min after administration d e m o n s t r a t e d two peaks o f radioactivity which c o r r e s p o n d e d to [~3~i]elastase bound by tt,-macroglobulin o r ttt-antitrypsin, respectively. In contrast, the elution pattern o f radioactivity in liver at 2 min showed a single peak c o r r e s p o n d i n g to the t~2-macroglobulin-[13tl]elastase complex. At 15 min after the injection, the fraction o f ~2macroglobulin-[13~I]elastase complex in liver was increased, but that in serum was m a r k e d l y decreased. In liver 60 rain after administration, the size o f the first peak which consisted o f the a2-macroglobulin-[ TM !]elastase complex was m a r k e d l y decreased and the second peak, the a~-antitrypsin-p3~l]elastase complex, was increased. The third peak which a p p e a r e d newly in liver 60 min after administration seems to he the n o n - p r o t e i n binding ~3~! that c o m e s f r o m the d e g r a d e d products o f [~S~l]elaslase, because the radioactivity in the peak was trichloroacetic acid soluble. It is suggested that the ~t-antitrypsin-[~3~l]elastase complex is slowly taken up by liver c o m p a r e d with the rapid a p p e a r a n c e o f the a z - m a c r o g l o b u l i n - [ TM l]elastase c o m p l e x in the tissue.

184

J~

;*:

2 m!.

,,-,

5,

01=J

.1000

....

~,

.__

o

•

'~

[. ,.

Fraction number Fig. 3. Sephadex G.200 gel filtration p a t t e r n s o f radioactivity in liver a n d s e r u m after intravenous administration ofO.3 mg [~3tl]elastase p e r kg in rats. Liver extract by 1% Triton X-tO0 from 0.67 g of liver o r serum (0.2 m | ) was fractionated with a Sephadcx G-200 c o l u m n (1.5 c m ~. 30 cm).

Fig. 4 shows the semilogarithmic plot of the hepatic concentrations of the azmacroglobulin-[t311]elastasc complex as p~li]elastase equiv./~g per g o f liver against time. The hepatic levels of the ,2-macroglobulin-[q~'l]elastase complex rapidly increased after injection, attained a maximum at 15 n~in and thereafter decreased exponentially. The half-life of disappearance from liver was estimated to be 7.2 rain. Plots of the residuals obtained by subtraction of the observed values from the extra30 A

o,

TM

2O 10 S

Q Ul

~'~~= Vl

7.2 rain

1

0.5

tlr2= 3.6 rain

~ ' ~

¢:

,<2_ ¢:

0.1 ~05

¢J

O.C Time (rain) Fig. 4. Liver concentration o f (t2-macroglobu|in-[ TM i]¢lastase c o m p l e x after intravenous a d m i n i s t r a tion o f [t~l|elastase in rats. M e a n concentration o f 3 e x p e r i m e n t s was represented as elastase equiv. ftg/g o f liver. Plots ot" (,~.) were the residuals o b t a i n e d by the s u b t r a c t i o n o f the o b s e r v e d values ( 0 ) f r o m the extrapolated values o f the terminal linear segment.

185 p o l a t e d values o f the terminal linear line, d e m o n s t r a t e d that the half-life o f liver u p t a k e o f a2-macroglobulin-[13~l]elastase c o m p l e x was 3.6 min.

Intracellular distribution of trichloroacetic acid-precipitable radioactivity in rat liver after intravenous administration of [~3tlJelastase In a preliminary e x p e r i m e n t , the a d s o r p t i o n o f [t3~l]elastase to particulate fractions w a s d e t e r m i n e d . A small a m o u n t o f [~3q]elastase in 0,15 M NaC1 or ir~ s e r u m w a s a d d e d to liver h o m o g e n a t e p r e p a r e d f r o m an u n t r e a t e d rat, and in a n o t h e r s a m p l e [t3ti]elastase in 0.15 M N a C I was a d d e d to liver h o m o g e n a t e containing serum. T h e specific radioactivity o f the particulate fractions collected by centrifugation is s h o w n in T a b l e ll. TABLE II ADSORPTION OF [t3tl]ELASTASE TO RAT LIVER PARTICLES 4 pg (7" 1(3"cpm) of ['3'l]¢lastas¢ were added to 3.5 ml of 25 % liver hornogenate, After 5 min at 4 ~'C, the mixture was centrifuged as described in the text. Each value of specific radioactivity (cpm/mg protein) or relative specific radioactivity (RSA, ratio of distribution percentage of radioactivity in each fraction to that of protein) is represented as the mean of 2 experiments, Added to liver homogenate pJil|Elastase in 0.5 ml saline 0.5 ml serum and p~ll]elastase, separately [l~ll]Elastase in 0.5 ml serum

900 >~g

27 000 x g

39 000 ,,

R

Supernatant

cpmjmg

RSA

cpmimg

RSA

cpm/mg

RSA

cpm/mg

RSA

452

1.25

122

0.34

581

1.61

320

0.88

65

O.16

8

0.02

69

0.18

718

1.79

23

0.06

3

0.01

51

O. 14

699

i .86

In the case o f the a d d i t i o n o f [t~ll]elastase in 0.15 M NaCI solution to liver h o m o g e n a t e , [13~l]elastase was a d s o r b e d t o all the fractions and 38-39'~;, o f the total radioactivity was recovered in the s u p e r n a t a n t fraction, in the samples in which the [l~tl]elastase-serum mixture was a d d e d to liver h o m o g e n a t e , or p ~ l ] e l a s t a s e in 0.15 M NaCI was a d d e d to liver h o m o g e n a t e - s e r u m mixture, however, a negligible a m o u n t o f radioactivity was a d s o r b e d o n t o the particulate fractions. T h e recovery in the final s u p e r n a t a n t w a s 9 5 - 9 6 ~ o f the total. T h e results s h o w n in Table I1 indicate that the a d s o r p t i o n o f [~3'1 ] e l a s t a s e - s e r u m protein c o m p l e x e s to hepatic particles is negligible. Fig. 5 s h o w s the time d e p e n d e n c e o f the relative specific radioactivity (ratio o f distribution p e r c e n t a g e o f trichloroacetic acid-precipitable radioactivity in each fraction to t h a t o f protein) in the particulate fractions o f liver during the first I h after i n t r a v e n o u s a d m i n i s t r a t i o n o f [t3ti]elastase, T h e relative specific r a d i o a c t i v i t y in the 3 9 0 0 0 / g fraction attained the highest level in all the fractions 2 mix after injection, maintained this level for a b o u t 5 min a n d thereafter decreased. T h e percentage o f radioactivity in the 39 000 y g fraction at 2 min was 1 3 . 4 % o f the total radioactivity in liver. The relative specific radioactivity in the 27 000 × g fraction rose rapidly, a t t a i n e d a m a x i m u m level at 15 min and then declined. T h e 27 000 × g fraction at the peak time c o n t a i n e d 64.3 %

186

.*t

i 0

0 fi

I5

3O 45 T i m e (rain)

60

Fig. 5. T i m e course o f the intracellular distributions o f trichloroacetic acid-precipitable radioactivity as relative specific radioactivity (ratio o f distribution percentage o f radioactivity in each fraction to that o f protein) . . . . . 9 0 0 ~ g; Q, 2"# 0 0 0 ~,. ~., ZS., 39 0 0 0 × g ; &, final supernatant+

of the total radioactivity in liver. In the 900 × g fraction, a m a x i m u m level o f relative specific radioactivity which was observed at 5 rain after injection was sustained during the subsequent 55 rain.

Nature of the 2 70(;0 ~: g fraction containing trichloroacetic acid-precipitable radioactivity As is shown in Fig. 5, the distribution o f radioactivity in the 27 000 × g fraction was the highest in all the fractions 5 rain after injection. Further fractionation and identification o f the 27 COO ~ g fraction was, therefore, performed using the liver hcmogenate 15 min after intravenous administration o f 0.3 mg o f 113~l]elastase per kg. Fig. 6 shows the hepatic intracellular distribution o f trichloroacetic acid(a)

(b)

(c)

r~

(d)

"d ¢*" q u

i

t,L

~ 0

2"

q

t

•

o,[..f-

,"1 -1 Per

cent

of total protein

Fig. 6, Distribution patterns of trichloroaoctic acid-precipitab|¢ radioacti,~ity tat, cytochrome oxidase (b), acid phosphatase (c) and glucosc-6-phosphatase (d) in liver homogenate 15 rain after intravenous administration of 0.3 mg [IJ'l]elastas¢ per kg in rat. Ordinate: relaLve specific activity

in each fraction. Abscissa: fractions were represented by their relative protein contents, in the order in w h i c h they were isolated, i.e. from left to right: 900 × g, 3300 >: g, 27 0 0 0 ~'~ g, 39 0(}0 ~: g and

final supernatant.

187 precipitable radioactivity, cytochrome oxidas¢, acid phosphatase and glucose-6phosphatase 15 min after injection. As indicated in Fig. 6a, the distribution of trichioroacetic acid-precipitable radioactivity in the 27 000 × g fraction was the highest and the recovery of radioactivity in this fraction was approximately 50% of the total in liven"homogenate. The distribution of acid phosphatase, a lysosomal marker, (Fig. 6c) was also fairly concentrated in the 27 000 x g fraction corresponding to the light mitochondrial fraction. The 39 000 × g fraction, in which 15.5% o f the total protein and 47.2% of the total glucose-6-phosphatase activity (Fig. 6d) were determined, was confirmed to contain mainly microsomes. Since the light mitochondrial fraction showed some extent of cytochrome oxidase activity, it might be contaminated with mitochondria, Accordingly, an attempt was made to analyse in a more detailed manner the distribution of radioactivity in the light mitochondrial fraction, with a help o f density gradient centrifugation. Fig. 7 shows the distribution of trichloroacetic acid-precipitab[e radioactnvity and acid phosphatase activity in the light mitochondriai fraction after sucrose density gradient centrifugation.

(a) o

(b) 0.2

2

1K

~0.I n C

E

1.3

gO

L2 ~, 1.? ~

Feaction volume(ml)

,.31

1.1 2.5 5 Fraction volume(rnl)

~o

Fig. 7. Sucrose density gradient centrifugation o f the light m i t o c h o n d r i a l fraction. 0.5 ml o f the light m i t o c h o n d r i a l fraction in Fig. 6 ( c o r r e s p o n d i n g to 0.5 g o f original liver) was layered on 4.5 ml o f a linear sucrose g r a d i e n t (density I.lO-1.32) a n d centrifuged. (a) Trichloroacetic acid-precipitable radioactivity. (b) A c i d phosphatase.

As dem.mstrated in Fig. 7, both trichloroacetic acid-precipitable radioactivity and acid phosphatase activity were distributed as sharp peaks in the same fraction of density 1.19-1.22. in order to confirm that the radioactivity in the light mitochondrial fraction exists in lysosomes, the light mitochondrial fraction was treated with Triton X-100 or by freezing-thawing. Fig. 8 shows the solubilization patterns of radioactivity, acid phosphatase and protein from the light mitochondrial fraction. 92.4% o f the total radioactivity were solubilized by 0.125°t;; Triton X-100 and the solubilization pattern of radioactivity in various concentrations of Triton X-100 (Fig. 8a) was observed to be in good agreement with that of acid phosphatase. Freezing and thawing exerts similar effects on the solubilization of radioactivity and acid phosphatase except for protein from the light mitochondrial fraction (Fig. 8b).

188 (u)

(a) 100

IOO

50

a~

0

~rM:m~trat~on o~ Triton X-lO0(%)

~

2

3

Freezing and thawing {times )

Fig. 8. Solubilization of radioactivity from the light mitochondrial fraction with Triton X-100 (a) and by freezing-thawing (b). Ordinate represented the percentage o f the solubilized a m o u n t to the total amount and each value was the mean o f 2 experiments. Q, radioactivity; ,~, acid phosphatase; ~ , protein. DISCUSSION

in the preceding paper [3], the disappearance of [~atl]elastase from serum after intravenous administration in rats was studied; it was found that [~3~l]elastase in serum circulates in the form of complexes a2-macrogiobulin-['J~l]elastase and ~-antitrypsin-[U~l]elastase, and the clearance o f the former complex from blood is more rapid than that of the latter, in the present study, the tissue and intraeellular distribution of injected ['3'l]elastase in rat were investigated. As shown in Fig. I, the time courses of tissue levels of trichloroacetic acidprecipitable radioactivity are divided into two patterns during the first I h after administration. One pattern in which the tissue levels a t t a i , e d the respective peaks 15 rain after administration and thereafter were rapidly decreased within I h, was observed in liver, spleen, bone marrow, kidney and adrenal gland, in this pattern, the contamination of tissues with serum was confirmed to be negligible, since the tissue level in perfused liver (Fig. 2) was in good agreement with that in the nonperfused liver (Fig. I ), The time courses of levels in such tissues as heart, lung. testis. stomach ~,nd intestine demonstrated the other pattern, in which the tis'~ae levels gradually inreased during the first 0.5 h or more and th"q declined monoe,~ponentially. These findings in tissue distribution suggest a close relationship between the disappearance of [~3~llelastase-serum protein complexes from blood and the uptake of radioactivity by tissue. This suggestion seems to be supported by the gel filtration patterns of radioactivity in serum and liver as shown in Fig. 3. The fraction of ~t:. macroglobulin-i~ati]elastase complex in serum rapidly decreased with time following intravenous injection of ['~' Ilelastase, while the fraction corresponding to the complex was increased markedly in liver at 15 rain. The non-protein bound radioactivity which seemed to originate from the degradation of the complex, also appeared in liver at 60 rain. In contrast, the fraction corresponding to the at-antitrypsin-[~3~llelastase complex in liver gradually increased with time, whereas the fraction corresponding to the complex in serum was gradually decreased. These results suggest that the two patterns observed in tissue distribution (Fig. l) result from the difference of ability in uptake of az-macroglobulin-['3~l]elastase complex by the tissue. Namely, the tissues

189 such as liver, spleen, bone marrow, kidney and adrenal gland rapidly take up the a 2macroglobulin-[lall]elastase complex, but they take up slowly ~L-antitrypsin-[~3q]elastase complex, being comparable with the manner of the uptake by the other tissues such as heart, lung, testis, stomach and intestine. The time course of concentration of the a,-macroglobulin-[~3q]elastase complex in liver shown in Fig. 4 can be fitted to a one-compartment open model with a first-order uptake and degradation. The rate constants in the uptake and degradation by liver were found to be 0.191 and 0.097 m i n - ' , respectively. The half-lives of the uptake and degradation of the a2-macroglobulin-[~a~i]elastase complex were also 3.6 and 7.1 min, respectively. The half-time for the hepatic uptake of the complex was shorter than that (9.4 mix, ref. 3) of transfer o f the complex to the site of degradation. However, since both half-lives were of the same order, it is suggested that liver contains the site of degradation of this complex. Furthermore, the fact that the amount o f a2-macroglobulin-[~3q]elastase complex taken up in liver 15 mix after administration attained 34 ~ of the dose, indicates that 74 °/~iof the complex formed in the body was taken up in the liver, calculated from the percentage of in vivo binding of [~a'l]elastase with th-macroglobulin ( 4 6 . 4 ~ of the dose [31). Accordingly, it could be emphasized that liver is the major site of degradation of the tta-macroglobulin-[l~l]elastase complex. This conclusion is in good agreement with the reports of Ohlsson et al. I! I, 12], which showed the biotransformation of trypsin injected in dog. However, further investigations on the identification o f the [~aq]elastase complex in liver are needed. As seen from the time course o f the intracellular distribution of trichloroacetic acid-precipitable radioactivity in liver (Fig. 5), the radioactivity in the 39 000 x g and supernatant fractions had already attained its maximal level at 2-5 rain, while the 27 000 ~ g fraction reached the maximum 15 mix after administration. The relative specific radioactivity in the 900 x g fraction was almost constant. These results are in agreement with those of Mego and McQueen [51 using formaldehyde-treated [~q]albumin. The difference between the peak time {5 mix) in the 39 000 × g fraction and that (I 5 mix) in the 27 000 x g fraction suggests that the tq-macroglobulin-[t3~l]elastase complex is taken up into the 39 000 ~ g fraction at first and then is transferred to the 27 000 ~ g fraction accompanied by an increase of particle size. Since the increase in size from the 39 000 ~'~:g particle to the 900 :~ g particle was of a slight degree, it is suggested that protein degradation occurs in the 27 000 -~ g fraction before incorporation into the 900 × g fractiom as has been discussed by Mego and McQueen [5]. Since the 39000 :-: g fraction has apparently no ability to degrade injected [t3'l]elastase as described in our following paper [13], the 39 000 .: g paritcte containing radioactivity may be formed by endocytosis and be represented as pinocytotic vacuoles (Mego and McQueen [5, 141) or phagosomes (Straus [15]). The protein-bound radioactivity detected in the 27 000 × g fraction of liver 15 mix after administration, as shown in Fig. 6, was confirmed to be present in lysosomes in the light mitochondrial fraction by sucrose density gradient centrifugation (Fig. 7) and by solubilization by Triton X-100 or freezing-thawing (Fig. 8). These results are in agreement with those of Bertini et al. [ ! 61 and suggest that the 27 000 ~ g particles correspond to phagolysosomes (Straus t lS]) or heterolysosomes (De Duve and Wattiaux [171). Thus, the t~2-macroglobulin~[~a'l]elastase complex might be taken up by endocytosis~ i.e. within a vacuole or phagosome arising from invagination

190 of the cell membrane and thereafter the phagosome can fuse with lysosome as described by De Duve and Wattiaux [17]. In the results reported herein, the biotransformation o f PS'l]elastase bound by a,-macroglobulin has been fully elucidated, but that o f [t3tl]elastase bound by atantitrypsin is still not completely clear. However, from the facts that the at-antitrypsin-[tstI]elastas¢ complex was taken up slowly by liver (Fig. 3) and the relative specific radioactivity in the 39 0C0 × g fraction was increased once again at 60 min (Fig. 5), it is suggested that the at-antitrypsin-[tStl]elastase complex is degraded in a similar pathway to the az-macroglobulin-['3q]elast ase complex. It is interesting that the tissue uptake of [tSq]elastase is mediated by two distinct binding proteins in serum. Studies on the role of the reticuloendothelial system involving Kupffer cells and/or parenchymal cells by the histological techniques of Ohlsson [12] and Straus [I 8] may be needed for the elucidation of the hepatic uptake of [~3tl]elastase-serum protein complexes. Further investigations on the degradation of injected pS~l]elastase in subcellutar fractions are presented in the following paper [13]. ACKNOWLEDGMENTS

The authors thank Dr A. J. Barrett, Strangeways Research Laboratory, Cambridge, and Dr S, Ohtake, Director, Section of Experimental Therapeutics Research for their helpful ad~ices, and also Mr T. Maito, Director, Research and Development Division for his support of our research. REFERENCES ! 2 3 4 5 6 7 8 9 lO I! 12 13 14 15 16 17 l~

Katayama, K. and Fujita, T. (1972) Biochim. Biophys. Acta 288, 172- 180 Katayama, K. and Fujita, T. (1972) Biochim. Biophys. Acta 288, 181-189 Katayama, K. and Fujita, T. (1974) Biochim. Bioph:,s. Acta 336, 165-177 Katayama, K. and Fujita. T. (1971) J. Labelled Compounds 7, 86-89 Mego, J. L. and MeQueen, J. D. (1965) Biochim. Biophys. Acta 100, 136-143 De Duve, C , Pressman, B. C~, Gianetlo, R., Wattiaux, R. and Appelnlans. F. (1955) Biochem. J. 60. 604-617 Lowry, O. H., Ro,,cbrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Wattiaux, R. and De Duvc, C. {1956) Biochem. J. 63, 606--608 Cooperstcin, S. J. and Lazarow, A. (1951) J. Biol. Chem. 189, 665-670 Harper, A. E. (1963) Methods of EnzymaHc Analysis, ed by Bergmeyer, pp. 788-792, Academic Press, New York and London Ohlsson, K., Gam'ol, P. O. and Laurell, C. B. (1971) Acta Chir. Scand. 137, i13-121 Ohlsson, K. (1971) Acta Physiol. Scand. 81, 269-272 Katayama, K. and Fujita, T. (1974) Biochim. Biophys. Acta 336, 191-200 Mcgo, J. L. and McQueen, J. D. (1967) J. Cell. Physiol. 70. 115-120 Straus, W. t1962~ J. Cell Biol. 12, 231-246 Bertini. F., Mego, J. L. and McQucen, J. D. (1967) J. Cell. Physiol. 70, 105- ll4 De Duve, C. and Wattiat:x. R. (1966) Annu. Rev. Physiol. 28, 435-492 Straus, W. (1964) J Cell Biol. 20, 497-507