Biochimica et Biophysica Acta, 385 (1975) 163--172 @) Elsevier Scientif ic Publishing Company , Ams te rdam -- Pr inted in The Nether lands

163

BBA 27634

METAL-ION EFFECTS ON PROTEOLYSIS AND STABILITY IN SECONDARY LYSOSOMES OF MOUSE KIDNEY*

SAMUEL JAMES DAVIDSON

Department o f Physiology, Tufts University Medical School, 136 Harrison Avenue, Boston, U ** Mass. 02111 ( . S . A . )

(Received October 24th, 1974)

Summary

In previous studies, in vitro digestion of [ 1 2 5 I] ribonuclease by lysosomes of mouse kidney was limited because breakdown, which was rapid at first, slowed markedly so that most of the labeled protein escaped degradation. We now describe incubation conditions which allow digestion to proceed until approximately 70% of the exogenous protein label is released in acid-soluble form, after 30--45 min at 37 ° C. Such activity is seen with either the addition of EDTA or incubation of concentrated cell particle suspensions. EDTA is effective in low concentrations and shows the same stimulation of digestion over a range of approximately 10-6--10 -3 M. Other chelating agents have simi- lar effects; dipyridyl and hydroxquinoline are as effective as EDTA, o-phenan- throline and diethyldithiocarbamate are slightly less effective. When the incuba- tion medium had been treated with a chelating resin, Chelex 100, dilute suspen- sions of lysosomes were as active as those in EDTA. These results lead to the conclusion that metal ions, present as contaminants in very small concentra- tions, inhibit the activity of mouse kidney lysosomes.

The effect of the metal ions is to diminish lysosomal stability, leading to release of intact labeled ribonuclease in non-sedimentable form. Interaction between lysosomes and metal, leading to inhibition of digestion upon heating, occurs at low temperature, but breakdown requires incubation at 37°C and may be autolytic. In contrast to chelators, mercaptoethanol is without marked effect on stability; the stimulation in digestion rate caused by this agent is due either to a direct effect on the lysosomal enzymes or to a non-destructive influence on the lysosomal structure.

* Parts o f this w o r k have b e e n repor ted at the m e e t i n g o f the Federa t ion o f A m e r i c a n So c i e t i e s for E x p e r i m e n t a l Biology, Atlantic City, N.J., April, 1973 [6].

** Address for c o r r e s p o n d e n c e ; B io -Research Ins t i tu te , 9 Commercial Avenue, Cambridge, Mass. 02138 (U.S.A.)

164

Introduct ion

Ribonuclease is rapidly and specifically absorbed from blood into kidneys [1--4]. The time course of kidney uptake is readily observed by injecting mice or rats intravenously with [ 1 : s I] ribonuclease. In this system, renal absorption of the protein is largely separate in time from breakdown, offering the oppor- tuni ty to study these phases of vacuolar function separately [4]. Work in this laboratory has been directed towards achieving a quantitative correlation be- tween the lysosomal processes observed in vitro and the in vivo kinetics of kidney labeling with exogenous ribonuclease. To this end, we have described observations upon lysosomal digestion of pinocytosed [12 S i]ribonuclease in vitro [5,6], using methods applied by Mego and associates to the study of lysosomal catabolism of denatured albumin [7,8]. The in vitro digestion pro- cess described in our earlier reports was initially rapid enough to account for the rate of kidney-label loss, but slowed greatly before a large proportion of the iodine label had been rendered acid-soluble. We now describe in vitro ribo- nuclease digestion which is extensive as well as rapid when precautions are taken to overcome the inhibitory effects of low concentrations of metal ions. These ions are shown to interact with the lysosomal membranes in a manner leading to destruction of the lysosomes. The in vitro process of lysosomal ribonuclease digestion, as described here, correlates well with the rapid decline in kidney radioactivity after uptake of ribonuclease.

Experimental procedure

Soluble chelating agents were obtained from Fisher Scientific Co. Chelex 100 chelating resin is a product of Bio-Rad Laboratories, Richmond, Va. Re- agent grade chemicals were used throughout. Glass-distilled water was also used, although similar results were obtained in a trial with water which had

.been purified by passage through "organic removal" and mixed-bed ion ex- changer columns manufactured by the Barnstead Sybron Corporation, Boston, Mass. Protein labeling and preparative methods have been described in detail previously [4,5]. In brief, mice were injected through the tail vein with 100 pg of 12 S i.labele d bovine ribonuclease, dissolved in isotonic saline. Mice were sacrificed 20 min after injection, and the kidneys removed and homogenized in isotonic sucrose, with a motor-driven teflon pestle. The homogenates were centrifuged in an International PR-2 centrifuge for 10 min at 250 X g to remove intact cells, nuclei etc. and the supernatant centrifuged again for 20 min at 2250 X g. The 2250 X g precipitates were suspended in 0.25 M sucrose, 0.025 M sodium acetate buffer, pH 5.5. When the 250 X g supernatant was stored ovemight before use, the 2250 X g precipitate obtained from it was resuspended in pH 5.5 buffer and recentrifuged at 1000 X g as previously described [5]. The repeated centrifugation removed some inactive material formed during storage. Unless otherwise described, incubations involved addi- tion of 0.1 ml of particle suspension to 4.9 or 9.9 ml of incubation medium at 37°C. The incubation medium usually included 0.25 M sucrose, 0.025 M so- dium acetate buffer, and 0.05 M mercaptoethanol, and was at pH 5.5. To determine the extent of digestion during incubations samples were withdrawn

165

and treated with phosphotungstic acid in hydrochloric acid, precipitating intact ribonuclease [4] . Digestion is expressed as the percentage of incubated label made soluble in phosphotungstic acid as a function of time at 37°C [5] . Protein concentrations were determined by the method of Lowry et al. [9] .

Buffers were treated in batches with Chelex 100 resin as described by Willard [10] . The resin was prepared according to Willard, except that 150-ml batches of 0.025 M sodium acetate buffer, pH 5.5, were used in place of phosphate for washing 10 g of the resin. During the first washing with buffer, the pH of the suspension was readjusted to 5.5 with NaOH. The resin was not washed with water after the washings in buffer.

Results

When particulate preparations are incubated at protein concentrations of abou t 8--20 mg/ml, lysosomes containing [ 12 si] ribonuclease digest the pro- tein actively for a longer period of time than in more dilute suspensions, leading to release of a greater proport ion of the label in acid-soluble form (Fig. 1). In these concentrated suspensions about 60--70% of the label is usual- ly rendered acid-soluble in the course of an incubation of 45--60 min. When the particulate preparations are repeatedly frozen and thawed prior to incuba- tion, rapid digestion in concentrated suspension is abolished, just as was previ- ously shown for dilute suspensions [5] . For example, a concentrated suspen- sion which released 42% of its radioactivity in acid-soluble form during 30 min of incubation solubilized only 11% during the same interval after repeated freezing and thawing. This shows that intact lysosomes are required for diges- tion in concentrated suspensions as well as dilute, and that the concentrat ion dependence of digestion is not due to alterations in concentrat ion of cathepsins released from broken lysosomes.

In media containing low concentrations of EDTA, dilute suspensions of these lysosome preparations also digested the ribonuclease more vigorously than did the dilute suspensions in EDTA-free medium (Fig. 2). Very low EDTA concentrations were effective: EDTA had its maximum stimulatory effect at concentrations varying from 10 -0 M at least up to 10 -3 M, the highest concen- tration tested. After addition of EDTA to the phosphotungstic acid reagent used to precipitate acid-insoluble label rather than to the incubation medium, the digestion curve was indistinguishable from that seen without EDTA, show- ing that the results obtained with EDTA are not due to an effect upon the assay chemistry. Table I presents a statistical comparison of incubations in dilute suspensions with and without EDTA, and in concentrated suspensions. Stimulation by EDTA, or in concentrated suspension, was consistantly seen and is significant. The chelator gives the greatest effect. For this reason, and since incubation in dilute medium gives the greatest control of medium compo- sition, incubations were routinely performed in dilute suspension with EDTA.

The results suggest that protein digestion by lysosomes of mouse kidney is inhibited by small amounts of metal ions. In our experiments, those ions were present as contaminants in the incubation medium. When a dilute suspension was incubated, enough metal was present relative to the quanti ty of protein to cause inhibition when scavenged and bound by the lysosomes. The inhibitory

166

70

6O

H 50

4o

(,q

(:3

10

c

v

MINUTES AT 3 7 ° C

70

°

a_

10

0 q 0

0 tO ZO 30 4 0 5 0

MINUTES AT 3 7 * C

Fig. 1. P ro t eo lys i s o f [ 12 s I] r i bonuc lease by rena l l y s o s o m e s was m o r e v igo rous in c o n c e n t r a t e d par t ic le

s u s p e n s i o n s t h a n in d i lu te ones. P r e p a r a t i o n s were m a d e as desc r ibed u n d e r E x p e r i m e n t a l p rocedure . The 2 2 5 0 X g p r ec i p i t a t e s were r e s u s p e n d e d in 0 .05 M m e r e a p t o e t h a n o l , 0 .25 M sucrose , 0 . 0 2 5 M s o d i u m

ace ta t e b u f f e r , pH 5.5. Fo r i n c u b a t i o n s , 0 .1-ml v o l u m e s of su spe ns ion were a d d e d to 0 .15, 0.9 or 9.9 ml

o f the s a m e m e d i u m at 3 7 ° C to give p r o t e i n c o n c e n t r a t i o n s of 8.96 m g / m l (o), 2 .24 m g / m l (v) and 0 .224 m g / m l (A). I n c u b a t i o n s a m p l e s were 25 k, 0.1 ml or 1 ml respec t ive ly , in p r o p o r t i o n to i n c u b a t i o n

v o l u m e . Wate r was added to b r ing all s amp le s up to the s a m e v o l u m e b e f o r e p r e c i p i t a t i n g i n t a c t r ibonu-

clease w i t h p h o s p h o t u n g s t i c acid as desc r ibed .

Fig. 2. L y s o s o m a l p r o t e o l y s i s o f [ 1 2 5 i ] r i bonuc lease was m o r e v i g o r o u s in the p resence of E D T A . Prepara-

t i on and i n c u b a t i o n were as d e s c r i b e d u n d e r E x p e r i m e n t a l p roc e du re . I n c u b a t i o n i n d i c a t e d by the t r ian-

gles inc luded 3 • 10 -5 M E D T A . Pro te in c o n c e n t r a t i o n d u r i n g i n c u b a t i o n was 0 .196 m g / m l .

e f f e c t o f the me ta l was n o t seen when a che la to r was presen t , or when the incuba t ion t o o k place in a small vo l um e , so t ha t the to ta l a m o u n t of me ta l ion was low. In order to tes t this exp lana t ion , e x p e r i m e n t s were done with a var ie ty o f chela t ing agents subs t i t u t ed for E D T A (Table II) . All che la tors were

T A B L E I

S T A T I S T I C A L C O M P A R I S O N O F E D T A A N D D I L U T I O N E F F E C T S U P O N L Y S O S O M A L P R O T E I N D E G R A D A T I O N

Labe led pa r t i cu l a t e f r ac t i ons , p r e p a r e d as desc r ibed u n d e r E x p e r i m e n t a l p r o c e d u r e , were s u s p e n d e d in

0 .05 M m e r c a p t o e t h a n o l , 0 .25 M sucrose , 0 . 0 2 5 M s o d i u m ace ta t e b u f f e r , p H 5.5. These suspens ions , w i t h p r o t e i n c o n c e n t r a t i o n s ave rag ing a b o u t 27 m g / m l , were i n c u b a t e d w i t h o u t f u r t h e r d i lu t ion to y ie ld the

va lues fo r " c o n c e n t r a t e d s u s p e n s i o n s " . 10-k s amp le s were t h e n r e m o v e d in the course of i n c u b a t i o n , m i x e d wi th 2 .35 m l of w a t e r and d i lu t ed wi th p h o s p h o t u n g s t i c acid r e a g e n t as p r e v ious ly desc r ibed to

d e t e r m i n e ac id-soluble r a d i o a c t i v i t y . F o r i n c u b a t i o n s in d i lu te suspens ion , 0.1 ml of the su spe ns ion desc r ibed above was a d d e d to 9.9 ml of the su spens ion m e d i u m at 37°C. 1 ml i n c u b a t i o n s a mp le s were

r e m o v e d , d i lu t ed wi th 1 .35 ml of w a t e r and t r e a t ed w i t h p h o s p h o t u n g s t i c acid r e a g e n t as desc r ibed . The table gives the p e r c e n t o f ac id-soluble r a d i o a c t i v i t y a f t e r 30 ra in of i n c u b a t i o n , less the value at the s t a r t o f i n c u b a t i o n . P robab i l i t i e s were c o m p u t e d b y t- test fo r u n p a i r e d m e a s u r e m e n t s .

C o n d i t i o n No. o f de te r - Acid-so luble 1251/30 min . m i n a t i o n s (% o f to ta l label + S.E.M.)

Di lu te s u s p e n s i o n 10 26.7 -+ 2 .0

C o n c e n t r a t e d s u s p e n s i o n 7 39.6 -+ 1 .5" Di lu te s u s p e n s i o n w i t h 3 • 10 -5 M E D T A 8 46 .8 + 1 .8"

• P < 0 .01 for the d i f f e r e n c e b e t w e e n the s t a r r ed values.

167

T A B L E II

T H E E F F E C T OF D I V E R S E C H E L A T I N G A G E N T S UPON P R O T E O L Y T I C A C T I V I T Y OF R E N A L LY- SOSOMES

Suspens ions were d i lu ted 1 : 1 0 0 in 0 . 025 M s o d i u m a c e t a t e , 0 .25 M sucrose , 0 .05 M m e r c a p t o e t h a n o l w i th the che la tor , if any , a t a c o n c e n t r a t i o n of 3 • 10 - s M. S o d i u m d i e t h y l d i t h i o c a r b a m a t e wa s dissolved in w a t e r to a c o n c e n t r a t i o n of 3 • 10 -2 M and d i lu ted in i n c u b a t i o n m e d i u m jus t p r io r to the e x p e r i m e n t . Chelex t r e a t m e n t was p e r f o r m e d as desc r ibed in E x p e r i m e n t a l P rocedure . The d i lu t ions were k e p t cold for 30 rain, ze ro - t ime samples were taken , and then the m i x t u r e s were i n c u b a t e d at 37°C for 30 rain. All i ncuba t ions in each e x p e r i m e n t were p e r f o r m e d in dupl ica te , and the dup l ica te resul ts averaged. Differ- ences b e t w e e n p h o s p h o t u n g s t i c acid soluble label at 30 and 0 m i n are given u n d e r A (30 rain).

A d d i t i o n or t r e a t m e n t A (30 m i n ) (-+ S.E.M.) (no. of expts ) %

N o n e (4) 20.2 (-+ 4 .33 ) E D T A (4) 44 .9 (+- 1 .28) Chelex 100 (4) 42.6 (-+ 1.76) D ipy r idy l (3) 42 .0 (-+ 3 .14) H y d r o x y q u i n o l i n e (3) 42 .6 (-+ 2 .15) o-Phenathro l ine (2) 36 .2 (+- 1 .07) D i e t h y l d i t h i o c a r b a m a t e (1) 37 .3

used at a concentration of 3 " 10 -s M. If the stimulation of digestion was due to the removal of metal ions by chelation, then diverse types of chelators should stimulate digestion in a similar fashion, and this occurred. Additions of either dipyridyl or hydroxyquinoline allowed digestion to proceed as vigorously as with EDTA. With o-phenanthroline or diethyldithiocarbamate, digestion was stimulated to a smaller extent. Trials with diethyldithiocarbamate gave varying results, and the stimulation given in the table is a minimum value, due to the instability of this reagent at low pH [11 ].

These results with chelators support the hypothesis that ~he inhibitory action of metal ions is responsible for inhibition of lysosomal digestion seen in the absence of chelator, but do not exclude the possibility of a direct interac- t ion between chelators and lysosomes. To test this possibility, incubation me- dium was treated with Chelex 100, a chelating resin. The resin was removed by centrifugation prior to incubation, so that no chelating agent was present at the time of incubation. The digestive activity observed with this medium was as great as that found with EDTA {Table II). It is established, therefore, that the chelators favor digestion by removing inhibitory metal ions, rather than by direct interaction with the lysosomes or their contents.

The differences between lysosomal action in dilute and concentrated me- dium, or with EDTA, do not appear or are much less marked in the first few minutes of incubation (Figs 1 and 2). This gives the impression that there are two stages of digestion, an earlier one which is insensitive to metal ions and a later, sensitive stage. This was shown to be false by diluting lysosomes in cold EDTA-free medium 45 min prior to warming to 37 ° C. Lysosomal digestion does not occur at the low temperature, but, even after warming, digestive activity in these samples was almost completely inhibited (Fig. 3). Parallel sam- ples, identically treated except that the dilution and incubation medium con- tained EDTA, were active. Thus, the entire course of lysosomal digestion is subject to inhibition by metal ions. An interval of time must elapse between

168

5 0

- 4 0

H

N 5 0

I¢1 J m

"J 2 0 O 0')

(9 <~ 10

0

o o

o o

i i

,'o 20 3'0 MINUTES AT 57 °

40

%-,.~ 20

m

i i I I

0 t0 20 3 0 4 0

MINUTES AT 37 °

Fig. 3. A f t e r p r e - i n c u b a t i o n a t 0 °C in the absence of E D T A , d iges t i on of [12 S i] r i bonuc lease by d i lu te

s u s p e n s i o n s o f l y s o s o m e s was a l m o s t c o m p l e t e l y inh ib i t ed . P r e p a r a t i o n s were as d e s c r i b e d u n d e r Exper i - m e n t a l p r o c e d u r e . F o r p r e i n c u b a t i o n , 0.1 ml a l iquo t s of labeled par t ic le su spe ns ion w e r e a d d e d to 9.9 ml

of i n c u b a t i o n m e d i a a t O°C. These d i lu t i ons were a g i t a t e d for 45 min a t a b o u t 150 r e v . / m i n on a

g y r o - r o t a r y s h a k e r ( N e w B r u n s w i c k Sc ien t i f i c Co., Inc. , N e w Brunswic k , N.J . ) in the cold r o o m , p r io r to

w a r m i n g to 3 7 ° C for i n c u b a t i o n as desc r ibed , o wi th 3 • 10 -S M E D T A , p r e i n c u b a t e d at 0°C; L w i t h o u t E D T A , p r e i n c u h a t e d at 0 ° C; e, w i t h o u t E D T A , no p r e i n c u b a t i o n .

Fig. 4. L y s o s o m a l b r e a k d o w n at 37 ° C, wi th re lease of i n t a c t [ 125 I] r ibonuc lease , was i n h i b i t e d in the p r e sence of E D T A . I n c u b a t i o n s were p e r f o r m e d as desc r ibed u n d e r E x p e r i m e n t a l p r o c e d u r e , in the

p r e sence and absence of 3 • 10 -5 M E D T A . 1 ml i n c u b a t i o n s a mp le s were r e m o v e d a t t he t i m e s s h o w n and m i x e d w i t h 0 .25 M sucrose on ice. When no E D T A was p r e s e n t d u r i n g i n c u b a t i o n , the cold suc rose

s o l u t i o n also i n c l u d e d 3 - lEtS m m o l of E D T A . These m i x t u r e s were c e n t r i f u g e d in the Sp inco No. 40 .3

r o t o r a t 8 9 0 0 X g for 15 ra in to b r ing d o w n in t ac t , labeled l y s o s o m e s [4] a n d the s u p e r n a t a n t s t r e a t e d wi th p h o s p h o t u n g s t i c acid to p r e c i p i t a t e i n t a c t r ibonuc lease . A c o r r e c t i o n was m a d e for q u e n c h i n g by p h o s p h o t u n g s t i c acid. R i b o n u c l e a s e re lease was the m e a s u r e of l y s o s o m e b r e a k d o w n and is g iven as ac id- inso luble 12 S I in t he 8 9 0 0 X g s u p e r n a t a n t , as a p e r c e n t a g e of the t o t a l label s p u n at 8 9 0 0 X g. The

t r i ang les r e p r e s e n t i n c u b a t i o n s w i t h o u t E D T A , and the circles, wi th E D T A . The o p e n s y m b o l s r e p r e s e n t

t he m e a n s o f t h r ee e x p e r i m e n t s w i t h mice sac r i f i ced 20 ra in a f t e r in jec t ion . Each e x p e r i m e n t invo lved

dup l i ca t e i n c u b a t i o n s b o t h w i t h and w i t h o u t E D T A . S t a n d a r d e r rors are g iven by the bars. The d a r k s y m b o l s r e p r e s e n t a single e x p e r i m e n t in w h i c h the mice were sac r i f i ced 5 rain a f t e r in jec t ion .

the exposure of the lysosomes to metal ions and the complet ion of processes leading to inhibition; during this interval, digestion may appear to be metal- insensitive. The slow processes could be binding of the metal ions to sites on the lysosomes, or structural changes subsequent to binding. By pre-incubation at 0 ° C, these processes can be separated from the digestive events.

Two sites of action are possible for inhibitors of lysosome function. Such inhibitors could act directly upon specific lysosomal enzymes, or indirectly by affecting lysosome structure. One effect of EDTA is to protect the structural integrity of the mouse kidney lysosomes, which break down more rapidly in dilute suspension at 37°C without EDTA. To show this, aliquots removed from dilute suspensions at 0-time and during incubation were diluted in cold, iso- tonic sucrose and centrifuged at 8900 X g, to bring down intact, labeled lyso- somes. The radioactivity remaining in the supernatant after such centrifugation includes both intact ribonuclease, released by particle breakage, and the labeled products of digestion. The intact ribonuclease was then brought down by addi-

169

tion of phosphotungstic acid to the supernatants. The amount of radioactivity precipitated by the acid was a measure of lysosome breakdown during incuba- tion. We have previously shown that, prior to incubation, labeled lysosome contents are entirely undegraded, by the criterion of phosphotungstic acid- solubility [5] . Release of undigested ribonuclease through particle breakdown in the course of a 45 min incubation was less than half as great when EDTA was present than otherwise (Fig. 4). The metal ions thus exert their inhibitory effect by damaging the lysosomal membrane. Breakdown during incubation was rapid, most of that observed occuring in the first 10 min of incubation. The significance of this fact is considered in the discussion. The figure also shows the results of a similar experiment with a kidney preparation from a mouse killed 5 min after injection. Digestive activity is low in these prepara- tions. The results are similar to those seen in preparations made after 20 min, showing that at least some of the pinocytic vesicles, those coming down at low speeds, are also subject to membrane damage due to metal ions.

The experiments just described allow a qualitative comparison of the fra- gility of vacuolar membranes in the presence or absence of chelator, but do not accurately measure total lysosome breakdown due to adsorption of ribo- nuclease onto components of the preparations. Accurate determination of breakage requires addition of 0.25 M NaC1 to the medium used for centrifuga- tion at 8900 × g, to prevent adsorption. Undamaged lysosome preparations are sedimented effectively in this medium [5] . When incubation samples were treated in this way, duplicate incubations in the presence of EDTA gave an average of 22% breakage in 45 min, and in the absence of chelator, 72% (Table III). Thus, most of the lysosomes are destroyed when no chelator is present. It should be noted that essentially all of the label in the EDTA-free incubation medium was either released in acid-soluble form by digestion, or intact by lysosome breakage, confirming that the limiting factor in digestion, in the absence of chelator, is membrane breakdown. In order to demonstrate this, Table III presents values for total digestion and breakdown at 45 min, uncor- rected by subtraction of 0-time values.

Verity and Reith [12] postulated that the destructive effect of Hg 2÷ upon lysosomes is due to interactions between the metal and the sulfhydryls of the

T A B L E II I

A B S O L U T E M E A S U R E M E N T OF L Y S O S O M A L B R E A K D O W N A F T E R 45 MIN AT 37°C, W I T H A N D W I T H O U T E D T A

E x p e r i m e n t was p e r f o r m e d as descr ibed for Fig. 4 e x c e p t that sa mple s w e r e ta ken on ly at 0 t i m e and af ter 45 ra in o f i n c u b a t i o n and the c e n t r i f u g a t i o n at 8 9 0 0 × g was p e r f o r m e d in 0 .25 M NaC1 plus 0 .25 M sucrose . D u p l i c a t e i n c u b a t i o n s w e r e p e r f o r m e d ; the results agreed wi th in -+ 2%. To ta l values, w i t h o u t sub- t rac t ing 0 - t ime values, are g iven for d iges t ion and b r e a k d o w n at 4 5 min .

To ta l l y s o s o m e breakage ( label u n s e d i m e n t a b l e in sa l t - sucrose m e d i u m ) (% o f to ta l labe l )

To ta l rib onuc lease d iges t ion (Ac id so luble label ) (% o f to ta l label)

S u m o f d ig e s t i o n and b r e a k d o w n (% o f t o ta l label)

3 • 10 - s M E D T A 22.2 59 .2 81 .4

No E D T A 72 .0 23.7 95 .7

170

5 0

H 4 0 u3

3 0 bJ _1

3 2o 0 (/1

--q 1o ¢j

o ' 6 ' fo ' 3'o ' , 6 MINUTES AT 37 °

l

w .J

Z O

rr

2 0 ~ " H hO

bA W ~r tL

Fig. 5. L y s o s o m e s t a b i l i t y is u n a f f e c t e d b y c o n c e n t r a t i o n s o f m e r c a p t o e t h a n o l w h i c h e n h a n c e p r o t e o l y s i s

b y l y s o s o m e s . I n c u b a t i o n s i n t h e p r e s e n c e a n d a b s e n c e o f 0 . 0 5 M m e r c a p t o e t h a n o l , were p e r f o r m e d as

d e s c r i b e d u n d e r E x p e r i m e n t a l p r o c e d u r e . Al l m e d i a i n c l u d e d 3 • 1 0 -s M E D T A . 1 m l i n c u b a t i o n s a m p l e s

were r e m o v e d a t t he t i m e s s h o w n a n d m i x e d o n ice w i t h 1 .5 m l o f 0 . 2 5 M suc rose . W h e n t he i n c u b a t i o n

m e d i u m l a c k e d m e r c a p t o e t h a n o l , 0 . 0 5 m m o l o f m e r c a p t o e t h a n o l were p r e s e n t i n t h e c o l d suc rose . T h e s e

s a m p l e s were t h e n c e n t r i f u g e d a n d t r e a t e d as d e s c r i b e d f o r Fig. 4. o, 0 . 0 5 M m e r c a p t o e t h a n o l ; A n o

m e r c a p t o e t h a n o l .

membrane. It was therefore of interest to determine whether the stimulatory effect of mercaptoethanol might partly reflect protect ion afforded to the mem- branes. Experiments upon stability in the presence and absence of mercapto- ethanol {Fig. 5) were performed, similar to those described for EDTA (Fig. 4). Mercaptoethanol stimulates lysosomal digestion in this system with an optimal concentrat ion of about 0.03--0.05 M, and inhibition at higher concentrations. If the sulfhydryl reagent is omitted, there is no effect on the fragility of the lysosomes during incubation. Thus, mercaptoethanol probably exerts its stimu- latory effects by way of the lysosomal proteases, although a subtle effect on lysosomal organization is not excluded.

D i s c u s s i o n

Renal absorption of ribonuclease is highly organ specific, rapid and mas- sive and is followed by rapid breakdown of the ribonuclease. Because the breakdown phase succeeds uptake, these processes can be studied separately [4,5] . In our previous studies of [1: Si]ribonuclease digestion within kidney lysosomes in vitro, we observed that the digestion process stopped after about 25% of the label was released in acid-soluble form. We now describe ribonu- clease digestion by kidney lysosomes which is extensive as well as rapid, and proceeds until about 60--70% of the particle-bound radioactivity is made acid- soluble, with rapid hydrolysis of ribonuclease for about the first 20 min of the reaction. This process has a rate constant comparable to that shown by the intact kidney in its loss of radioactivity after [ 12 s I] ribonuclease absorption, as will be described subsequently (manuscript in preparation). Thus, the rapidity and extent of the in vitro lysosomal reaction confirm that the reaction ob- served in v~tro can also account for ribonuclease breakdown and the very rapid loss of label from kidney in vivo.

171

In making these observations, we found that the lysosomes are sensitive to very low concentrations of a metal ion contaminant. Since 10 -6 M chelator protec ted the lysosomes it follows that metal concentrations of the order of 10 -6 M caused damage to the lysosome membranes, leading eventually to lysis. This was shown by the increased extent of lysosomal digestion, either in the presence of chelators with diverse structures, in the absence of chelator when the medium was first treated with a chelating resin (Table II), or in a very concentrated particle suspension without chelator, in which proteins and possi- bly other consti tuents bound the metal (Fig. 1). The metal effect was exerted at the membranes, as shown by the increased breakdown of lysosomes when these were not protected by chelator (Fig. 4). After 45 min of exposure to the metal ions in this fashion, about 70% of the labeled particles were subject to lysis (Table III).

Verity and Reith [12] and Lauwerys and Buchet [13] have described labilization of lysosomes by Hg 2÷. Media for the experiments described here included 0.05 M mercaptoethanol, which would have bound Hg 2÷ and opposed their interaction with membrane sulfhydryls [12] . Chvapil et al. described lysosome labilization by Cu 2÷ [14] . Dipyridyl has a relatively low association constant for copper [15] and its effectiveness in stabilizing the renal lysosomes makes copper an unlikely cause of the labilization described here. Desai et al. reported a labilizing effect of Fe 2~ and Fe 3÷ [16]. The stabilizing effect of dipyridyl, which is an effective chelator of Fe 2÷ and Fe ~÷, suggests that these ions may be causing the inactivation. The lesser stabilization conferred by o-phenanthroline accords with this interpretation, due to its smaller association constant with iron [15] . A likely mechanism for this effect was postulated by Desai et al. [16] . They suggested that a mixture of Fe 2~ and Fe 3+ initiated peroxidation of membrane lipids, leading to the lysosomal disruption which they observed in this medium.

Although pre-incubation at 0°C in the absence of chelator causes loss of proteolyt ic activity by mouse kidney lysosomes, these particles do not lyse prior to incubation at 37 ° C. In preparations stored for 30 min in chelator-free medium at 0°C, but not heated thereafter, about 15% of the radioactivity remained in the superna tan ta f t e r centrifugation at 8900 × g for 15 min; much less than the amount found (40%) after a further incubation of 30 min at 37 ° C. Clearly, the lysosomes were still, largely intact before the temperature was raised. Similarly, Lauwerys and Buchet found that the labilization of rat liver lysosomes by Hg 2÷ also proceeded in two steps; first the metal ion interacted with lysosomes and the second step required elevated temperature and pro- ceeded even after removal of the metal [13] . Lauwerys and Buchet interpreted their results as showing that lysis was in fact autolysis, and required the action of the lysosomal enzymes themselves, after the initial labilization caused by the action of the metal. The present results are in accord with this interpretation. The metal ions appear to destroy the relative immunity which lysosomes enjoy to the destructive effects of their own complement of enzymes. Perhaps this effect may prove useful in elucidating the nature of this immunity against autolysis.

It is striking that most of the breakdown of labeled particles which oc- curred during incubation, with or without EDTA, came during the first 10 min

1 7 2

of incubation, and that breakdown thereafter was very slow (Fig. 4). The phase of lysosome disruption is quite rapid. The results with EDTA suggest that events prior to incubation, perhaps in the preparation process, had primed some of the particles for breakdown during incubation. An alternative hypothe- sis is that some of the labeled particles are intrinsically susceptible to lysis during incubation under these conditions. It is clear, however, that the bulk of the particles are quite stable during incubation, when certain metal ions are excluded.

Despite the postulated role of membrane sulfhydryls in lysosomal stability [12] , the mercaptoethanol in these systems was without apparent effect on membrane stability. This supports the suggestion of Mego and McQueen [17] that the sulfhydryl compounds act to enhance catheptic activity in these systems.

Acknowledgements

The author is grateful to Lorraine Wolfe and Sheila Song for skilled technical assistance. Thanks are due to Dr Walter L. Hughes for most helpful discussions. This work was partly supported by Research Grant BC-41 from the American Cancer Society.

References

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