853

Biochimica et Biophysica Acta, 444 (1976) 853--862

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 28048

LOCALIZATION AND SOME PROPERTIES OF LYSOSOMAL DIPEPTIDASES IN RAT LIVER

J.M.W. BOUMA, A. SCHEPER, ANNEKE DUURSMA and M. GRUBER

Biochemisch Laboratorium, The University, Groningen (The Netherlands) (Received April 8th, 1976)

Summary 1. The rates of hydrolysis of 26 synthetic dipeptides by extracts from highly purified lysosomal fractions from rat liver at pH 5.0 and by whole liver homogenates at pH 7.4 have been determined. Extracts from the lysosomal fractions hydrolysed most peptides at a lower rate per mg protein than the homogenates, and some peptides n o t at all. 2. Properties of two dipeptidases present in the extracts from the lysosomal fractions, splitting Ile~31u and Leu-Gly, respectively, were studied in greater detail. The enzyme that hydrolysed Ile~31u was strongly activated by dithiothreitol, showed optimal activity at pH 4.5 and had a molecular weight of about 120 000. Leu-Gly dipeptidase did apparently n o t contain an essential thiol group and had a molecular weight of approx. 90 000. It showed maximal activity at pH 6.5. 3. After differential centrifugation of liver homogenates, Ile-Glu and LeuGly-splitting activities were determined in the fractions, under the optimal conditions mentioned above. The Ile~lu-hydrolysing enzyme activity showed about the same distribution as the lysosomal marker enzyme acid phosphatase. Leu~ly~splitting activity, however, was largely present in the cytosol fraction, with only a small peak in the lysosomal fraction. We obtained evidence that the activities present in the lysosomal fraction and in the cytosol fraction were due to different enzymes, and that one of these enzymes was localized exclusively in lysosomes. 4. It is concluded that some dipeptides originating from intralysosomal proteolysis might be split by lysosomal dipeptidases, whereas others are probably hydrolysed only in the extra-lysosomal compartment of the cell.

Abbreviation:

HEPES, N.2-hydroxycthylpiperazine-N'-2.ethane s u l f o n i c

acid.

854 Introduction Extracts from purified lysosomes are capable of extensive hydrolysis of many proteins at acid pH [1--3]. It has been shown [1,2] that products of this hydrolysis consist predominantly of amino acids and dipeptides. Coffey and de Duve [ 1] have shown that the extent of degradation of globin by lysosomal extracts can be increased by subsequent incubation of the digest with enzymes from the cytosol fraction of liver at pH 8. These results suggested that some dipeptides, which are resistant to lysosomal enzymes, might pass the lysosomal membrane and he split near neutral pH by enzymes present in the cytosol. Subsequent experiments by Lloyd [4], Goldman [5], and ourselves (unpublished results) have shown that some dipeptides can indeed readily diffuse through the lysosomal membrane. We have now measured the rate of hydrolysis of 26 synthetic dipeptides of widely different composition by whole liver homogenates at physiological pH and by extracts from purified lysosomal fractions at pH 5.0. The latter pH is within the range where maximal proteolysis of several substrates by lysosomal enzymes occurs [1,2,6]. Some of the dipeptides were hydrolysed quite rapidly by enzymes present in the lysosomal preparation. Peptides that were n o t hydrolysed by the lysosomal enzymes were readily split by enzymes present in the homogenate near neutral pH, in agreement with the hypothesis mentioned above. Materialsand Methods

Materials Ala-Pro, G l y ~ l u , GlyoLys. HC1, Gly-Pro and Gly-Ser were obtained from Fluka AG, Buchs, Switzerland; Ala-Leu, Gly-Phe, Gly-Trp, Gly-Tyr, Leu-Gly and Leu-Trp from Mann Research Laboratories Inc., New York, N.Y.; a-AspGly, a-Glu-Ala, Gly-Gly, Lys-Ala. 2HBr and Lys-Asp from Sigma Chemical Co., St. Louis, Mo.; His-Leu, His-Lys, Lys-Lys • 2HC1 and Pro-Phe from Miles Laboratories Inc., Kankanee, Ill.; Ile-Asp, I l e ~ l u , Ile-Lys. HC1 and Ile-Pro from Calbiochem AG, Lucerne, Switzerland; P r o ~ l y from Mann or Sigma; Gly-Leu from Fulka or Mann; Leu-Phe from K and K Laboratories Inc., New York, N.Y., U.S.A. 2,4,6-Trinitrobenzene sulfonic acid was purchased from K and K Laboratories or from J.T. Baker Chemicals, Deventer, The Netherlands. Sucrose (Analar grade) was obtained from BDH Chemicals Ltd., Poole, England. Pepstatin, leupeptin and antipain were generous gifts of Professor H. Umezawa, Institute of Microbial Chemistry, Tokyo, Japan. Preparation of subcellular fractions Adult Wistar rats of either sex were used. Triton WR-1339-filled rat liver lysosomes were purified by a modification of the method of Trouet [7] as described previously [2]. The specific activity of acid phosphatase in the lysosomal fractions was 55-+ 4 (mean-+ S.E., 10 isolations) times that of the hornogenates. Lysosomal fractions were frozen in liquid nitrogen and stored at --20°C. Before use, Triton X-100 was added to the preparations to a final

855

concentration of 0.25% (v/v). Differential fractionation was performed according to Bouma and Gruber [8]. Sucrose solutions used for tissue fzactionations did n o t contain EDTA.

Determination of dipeptidases For the determination of I l e ~ l u dipeptidase, 200/~1 enzyme solution, 100 /~1 0.5 M sodium acetate buffer pH 4.5, and 100/~1 5 mM dithiothreitol were preincubated at 37°C for 30 rain. Then, 100/~1 50 mM Ile-Glu was added and the mixture was incubated at 37°C for 1 h. To blanks, water was added instead of Ile-Glu solution. At the beginning and the end of the incubation period, samples of 200/~1 incubation mixture was added to 200/~1 10% trichloroacetic acid and the precipitate was removed by centrifugation. The increase in free amino groups caused by hydrolysis of the dipeptide was measured by the m e t h o d described by McDonald et al. [9]. In this method, trinitrobenzene sulfonic acid reacts with amino groups from amino acids, whereas reaction with dipeptides is suppressed by Cu 2÷ ions. In our modification of this method, triplicate samples of 100 /~1 of the trichloroacetic acid supernatant were first mixed with 200/~1 150 mM borate buffer containing 25 mM HEPES pH 9.5 (in recent experiments it was found that HEPES could be omitted). Subsequently, 500 /~1 4.1 mM CuSO4 in 155 mM borate buffer pH 8.4 was added. The final pH was 8.3. After 15 min at room temperature, 100 /~1 31 mM trinitrobenzene sulfonic acid was added and the mixture was kept at 37°C for exactly 30 rain. The reaction with trinitrobenzene sulfonie acid was stopped by adding 200/~1 1.2 M HC1 and the absorbancy at 420 nm was read in a 1 cm cuvette. Reference curves containing mixtures of the dipeptide and its constituent amino acids in proportions corresponding to 0, 10, 20, 30 and 40% hydrolysis, and including the appropriate amounts of buffer, dithiothreitol and trichloroacetic acid, were prepared for each set of assays. For the determination of Leu-Gly dipeptidase, 200/~1 enzyme solution, 100 /~1 0.5 M HEPES buffer pH 6.5, 100/~1 5 mM iodoacetic acid and 100/~1 50 mM Leu431y were incubated at 37°C for 2 h. To blanks, water was added instead of Leu-Gly solution. At 0 and 120 min, samples of 200/~1 incubation mixture were added to 200/~1 10% trichloroacetic acid. T h e precipitate was removed by centrifugation and the degree of hydrolysis was determined by the method described above.

Other determinations Protein was determined by the method of Lowry et al. [10]. Acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) was measured according to Gianetto and de Duve [11]. Malate dehydrogenase (L-malate: NAD ÷ oxidoreductase, EC 1.1.1.37) was determined as described by Beaufay et al. [12]. Glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) was assayed according to de Duve et al. [13], with minor modifications. Cathepsin BI (EC 3.4.22.1) was determined using Method I given by Barrett [14] with minor modifications. Results

Hydrolysis of dipeptides by lysosomal extracts and homogenates The rate of hydrolysis of dipeptides by extracts from the lysosomal fraction

N-terminal Ala o r GIy Ala-Leu Ala-Pro GIy-GIu GIy-GIy Gly-His Gly-Leu Gly-Phe Gly-Pro Gly-Ser GIy-Trp GIy-Tyr

N-terminal hydrophobic lie-Asp IIe-Glu IIe-Lys He-Pro Leu-Gly Leu-Trp

Substrate

14

1 5 4 4 20 -3 20 18

--

+ -38 I00

31 31

Lysosomes

340 + 35 29 39 220 420 + 120 450 170

18 150 71 + 140 ST0

Homogenate

Dipeptidase activity (nmol/min per mg protein)

C-terminal Ala, G I y o r Ser Glu-Ala Lys-Ala Agp-GIy GIy-GIy Leu-Gly Gly-Ser Pro--GIy

C-terminal hydrophobic Ala-Leu Gly-Leu His-Leu Gly-Phe Pro-Phe Gly-Trp Leu-Trp GIy-Tyr

Substrate

+ 3 5 38 3 2

21

14 4 46 20 2 20 I00 18

Lysosomes

130 + 32 29 140 120 22

340 220 37 420 57 450 270 170

Homogenate

Dipeptidase activity (nmol/min per mg protein)

2 2

3 21

N-terminal acidic Asp-GIy Glu-Ala

N-terminal proline Pro-Gly Pro-Phe

46 + + + +

Lysosomes

•

22 57

32 130

37 140 + 47 +

Homogenate

Dipeptidase activity (nmol/min per mg protein)

N-terminal basic His-Leu l-Iis-Lys Lys-Ala Lys-Asp Lys-Lys

Substrate

C-terminal proline Gly-Pro Ala-Pro He-Pro

C-terminal acidic lie-Asp IIe-Glu Lys-Asp GIy-GIu

C-terminal ba~c Gly-His Hi~Lys Ile-Lys Lys-Lys

Substrate

m

1

31 +

31

+ + 4-

4

Lysosomes

18 150 47 35

39 140 71 4-

Homogenate

Dipeptidase activity (nmol/min per mg protein)

F o r these e x p e r i m e n t s T r i t o n W R - 1 3 3 9 - f i l l e d l y s o s o m a l f r a c t i o n s , a n d liver h o m o g e n a t e s f r o m f a s t e d r a t s in 1 0 m M H E P E S b u f f e r p H 7.4 w e r e u s e d . A c t i v i t i e s o f l y s o s o m a l e x t r a c t s w e r e d e t e r m i n e d b y i n c u b a t i n g 0 . 5 m g l y s o s o m a l p r o t e i n in a v o l u m e o f i m l c o n t a i n i n g 1 0 m M d i p e p t i d e , 1 m M d i t h i o t h r e i t o l , a n d 1 0 O m M s o d i u m a c e t a t e b u f f e r (final p H 5 . 0 ) a t 3 7 ° C f o r p e r i o d s u p t o 3 h. H y d r o l y s i s o f d i p e p t i d e s b y h o m o g e n a t e s w a s m e a s u r e d i n m i x t u r e s ( I m l ) c o n t a i n i n g 0 . 5 m g p r o t e i n , 10 m M d i P e p t i d e , a n d 1 0 0 m M H E P E S b u f f e r , final p H 7 . 4 . A f t e r v a r i o u s p e r i o d s o f t i m e , a l l q u o t s o f 0 . 3 m l w e r e a d d e d t o 0 . 2 m l 1 0 ~ ( w / v ) t r i e h l o r o acetic acid and t h e d e g r e e o f h y d r o l y s i s w a s d e t e r m i n e d a f t e r r e a c t i o n o f f r e e a m i n o g r o u p s w i t h t r i n l t r o b e n z e n e s u l f o n i c acid in t h e p r e s e n c e o f c o p p e r i o n s as d e s c r i b e d in t h e M e t h o d s s e c t i o n . V a l u e s g i v e n are a v e r a g e s o f initial r a t e s o f h y d r o l y s i s o b t a i n e d w i t h t w o d i f f e r e n t e n z y m e p r e p a r a t i o n s . H y d r o l y s i s o f t h e m a j o r i t y of t h e d i p e p t i d e s h a s also b e e n f o l l o w e d b y t h i n l a y e r c h r o m a t o g r a p h y o n silica gel G w i t h a s o l v e n t s y s t e m c o n t a i n i n g n - b u t a n o l ~ p y r i d i n e / a c e t i c a c i d / w a t e r ( 8 0 : 20 : 6 : 24, v / v ) . T h e s p o t s w e r e c o l o u r e d t o M o f f a t a n d L y t i e [ 1 5 ] . L y s o ~ o m a l f r a c t i o n s u s e d f o r t h e s e e x p e r i m e n t s w e r e p r e v i o u s l y d i a l y z e d a g a i n s t 0.1 M a c e t a t e b u f f e r pH 5.0 c o n t a i n i n g 1 m M d i t h i o t h r a i t o l . H y d r o l y s i s o f p e p t i d c s c o n t a i n i n g a n h n i n o b o n d a n d o f s o m e o f t h e l y s i n c - c o n t a l n i n g p e p t i d e s w a s e s t i m a t e d b y t h i n l a y e r c h r o m a t o g r a p h y o n l y (results i n d i c a t e d b y + o r --).

H Y D R O L Y S I S O F D I P E P T I D E S BY L Y S O S O M A L E X T R A C T S ( A T p H 5 . 0 ) A N D BY L I V E R H O M O G E N A T E S ( A T p H 7 . 4 )

TABLE I O0 01

857 at pH 5.0 and by whole homogenates at pH 7.4 is shown in Table I. Among 26 dipepfides tested, about 15 were h y d r o l y ~ at an appreciable rate by enzymes present in the lysosomai fraction. Most of the peptides containing hy&ophobic or basic amino acid residues were quite rapidly split, whereas five peptides containing proline either as the amino or the carboxyl component, were not. All peptides were hydrolysed by the homogenates at pH 7.4. Some dipeptidases isolated from animal tissues and active above neutral pH are known to be activated by divalent metal ions [16--18]. We made our determinations on diluted homogenates without addition of metal ions and it is, therefore, possible that the activities of the enzymes are still higher in vivo. The HEPES buffer used for the preparation of the homogenates and the enzyme determinations has negligible affinity towards the metal ions concerned. The homogenates did contain lysosomes. It is, however, unlikely that the activities measured in homogenates at pH 7.4 were due to lysosomal enzymes. The specific activities measured in the lysosomal fraction at pH 5 were generally much lower than the activities measured in the homogenates at pH 7.4, and if the activities of the lysosomal fraction were due to t n d y lysosomal enzymes, their activities near neutral pH would be expected to be still lower, for nearly all known lysosomal enzymes show acid pH optima. In addition, homogenates contain only about 2% lysosomal protein. Our lysosomal fractions, on the other hand, contained significant amounts of mitochondria and microsomes. Specific activities of marker enzymes for these particles in the highly purified lysosomal fraction were still about 50% of those of homogenates [2]. From the data in Table I it is clear that hydrolysis by the lysosomal fraction of peptides like Ile-Glu or Leu-Gly (which were rapidly split by homogenates) might easily be due to enzymes present in contaminating mitochondria or microsomes. In order to check this possibility, we have studied the localization of enzymes splitting these substrates more carefully.

Ile-Glu dipeptidase Optimal conditions for determining this dipeptidase were studied using extracts from purified Triton WR-1339-filled lysosomes as enzyme preparations. I l e ~ l u dipeptidase was strongly activated by 1 mM dithiothreitol. Raising the concentration of the activator to 5 mM did n o t increase the activation. In the presence of 1 mM dithiothreitol, 1 mM monoiodoacetate caused some inhibition. Activation of the dipeptidase by dithiothreitol was n o t instantaneous. In routine experiments, enzyme was preincubated with activator for 30 min. Addition of 100 mM NaCl had no effect. Pepstatin (5.7/~Vl), leupeptin (42/~M) and antipain ( 3 0 / ~ I ) , peptide-like antibiotics that strongly inhibit proteolysis by lysosomal enzymes in vitro [19], did not change the activity. The enzyme showed an optimum at about pH 4.5, with a sharp decline in activity towards lower pH values (Fig. 1). Under the conditions described in the Methods section linearity with enzyme concentration and incubation time was obtained up to 30% hydrolysis and 120 rain incubation. The distribution of I l e ~ l u dipeptidasc after differential centrifugation is shown in Fig. 2. The pattern obtained is very similar to that of the lysosomal marker enzyme acid phosphatase and differs from those of the microsomal

858 100-

Zte-GL.u

idQse

100-

80-

80-

60-

60-

40-

40-

20-

20-

Leu-GLy dip' ~

~

?

~ >

~

~

~

~

o pH

pH

Fig. 1. Hydrolysis o f ile-Glu and Leu-Gly by extracts f r o m a Triton WR-1339-fllled lysosomal fraction as f u n c t i o n of pH. For determinations o f ne-Glu dipeptidase, 0.1 M acetate buffers were used up to pH 5.5; 0.1 M m s / a t e buffers were used at pH 6 and 7. For determinations of Leu-Gly dipeptidase, 0.1 M acetate buffers were used up to pH 5.5 and 0.1 M HEPES buffers were used at higher pH values. All buffers were b r o u g h t to t h e same ionic strength b y adding appropriate a m o u n t s of NaCl. Other conditions, and methods o f determination are given in Materials and Methods.

Leu -Gty

Leu=GLy

(acid-stobLe) (totaL) (4)

d dipeptidese (3) (5) [~ I

n

b

'

,e

~ ' l& b ' ~ Percentage of toteL protein

'

Fig. 2. Distribution patterns o f e n z y m e s after dlffcrentia| fraetionation. Liver h o m o g e n a t e s were fractionated in nuclear, heavy and l i g h t mitochondrial, microsomal and cytosol fractions. Protein and enz y m e s were measured as described in Materiak and Methods. Acid-stable Leu-Gly dipeptidase was determ i n e d after preincubation o f fractions at pH 5.0 as described in Table' II. E n z y m e aetiv/ties are given as blocks. The first block from t h e left represents t h e nuclear fraction, t h e second t h e heavy mitochondrial fraction, ete. The width of each block is proportional to the percentaJe of protein found in the fraction and t h e height gives t h e relative specific activity (percentage o f e n z y m e div/ded by percentage of protein). Each graph gives the m e a n result of the n u m b e r of fraetinnations given in brackets. Average e n z y m e aetivities ~umol of substrate per rain) pe r g fresh weight (~S.E.) were: acid4~able Leu-Gly dipeptidase, 0.73 + 0.04; total Leu-Gly dipeptidase, 6.4 ± 2.4; lle-Glu d i p e p t i d u e , 0.74 ± 0.10; malate dehydrogenase, 42.6 ± 1.4; acid phosphatase, 5.05 + 0.25; giucose-6-phosphatase, 9.1 ± 1.1. AvereEe p r o t e i n c o n t e n t was 178 -+ 6 mg per g fresh weight. Averaged recoveries from the h o m o g e n a t e s ranged between 85 and 111%;

859 T A B L E II SOLUBILIZATION OF ACID PHOSPHATASE; IIe4SIu:DI1PEPTIDASE AND Leu-Gly DIPEPTIDASE FROM A MITOCHONDRIAL-LYSOSOMAL FRACTION A m i t o c h o n d r i a l - l y s o s o m a l f r a c t i o n ( M L - f r a c t i o n ) , p r e p a r e d f r o m 8 g o f liver, w a s s u s p e n d e d i n 3 0 m l s u c r o s e . T r i t o n X - I O 0 w a s a d d e d t o a p o r t i o n o f t h e M L - f r a c t i o n t o a f i n a l c o n c e n t r a t i o n o f 0 . 2 5 % (v/v), a n d e n z y m e a c t i v i t i e s w e r e m e a s u r e d in t h e t o t a l s u s p e n s i o n a n d in t h e s u p e r n a t a n t o b t a i n e d a f t e r c e n t r l f t t g i n g f o r SO mill a t S 2 0 0 0 X g. A n o t h e r p o r t i o n o f t h e M L - f r a c U o n w a s f r o z e n i n l i q u i d n i t r o g e n a n d thawed on a water bath at 20°C three times, and subsequently also centrifuged. A third sample of the MLfraction was not treated, but centrifuged immediately. N o n - s e d i m e n t a b l e a c t i v i t i e s (%)

ML-fraction containing 0.25% Triton X-100, not centrifuged S u p e r n a t a n t s o f MI.,-fraction t r e a t e d as f o l l o w s : 0.25% Triton X-100 3 X frozen and thawed untreated

Acid phosphatase

Ile-Glu dipeptidase

Leu-Gly dipeptidase

100

100

100

82 54 3

83 70 6

84 73 25

marker glucose-6-phosphataseand from malate dehydrogenase, which is located in mitochondria. Additional evidence for the presence of the enzyme within membrane-bound subcellular particles was the solubilizationof the enzyme from a mitochondrial-lysosomalfractionby treatment with Triton X-100 or by repeated freezingand thawing (Table II). A n estimation of the molecular weight of the enzyme was made by gelfiltration on Sephadex G-150 (Fig. 3). Allenzyme activitywas present in one peak, that was eluted shortly after the dimer of albumin. A graph of the elutionvolume against log molecular weight as described by Andrews [20] indicated a molecular weight of approx. 120 000.

Leu-Gly dipeptidase Hydrolysis of this substrate by extracts from purified lysosomal fractions was not affected by 1 mM monoiodoacetate and even somewhat inhibited by 1 mM dithiothreitol. Pepstatin (5.7/~M) and leupeptin ( 4 2 / ~ I ) did not change the activity and antipain (30 /~M) had only a small effect. The enzyme was slightly inhibited by 1 mM EDTA (Table III), but none of the metal salts tested (1 mM CaC12, MgC12, ZnSO4, COSO4, CdC12 and MnCI2) caused activation, whereas some (CdC12 and COC12)gave strong inhibition. Addition of 100 mM NaC1 had no e f f e c t . L e u ~ l y dipeptidase had a pH optimum of about 6.5 (Fig. 1). Hydrolysis of the substrate was linear with enzyme concentration and incubation time up to 2 h and 15% hydrolysis. After differential fractionation, the enzyme activity was largely presen t in the cytosol fraction, with a small peak in the lysosomal fraction (Figi 2). Subsequent experiments showed that the activities present in the two fractions can be attributed to two different enzymes (Tabl e Ill). The cytosol enzyme was strongly activated by MnCI2

860 T A B L E lII COMPARISON CYTOSOL

OF

Leu-Gly DIPEPTIDASE

ACTIVITIES

FROM

LYSOSOMAL

FRACTIONS

AND

T r i t o n W R - 1 3 3 9 - f l l l e d l y s o s o m a l f r a c t i o n s a n d c y t o s o l f r a c t i o n s w e r e p r e p a r e d as d e s c r i b e d i n t h e M e t h o d s s e c t i o n . P r e i n c u b a t i o n w a s d o n e a t 3 7 ° C in m i x t u r e s o f 0 . 5 m l l y s o s o m a l f r a c t i o n ( a b o u t 0 . 6 m g p r o tein) or 0.5 ml cytosol fraction (about 1.3 mg protein) and 1.0 ml 0.1 M sodium acetate buffer pH 5.0. w h i c h c o n t a i n e d 1 . 5 m M m o n o i o d o a c e t a t e a n d 5 . 7 /~M p e p s t a t i n in o r d e r t o i n h i b i t a u t o l y s i s . P r e i n c u b a tion was stopped by adding I ml 0.1 M HEPES buffer pH 7.3. For controls and for incubation mixtures c o n t a i n L n g E D T A o r M n C I 2, 0 . 5 m l l y s o s o m a l o r c y t o s o l f r a c t i o n w a s a d d e d t o a m i x t u r e o f t h e a c e t a t e a n d H E P E S b u f f e r s d e s c r i b e d a b o v e a n d k e p t at 0 ° C . Preincubation time a t p H 5 (h)

0 0 0 1 2

Addition during enzyme assay

None 1 mM EDTA 1 mM MnCl 2 None None

A c t i v i t y (%) Lysosomal fraction

Cytosol fraction

100 71 62 100 107

100 33 436 4 9

15-

10-

t-

O Eiution voturne (mL) Fig. 3. E l u t i o n p r o f i l e s o f Ile-Glu d i p e p t i d a s c ( e ) a n d L e u - G l y d / p e p t i d a s e (o) a f t e r gel f i l t r a t i o n o f a n extract from a Triton WR-1339-fllled lysosomal fraction on Sephadex G-150. About 5.5 ml of a lysosomal extract containing 15.5 mg protein was applied on a column (63 × 3,2 cm) of Sephadex G-150 (superfine), a n d e l u t e d w i t h 1 0 m M a c e t a t e b u f f e r p H 5 . 7 c o n t a i n i n g 0 . 5 m M d i t h / o t h r e i t o l a n d 0.1 M N a C L F r a c t i o n s o f a b o u t 4 . 5 m l w e r e c o l l e c t e d a t a r a t e o f a p p r o x . 7 m l / h a n d d i p e p t i d a s c s w e r e d e t e r m i n e d : as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . R e c o v e r i e s w e r e 1 2 2 % f o r t h e Ile-Glu d i p e p t i d a s c a n d 93% f o r L e u GIy dipeptidase. The column was eanbmted by detet.m/ning elut/on volumes of the d/met and monomer of bovine serum albumin (moL wt. 134000 and 67 000, respect/vely), trypsin inhibitor from soya b~an (tool. w t . 21 5 0 0 ) a n d h o r s e h e a r t c y t o c h r o m e c ( m o l . w t . 1 2 4 0 0 ) . C a t h e p s i n B1 ( m o l . w t . a p p r o x . 2 5 0 0 0 ) , p r e s e n t in t h e l y s o s o m a l e x t r a c t , w a s d e t e r m i n e d b y m e a s u r i n g h y d r o l y s i s o f b e n z o y l - L - A r g - p nitroanilide.

861

and almost completely inactivated by preincubation at pH 5. The lysosomal

enzyme was inhibited by MnCI2 andstable atpH 5. The acid-stability of the latter enzyme offered an opportunity to determine its distribution among fractions obtained by differential centrifugation. The distribution profile of the acid-stable enzyme was very similar to that of acid phosphatase (Fig. 2). Like acid phosphatase and Ile~:~lu dipeptidase, Leu-Gly dipeptidase could be solubilized by treatment of a mitochondrial-lysosomal fraction with Triton X-100 or freezing and thawing (Table II). Most lysosomal enzymes show latency if intact particles are incubated with substrate. The lysosomal membrane is however, permeable to dipeptides [4], and Goldman [5] has shown that dipeptides that can be split by lysosomal enzymes, cause lysis of the particles. In agreem e n t with these results, Dr. M.F. Zuretti (personal communication) has demonstrated lysis of liver lysosomes by 10 mM Leu-Gly in the presence of 0.25 M sucrose.

After gelfiltration, lysosomal Leu-Gly dipeptidase was eluted in one peak, corresponding to a molecular weight of approx. 99 000 (Fig. 3). Discussion We found no significant breakdown by lysosomal extracts of a number of our dipeptides. This result is in agreement with the work of Coffey and de Duve [1] and by ourselves [2]. In our previous study [2], we identified several dipeptides after extensive hydrolysis of the B-chain of insulin by lysosomal extracts at pH 5. Among the dipeptides present in significant amounts were GlyGlu and Gly~Ser, which were also found to be resistant to lysosomal enzymes in the present experiments. We also found a large a m o u n t of Thr-Pro, in agreem e n t with the resistance to hydrolysis of dipeptides containing iminobonds shown in Table I. A small amount of Ala-Leu in the hydrolysate of the B~hain had perhaps escaped hydrolysis owing to the low substrate concentration used in those experiments. We have shown the presence within lysosomes of two enzymes hydrolysing L e u ~ l y and I l e ~ l u respectively. Few other papers on lysosomal dipeptidases have appeared. The existence of a dipeptidase active at pH 5 (and, therefore, probably of lysosomal origin) was first demonstrated by Loughlin and Trikojus [21] in a preparation from thyroid tissue. This enzyme was tested on Cys-Tyr, but a number of other dipeptides {among them Leu-Gly) was also highly susceptible. The enzyme differed from our L e u ~ l y dipeptidase by being activated by Zn 2÷, Mn 2* and Co 2÷. A dipeptidase specific for Ser-Met and a number of related dipeptides, was discovered by McDonald et al. [22] as a contamination of cathepsin C preparations from bovine spleen and rat liver. The enzyme, which was also demonstrated in lysosomal fractions from bovine spleen, was optimally active at pH 5.5 and fully inactivated by 0.5 mM EDTA. Taylor and Tappel [23] recently described the subceUular localization of dipeptidases splitting Trp-Leu, Arg~:~ly and Arg-Phe in rat liver. Like our Leu-Gly dipeptidase, these enzyme activities were present both in the cytosol and in the lysosomal fraction and they were optimally active at pH 6.5. The authors found, at the pH optimum, some activation by very high concentrations of dithiothreitol. McDonald et al. [22] probably isolated a true dipeptidase, for their enzyme

862 failed to split Ser-Met-NH2, Z-Met-Ser and Ser-Met-Glu, but it should be remembered that enzymes splitting dipeptides are not necessarily specific for these substmtes. Highly purified preparations of an aminopeptidase from kidney, for instance, hydrolysed numerous dipeptides [24]. Our I l e ~ l u and LeuGly-splitting enzymes differ from the classical cathepins B1, B2, C and D with respect to their molecular weights and several other properties. One of the multiple forms of cathepsin A (a carboxypeptidase that might be active on dipeptides) has a molecular weight of about 100 000, but this enzyme is, in contrast to our dipeptidases, not at all influenced by 1 mM glutathione, iodoacetate, EDTA, MnC12 or CdCI2 [25]. A study of the specificities of the I l e ~ l u and Leu~ly-splitting enzymes must await further purification. Acknowledgements Our thanks are due to Dr. J. Ken McDonald for providing us with details of the dipeptidase assay, to Mr. Th. de Boer for adapting the method to our purposes, and to Dr. M.F. Zuretti for her valuable criticism of the manuscript. The present investigations have been carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organization for the Advancement of Pure Research

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