Studies on canavanine and its derivatives

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BBA 26 034 LYSOSOMAL ENZYME ACTIVITY IN RAT AND B E E F S K E L E T A L MUSCLE N. STAGNI AND B. DE BERNARD Institute of Biochemistry, University of Trieste, Trieste (Italy) (Received June I9th, I968)

SUMMARY I. Acid hydrolases (fl-glucuronidase EC 3.2.1.31 ; cathepsin; fl-galactosidase EC 3.2.1.23; ribonuclease EC 2.7.7.17) of rat and beef skeletal muscle are associated with cytoplasmic particles and are poorly reactive towards external substrates; however, their activity is enhanced or even fully displayed by injuring the particles with a variety of treatments (osmotic shock, sonication, alternate freezing and thawing, addition of Triton X-Ioo). 2. By gradually increasing the concentration of Triton X-zoo in the enzyme assays, acid hydrolases are gradually liberated from the particles, more homogeneously from those of rat than of beef muscle. 3. Osmotic and thermal treatments of beef skeletal lysosomes reveal a higher stability of the particles as compared to those from liver and kidney. 4. The 4 hydrolytic enzymes display in the sedimented fractions a very similar distribution pattern, different from that of cytochrome c oxidase and NAD glycohydrolase. Highest relative specific activity of lysosomal enzymes was found associated to a post-mitochondrial fraction. 5. By isopicnic centrifugation of a post-mitochondrial fraction, both from rat and beef muscle, acid hydrolases were broadly distributed throughout the water sucrose gradient (d = 1.1o-1.2o); cytochrome c oxidase was in a narrow band and NAD glycohydrolase showed a bimodal distribution. 6. On the basis of sedimentation coefficient and equilibrium density acid hydrolases of skeletal muscle are associated to particles different from mitochondria and microsomes. The homogeneous properties of these enzymes suggest that they are associated with a single functional form of lysosome-like particles.

INTRODUCTION In a previous paper 1 we have provided evidence that lysosomes occur in heart muscle. This conclusion was drawn also by WHEATs on the basis of histochemical and electron microscopic analysis of myocardial cells. Lysosomal hydrolases, such as cathepsin, acid ribonuclease, fl-glucuronidase, arylsulphatase have been studied also in skeletal muscle3, 4 where a low specific activity of the enzymes was found, as compared to organs such as liver and spleenL SMITHs has reported that acid phosphatase is scarcely detectable histochemieally in normal rabbit muscle and confined to blood vessels. PELLEGRINO AND FRANZINI7 state that in "normal muscle no lysosomes have Biochim. Biophys. Actal 17o (1968) 129--139

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N. STAGNI, B. DE BERNARD

been detected by electron microscopy". They draw, on the contrary, a correlation between the disappearance of the muscle components during atrophy and the appearance of these organelles. PELLEGRINO, VILLANI AND FRANZINI 8 have observed increase of activity of 2 lysosomal enzymes during the course of atrophy of skeletal muscle. A large increase in lysosomal enzymes have been reported, also, in vitamin E dystrophy of the rabbit, by ZALKIN et al. 9 and in genetic muscular dystrophy by TAPPEL5 a n d TAPPEL et al. 1°. The presence of lysosomes, in normal muscle tissue is therefore still doubtful. It appeared then to us that a biochemical study, as complete as possible, of lysosomal enzymes in skeletal muscle was interesting to be carried out. The properties of flglucuronidase, fl-galactosidase, cathepsin and acid ribonuclease in rat and beef skeletal muscle are here reported. MATERIALS AND METHODS

Phenolphthalein glucuronide and cytochrome c were supplied by Sigma (U.S.A.) ; o-nitrophenyl-fl-D-galactopyranoside by Koch-Light (Great Britain); hemoglobin by Gianni (Milano, Italy); RNA by Boehringer (Germany); NAD by DE.BI. (Varese, Italy) ; Triton X-Ioo by Nymco (Milano, Italy) ; heparin by Evans Med. Ltd. (Great Britain); sucrose by Mallinckrodt (U.S.A.); all other chemicals by Erba (Milano, Italy). Fractionation of the constituents of the tissue Beef and rat skeletal muscle, devoid of fat and connective tissues, was cut into small pieces. Dices of the tissue were ground in a commercial meat grinder and washed with ice-cooled 0.25 M sucrose solution. Portions of about 30 g of the ground tissue were homogenized for 4 ° sec in 12o ml of 0.25 M sucrose solution with a commercial Bamix homogenizer. The pH of the suspension was kept at neutrality by adding drops of 6 M KOH. After filtration, first through 4, and then through 8 layers of cheesecloth, the cellular extract was centrifuged at 2500 × g for IO min (Servall model SSI or Lourdes model AX centrifuges). The sediment was resuspended in 0.25 M sucrose (half volume of the initial) by means of a hand-moved Potter homogenizer with a teflon pestle. The new suspension was centrifuged again at 800 × g for io min, and the precipitate was considered as the nuclear fraction (N). Its supernatant was further centrifuged at 8500 × g for IO min and the pellets, formed mainly by mitochondria, named M 2 fraction ; the supernatant obtained after the first centrifugation (2500 × g) was also centrifuged at 8500 × g for IO rain. This precipitate represented the M1 mitochondrial fraction. Unless otherwise stated, we will indicate as mitochondrial suspension a mixture of the M1 and M 2 fractions in 0.25 M sucrose. For the experiments of differential centrifugation, rat skeletal muscle homogenates (20%) were prepared in 0.25 M sucrose + 500o0 I.U./1 heparin, beef skeletal muscle homogenates (20%) in 0.25 M sucrose + 50 mM KC1, homogenization time being extended to 60 sec. Furthermore the supernatant relative to M 1 fraction was centrifuged at 14 ooo × g for 20 rain to obtain a post-mitochondrial fraction (L fraction). All the fractionation operations were effected at a temperature between o ° and 4 °, in the cold room. Biochim. Biophys. Acta, 17o (1968) I 2 9 - I 3 9

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I sopicnic centrifugation Sucrose-H,O (containing 50 mM KCI) density gradients were prepared using the device described by DE DUVE, BERTHET AND BEAUFAY 11, the density range being 1.10-1.20.

Post-mitochondrial fraction (L) obtained from rat or beef muscle homogenates, prepared in 0.25 M sucrose + 50 mM KC1 (initial homogenization time 60 sec), was generally used. The sediment, washed once, was suspended in the same medium. Aliquots of 0. 4 ml (0.5-0.7 mg of protein nitrogen) were layered on top of the gradient and the centrifugation was performed at IOOOOO × g for 3 h, using the SW 39 rotor of the preparative Spinco centrifuge model L-5o. The material was fractionated by means of the Buchler piercing unit: 33 ° :~ 6 drops were collected in about 8 fractions. Aliquots from each fraction were taken for determination of nitrogen and density, as previously described 1, and for enzymatic analysis.

Enzyme assays fl-Glucuronidase (fl-D-glucuronide glucuronohydrolase, EC 3.2.I.3I ), fl-galactosidase (fl-D-galactoside galactohydrolase, EC3.2.I.23) and hemoglobin-splitting cathepsin were assayed according to the procedure described by ROMEO et al. x. Acid ribonuclease (ribonucleate nucleotido-2'-transferase, EC 2.7.7.17) activity was determined as previously reported 1. RNA concentration in the assay mixture was o.o75 % when beef homogenate and rat mitochondrial suspensions were texted, and o.I % for beef mitochondrial suspensions and rat homogenate. Enzyme assays were run at 37 ° for different times. Total enzyme activity was measured by adding Triton X-ioo to the assay mixture (final concentration o.I %, v/v). In the gradient experiments the incubation time was I h in the presence of 0.025 % (v/v) Triton X-Ioo. In the experiments of intracellular distribution, cytochrome c oxidase (EC 1.9.3.1) activity, as marker of mitochondria, was measured polarographically at 3 °0 using a Clark oxygen electrode according to SOTTOCASAet al. 1~. In is0picnic centrifugation experiments, cytochrome c oxidase was determined according to the procedure previously reported 1. NAD glycohydrolase (EC 3.2.2.5), as marker of microsomesTM, was determined according to KAPLAN14. The reaction mixture consisted of: enzyme; 0.05 M phosphate buffer (pH 7.2) ; 0.9 mM NAD in a total volume of 0.4 ml. The reaction was blocked, after 15 min of incubation, by adding 2.5 ml of 0.5 M KCN and absorbance was recorded at 325 mtz, against a blank, where NAD was added at the end of the incubation time, after KCN. Protein determination Protein nitrogen was determined according to the procedure previously reported 1. RESULTS AND DISCUSSION

Latency of hydrolytic enzymes One of the main criteria for the presence of lysosomal particles in a tissue is that acid hydrolases, when present in these particles, are poorly reactive towards Biockim. Biophys. Acta, I7o (1968) 129-139

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external substrates. The enzymic activity should, therefore, either be fully displayed or strongly enhanced by a variety of treatments that injure the particles. Results reported in Table I confirmed this fact. When enzyme activities were determined in untreated homogenates or mitochondrial suspensions of rat muscle, the amount of hydrolyzed substrate was always lower than that obtained when the same material was subjected to membrane-disrupting treatments. The activity of the untreated material corresponds to that defined as free activity: i.e. indicates that portion of soluble enzymes, available to their specific substrates. As it appears clearly from the data collected in Table I, this free activity, if compared to the activity of the same material additioned with Triton X-ioo, notoriously the most efficient unmasking agent of bound enzyme activity 15, is always higher in homogenates than in mitochondrial suspensions. TABLE I ENZYMIC ACTIVITIES IN RAT SKELETAL MUSCLE HOMOGENATES AiND MITOCHONDRIAL SUSPENSIONS S U B J E C T E D TO D I F F E R E N T T R E A T M E N T S

/~-Glucuronidase,/~-galactosidase, cathepsin, acid ribonuclease specific activities of r a t h o m o g e n a t e s and mitochondrial suspensions subjected to different activating t r e a t m e n t s (average values of at least 4 e x p e r i m e n t s 4- S.E.). T r i t o n X - I o o = o.1% (v/v) in the assay mixtures. H y p o t o n i c i t y ; o.o625 M sucrose instead of o.25 M in the assay mixtures. Sonic irradiation; h o m o g e n a t e s and mitochondrial suspensions subjected twice in aliquots of 3.5 ml to sonic oscillation at 3 A for 3 ° sec (Branson Sonifier). F r e e z i n g - t h a w i n g ; h o m o g e n a t e s and mitochondrial suspensions frozen (--24 °) and t h a w e d (room temperature) 3 times before enzyme assays.

Treatment

Enzyme fl-Glucuronidase fl-Galactosidase

Cathepsin

Ribonuclease

(nmoles of hydrolyzed substrate per mg of nitrogen per rain) Homogenate None Triton X - i o o Hypotonicity Sonic irradiation Freezing-thawing

0.77 i.io o.91 1.o2 1.o 4

i 44+ 4-

0.06 0.08 0.06 o.15 0.09

1.72 2.82 2.3 ° 2.25 2.3 °

4± 4± 4-

o.12 0.22 o.21 0.34 0.24

0.46 i.oo 0.67 0.62 0.83

i 444:j_

0-03 0.06 0.08 o.io 0.09

17.34 38.66 28.93 36.13 26.37

± ± 4± 4-

2.43 2.33 4.52 3.26 3.19

0.40 2.06 I.OI 1.33 o.89

4:L ± :j_ 4-

0.o5 o.16 o.o9 o.07 0.08

2.30 8.21 5.37 6.Ol 6.36

~2 :L 4± 4-

0.23 0.43 0.29 0.55 0.47

1.91 6.80 5.12 5 .o8 5.43

44444-

0.09 0.33 0.47 0.38 0.35

32.67 94.4 ° 84.00 58.54 76.59

444± 4-

1.3o 4.o9 0.33 3.33 6.16

Mitochondrial suspensions None Triton X-ioo Hypotonicity Sonic irradiation Freezing-thawing

This fact is due to the presence, in homogenates, of soluble acid hydrolases, either derived from the cell cytoplasm or released from the particles, during the preparation of the homogenate. The other treatments, used in these experiments enhanced the availability of all the enzymes to their substrates, but none of them was as good as Triton X-Ioo. If specific enzyme activity of homogenates, measured in presence of Triton X-Ioo, is compared with that of mitochondrial suspensions, one realizes that the latter is higher than the former, which indicates that hydrolases tend to concentrate in the sedimented fractions. Biochim. Biophys. Acta, 17 ° (1968) I20-130

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Identical results were obtained with homogenates and mitochondrial suspensions prepared from beef muscle. Activation of latent hydrolases by means of Triton X-too According to DE DuvE le, all lysosomal hydrolases should be liberated in the same proportion, if unmasking of the enzymes is brought about gradually, so that a partial liberation of enzyme is obtained. A suitable treatment, in this respect, is the addition of a detergent, such as Triton X-Ioo, to the suspensions of particles. In order to know the relationship between the detergent concentration and the percentage of each enzyme made available, a series of enzyme assays was run, in which the mitochondrial suspensions, both from rat and beef skeletal muscle, were incubated with increasing concentrations of Triton X-Ioo. Results are reported in Fig. I.

100-

,o.o

/~-

Oalact0sidase~~athepsin

50"

o

o

100

~-Glucuronldase

Y. o

nuctease

50"

,/

o

'

o

~t of Triton X-lO0 per mg of nitrogen

Fig. I. R e ] a t i o n s h i p b e t w e e n t h e a m o u n t of T r i t o n X - i o o a n d p e r c e n t of e n z y m e a c t i v a t i o n in r a t ( × I x ) a n d beef ( O - - O ) skeletal m u s c l e m i t o c h o n d r i a l s u s p e n s i o n s . P r o t e i n nitrogen in t h e a s s a y m i x t u r e s : o.4--o. 5 m g / m l .

With rat particles the free activity (Triton X-ioo/nitrogen-~ o) is, for all enzymes, about 30% ; by increasing the concentration of Triton X-ioo in the assays media, the enzymes availability becomes higher, the maximum falling at 1-1.2/A of Triton per mg of nitrogen. Enzyme activation curves vs. Triton concentration are very similar for the 4 enzymes considered. With beef particles these curves, on the contrary, are less homogeneous. Either they start at different levels of basic free activity (20% for fl-glucuronidase, ribonuclease and cathepsin, 30% for fl-galactosidase) or they reach the maximum at different Triton X-Ioo/nitrogen ratios (I.I for fl-galactosidase, 1.5 for fi-glucuronidase and cathepsin, 2.o for ribonuclease). Bioehim. Biophys. Ac2a, i 7 o (I968) 1 2 9 - I 3 9

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Osmotic and thermal activation One of the characteristics of lysosomes is their response to osmotic and thermal activation. This property has been studied, for instance, with hearO, liver ~7,1s, kidney 19 and, more recently, with Ehrlich ascites tumor cells ~°. Fig. 2 illustrates the results obtained with beef skeletal muscle. From Fig. 2A it appears that, at least under the experimental conditions reported,/~-glucuronidase and ribonuclease increase their availability to the substrates by decreasing the osmolarity of the preincubation medium. 100-

100-

A

B

*,~- Gtucuronidase o Ribonuc[ease

#

"6 v ~-~ 5 0 -

~,f

{

50.

./-~''~/ ,

g

o

6

o'.~ ~

Sucrose (M)

~

.~-G[ucuronidase o~-Gatactosidase

I~O

I~O

2~o

Preincubation time (rain)

Fig. 2. A. Osmotic activation of beef muscle lysosomes. The fraction used was obtained b y centrifuging the nuclear s u p e r n a t a n t at I4OOO x g for 2o rain; the sediment was t h e n directly suspended in the sucrose solution at different concentrations. After 30 rain at o °, the sucrose concentration was adjusted to 0.25 M and free and total activities of/~-glucuronidase a n d ribonuclease determined as described u n d e r METHODS. B. T h e r m a l activation of beef muscle lysosomes. Aliquots of the fraction in 0.25 M sucrose were i n c u b a t e d for different times (abscissa) at 37 ° and p H 5 (0.o5 M acetate buffer). At the times indicated b y the s y m b o l s of the curves, aliquots were t a k e n in order to determine free and total activities of/~-glucuronidase and/~-galactosidase, as described u n d e r METHODS.

This fact becomes evident at sucrose concentrations lower than o.2 M. But even when the sediment is resuspended in distilled water the ]ysosomal enzymes are not completely unmasked. This result indicates that muscle lysosomes, in contrast to liver lysosomesm is and similarly to ascites t u m o r lysosomes 2°, are rather resistant to osmotic activation. In the case of ]ysosomes disruption by thermal treatment (Fig. 2B), a full activation of the two lysosoma] enzymes has never been achieved, the highest availability being 80-85% of the complete. Even in these experiments no decrease of total activity has been observed following the thermal treatment. This result again underlines the good stability properties of muscle lysosomes as compared to liver 17,18 and kidney 19 lysosomes.

Differential centr~[ugation Since it is well known that fractionation of striated muscle homogenate is a difficult task due to aggregation phenomena, a preliminary study was carried out in order to find a suitable dispersing medium. A satisfactory fractionation of rat muscle homogenate was obtained b y using 0.25 M sucrose, supplemented with heparin (50000 I.U./]). Beef muscle homogenate was, on the contrary, fractionated in o.25 M sucrose, 5 ° mM with regard to KC1. Both heparin and KC1, at the concentrations indicated above, were without effect on the free and total activity of acid hydrolases. Four fractions have so been prepared from the homogenate: a nuclear, two mitochondrial and a post-mitochondrial one. Enzyme activity and protein collected Biochim. Biophys. ,~cta, 17o (1968) 129--139