Comp. Biochem. Physiol., 1978. I ol. 60B. pp. 35 to 40, Pergamon Press. Printed in Great Britain

LYSOSOMES IN ERYTHROCYTES FROM THE FLOUNDER (PLATICHTHYS FLESUS)

TONE VISLIE* Institute of Zoophysiology, University of Oslo, Oslo, Norway

(Received 18 July 1977)

Abstract--l. Activities of the lysosomal enzymes fl-N-acetylglucosaminidase, cathepsin D and fl-glucur- onidase, were found in hemolyzates of purified erythrocytes from the flounder Platichthys flesus.

2. Latent enzyme activities were found for cathepsin D and fl-giucuronidase in homogenates of the flounder erythrocytes, but no consistent latent activity was found for fl-N-acetylglucosaminidase.

3. Neutral red was shown to be taken up by the erythrocytes from the flounder. 4. The possibility that lysosomes are present in flounder erythrocytes is discussed.

INTRODUCTION

In all vertebrates except mammals, the mature eryth- rocytes are nucleated (Kindred, 1972). During matur- ation the cellular hemoglobin content increases and most of the cytoplasmic organelles disappear. In intermediate stages of erythrocytes from lower verte- brates (Tooze & Davies, 1965, 1967; Scorza et al., 1970; Sekhon & Maxwell, 1970), as in- mammalian reticulocytes (Lessler & Pack, 1964), lysosomes have been shown to be present by histological techniques. It is assumed that lysosomes are involved in degrada- tion of cytoplasmic organelles during cell maturation (Tooze & Davies, 1965; Sekhon & Maxwell, 1970). It is a matter of discussion whether or not lysosomes are present also in mature erythrocytes from lower vertebrates, although it seems accepted that mature mammalian erythrocytes contain no cytoplasmic organelles (Bloom & Fawcett, 1968).

A protein-splitting enzyme with an optimal activity in the acid range has, however, been demonstrated in hemolysates of mammalian erythrocytes (Goetze & Rapoport, 1954; Haschen et al., 1964, 1965; Haschen, 1965; Reichelt et al., 1973). This acid pro- teinase shows properties which suggest its classifica- tion as cathepsin D, but has been found attached to the plasma membranes of these cells (Bernacki & Bos- mann, 1972; Reichelt et al., 1974; T6k6s & Chambers, 1975). Different glycosidases, among them fl-N-ace- tylglucosaminidase, are also found in human erythro- cyte plasma membrane fractions and these glycosi- dases are quite similar to those of lysosomal origin in other tissues (Bosmann, 1971). Both cathepsin D and fl-N-acetylglucosaminidase are typical lysosomal enzymes (de Duve, 1963). The observed activities of the lysosomal enzymes in mammalian erythrocytes cannot be explained by contamination of reticulocytes or leukocytes in the erythrocyte suspensions analysed (Reichelt et al., 1973).

To our knowledge, the activities of lysosomal enzymes have not been determined in red blood cells from lower vertebrates, and the presence of lysosomes has not been studied by examining the properties of

* Present address: Zoological Institute, University of Oslo, Box 1050, Blindern, Oslo 3, Norway.

35

iysosomal enzymes in such cells. Earlier studies on lysosomes in erythrocytes from lower vertebrates have been based on histological techniques (Tooze & Davies, 1965, 1967; Scorza et al., 1970; Sekhon & Maxwell, 1970).

The purpose of the present investigation was to find out if lysosomal enzymes and/or lysosomes can be demonstrated in erythrocytes from a teleost, the flounder Platichthys flesus.

MATERIAL AND METHODS

Materials

Flounders, Platichthys flesus L., weighing approximately 300-400 g, were caught in fishing nets in the inner part of Oslot~ord and were kept in a refrigerated seawater aquarium (temp 6-8°C, f.p. -1.88°C) for at least 7 days before being used. The fish were not fed during the period of captivity.

Preparation of red blood cells Blood was obtained by puncturing the bulbus arteriosus

with a heparinized syringe. White and red blood cells were separated by layering blood above a solution of high den- sity containing an agglutinating agent (Boyum, 1964). The erythrocytes, after aggregating at the interface, sedimented to the bottom of the tube. A 2% solution of methylcellulose (Methocel MC cP, Fluka AG) and a sodium metrizoate solution (Isopaque, Nyco Ltd, Norway) with a specific gra- vity of 1.20 (33.9%) were mixed in a ratio of 1.5:1 to get a separation solution with a specific gravity of 1.08 (Boyum, 1964; HuUiger & Blazkovec, 1967). Different con- centrations of the components in the separation fluid were tested, but the one used was found to give the most satis- factory result. The separation fluid was pipetted into test- tubes to give a column 2-3 cm high. The blood was care- fully layered above this separation fluid without mixing at the interphase, to give a column about I cm high. The tubes were left at 0°C in a vertical position until the separ- ation was completed (about 2 hr). The top layer containing the leukocytes was carefully removed with a Pasteur pipette (Boyum, 1964).

After the sedimentation of erythrocytes was completed, the packed cells were washed 3 times by centrifugation in 1 ml cold 0.9% NaCl solution. The cells were first centri- fuged twice for only 20 sec at 600g to avoid sedimentation of remaining leukocytes. The suspension was finally centri- fuged at 4000@ for l0 min.

36 TONE VISLIE

The contamination of leukocytes in the erythrocyte frac- tions was tested by counting the number of white and red blood cells.

Counting of blood cells

Leukocytes and erythrocytes were counted in a Biirker counting chamber after staining the cells with a solution containing both neutral red and crystal violet (Hesser, 1960). The differentiation between erythrocytes and leuko- cytes in the microscopic preparations was based on studies done by Catton (1951), Watson et al. (1956), Weinreb (1958), Boyar (1962), Saunders (1966, 1968) and Kindred (1972). Table 1 shows the number of leukocytes and eryth- rocytes in whole blood and in the purified erythrocyte frac- tions after separation. The fraction of leukocytes was reduced from 1.3 to 0.2% by using the method for purifica- tion described above.

Hemolysis of red blood cells

A known amount of purified erythrocytes was hemo- lysed in distilled water to give a concentration of about 5% (w/v). For measurements of total enzyme activities, the hemolysate was treated with Triton X-100 to a final con- centration of 0.1% (v/v). The hemolysate was centrifuged at 10,000 g for 20 min and the supernatant removed for enzyme assays.

Latent enzyme activity

To study structural bound latency of acid hydrolases in erythrocyte homogenates, a known amount of separated red blood cells was resuspended in ice-cold buffer contain- ing 10 mM Tris-HCl, 2 mM MgC12, 3 mM EGTA pH 7.45 (Berg et al., 1975). The cells were homogenized by four strokes in a Dounce homogenizer with a tight-fitting pestle. to give approximately 8-12% (w/v) homogenates. The homogenizing vessel was surrounded by an ice slurry. After 5 min, ice-cold sucrose solutions were added to the homo- genates to give a final sucrose concentration of 0.25 M. The homogenates were centrifuged for 4 min at 600 g. The supernatant was carefully pipetted out with a Pasteur pipette and Triton X-100 (0.1% v/v) was included in one portion of the supernatant for measurements of total enzyme activities. In the other part of the supernatant, enzymes were determined without further treatment.

Enzyme assays

Cathepsin D (E.C. 3.4.4.23) was determined by the method of Anson (1937) as modified by Barrett (1972). The assay mixture contained, in a total volume of 0.5 ml, 2% (w/v) bovine hemoglobin, 0.25 M formic acid/sodium for- mate buffer and 0.25 ml homogenate/hemolysate. The tem- perature during incubation was 37°C and the pH of the incubation solution usually 3.15.

//-N-acetylglucosaminidase (fl-AGA) (E.C. 3.2.1.30) was determined by the method of Barrett (1972). The assay mixture contained, in a total volume of 1.5 ml, 5mM p-nitrophenyl-N-acetyl-fl-n-glucosaminide, 0.1 M sodium

citrate/citric acid buffer, 0.1 M NaCI and 0.5 ml homogen- ate/hemolysate. The temperature during incubation was 37°C. Two different incubation mixtures, one with pH 4.3 and the other with pH 5.7, were used in most of the deter- minations. In some of the assays, sucrose was added to a final concentration of 0.25 M in the incubating mixture.

fl-glucuronidase (E.C. 3.2.1.31) was measured according to Barrett (1972). The assay mixture contained, in a total volume of 0.5 ml, I mM phenolphthalein glucuronic acid, 0.08 M acetic acid/sodium acetate buffer, pH 5 and 0.25 ml homogenate/hemolysate. The incubation temperature was 37°C.

The enzyme substrates used were from Sigma Chemical Co., St. Louis, Missouri, U.S.A.

The accuracy of the determination of cathepsin D and fl-AGA in erythrocyte hemolysates was about 7 and 4% respectively.

Neutral red uptake Washed erythrocyte fractions were incubated at 10°C

in a solution containing neutral red. A stock solution of neutral red was made by dissolving neutral red in 0.9% NaC1 to a final concentration of 0.3 mg/ml. The neutral red solution was filtered just before use. The concentration of neutral red in the incubation solution was between 1 and 8 mg/g cells. The incubation was carried out in dark- ness and the tubes were continuously shaken. Control cells were incubated in 0.9% NaCI solution without neutral red. Immediately after the incubation, the erythrocytes were centrifuged at 4000 g for 10 min, the supernatant discarded and the ceils washed 5 times by centrifugation in 0.9% NaCI solution. 0.1 M HCI was added to the final cell pellet for extraction of neutral red. After 30 min, TCA (trichlor-acetic acid) was added to a final concentration of 2.5%. After centrifugation at 1000g for 25 min, the supernatant was carefully removed and its optical density (OD) determined at 530 nm in a Beckman spectrophot- ometer. The amount of neutral red in the supernatant cor- responds approximately to the amount of dye taken up by the erytbrocytes during incubation.

• RESULTS

Acid hydrolases

Table 2 shows the total activities of the two enzymes, ~-N-acetylglucosaminidase and cathepsin D, in hemolysates from purified flounder erythrocytes. By measur ing the activities of these two enzymes in leukocyte fractions with a known number of leuko- cytes, it was possible to estimate tha t the contami-

Table 2. Total activity of fl-N-acetylglucosaminidase and cathepsin D in hemolysates of erythrocytes from the

flounder Platichthys flesus

Table I. Number of erythrocytes and leukocytes in un- Fish treated blood and in erythrocyte fractions after separation number

of the blood from the flounder Platichthys flesus 1

Number of Number of % Leukocytes of 2 erythrocytes leukocytes total cell 3

per mm 3 per mm 3 number 4 5

Untreated 2.41 x 106 3.19 x 104 1.3 6 blood Erythrocyte 9.01 x 1 0 6 1.86 x 104 0.2 fraction

The values are based on two separate countings of blood from two different fishes.

Total activity* fl- N-acetylglucosaminidaset

pH 4.3 pH 5.7 Cathepsin D~:

117.87 _ 6.34 296.79 ___ 19.52 198.44 + 12.03 217.38 __+ 6.91 150.50 + 3.19 214.32 + 19.14 93.33 +_ 6.33 149.93 + 7.86 182.43 + 10.13

154.89 + 2.21 182.34 + 5.11 138.66 _ 5.23 184.96 _ 2.01 181.81 + 8.35

* Values represent mean enzyme activities + S.D. for at least 3 determinations of blood from the same fish.

t Enzyme activity expressed as nmoles tyrosine/min g cells.

:~ Enzyme activity expressed as nmoles/min g cells.

Lysosomes in erythrocytes from flounder 37

nation of leukocytes in the purified erythrocyte frac- tions could not possibly contribute more than about 1~o of the total enzyme activities measured, It is there- fore unlikely that the enzyme activities measured can be derived from contaminating leukocytes.

Valid assay conditions were established for the enzymes by exploring such variables as enzyme con- centration, incubation time, incubation temperature and pH. The activities of both cathepsin D and f-N- acetylglucosaminidase were linear with the time of in- cubation and the enzyme concentration (expressed as ~o hemolysates) under the conditions adapted. Figure 1 shows that the enzyme activites increased with in- creasing temperature, but the correlation is not linear. The activity of cathepsin D as a function of pH is shown in Fig. 2; pH optimum is at about pH 3. Two distinct peaks on the pH curve were observed for fl-AGA, one at pH 4.34.4 and another at approxi- mately pH 5.7 (Fig. 2). The activity of #-AGA was found to be higher at pH 5.7 than at pH 4.3 (Fig. 2 and Table 2).

Table 2 shows a considerable variation in the ac- tivities of both cathepsin D and fl-AGA in erythro- cytes from different fishes.

Latent activity of the hydrolytic enzymes Total enzyme activity in homogenates from

flounder erythrocytes was measured in the presence of Triton X-100 and "free" (non-latent) activity in its absence. The difference between these two activities gives a measure of the latent enzyme activity and was expressed as percentage of total activity. Six different preparations of erythrocytes gave latent activities of cathepsin D varying from 6.0 to 19.2~o. The mean (+S.D.) was 10.5 (+5.1)% (Table 3). The total and free activities of cathepsin D were found to be signifi- cantly different (P < 0.05) (Table 3). For fl-AGA, total and free activities (measured at pH 4.3 and pH 53) were not significantly different (P > 0.05) (Table 3). It was noticeable, however, that relatively high latent activities were observed in two experiments (10.3 and 17.9%). Sucrose added to the incubation mixture (to offer osmotic protection to the lysosomes) did not reduce the free activity.

Total and free activities of fl-glucuronidase were also measured in homogenates of flounder erythro- cytes and were found to be significantly different (P < 0.05) (Table 3). Four separate preparations gave latent activities varying from 4.4 to 14.8%. The mean (+S.D.) was 7.6 (___4.8)% (Table 3). In two of the homogenates prepared for determination of latent ~-glucuronidase activity, the erythrocytes were hom- ogenized in 0.9% NaCI solution and not in hypotonic Tris buffer. This treatment did not increase the latent activity consistently. Only a small fraction of the cells was destroyed in the isotonic NaC1 solution in spite of a rough homogenization with a Potter-Elvehjem homogenizer equipped with a Teflon pestle. The results for fl-glucuronidase indicated, however, that neither the solution used for homogenization nor the homogenization procedure had any influence on the amount of latent enzyme activities in the erythroeytes.

Uptake of neutral red A considerable uptake of neutral red was found

in the erythrocytes from the flounder Platichthys

0.5-- o ,(~- N- Acetylglucosaminidase • Cathepsin O

o5

r-~

OI

, I , J 20 40

Temperature, °C

Fig. 1. Activity of erythrocyte acid hydrolases as a function of incubation temperature. OD --- optical density. Assay conditions were as described in the text. fl-N-acetyglucosa-

minidase was assayed at pH 4.3.

flesus. Figure 3 shows that the uptake of dye increased with increasing concentrations of dye in the incuba- tion medium. Microscopic examination of the eryth- rocytes exposed to neutral red indicated that dye accumulated in cytoplasmic granules in the perinuc- lear region of the cells.

Activities of the lysosomal enzymes, fl-N-acetylglu- cosaminidase (fl-AGA), cathepsin D and fl-glucuroni- dase, were in the present report found in hemolysates of flounder erythrocytes. A considerable variation in the enzyme activities was observed for fl-AGA and cathepsin D in erythrocytes from different fish (Table 2).

The enzyme fl-AGA present in hemolysates from flounder erythrocytes was found to have two distinct peaks on its pH curve, and the activity was always

0 3

02--

I , I , I , I F 3 5

pH

Fig. 2. Activity of erythrocyte acid hydrolases as a function of pH in the incubation solution. OD = optical density.

Assay conditions were as described in the text.

I o /~- N - Acetylglucosarninidase • Cethepsin D

DISCUSSION

04

38 TONE VISLIE

Table 3. Latent activity of acid hydrolytic enzymes in erythrocytes from the flounder Platichthys flesus

Latent activity n* o(, of total activityt P$

Cathepsin D 6 10.5 ___ 5.1 < 0.05 fl-N-acetylglucos- pH 4.3 7 2.6 + 4.7 > 0.05

aminidase pH 5.7 5 5.4 __+ 9.3 > 0.05 ~-glucuronidase 4 7.6 + 4.8 <0.05

* Number of determinations. t Values are mean _+ S.D.

The level of significance for the difference between total and free activity.

higher at pH 5.7 than at pH 4.3 (Table 2 and Fig. 2).

Both ]~-AGA and cathepsin D which are known as typical lysosomal enzymes (de Duve, 1963) are, in mammalian erythrocytes, assumed to be associated with the plasma membranes (Morrison & Neurath, 1953; Bosmann, 1971; Bernacki & Bosmann, 1972; Reichelt et al., 1973, 1974; Tfk~s & Chambers, 1975). In the work of Morrison & Neurath (1953), where acid proteinase activity (cathepsin D) was found in human erythrocytes, the cells were hemolysed in a hypotonic solution. If the human erythrocytes do con- tain lysosomes, at least some of them would have burst during the exposure of the cells to the hypotonic solution, so that lysosomal enzymes should have been found in the supernatant. Following centrifugation of the hemolysates, Morrison & Neurath (1953) found that all the acid proteinase activity sedimented.

Lysosomes have been identified in mammalian reti- culocytes (Lessler & Pack, 1964) and it has been shown that both reticulocytes and circulating erythro-

Ol5

~010 0

~g

c

005

, I I I i I ~ I 2 4 6 8

Concentration of neulrdl red, mg/g cells

Fig. 3. Uptake of neutral red in flounder erythrocytes (expressed as OD/min g cells) as a function of neutral red concentration per g cells in the incubation solution (total volume 4 ml). OD = optical density. Each point in the

figure represents one determination.

cytes from mammals contain membrane-enclosed bodies displaying acid phosphatase activity (Kent et al., 1966). Somewhat opposed to this observation, and more in accordinace with the general opinion, are the results of experiments carried out by Lutzner (1964) who found that it was impossible to isolate acid phos- phatase-positive organelles from the erythrocytes of guinea-pig. This is also in agreement with Haschen (1975) who suggested that, in immature erythrocytes, the acid proteinase (cathepsin D) is predominantly located in the lysosomes, but in mature cells is pre- dominantly membrane-bound.

Histological studies have been done on circulating nucleated erythrocytes of lower vertebrates. Lyso- somes (cytolysosomes) have been described in the cir- culating erythrocytes from adult urodeles, but not in such cells from adult frogs (Tooze & Davies, 1965, 1967). A possible interpretation of these contradictory findings is given by Tooze & Davies (1965). They sug- gested that adult anuran amphibia erythrocytes are liberated from the erythropoietic organs after the degradation of their cytoplasmic organelles is com- pleted, whereas urodele erythrocytes are liberated at an earlier stage, before this process is completed. In lizards, lysosomes have been described in erythro- blasts (Scorza et al., 1970). Observations on Myxine blood cells also indicate that only immature erythro- cytes possess lysosomes (Sekhon & Maxwell, 1970).

Somewhat opposed to the histological works men- tioned above, which seem to imply that no lysosomes are present in the circulating erythrocytes of non- mammalian vertebrates, are the results reported in the present paper. Not only are activities of typical lysosomal enzymes shown in hemolysates of flounder circulating erythrocytes, but the present results indi- cate that at least cathepsin D and fl-glucuronidase display latency. On the other hand, no consistent latent activity was found for ]~-AGA. Possibly the substrate used in the assay may diffuse into the lyso- somes and thereby increase the free activity.

Because one fundamental property of enzymes enclosed in lysosomes is their structure-linked latency (de Duve, 1963), one conclusion in the present report might be that lysosomes are present in the circulating erythrocytes of the flounder Platichthys flesus.

The latency shown for fl-glucuronidase and cathep- sin D was relatively low. This is not unexpected in the present investigation. A rough treatment was necessary to disrupt the nucleated erythrocytes of the flounder, and cells were homogenized in a hypotonic solution. When erythrocytes are exposed to the hypo- tonic solution it is likely that a great number of lyso-

Lysosomes in erythrocytes from flounder 39

somes, in addition to the cells, will disrupt and the latency of lysosomal enzymes thereby be reduced.

The data showing that neutral red is taken up by the erythrocytes also indicate that lysosomes are present in the circulating erythrocytes from the flounder. By microscopic examination of erythrocytes which had been exposed to neutral red, it seems that the dye is concentrated in cytoplasmic granules in the perinuclear region of the cells--most probably in lysosomes (Byrne, 1964). It is well known that basic dyes such as neutral red (Conn, 1969) are taken up and accumulated in the lysosomes of the cells (Allison & Young, 1964; Byrne, 1964; Allison & Mallucci, 1965, de Duve et al., 1974).

In conclusion, the results in the present report indi- cate that the enzymes fl-AGA, cathepsin D and fl-glu- curonidase are most probably enclosed in lysosomes in the circulating erythrocytes from the flounder. These results are somewhat opposed to earlier works which seem to indicate that no lysosomes are present in mature erythrocytes, even from lower vertebrates (Tooze & Davies, 1965, 1967; Sekhon & Maxwell, 1970). One possible explanation of these contradic- tory findings might be that the circulating blood from the flounder contains a great number of immature red blood cells. On the other hand, it was impossible to distinguish any circular immature red blood cells from the oval mature cells during microscopic exam- ination of blood from the flounder. This seems to be in agreement with Catton (1951) who found that reticulocytes represent only about 1-29/o of the eryth- rocyte population in blood from teleosts. It is then reasonable to assume that lysosomes are present in the circulating, mature erythrocytes of the flounder Platichthys flesus.

Acknowledgements--I wish to thank Dr Trond Berg and Kjell Fugelli for their interest and support during this work.

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