J. Biosci., Vol. 3 Number 3, September 1981, pp. 285-291. © Printed in India.

Binding of arylsulphatase Β to isolated rat liver lysosomal membranes

H. R. ADHIKARI, ATEEQ AHMED* and U.K. VAKIL Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085

MS received 4 May 1981; revised 26 June 1981

Abstract. Binding of arylsulphatase Β to isolated rat liver lysosomal membrane has been studied at 37°C. The binding is strongly pH-dependent and is governed by ionic strength of the medium. Experimental evidence is given for the ability of the to dissociate from the firmly formed membrane-enzyme complex. The dissociation rate is greatly accelerated by raising the buffer molarity. -treatment of the membrane causes significant reduction in its binding ability to the enzyme. This suggests that groups participate, presumably by maintaining surface negativity of the membrane, at a stage of enzyme- membrane interaction process which precedes the internalization of the lysosomal in the lysocomes.

Keywords. Rat liver; lysosomal membrane; arylsulphatase Β; arylsulphatase B-lysosomal membrane complex; dissociation; sialic acid.

Introduction

Receptor-mediated endocytosis is employed to internalise the lysosomal enzymes into the lysosomes from the cells following the formation of secondary lysosomes with high selectivity (von Figura, 1977). This mode of assimilation of enzymes into the lysosomes depends on the phosphomannosyl recognition markers on lysosomal enzymes (Kaplan et al., 1977) and high affinity pinocytic receptors on the cell surface (Sando and Neufeld, 1977), forming a ligand-receptor complex. The binding is highly specific and once bound, the enzyme is rapidly endocytosed and incorporated into lysosomes (von Figura, 1977). Though most of the studies have been limited to intact hepatocytes (Tolleshaug, 1981) or to cultured fibroblasts (Hickman et al., 1974; Ullrich et al., 1978), a direct membrane binding of α-L- induronidase (Rome et al., 1979) and β-glucuronidase (Fischer et al., 1980) to isolated fibroblast membrane receptors have been reported. The binding is reversible and depends mainly upon their immediate environment. Although a plasma membrane receptor for binding of lysosomal has been indirectly demonstrated (Kaplan et al., 1977), the presence of such a receptor on lysosomal membrane is not well known.

* Biophysics Division, Central Drug Research Institute, Lucknow 226 001.

285 286 Adhikari et al.

In the present investigation, we have described an in vitro interaction between partially purified arylsulphatase Β and highly purified lysosomal membrane. Association and dissociation of the complex are strongly dependent upon pH and ionic strength. Pre-incubation of the membrane with proteolytic enzymes has no inhibitory effect; however, neuraminidase or sodium periodate partially or completely impair its binding ability.

Materials and methods p-Nitrocatechol sulphate, trypsin, chymotrypsin and papain were from Sigma, Chemical Co., St. Louis, Missouri, USA. Neuraminidase (Vibrio cholarae) was obtained from Grand Island Biological Co., New York, USA. Concanavalin A- Sepharose 4B was purchased from Pharmacia, Uppsala, Sweden. All other chemicals were of analytical grade.

Isolation of lysosomal membrane

Normal male albino rats of Wistar strain (body weight, 220-250 g), fed on laboratory stock diet, were used. The animals were sacrificed by decapitation, livers excised quickly, washed and homogenized in 8 vol of 0.25 Μ sucrose/1.0 mM EDTA. Lysosomes were isolated by the modified differential centrifugation method (Ragab et al., 1967). The final membrane preparation was obtained essentially as described earlier (Pappu et al., 1978) by leaching out the membrane-bound and intralysosomal enzymes by incubating lysosomes with 0.15 Μ NaCl followed by washing with 0.12 mM and 0.24 mM sodium deoxycholate solutions.

Purification of arylsulphatase Β

Rat liver lysosomes were incubated in 0.15 Μ NaCl for 1 h at 37°C and centrifuged at 27,000 g for 10 min. The clear supernatant was concentrated to a small volume using an Amicon PM 10 membrane. Arylsulphatase Β was purified by affinity chromatography on a concanavalin A-Sepharose 4B column (1×10 cm) followed by gel filtration on Sephadex G-200 column (2.5×75 cm) as described earlier (Adhikari and Vakil, 1980). In preliminary experiments, it was also ascertained that arylsulphatase A did not bind with the membrane under the experimental conditions employed.

Enzyme assay

Arylsulphatase Β activity was estimated using 12 mM.p-nitrocatechol sulphate in 0.1 Μ acetate buffer, pH 5.5, as the substrate (Roy, 1953). One unit of the enzyme activity was expressed as 1 nmol of the substrate hydrolyzed/min.

Protein was estimated as described by Miller (1959).

In vitro binding of arylsulphatase Β with lysosomal membrane

In all the experiments, purified membrane preparation (≡1 mg ) was incubated with arylsulphatase Β(≡250 units) for 10 mmat37°C in 10 mM acetate buffer (pH 4.5 or 5.0). The incubation mixture was then chilled, centrifuged (27,000 g, 20 min) and the pellet was washed with the same buffer to remove non-

Arylsulphatase B binding to lysosomal membranes 287 specifically adhered enzyme. The pellet was resedimented, suspended in water and the enzyme activity bound to the membrane was determined.

To study the effect of ionic strength on the association of the enzyme-membrane complex, the ionic strength of the acetate buffer (pH 4.5 and 5.0) added to the incubation mixture, was adjusted to molarity varying between 10 and 100 mM.

For dissociation studies, the enzyme-membrane complex was first prepared by incubating the membrane with the enzyme in presence of 10 mM acetate buffer, pH 5.0. The pellet was centrifuged, washed with the same buffer and resedimented. The final pellet was suspended in acetate buffer (pH 4.5 and 5.0) of ionic strength increasing from 10 to 100 mM and incubated for 5 min at 37°C. The mixture was centrifuged, the pellet washed with water and the residual activity bound to the pellet was measured.

Pre-digestion of the membrane with hydrolytic enzymes

For proteolytic digestion, the membrane preparation (about 0.3 mg protein) was incubated with 1 mg papain in 0.1 Μ tris-HCl buffer, pH 7.6 containing 5 mM cysteine and 1 mM EDTA, or with 1 mg of trypsin or chymotrypsin in 0.01 Μ tris- HCl buffer (pH 8.1) containing 10 mM CaCl2. Another aliquot of the membrane (≡0.3 mg protein) was incubated with increasing concentrations of neuraminidase in 0.1 Μ tris-acetate buffer (pH 6.5) containing 5 mM CaCl2. All the samples were incubated at 37°C for 1 h and centrifuged (27,000 g, 20 min), the pellets were washed once with 0.01 Μ acetate buffer pH 5.0 and used for the binding studies.

For periodate oxidation, the membrane suspensions in distilled water were mixed with an equal volume of 0.02 Μ sodium-meta-periodate (in 0.1 Μ tris-HCl buffer, pH 7.4) and the mixture was kept at 4°C. Suitable aliquots were withdrawn at regular time intervals and centrifuged (27,000 g, 20 min). The membrane pellet was washed once with water, resedimented and used for the binding studies.

Results and discussion

Purity of lysosomal membrane

Information on the intralysosomal distribution of the lysosomal hydrolases is fragmentary. Although some enzymes are known to be loosely or tightly bound to the membrane (Baccino and Zuretti, 1975), it is not known whether any of them is the structural component of the membrane. Since arylsulphatase Β is loosely bound on the lysosomal membrane, most of it can be easily removed by washing the isolated membrane with 0.15 Μ NaCl (Pappu et al., 1978). Further washing with tris-HCl buffer of high molarity (0.1 Μ) and alkaline pH (8.0) and subsequent washing with distilled water resulted in the lysosomal membrane preparation, completely free of arylsulphatase Β.

Binding of arylsulphatase to lysosomal membranes

In preliminary experiments, the time course and temperature dependence of arylsulphatase Β binding to the membrane were carried out (results not shown) The binding was almost instantaneous and the amount of enzyme bound reached a

288 Adhikari et al. plateau within 10 min. Binding was maximum at 37°C. Fischer et al., (1980) have shown that most of the lysosomal enzymes bind to the membrane with equal efficiency at 3 7°C .

It was reported earlier (Adhikari and Vakil, 1980) that the binding was pH dependent; the maximum binding took place at pH 5.0. Though at pH values lower than 5.0. binding was not affected, it declined rapidly at higher pH.

Influence of buffer molarity

The ionic nature of arylsulphatase Β binding to lysosomal membrane has been demonstrated by studying the effect of increasing buffer molarity, at pH 4.5 and 5.0. on the association and dissociation of the membrane - enzyme complex. At both pH. maximum association was observed at 10 mM and by increasing the molarity of the buffer, a progressive decrease in the binding was observed. How- ever, the inhibitory effect was more at pH 5.0 (figure 1). Similar dependence on ionic strength of the binding of β-glucuronidase has been reported using the model system of a strong cationic exchanger and with tritosomes (Henning et al., 1973).

Figure 1. Effect of ionic strength on in vitro binding of arylsulphatase Β. ph 4.5,(●); pH 5.0. (Ο).

The enzyme was incubated with the isolated membrane in acetate buffer (pH 4.5 or pH 5.0), with increasing ionic strength. Binding at 10 mM was taken as 100%,

Dissociability of binding is shown in figure 2. The isolated membrane-enzyme complex was suspended in acetate buffers (pH 4.5 and 5.0) of different molarity and incubated for 5 min at 37° C. No dissociation of pre-bound enzyme from the lysosomal membrane was detected at lower (10 mM) buffer molarity and all the enzyme activity was retained in the complex. However, the dissociation of the membrane-bound arylsulphatase Β was rapid at higher ionic strength and at 100 mM, about 85% of the bound enzyme was displaced from the membrane within 5 min. Though the pattern of enzyme release as a function of ionic strength of buffer was comparable, it is always higher at pH 5.0. Gonzales-Noriega et al. (1980) have demonstrated that enzymes bound to fibroblast monolayers dissociate minimally at neutral pH but maximally at pH 5.0, which is expected in

Arylsulphatase B binding to lysosomal membranes 289

Figure 2. Dissociation of membrane-bound arylsulphatase Β at different ionic strengths of the buffer, pH 4.5(●); 5.0(O).

After the membrane was incubated (0.01 Μ acetate buffer pH 5.0) with arylsulphatase Β for 10 min at 37°C, the pellet was washed and suspended in acetate buffer (pH 4.5 and 5.0) of varying ionic strengths. The enzyme activity retained on the membrane after incubation at 37°C for 5 min was assayed.

lysosomes. This acidic pH may facilitate the enzyme-receptor dissociation following the delivery of acid hydrolases to lysosomes (Shoji and Poole, 1978). The foregoing results suggest that alterations in the equilibrium of cations and anions affect the enzyme binding to the lysosomal membrane, presumably, by influencing the charges on the cell surface.

Specificity and chemical nature of the binding

Lysosomal membranes contain negatively charged sialic acid groups as well as carboxylic groups of amino acids of the membrane , both of which contribute to the cell surface negativity (Dean and Barrett, 1976). Pretreatment with proteolytic enzymes (table 1) such as trypsin, chymotrypsin or papain had no

Table 1. Effect of proteolytic digestion of the membrane on the binding of arylsulphatase Β.

Lysosomal membrane, was pre-treated with the proteolytic enzyme and then used for binding studies. 290 Adhikari et al. appreciable effect on the binding ability of the membrane indicating non-involve- ment of acidic groups from amino acids in the process. However, when the membrane was pre-treated with different concentrations of neuraminidase (figure 3A) and then incubated with the enzyme, the binding ability was decreased with a

Figure 3. Effect of preincubation of rat liver lysosomal membrane with neuraminidase (A) or with sodium metaperidate (B) on in vitro binding of arylsulphataseB. Arylsulphatase Β binding, (●) sialic acid, (Δ).

Lysosomal membrane was preincubated with increasing concentrations of neuraminidase at 37°C for 1 h or with 0.01 Μ (final concentration) Na-metaperiodate at 4°C for different time intervals, washed and used for binding studies. Binding capacity of the untreated membrane was taken as 100%. concomitant release of sialic acid from the membrane. Thus, enzymic removal of sialic acid residues resulted in loss of the binding potency of the membrane. However, Neufeld and Ashweil (1980) have shown that the destructive action of neuraminidase is a repairable lesion in plasma membrane and resïalylation can restore its binding ability. The residual binding activity may be attributed either to incomplete enzymic cleavage of sialic acid residues from the membrane or to the fact that perhaps arylsulphatase Β binds to the membrane also through some other ligand.

In comparison with the neuraminidase treatment, (figure 3B) pretreatment of the membrane with 0.01 Μ (final concentration) sodium metaperiodate reduces the binding ability of the membrane and the effect is time-dependant. Incubation for 1 h destroyed about 85% of the binding ability of the membrane. Since periodate can oxidise non-specifically all the sugar moieties including sialic acid (Hickman et al., 1974), the possibility of the involvement of other sugar receptor residues on the membrane in enzyme binding processes may not be ruled out. Recently, the presence of such receptors that can recognise carbohydrate residues has also been demonstrated on the lysosomal membrane (Burnside and Schneider, 1980). These may be vitally necessary to ensure the integrity of the lysosomal system during repeated binding events. Arylsulphatase Β binding to lysosomal membranes 291

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