Enzymatic properties of microsomal membranes from the protozoan Acanthamoeba castellanii

Experimental Cell Research 68 (1971) 106-l 12

ENZYMATIC

PROPERTIES

THE PROTOZOAN

OF MICROSOMAL ACANTHAMOEBA

MEMBRANES

FROM

CASTELLANII

J. E. THOMPSON and T. M. G. SCHULTZ Department of Biology, Unir;ersity of Waterloo, Waterloo, Ontario, Canada

SUMMARY Analyses of microsomes from Acanthamoeba castellanii have revealed certain fundamental similarities in enzymatic organization between microsomal membranes from this cell, a protozoan, and corresponding membranes from mammalian cells. Separation of the microsomes into their rough and smooth subfractions was achieved by density gradient centrifugation of a microsomal suspension. The subfractions were identified by chemical analyses. Enzymatic determinations showed that the rough and smooth membranes possess certain enzymes in common but distinctive differences in enzyme activities were also apparent. Substantial levels of two phosphatases, glucose-6-phosphatase and 5’-nucleotidase, were present in both rough and smooth subfractions. 5’-Nucleotidase was approximately equally distributed between the two, but the specific activities of glucose-6-phosphatase were consistently 2-3 fold higher in the rough membranes than in the smooth. On the other hand, NADPH-cytochrome c reductase and rotenoneinsensitive NADH-cytochrome c reductase displayed a distinctly non-random distribution in that they were primarily associated with smooth surfaced membranes and showed only low activities in the rough subfraction. Since the major component of a microsomal fraction is fragmented endoplasmic reticulum, it would appear that in Acanthamoeba the rough and smooth membranes of this organelle have diverse functions.

It has been previously reported that purified plasma membrane from Acanthamoeba castelZanii is enriched relative to corresponding homogenates in ATPase (EC 3.6.1.3) and S-nucleotidase (EC 3.1.3.5) activities [21]. Enrichments of ATPase were not as great as those of 5’-nucleotidase, but this is to be expected since ATP-hydrolysing activity is also featured in mitochondria from this organism [ 151. In the course of elucidating properties that specifically characterize other types of membrane present in vegetative cells of Acanthamoeba, it was observed that the enzymes glucose-6-phosphatase (EC 3.1.3.9), 5’-nucleotidase, rotenone-insensitive NADHcytochrome c reductase (EC 1.6.99.3) and Exptl Cell Res 68

NADPH-cytochrome c reductase (EC 1.6.99. 1) were consistently present in microsomal fractions. Klein [15] has demonstrated that ATPase is also characteristically found in microsomal fractions from these amoebae. Acanthamoeba cells, like other typical eucaryotes, possess two morphologically distinguishable types of endoplasmic reticulum, rough and smooth [l], and in order to determine if there is a corresponding heterogeneity of function, experiments were carried out to establish the distribution of selected enzymes between rough and smooth microsomes. Of the enzymes known to be present in microsomal fractions from these cells, glucose-6-phosphatase, rotenone-insensitive NADH-cytochrome c reductase and

Microsomal enzymes of Acanthamoeba NADPH-cytochrome c reductase were chosen because, at least in mammalian tissue, they are well established as being bound to the membranes of endoplasmic reticulum [ 1I, 181. 5’-Nucleotidase was selected because in both Acanthamoeba [21] and several mammalian tissues [2, 31 it is found in both microsomal fractions and preparations of purified plasma membrane. Its distribution was of interest since its presence in microsomal fractions is thought to be due at least in part to fragmentation of portions of plasma membrane into vesicles of microsomal size upon homogenization [3]. This communication describes the results of these distribution studies and, in addition, discusses similarities between the enzyme complement of microsomes from this cell, a protozoan, and that of corresponding membranes from mammalian cells.

107

moeba gave scope for determining which conditions provided for the best separation of rough and smooth microsomes. Secondly, a comparative analysis of the effects of these three treatments on the distributions of enzymes between the subfractions revealed trends which were of assistance in making conclusions about the native distributions of these enzymes between rough and smooth endoplasmic reticulum. Details of the three separation procedures are as follows: Procedure I

Untreated 10000 g supernatant was centrifuged through a barrier of 1.3 M sucrose, adjusted to pH 7.5 with 1 mM NaHCO,,, by layering 6.0 ml of suspension over 4.0 ml of sucrose and spinning for 210 min at 124, 244 g in a Spinco 50 angle rotor. Procedure 2

The microsomes were nretreated with CsCl before subfractionation, as described previously by Dallner et al. 171.The 10000 g suuernatant was mixed with CsCl to give a final conceniration of 1.5 mM and the suspension centrifuged as in procedure 1. The barrier layer of sucrose contained a matching concentration of CSCI. Procedure 3

In this case subfractionation was carried out exactly as described in procedure 2 except that CsCl was present at a concentration of 15 mM. In all three procedures, centrifugation produced an interface layer as well as a layer of loose tan-coloured MATERIALS AND METHODS material immediately overlying a hard packed, colourless pellet at the bottom of the tube. The interface Preparative procedures layer was removed with a syringe, diluted with distilled water and pelleted by centrifugation at Vegetative cells of Acanthamoeba castellanii (Neff strain) were cultured as nreviouslv described 1211. 133 500 g for 2 h; the resulting pellet was resuspended in 0.3 M sucrose-bicarbonate buffer. The loosely When the cultures had attained a population de&y packed layer at the bottom of the tube was separated of 50-100 x lo1 cells ner ml. the amoebae were haraway from the hard packed pellet by gentle agitation, vested by centrifugatibn at SO0g for 10 min at 4°C. decanted and diluted to a sucrose molarity of 0.3 (All subseauent urocedures were also carried out at with distilled water. This separation occurred easily 4°C.) The cells were resuspended (25 % w/v) in preand did not disturb the hard packed pellet; subsecooled 0.3 M sucrose. adiusted to DH 7.5 with 1 mM NaHCO,, and homogemzed with’ 15 strokes of a quently this pellet too was resuspended in 0.3 M sucrose-bicarbonate solution. Samples of the homoPotter-Elvehjem homogenizer rotating at 1 700 rpm at a clearance of 0.13-0.18 mm. The homogenate was genate and all fractions were stored at -10°C until required for analysis. centrifuged at 10 000 g for 20 min in order to sediment non-microsomal particulate material and the supernatant, which was essentially a suspension of microChemical analyses somes and free ribosomes, was retained. The microsomes were routinely subfractionated by Levels of RNA-P and phospholipid-P were deterthree procedures, one involving no chemical treatmined as described by Schneider [20] and Dallner et al. ment prior to densitv gradient centrifugation and two for both procedures, 171, respectively. Initially, involving pretreatment with different concentrations aliquots of the microsomal suspensions (1 or 2 ml) of CsCI. This was done for two reasons. First. bound were treated with 2.5 ml of 10 % trichloroacetic acid cations are known to influence the sedimentation and the precipitates pelleted bv centrifugation. Lipid properties of microsomes 16. 71. Since the amoebae was extracted from- the pellets with chloroformwere cultured in medium containing MgZ+, Ca2+ ethanol and the extracts evaporated to dryness at and K- at concentrations of I mM, 0.05 mM and 100°C in Kjeldahl digestion flasks. Digestions and 2 mM respectively, it seemed quite possible that the determinations of P were carried out as previously method of Dallner et al. 171 for subfractionating rat described by King [14] with the modification that liver microsomes after pretreatment with 15 mM the samples were predigested with 0.3 ml of conCsCl might not be equally well suited for Acanthacentrated nitric acid prior to the final perchloric moeba. Thus the use of three procedures for Acanthaacid digestion. Levels of RNA-P remaining in the Exptl Cell Res 68

108

J. E. Thompson & T. M. G. Schultz

residue after the chloroform-ethanol extraction were measured exactly as described by Schneider [20]. Protein was routinely measured by the method of Lowry et al. [16].

Table 2. Glucose-6-phosphatase homogenates and microsomal ,from Acanthamoeba castellanii

activities in subfractions

Enzyme determinations

/lg P/mg protein/h CSCI Assays for glucose-6-phosphatase [13], rotenoneSM/RM Nom SM RM insensitive NADH cvtochrome-c reductase 1221. treatment Expt NADPH cytochrome . c reductase [22] and 5’: nucleotidase [ 171 were carried out on homogenates 0.30 12.0 15 mM A 10.2 33.9 and all isolated fractions. The inclusion in the glucose19.0 0.54 B 16.3 10.2 6-phosphatase assay of KF and ethylene diaminete0.30 A 27.7 14.0 47.2 1.5 mM traacetic acid as inhibitors of alkaline and acid 0.26 B 21.2 11.9 46.0 phosphatases helped to ensure that the substrate was None 65.0 0.39 A 27.1 25.4 being hydrolyzed only by glucose-6-phosphatase. The 0.23 B 21.2 13.3 57.2 methods of Sottocassa et al. [22] for assaying the two reductase enzymes were modified in that rotenone Horn, Homogenate; SM, Smooth microsomes; RM, and KCN were present at final concentrations of Rough microsomes 30 /cM and 1 mM respectively rather than 1.5 /tM and 0.3 mM.

RESULTS Measurements of RNA and phospholipid in the microsomal subfractions indicated quite clearly that the upper interface layer from the sucrose gradient was predominantly smooth surfaced vesicles whilst the lower loosely packed layer contained primarily rough surfaced membrane. As can be seen from table I, the ratio RNA-P: phospholipid-P was markedly greater in the rough microsomal fraction than in the smooth, both in the presence and absence of CsCl. In most cases, phospholipid was not detectable in the hard packed pellet which formed at the very Table

1. RNA-P: phospholipid-P ratios in microsomal subfractions from Acanthamoeba

castellanii CSCI treatment 15 mM 1.5 mM None

Expt

Smooth microsomes

Rough microsomes

A B C A B C A B C

0.57 0.26 0.26 0.29 0.18 0.16 0.31 0.36 0.20

3.6 1.0 1.9 6.4 5.2 4.8 1.5 4.4 6.3

Exptl Cell Res 68

bottom of the centrifuge tube, yet levels of RNA were substantial. This suggests that free ribosomes were the predominant feature of this subfraction, although glycogen granules may have been present as well. For both the smooth and rough subfractions there were no significant changes in levels of RNA corresponding to the presence or absence of CsCl (table 1). The variation in amount of RNA detected in the rough subfraction is probably attributable to some cross contamination with the hard packed pellet of ribosomes and glycogen granules. Comparisons of RNA: protein ratios confirmed that the interface layer from the gradient was essentially smooth surfaced membrane and the lower loosely-packed layer rough surfaced vesicles. Values for this ratio in the smooth surfaced portion averaged 0.06 and corresponding values in the rough subfraction were consistently 2-5 fold higher. Neither ratio showed any significant response to pretreatment with CsCI. Substantial levels of glucose-6-phosphatase (table 2) and 5’-nucleotidase (table 3) were detectable in both microsomal subfractions. The specific activities of glucose-6-phosphatase were consistently 223 fold higher in the rough subfraction whilst 5’-nucleotidase

Micvosonlal

Table 3. 5’-Nucleotidase genates

and

activities in hornosubfractions from

microsomal

Acanthamoeba castellanii /lg P/mg protein/h CSCI treatment 15mM 1.5 mM None

Expt

Horn

SM

RM

SM/RM

A B A B A B

21.8 23.5 34. I 46.0 34.1 46.0

26.2 28.3 29.4 51.6 48.9 59.5

29.8 24.6 45.2 48.0 49.9 47.0

0.88 1.19 0.65 1.07 0.98 I .27

Horn, Homogenate; Rough microsomes

SM, Smooth

microsomes;

RM,

was in most cases approximately equally distributed between the two. Pretreatment with CsCl did not significantly alter these proportions for either enzyme. Distributions between rough and smooth membranes of the two redox enzymes NADHcytochrome c reductase (table 4) and NADPH-cytochrome c reductase (table 5) were distinctly non-random. Both were primarily associated with smooth surfaced membranes to an extent that was considerably more pronounced for preparations in which the original microsomes had either received no chemical pretreatment or had been treated with the lower concentration of CsCI. For example, the smooth subfraction was enriched in NADH-cytochrome c reductase by 2-3 fold relative to the rough in preparations pretreated with 15 mM CsCl and by better than 40 fold in preparations receiving no pretreatment (table 4). Similarly, higher enrichments of NADPH-cytochrome c reductase were obtained at the lower concentration of CsCl and when pretreatment was omitted entirely (table 5).

pretreatment with 15 mM CsCl is highly effective for rat liver [5]. The separation has been attributed to a preferential aggregation of rough microsomes which in turn increases their sedimentation velocity. Dallner & Ernster [5] have reported, however, that for rotors with tube angles of less than 35’ convection leads to some cross contamination. In this study a spinco 50 rotor with a 20“ tube angle was used and thus undoubtedly some cross contamination did occur. Nevertheless, the chemical analyses of RNA, protein and phospholipid indicate that the subfractions were predominantly rough and smooth surfaced membrane respectively. A more effective separation of smooth from rough surfaced membranes was obtained following either no pretreatment or treatment with 1.5 mM as opposed to 15 mM CsCI. Under these conditions apparently fewer smooth vesicles were pulled down into the rough surfaced fraction. This is apparent from two lines of evidence. First, NADHcytochrome c reductase and NADPHcytochrome c reductase, which appear to be associated primarily with smooth surfaced membrane in this organism, showed markedly lower specific activities in rough subfractions prepared under these conditions (tables 4 c reductase Table 4. NADH-Cytochrome activities in homogenates and miuosomal subfractions from Acanthamoeba castellanii /lmoies cytochrome protein/h CSCI treatment 15mM 1.5 mM

DISCUSSlON It is well documented that separation of rough and smooth microsomes following

109

enzymes of Acanthamoeba

None

c’/mg

Expt

Horn

SM

RM

SM/RM

A B A B A B

3.28 2.99 3.24 6.43 3.24 6.43

3.86 8.55 9.70 15.4 8.19 13.0

I .22 3.25 0.22 0.54 0.12 0.27

3.17 2.63 44.2 28.5 68.2 48.2

Horn, Homogenate; Rough microsomes

SM, Smooth

microsomes;

RM,

Exptl Cell RES 68

110 J. E. Thompson & T. M. G. Schultz and 5). In contrast, smooth membrane: rough membrane ratios of specific activities for glucose-6-phosphatase and 5’-nucleotidase were essentially unaffected by CsCl (tables 2, 3). This is to be expected for these two enzymes are present in both rough and smooth subfractions. Secondly, the RNA: protein and RNA: phospholipid ratios of the smooth subfraction remained essentially constant among treatments (table 1) implying that its membranous composition had not been altered whilst that of the rough subfraction had. One would expect to find a corresponding decreased contamination of the rough subfraction by smooth membranes but this did not significantly register in the RNA: protein or RNA: phospholipid ratios of this subfraction. However, such changes could very well have been masked by the variable levels of RNA in the rough subfraction thought to be due to cross contamination with the ribosomal pellet. It would appear, therefore, that conditions rendering the best separation of rough and smooth microsomes for this cell, a protozoan, differ from those previously established for mammalian liver. This may in part be due to differences between the two systems in endogenous levels of cations bound to the microsomes before subfractionation is undertaken. There have been several reports of nonrandom distributions of enzymes between rough and smooth microsomes from mammalian tissue [4, 5, 8, 10, 121,but the patterns of distribution have proven to be variable for the tissues and species examined. For example, Fouts et al. [lo, 121 have demonstrated for mature rabbit liver tissue that NADPH oxidase as well as several drug metabolizing enzymatic activities are about 5 times more concentrated in smooth surfaced microsomes than in rough. On the other hand, Dallner [4] has reported that for liver microsomes from mature rats, Exptl Cell Res 68

Table 5. NADPH-Cyrochrome c reductase activities in homogenates and microsomal subfractions from Acanthamoeba castellanii prnoles cytochromec/mg protein/h CsCl

treatment

Expt

Horn

SM

RM

SM/RM

15mM

A

1.5 mM

:

None

: B

1.09 1.61 I .40 I .62 I .40 1.62

0.46 0.40 0.61 0.28 0.62 0.45

0.35 0.41 0.10 0.07 0.08 0.08

1.32 0.97 5.89 3.96 7.88 5.78

Horn, Homogenate; SM, Smooth microsomes;RM, Rough microsomes NADPH-cytochrome c reductase as well as glucose-6-phosphatase and NADH-cytochrome c reductase are rather evenly distributed between the two subfractions. This does not, however, appear to be the case for liver from young rats. More recently, Dallner et al. [8] have demonstrated that at birth the concentration of glucose-6-phosphatase is higher in the rough subfraction than in the smooth; at 3 days after birth the same enzyme is still about twice as concentrated in rough vesicles. Similarly, NADPHcytochrome c reductase is also more enriched in rough microsomes from young rats than in smooth [8]. This investigation has demonstrated that for the protozoan Acanthamoeba there are also differences in enzymatic composition between rough and smooth microsomes. Glucose-6-phosphatase is located on both rough and smooth membranes, although it appears to be 2-3 fold more concentrated in the rough subfraction. Conversely, the two cytochrome c reductases seem to be almost completely localized on smooth surfaced membrane. This interpretation is supported by their substantial enrichments in the smooth subfraction and also by the dramatic effect of pretreatment with CsCl on their-

Microsomal enzymes of Acanthamoeba distribution between the two fractions. For example, if these enzymes were also present to a significant extent on rough microsomes, their activities in this subfraction would presumably not have been so severely decreased when CsCl was either omitted or used at the lower concentration. The presence of 5’-nucleotidase in both subfractions may in part reflect contributions by plasma membrane. This enzyme is well established as a plasma membrane marker for several mammalian tissues [2, 3, 91 and has recently been shown to be enriched in a purified plasma membrane fraction from Acanthamoeba [21]. However, the possibility that the enzyme also represents a true activity of endoplasmic reticulum is not precluded. Microsomal fractions from mammalian tissue possess substantial levels of 5’-nucleotidase activity [23] and it is not yet clear whether or not this simply reflects contamination by plasma membrane fragments. At this stage, only the same can be said for Acanthamoeba. In any event the enzyme is clearly a property of both microsomal subfractions for this organism. A microsomal fraction by its very nature contains membranous vesicles derived from several organelles. Nevertheless, its major constituent is thought to be fragments of reticulum. Hence it seems endoplasmic likely that the different enzymatic properties of rough and smooth microsomes from Acanthamoeba in turn reflect differences in function between the membranes of rough and smooth endoplasmic reticulum. To some extent, the biological functions of microsomal enzymes have been elucidated for mammalian tissue. Glucose-6-phosphatase is involved in carbohydrate metabolism [19]. It has been demonstrated for rat liver tissue that rotenone-insensitive NADH-cytochrome c reductase and NADPH-cytochrome c reductase are each component

111

enzymes of two separate electron transport chains present on endoplasmic reticulum [8]. The latter enzyme, in conjunction with cytochrome P-450, has been shown to be involved in a variety of hydroxylation reactions including those leading to drug detoxification [18]. The functional significance of these enzymes in Acanthamoeba is unknown at the present time. Nevertheless, the very presence of enzymes characteristically featured in preparations of mammalian microsomes in a corresponding fraction from Acanthamoeba suggests a fundamental similarity in the enzymatic organization of membranes between organisms of widely differing complexity. This similarity also extends to plasma membrane. It has been previously reported [21] that purified surface membrane from Acanthamoeba possesses enzymatic properties in common with corresponding preparations from mammalian tissues. We are grateful to the National Research Council of Canada for a grant-in-aid of this research.

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112 J. E. Thompson & T. M. G. Schultz 17. Michell, R H &Hawthorne, J N, Biochem biophys res commun 21 (1965) 333. 18. Orrenius, S, Ericsson, J L E & Ernster, L, J cell biol 25 (1965) 627. 19. Reid, E, Enzyme cytology (ed D B Roodyn) p. 321. Academic Press, New York (1967). 20. Schneider, W C, Methods in enzymology (ed S P Colowick & N 0 Kaplan), vol. III, p. 680. Academic Press, New York (1957).

Exprl Cdl Res 68

21. Schultz, T M G & Thompson, J E, Biochim biophys acta 193 (1969) 203. 22. Sottocassa, G L, Kuylenstierna, B, Ernster, L & Bergstrand, A, J cell biol 32 (1967) 415. 23. Thines-Sempoux, D, Amar-Costesec, A, Beaufay, H & Berthet, J, J cell biol 43 (1969) 189. Received January 20, I97 I Revised version received April 5, 1971