Biochem. J. (1963) 87, 354

Mammalian Glycosidases4. THE INTRACELLULAR LOCALIZATION-OF P-GALACTOSIDASE, cc-MANNOSIDASE,fi-N-ACETYLGLUCOSAMINIDASE AND OC-L-FUCOSIDASE IN MAMMALIAN TISSUES*

BY J. CONCHIE AD A. J. HAYRowett Research Inatitute, Buccksburrn, Aberdeen

(Received 26 October 1962)

The lysosome theory, whether it is true or not, certain mouse tumours, however, had a consider-embodies biological,features of general importance. able proportion of their P-glucuronidase activity inIn its original form (de Duve, Pressman, Gianetto, the soluble fraction. No appreciable fraction of theWattiaux & Appelmans, 1955) this theory favours enzyme activity appeared to be in the nucleus.the view that 'lysosomes form a single population The observation that P-glucuronidase in animalof enzymically homogeneous granules', within tissues is not always particle-bound, or if particle-which the hydrolytic enzymes display structure- bound is not necessarily latent to any markedlinked latency, becoming active onlyon the death degree, has led to the present investigation on theof the cell. This theory has become considerably intracellular localization ofthose mammalian glyco-more elastic, as for instance in the statement that sidases asociated with ,-glucuronidase, namely,'individual lysosomal particles may differ quite ,B-galactosidase, x-mannosidase, f-N-acetylglucos-widely from each other in a number of properties aminidase and a-L-fucosidase (Conchie, Findlay &such as size, structure, relative enzymic equipment, Levvy, 1959; Levvy & McAllan, 1961; Levvy,density in various media and sediimentation co- McAllan & Hay, 1961). It was considered that suchefficient' (de Duve, 1962). Nevertheless, cyto- a study would prove of value in further elucidatinglogists have used acid-phosphatase activity as the the properties and function of the lysosomalsole and sufficient enzymic criterion in stu-dies in enzymes.which they claim to observe lysosomes as a new The intracellular localization of fi-galactosidasetype of subcellular particle (e.g. Holt, 1959; and ,B-N-acetylglucosaminidase in rat liver has alsoNovikoff, 1961; Holt & Hicks, 1962; Bitensky & been studied by Sellinger, Beaufay, Jacques,Gahan, 1962). So far as histochemical work is Doyen & de Duve (1960), who also mentioned aconcerned, another important factor has been few preliminary experiments with cx-mannosidase.ignored in attempts to study the lysosome, namelythat the original work on which the definition isbased dealt with gross differences in subcellular EXPERIMENTALlocalization as measured by centrifugal fraction- Substratesation. Fractions containing small amounts, of o-Nitrophenyl ,-D-galactoside, prepared by the method ofenzyme activity were ignored. Such fractions, how- Seidman & Link (1950), had m.p. 1930 and []16-51"ever, may represent the only 'free' or easily (c 1-0) in water. p-Nitrophenyl x-D-mannoside was pre-accessible enzyme in the cell, and would thus be pared as described by Conchie et al. (1959). p-Nitrophenylthe most readily identified by histochemical N-acetyl-fiD-glucosaminide was prepared as described bytechniques. The lysosomal enzyme by definition is Findlay, Levvy & Marsh (1958). p-Nitrophenyl oc-L-fucos-not readily accessible to substrate. ide was prepared as described byLevvy & McAllan (1961).Rodent liver was used almost exclusively in The yield from the tri-0-acetate was greatly improved by

developing the lysosome theory. However, the using the method of deacetylation desoribed by Leabackintracellular localization of fi-glucuronidase in other (1960).mammalian tissues has been investigated (Conchie, Preparation of homogenatesHay & Levvy, 1961). In most of the tissues Tissues were homogenized in a cooled Potter-Elvehjemexamined, the bulk of the enzyme was associated all-glass homogenizer, with a diameter clearance of

with thecytoplasmic granules, within .which, 0-23-0-43 mm., driven by an electric motor (average speedwith the cytoplasmic granules, within which, 1500 rev./min.). For the preparation of sucrose homo-in sucrose homogenates, only a fraction of the genates, tissues were washed with ioe-cold 0-25m-sucrose,potential activity was observed: the actual degree pressed between pieces of hardened filter paper, weighedof latency displayed by the particulate enzyme and homogenized in 0 25x-sucrose. Periods of homo-varied from tissue to tissue. Mouse spleen and genizing were as stated in the text. Enzyme concentrations

* Part 3: Conchie, Hay & Levvy (1961).were adjusted so that not more than 10% of each substratewas hydrolysed during the period of assay.

354

INTRACELLULAR LOCALIZATION OF GLYCOSIDASES

Fractionation of homogenatesHomogenates were fractionated as described by Conchie

et al. (1961). Fraction I, obtained by layering the 0-25m-sucrose homogenate over 0-34M-sucrose and centrifugingat 700g for 10 min., consisted. mainly of unbroken cells andnuclei. Fraction II, obtained by centrifuging the super-natant and washings from fraction I at 5000g for 10 min.,contained the larger cytoplasmic particles. Fraction III,which contained the small cytoplasmic particles, was

obtained by centrifuging the supernatant from fraction IIat 70000g for 1 hr. In some experiments (see below) aceticacid-NaOH buffer, pH 5-2, was used to obtain fraction III.The final supernatant and washings, which were particle-free, formed fraction IV.

Enzyme assays

In experiments involving the measurement of enzymeactivity of untreated homogenates in 0 25M-sucrose, theincubation buffers for all assays contained 0-25M-sucrose(final concentration). In osmotic-activation experiments,sucrose of the appropriate concentration was present in theincubation buffers. For measurement of the total activityof sucrose homogenates Triton X-100 (0-1 %, w/v) wasincluded (Walker, 1952).

ac-MannoWidase and fl-galaCtoidase. Assay conditions wereas described by Conchie & Hay (1959), but to diminishenzyme blanks assays were stopped by the addition of 2 ml.of 5% (w/v) trichloroacetic acid. The mixture was centri-fuged at 1500g for 10 min., 4 ml. of the supernatant wasadded to 4 ml. of 0 4M-glycine-NaOH buffer, pH 11-2, andthe colour intensity of the liberated nitrophenol wasmeasured on the Spekker photoelectric absorptiometerwith Ilford no. 601 violet filters (peak transmission430 mix). When the ,B-galactosidase activity of sucrose

homogenates was being measured, assays were done atpH 5, since at pH 3 there was loss of latency (see below).

P-N-Acetylgluco8aminida8e. Liberation of p-nitrophenolfrom p-nitrophenyl N-acetyl-fl-D-glucosaminide was mea-

sured as described by Findlay et al. (1958).CX-L-Fucos0da8e. Assay conditions were as described by

Levvy & McAllan (1961), but the reaction was stopped bythe addition of trichloroacetic acid and the colour of theliberated p-nitrophenol developed as described above forcx-mannosidase.

RESULTS

Distribution of glycosidase activities after fractiona-tion. Studies on the intracellular localization ofP-glucuronidase had shown that to compare thedistribution of enzyme activity in the fractionsobtained from various tissues it was necessary tocontrol the clearance in the homogenizer and theperiod of homogenization (Conchie et al. 1961). Themost satisfactory procedure for f-glucuronidaseand the related glycosidases studied in the presentwork was to homogenize four times for 15 sec. Thisgave maximum disintegration of the cells withminimum disruption of the cell particles. Thedistribution of P-galactosidase, cx-mannosidase andf-N-acetylglucosaminidase obtained by this pro-

cedure for a number of mouse and rat tissues isshown in Table' 1.

Fraction I comprises mainly unbroken cells andnuclei, fraction II the usual mitochondrial fraction(large granules), and fract. on III the microsomalfraction (small granules). Fraction IV, the clearsupernatant, is defined as soluble enzyme. Forassay, all fractions were suspended in 0-25M-sucrose,and enzyme activities were measured in the pre-sence of Triton X-100, the results being expressed aspercentages of the activities of the whole homo-genate in 0-25M-sucrose, also assayed in thepresence of Triton X- 100. In many cases all threeenzymes were assayed in the same enzyme prepara-tion. Glycosidase activities of sucrose homogenatesin the presence of Triton X-100, after correction forany small inhibition caused by sucrose, were alwaysequal to those of the corresponding water homo-genates. Sucrose never caused more than 10%inhibition of any enzyme.The distribution of fl-galactosidase, oc-mannosid-

ase and P-N-acetylglucosarninidase activities inmouse liver was similar to that observed for ,B-glucuronidase in that the bulk of the enzymeactivity was confined to the two granular fractions.Among the different enzymes there was evidence ofsmall variations. The same general pattern was alsoobserved in rat liver and in mouse kidney. A dif-ferent pattern was observed in mouse spleen andcertain mouse tumours, and qualitative differencesbetween enzymes were marked. In both of thelatter types of tissue there was a large proportionof f-galactosidase and a-mannosidase activities inthe soluble enzyme fraction, with very littleactivity in the mitochondrial fraction. In contrast,fl-N-acetylglucosaminidase displayed little solubleactivity in mouse spleen and tumours, and the bulkof the enzyme was in the microsomal fraction.Ehrlich tumours gave results similar to thoseshown in Table 1 for T 2146 tumours. Rat spleenresembled mouse spleen, except in that it lackedsoluble oc-mannosidase. [,-Glucuronidase in ratspleen followed the pattern described for mousespleen by Conchie et al. (1961), being divided mainlybetween the microsomal and soluble fractions.]Enzyme activities and distribution patterns forthe livers of C3H mice, previously examined be-cause of their low fl-glucuronidase activities,resembled those for white mice in the present work.

In some furthe 'fractionations of rat-liver homo-genates, the particulate glycogen was separated bythe procedure of Drochmans (1962). Fraction IIIwas obtained by adding acetic acid-sodium hy-droxide buffer, pH 5-2, and centrifuging at 110Ogfor 5 min. Particulate glycogen was then separatedfrom the supernatant by centrifuging at 20000gfor 50 min. None of the glycosidases showed anyactivity in the glycogen fraction.

23-2

VoL 87 355

J. CONCHIE AND A. J. HAY

Table 1. Ditribution of glycosida8e activities in sucrose homogenate8 of mammalian ti8sue8Details ofhomogenization, fractionation and assays are given in the text. Results are expressed as percentages

of the activity of a whole sucrose homogenate: all assays were done in the presence of Triton X-100 (0-1%).Typical total activities for the various tissues are shown in Tables 3-5. The values in parentheses were obtained inexperiments in which fraction III was agglutinated with 0 1x-acetic acid-NaOH buffer, pH 5-2, and sedimentedat 15OOg for 10 min. The fractions had the following compositions: I, unbroken cells and nuclei; II, mito-chondria; III, microsomes; IV, clear supernatant (soluble enzymes).

Mouse liver

Mouse kidney

Mouse spleen

Mouse T2146 tumour

Rat liver

Rat spleen

FractionIIIIIIIV

RecoveryIIIIIIIV

RecoveryIIIIIIIV

RecoveryIIIIIIIV

RecoveryIIIIIIIV

RecoveryIIIIIIIV

Recovery

f-Galacto-sidase*0 (0)

61 (61)23 (23)9 (9)

93 (93)9 (3)

39 (45)22 (33)22 (16)92 (97)7 (3)10 (13)17 (29)72 (50)

106 (95)11 (2)12 (5)39 (43)42 (46)104 (96)

2 (1)53 (64)34 (27)6 (12)

95 (104)1 (4)8 (16)36 (37)52 (41)97 (98)

* Assays at pH 3-0.

oc-Manno-sidase1 (1)

37 (37)47 (47)14 (14)99 (99)11 (11)41 (41)44 (28)16 (23)

112 (103)9 (0)

20 (8)32 (51)44 (35)105 (94)13 (9)15 (9)34 (34)44 (49)106 (106)

1 (1)41 (45)50 (48)8 (7)

100 (101)10 (3)27 (13)50 (71)0 (1)

87 (88)

,B-N-Acetyl-glucos-

aminidase2 (2)56 (58)31 (28)6 (8)

95 (96)11 (5)37 (41)45 (33)6 (20)99 (99)21 (1)15 (6)46 (79)12 (12)94 (98)15 (5)11 (6)48 (74)18 (11)92 (96)2 (2)

52 (62)32 (36)4 (3)

90 (103)17 (14)33 (23)37 (49)10 (8)97 (94)

The distribution of OC-L-fucosidase in rat liver andrat spleen is given in Table 2; it resembled that ofP-galactosidase in the rat. Studies of OC-L-fucosidasein the mouse were not possible owing to its lowactivity in mouse tissues.

Use of acetate for fractionation. In initial experi-ments, agglutination by means of acetic acid-sodium hydroxide buffer (Conchie et al. 1961) wasused, both for complete fractionations and forobtaining non-sedimentable fractions from sucroseand water homogenates. This method is con-venient, and in general gives results identical withthose obtained by the high-speed-centrifuging pro-cedure (see Table 1). In a few specific cases, how-ever, higher soluble activities were obtained by theacetate separation method, as a result of extractionof particle-bound enzyme by the buffer in sucroseor water homogenates.

Glycoidase activties in sucrose homogenates.Tissue homogenates in 0 25m-sucrose display only

Table 2. Distribution of OC-L-fucosidase activitiesin sucrose homogenates of rat liver and rat spleenDetails of homogenization, fractionation and assays are

given in the text and Table 1. Results are expressed aspercentages of the activities of a sucrose homogenate. Allassays were done in the presence of Triton X-100 (0.1 %).Total activities, expressed as tog. of p-nitrophenol liber-ated/g. wet wt. of tissue/hr., were: liver, 1190; spleen, 2130.

Activity (%)

Fraction Rat liver Rat spleenI 0 3II 50 17III 33 45IV 10 34

Recovery 93 99

a fraction of their total hydrolase activity asmeasured in the corresponding water homogenates(Berthet & de Duve, 1951; Walker, 1952; Gianetto

356 1963

8INTRACELLULAR LOCALIZATION OF GLYCOSIDASES& de Duve, 1955; Conchie et al. 1961). Further,though the water homogenates are fully active,only a proportion of the enzyme activity is non-sedimentable, the remainder still being bound tothe cell fragments. Analysis of these variables wasmade for the glycosidases and tissues studied in thepresent work. Tables 3-5 show the enzyme activ-ities of iso-osmotic (0-25M) sucrose homogenatesof some mouse and rat tissues expressed as percent-ages of their total activities, together with thepercentage activities in the soluble fractions fromboth sucrose and water homogenates.The optimum pH of ,-galactosidase in the tissues

studied is 3-0. At thispH, however, the ,-galactosid-ase activities of iso-osmotic sucrose homogenateswere equal to those of corresponding water homo-genates, i.e. there was instantaneous and completeactivation of the sucrose homogenates at this pH.

When assays were done at pH 5-0, ,B-galactosidaseresembled the other glycosidases in that theactivities of iso-osmotic sucrose homogenates wereonly a fraction of those of corresponding waterhomogenates measured at pH 5-0. Accordingly, all,-galactosidase assays concerned in Table 3 werecarried out at pH 5-0. Tissue activities at pH 3-0are given by Conchie et al. (1959).In all cases the percentage enzyme activities

observed in whole iso-osmotic sucrose homogenateswere considerably greater than those in the derivedsoluble fractions. The difference between the twovalues measures the activity of the enzyme withinthe granules, together with the contribution of anysoluble enzyme adsorbed on their surface. Thesedifferences, which are an inverse measurement oflatency, varied widely from tissue to tissue witheach enzyme.

Table 3. fl-Galactosidase activities of tisseq homogenates in 0-25M-sucrose, and of the 80oluble fractionsfrom sucrose and water homogenates

Tissues were homogenized for the single periods shown. The whole sucrose homogenates were assayed in theabsence of Triton X-100, and the results are expressed as percentages of the total activities determined in thepresence of Triton X-100. The soluble fractions were prepared by removing all particulate matter by centri-fuging at 70000g for 1 hr. Results for the sucrose-homogenate soluble fractions are expressed as percentages ofthe activities of the whole homogenates when treated with Triton X-100. Those for the water-homogenatesoluble fractions are expressed as percentages of the activities of the whole water homogenates. Total activitiesare expressed as pg. of o-nitrophenol liberated/g. wet wt. of tissue/hr. Details of assay conditions are given inthe text. Activity (%)

Sucrose homogenatePeriod of ,_A

Mouse liverMouse kidneyMouse spleenMouse T2146 tumourRat liverRat spleen

homo-genizing

(sec.)153015301515

Wholehomo-genate

717480852974

Solublefraction

91852419

53

Waterhomo-genate.Solublefraction

515182737073

Totalactivity*(/Ag./g./hr-)

58825252810149028004840

* Measured at pH 5.0.

Table 4. o-Mannosidase activities of tissue homogenates in 0-25M-sucrose, and of the solublefractionsfrom sucrose and water homogenates

The preparation of homogenates and fractions was as given in Table 3. Total activities are expressed as Hg. ofp-nitrophenol liberated/g. wet wt. of tissue/hr. Details of assay conditions are given in the text.

Mouse liverMouse kidneyMouse spleenMouse T2146 tumourRat liverRat spleen

Period ofhomo-genizing

(sec.)153015301515

Activity (%)

Sucrose homogenate Water_________A_______ homo-Wholehomo-genate

687380806172

Solublefraction

151047361213

genate.Solublefraction

551957491823

Totalactivity

(,ug./g./hr.)544072804660290052101840

Vol. 87 357.

Table 5. P-N-Acetylglucosaminidase activities of tisue homogenates in 0 25M-s8ucrose, and of the solublefractionq from sucrose and water homogenates

The preparation of homogenates and fractions was as given in Table 3. Total activities are expressed as Hg. ofp-nitrophenol liberated/g. wet wt. of tissue/hr. Details of assay conditions are given in the text.

Mouse liverMouse kidneyMouse spleenMouse T 2146 tumourRat liverRat spleen

Period ofhomo-

genizing(sec.)153015301515

Activity (%)

Sucrose homogenate Water_ ,A I homo-

Wholehomo-genate688889848279

Solublefraction

35

201117

genate.Solublefraction

135

34167

16

Table 6. Glyco8idasin 0-25M-sucrose afti

various periods

Tissues were homogeiperiods shown. Activitafter incubation with si

results are expressed aa

determined by assayinmDetails of other assay c

Pehge

oa-MannosidaseMouse liverMouse T2146tumour

,B-GalactosidaseMouse kidneyMouse spleen

fl-N-Acetylglucosamini(Mouse spleenMouse T2146tumour

All results quotEvalues, and some a

during the period ofassay periods of lesslinear portion of th4liable to error the si

After 1 hr. the slopconstant, the absoluland comparison beimade more accurat4the assay is illustrataken into account ixdifferent enzymes adassay values. Errorenter only into me

distribution.

S activities of tissue homogenates The fact that the percentages of non-sediment-er incubation with sub8trate for able P-N-acetylglucosaminidase (Table 5) in water

homogenates of all the tissues were appreciablynized in 0-25m-sucrose for the single

lower than the corresponding values for the other

ies of homogenates were measured glycosidases (Tables 3 and 4) could conceivablyubstrate for the periods shown and have been due to the use of citric acid-sodium3 percentages of the total activities hydroxide buffer in the assay instead of aceticg in the presence of Triton X-100. acid-sodium hydroxide buffer. There was, how-%onditions are given in the text. ever, no change in the percentages of non-sediment-

niod of Duration of assayable P-galactosidase and oc-mannosidase in sucrose

Lomo- (min.) homogenates when assays were done in 0-2 M-)nizing, disodium hydrogen phosphate-citric acid buffer.(sec.) 15 30 60 Effect of increasing sUbstrate concentration on

15 67 70 72 fi-galactosidase activity. Walker (1952) found that30 69 73 86 the P-glucuronidase activity of iso-osmotic sucrose

homogenates of mouse liver could be increased byincreasing the substrate concentration beyond the

30 43 55 73 optimum for the enzyme, although the activated15 51 79 88 enzyme is subject to inhibition by excess of sub-

1dase strate. A similar effect was observed when the15 75 84 85 ,-galactosidase activity of an iso-osmotic sucrose30 56 78 83

homogenate of rat liver was measured withincreasing concentrations of o-nitrophenyl f-galac-toside (Table 7). At a substrate concentration of

ad are based on 1 hr. assay 30 mM, which caused considerable inhibition in an

Lctivation of the homogenates activated preparation, there was greatly increasedf assay must be expected. For activity of the untreated iso-osmotic sucrose

than 1 hr., values lie on a non- homogenate. It was evidently possible to increasee velocity curve, and are more the access of the enzyme to the substrate by in-iorter the period of incubation. creasing the substrate concentration.me of the curve becomes fairly Owing to the limited solubility of p-nitrophenylte experimental error is smaller, oc-mannoside,p-nitrophenyl N-acetyl-p-glucosamin-tween different tissues can be ide and p-nitrophenyl OC-L-fucoside, this type ofely. The effect of activation in experiment could not be repeated with these.ted in Table 6, and has been substrates.n drawing comparisons between Activation of sucrose homogenates. Iso-osmoticnd tissues on the basis of 1 hr. sucrose homogenates of mouse and rat tissuess from activation in the assay showed a progressive activation of all four enzymesasurements of latency, not of (P-galactosidase, oc-mannosidase, P-N-acetylglucos-

aminidase and oc-L-fucosidase) as the suspending

Totalactivity

(,ug./g.hr.)33000100400135300372004600022500

J. CONCHIE AND A. J. HAY 1, .1 1963358

INTRACELLULAR LOCALIZATION OF GLYCOSIDASESmedium was made increasingly hypo-osmotic. Theosmotic activation of a rat-liver homogenate isshown in Fig. 1.As observed above, TritonX-100 (0- 1 %) added to

iso-osmotic sucrose homogenates made them fullyactive. It did not, however, make the enzymes

Table 7. Effect of increasing substrate concentrationson the f-galactosidase activity of a rat-liver homo-genate in 0-25M-s8crose, before and after activationwith Triton X-100

The tissue was homogenized in 0-25M-sucrose for a singleperiod of 15 sec. Details of the concditions of assay aredescribed in the text, the pH of assay being 5-0 and thesubstrate concentrations as shown in the Table. Activationwas obtained by assaying in the presence of Triton X-100(0-1%). Activities are expressed as pg. of o-nitrophenolliberated/g. wet wt. of tissue/hr.

Concn. ofsubstrate(mM)

2-57-515-0300

Sucrose homogenateactivity (yg./g./hr.)Before After

activation activation992 25151155 25751248 23101410 2070

100

90 A

90~~~~~~

80~~~

o60 -

50

40- ~ ~ A

30 IIL0 05 0.10 0-15 0-20 0-25

Conon. of sucrose (m)

Fig. 1. Enzyme activities in a rat-liver homogenatesuspended in various concentrations of sucrose. 0, p-Galactosidase; *, a-mannosidase;, A, fl-N-acetylglucos-aminidase; A, M-L-fucosidase. Activities are expressed aspercentages of the total activities measured in a waterhomogenate of the liver. DetaiLs of the conditions of assayare given in the text. Total activities, expressed as ug. ofo- or p-nitrophenol liberated/g. wet wt. of tissue/hr., were:,-galactosidase, 3110; c-mannosidase, 4500; P-N-acetyl-glucosaminidase, 34700; O-L-fucosidase, 1130.

completely soluble, the proportion of solubleenzyme being in general only a little higher thanthat in a water homogenate; neither did treatmentof a water homogenate with Triton X-100 make theenzymes fully soluble.

DISCUSSIONThe hydrolytic enzymes discussed in the present

paper all belong to the group known as 'lysosomalenzymes'. All of them are to some extent particle-bound in the tissues examined, though there isconsiderable variation in the actual pattern, de-pending on the enzyme and the tissue. Under theexperimental conditions described, some enzymeactivity is always found in the final supematantafter fractionation, although this fraction is fre-quently small. Though such activity is generallyregarded as coming from the extragranular cyto-plasm, it is possible that some of the activity wasoriginally present behind highly permeable mem-branes, which may or may not rupture when thecell is homogenized, but which allow immediatediffusion, to reach equilibrium with the artificialmedium. Conversely, there will be immediate dif-fusion of substrate through such membranes.Consequently, it is just such a 'cytoplasmic'fraction that would be prominent in a histochemicalstudy. It has been shown that liver nuclei pre-pared in sucrose solution by several methods aredevoid of certain water-soluble enzymes, such asadenosine deaminase and nucleoside phosphorylase,which are concentrated in nuclei prepared by non-aqueous methods (Stem & Mirsky, 1953; Stern,Allfrey, Mirsky & Saetren, 1952; Allfrey, Stem,Mirsky & Saetren, 1952).For any given tissue, the differences in the

patterns of distribution for the glycosidases couldbe considerable. Since the enzymes segregatedifferently, the particles with which they areassociated must be heterogeneous both in func-tion and size. Previous workers have observedthat there is considerable f-glucuronidase in themicrosomal fraction of liver, as well as in the 'mito-chondrial' or 'lysosomal' fraction. It has beenargued by de Duve (1962) that, compared withother members of the lysosomal group, ,B-glucuron-idase is atypical in this respect. Examination of theresults described above for other glycosidases,however, suggests that such a distribution may bethe rule rather than the exception. Other workershave also commented on the heterogeneity both inenzymic composition and size of the particles withwhich the lysosomal enzymes are associated(Whittaker, 1959; Roy, 1960; Franklin, 1962;Zalkin et al. 1962). Moreover, when acid-phosphat-ase activity in fractions from mammalian-liversucrose homogenates is assayed with substrates

Vol. 87 359

360 J. CONCHIE AND A. J. HAY 1963

other than P-glycerophosphate, such as p-nitro-phenyl phosphate, the distribution pattern ismarkedly different, there being considerableactivity in the final supernatant (Neil & Homer,1962); nitrophenol derivatives are often used inlysosomal studies. There appears to be no justifi-cationfortheuseofacidphosphatase,withp-glycero-phosphate as the only substrate, as the markerenzyme in tissue sections for identifying unknownorganelles that are assumed to be the sole depot,not only of acid phosphatase, but also of certainother hydrolytic enzymes (see above).Under suitable conditions all of the glycosidases

investigated exhibited the phenomenon of latencyin sucrose homogenates of all the tissues examined,though to different degrees, whereas water homo..genates displayed full activity. Sometimes thedegree of latency displayed by a particle-boundenzyme was small, i.e. the activity in the sucrosehomogenate was too high to be explained by thesoluble enzyme present. In certain tissues, such asspleen and tumours, some enzymes displayedconsiderable activity in the supernatant fractionfrom the sucrose homogenates. In ox liver, 80% ofthe fl-galactosidase activity was found to be soluble(i.e. 'cytoplasmic') on centrifugal fractionation ofhomogenates made in 0 25M-sucrose solution(Levvy, McAllan & Hay, 1962).Such variability in the distribution of acid-

hydrolase activity in the examples discussed abovemakes it difficult to accept the concept of the lyso-some as originally envisaged by de Duve. If theenzymes dealt with in the present study aresituated in special particles distinct from mito-chondria and microsomes, then individually theymust differ in sedimentation properties and stabil-ities in different tissues, and must differ betweeneach other in stability and situation within a singletissue. What is required is a new definition of theterm that will embrace all the different shades ofmeaning and reservations attached by differentauthors to the term. At one extreme of opinion,the mere suggestion that the enzyme has an auto-lytic or catabolic function is enough to label it'lysosomal'; at the other, the original 'lethal-bag'theory is adhered to strictly. The lysosome theorydoes embrace phenomena ofgreat importance in theregulation of cell metabolism, but it may be thatthese do not apply to hydrolytic enzymes only.Such enzymes may merely more convenientlymeasure factors that apply equally to other en-zymes in all types of subcellular particles. Withoutseparation of the different anabolic and catabolicactivities of the cell it could not exist, and 'mem-branes' of some type or other are an essentialpostulate. The term 'lysosomal', in contrast with'latent', however, becomes meaningless unlessassumptions about the function of the enzymes are

introduced. It is difficult to see what distinctioncan be drawn on grounds of function between'lysosomal' enzymes and those numerous otherhydrolytic enzymes that are not particle-bound orare devoid of latency. Nevertheless, however thelysosome theory is interpreted, changes in the sedi-mentation pattem of an enzyme under pathologicalconditions may still be biologically valid, as in ourfindings for glycosidases in tumours.

SUMMARY

1. The intracellular localization of ,-galacto-sidase, a-mannosidase, P-N-acetylglucosaminidaseand oc-L-fucosidase in some tissues of the mouse andthe rat has been studied by centrifugal fractiona-tion of homogenates in 0 25M-sucrose.

2. All the glycosidases were particle-bound tovarious degrees, the extent depending on theglycosidase and the tissue.

3. In some tissues there was considerableheterogeneity in the patterns of distribution for theenzymes.

4. The degree of latency exhibited in theparticles by each enzyme varied from tissue totissue.

5. The significance of these results with re-ference to the lysosome theory is discussed.

The authors thank Dr G. A. Levvy for his advice andencouragement.

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Mammalian Fucosidases3. P-D-FUCOSIDASE ACTIVITY AND ITS RELATION TO g-D-GALACTOSIDASE*

BY G. A. LEWVY Aim A. McALLANRowett Research In8titute, Buck8burn, Aberdeen

(Received 10 October 1962)

During a study of mammalian tissues for Oa-L-fucosidase activity (Levvy & MeAllan, 1961), thepreparations were seen to cause rapid hydrolysis ofp-nitrophenyl P-D-fucoside, and the present paperdeals with attempts to discriminate between theenzyme responsible and the extensively studiedP-D-galactosidase in mammalian tissues (Conchie,Findlay & Levvy, 1959; Conchie & Hay, 1959;Levvy, McAllan & Hay, 1962). ,B-D-Fucosidase and,B-D-galactosidase activity in the limpet, PateUavulgata, have been shown to be due to two differentenzymes (Levvy & McAllan, 1963). Levvy et al.(1962) have also shown that rat-epididymis P-D-galactosidase is powerfully inhibited by solutionsof galactonolactone, but not of fuconolactone,whereas ox-liver P-D-galactosidase is powerfullyinhibited by fuconolactone solutions, and onlyrelatively feebly by galactonolactone solutions: inthe course of this same work it was found that thepotency of both inhibitors was dependent upon theproportion present in the solution in the form ofthe (1-+5)-lactone.

EXPERIMENTAL

Enzyme preparations. The freshly dissected tissues werehomogenized in water, incubated for 1 hr. at pH 5-2 inacetate buffer, and precipitated from the clear extract with(NH4)2SO4 between 20 and 80% saturation (Levvy &McAllan, 1961). As with P-D-galactosidase (Levvy et al.1962), incubation was not really necessary for extraction ofthe f-D-fucosidase activity in water homogenates of ratepididymis and ox liver, but to retain complete rat-epidi-dymis f-D-fucosidase activity it was necessary to add NaClto the preparation at a final concentration of 0 1M (seebelow).

Substrates and inhibitors. The syntheses ofp-nitrophenyl,B-D-fucoside and o-nitrophenyl f-D-galactoside, and thepreparation of solutions of fuconolactone and galactono-lactone containing maximum amounts of the inhibitory(1--5)-1actones, are described by Levvy & McAllan (1963).p-Nitrophenyl fl-D-galactoside tetra-o-acetate was made bythe general method of Glaser & Wulwek (1924), and de-acetylated to give p-nitrophenyl P-D.galactoside by theprocedure ofLeaback (1960). Because earlier constants forthese well-known compounds are not easily found in theliterature, they are given in Table 1, together with our ownand other recent values. Apart from Goebel & Avery(1929), who condensed the acetobromo-sugar with silver

Table 1. Constants for p-nitrophenyl P-D-galactoside and its tetra-O-acetate

Galactoside Tetra-0-acetate

[a]D in [r]D inReference m.p. water m.p. CHC13

Goebel & Avery (1929) 181-1820 144 1450 - 8.30Aizawa (1939) 170 - 750 138Snyder & Link (1952, 1953) - -85 -9Beiser, Burke & Tanenbaum (1960) 173-175 139-140Heyworth & Walker (1962) 181-182 - 84 138 -12This paper 183-184 147-148 - 9

* Part 2: Levvy & McAllan (1961).