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  • 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, wasobtained 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 assaysIn experiments involving the measurement of enzyme

    activity 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 sucrosehomogenates 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 byLevvy & 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-glucur