Download The Permeability Properties of Rat Liver Lysosomes to Nucleosides

559th MEETING, CORK
1251
Headon, D. R. & Duggan, P. F. (1971) in Methodologicnl Developments in Biochemistry (Reid,
E., ed.), vol. 1, pp. V-2.1-V-2.12, Wolfson Bioanalytical Centre, University of Surrey
Headon, D. R., Barrett, E. J.,Keating, H. &Duggan,P. F. (1974)inMethodologiculDeuelopments
in Biochemistry (Reid, E., ed.), vol. 4, pp. 279-291, Longman, London
Joyce, N. & Headon, D. R. (1975) Biochem. SOC.
Trans. 3,1246-1248
O’Flaherty, J., Barrett, E. J., Bradley, D. P. & Headon, D. R. (1975) Biochim. Biuphys. Acta
401.177-183
The Permeability Properties of Rat Liver Lysosomes to Nucleosides
ROBERT BURTON, CHERYL D. ECK and JOHN B. LLOYD
Biochemistry Rcsearch Unit,University of Keele, Keele, Staffs. ST5 SEG, U.K.
Lysosomes contain ribonuclease and deoxyribonuclease activities, which are doubtless
employed in the intralysosomal degradation of RNA and DNA. The products of such
digestion are the mononucleotides, which could be further degraded within lysosomes
by an enzyme or enzymes of the acid phosphatase complex to yield the nucleosides
(Arsenis e t ul., 1970). The fate of nucleosides arising within lysosomes in this way is not
known. One possibility is the penetration of intact nucleosides through the lysosomal
membrane out into the cytosol, where anabolic or further catabolic reactions could take
place. A second possibility is that nucleosides are degraded intralysosomally to base and
pentose before release from the lysosome is possible. Neither of these explanations
appears immediately plausible. There is a molecular-weight ceiling of about 200, beyond
which water-soluble substances such as carbohydrates and oligopeptides apparently
cannot penetrate the lysosomal membrane (Lloyd, 1973); nucleosides exceed that value.
And there is no report of nucleoside hydrolase or phosphorylase activity within lysosomes.
A liver fraction rich in lysosomes, prepared by the method of Lloyd (1969), was incubated overnight at 37°C in 0.1 M-sodium acetate-acetic acid buffer, pH5.0, containing
0.1 % Triton X-100 and either uridine or cytidine. Paper chromatography of the incubation mixture in butan-1-ol-pyridine-water(1 :5:4, by vol.) revealed no trace of uracil or
cytosine; unchanged nucleoside was the only detectable component. Similar experiments
in which UMP was incubated with liver lysosomes revealed the rapid production of
uridine.
The ability of substances to penetrate into intact lysosomes may be assessed by
measuring their capacity to afford osmotic protection to isolated intact lysosomes.
Lloyd (1969, 1971) has explained the rationale of such experiments and applied the
method to a wide range of carbohydrates to amino acids and small oligopeptides.
In the present work it is applied to nucleosides and their parent bases. The method,
described fully by Lloyd (1969), is applicable only to substances sufficiently soluble in
water to produce a 0 . 2 5 ~solution, and so only the pyrimidine nucleosides and a nonphysiological base, purine, could be tested.
Table 1 shows the percentage free activity, in a lOmin assay, of p-nitrocatechol sulphatase of rat liver lysosomes incubated at 25°C for 0,30or 60min in 0 . 2 5 solutions
~
of
mannitol, glucose, uridine, thymidine, cytidine or purine. Table 2 shows similar data
for a second lysosomal enzyme, maltase. In agreement with earlier data (Lloyd, 1969),
mannitol gave prolonged, but glucose only transient, osmotic protection. Purine failed
even to give initial protection, indicating a very rapid rate of entry into lysosomes. This
is consistent with its low molecular weight (120). The nucleosides all afforded good initial
osmotic protection, but this was lost on incubation for 60min. The rate of loss of latency
was relatively slow for lysosomes incubated in 0.25~-cytidineor -midine, indicating a
low rate of penetration into the lysosome. Lysosomes incubated in thymidine lost their
latency more quickly, indicating a more rapid penetration of this substance. As indicated
above, these results are somewhat surprising, as the nucleosides have n~olecularweights
VOl. 3
1252
BlOCHEMlCAL SOClETY TRANSACTlONS
Table 1 . 'Free' nitrocatechol srrlphatase of rat liver lysosomes preincubated at 25°C in
various media
Each value ( m e a n k s . ~of
. fourexperiments) isexpressed as a percentage of total activity.
.
Activity (% of total activity)
Medium
( 0 . 2 5 ~ ) Preincubation
I
. ..
Mannitol
Glucose
Uridine
Thymidine
Cytidine
Purine
Omin
14.9 k 2.9
16.3 f 6.0
18.6f 5.1
20.2k 5.6
17.8 f7.4
84.55~12.7
30min
15.2k 4.5
35.1 k 6.3
29.2+ 5.2
77.1 f 15.7
22.8 f 5.2
91.22 14.3
6Omin
20.0 f4.0
55.1 k 5.3
48.3 2 4.5
89.7f 5.4
35.9 5.3
96.7k5.4
*
greater than 200. The explanation possibly lies in the considerably hydrophobic nature
of the aglycone, particularly in the case of thymidine. There is good evidence (see Lloyd,
1973) that iodotyrosine, whose molecular weight is 307, can penetrate out of lysosomes,
whereas some smaller but less hydrophobic molecules probably cannot.
It seems likely therefore that nucleic acids are broken down by lysosomal enzymes to
the nucleosides, which then escape intact from the lysosome. It is extremely unlikely that
nucleotides could pass across the lysosomal membrane [much smaller charged molecules
cannot (Lloyd, 1969, 1973)] and the lysosome appears to have no enzymic mechanism
for nucleoside degradation. Presumably, however, inorganic phosphate from nucleotide
hydrolysis can pass across the membrane.
In these experiments and previous ones (Lloyd, 1969, 1971) of the same type, rat
liver lysosomes incubated in 0 . 2 5 ~solutions lost their latency progressively over a
60min incubation at 25"C, the rate varying with the nature of the solute. That the loss
of latency was progressive, rather than abrupt, deserves comment. One explanation is
that, as lysosomes swell, their membrane becomes progressively more leaky: substrates
may enter, but not rapidly enough to achieve an intralysosomal concentration of substrate equal to that in the bulk assay medium. As explained by de Duve (1965), it should
be possible, if such were the case, to demonstrate a higher free activity by raising the
substrate concentration used in the assay to values above the K,,,. No alteration in the
percentage free activity of maltase (K,,,approx. 2 m ~in) lysosomes incubated for 30rnin
in 0.25~-cytidineor -uridine was found on assaying at maltose concentrations from 1 to
10mM. A second, and more plausible, explanation is the heterogeneity of lysosomes in
Table 2. 'Frcc' rnoltasc of rat fiver lysosorncspreiricirbafrrfat 25°C in varioirs media
Each value (mean +s.D. of four experiments) is expressed as a percentage of total activity.
Activity
Medium
( 0 . 2 5 ~ ) Preincubation
Mannitol
Uridine
Thymidine
Cy t idine
Purine
...
(xof total activity)
I
Omin
30min
60min
13.9k2.7
18.5 ? 4.2
26.4f 8.2
23.2k3.0
77.9 f9.5
20.2 k 5.1
38.3 f 5.1
71.42 5.3
35.4f 4.6
67.9f 11.9
17.0f 3.5
42.1 f 2.8
86.8 f 2.9
41.7 k 3.1
81.0+ 12.2
1975
559th MEETING, CORK
1253
the rat liver preparation. Lysosonies have a spectrum of sizes and would respond at
differing rates to an osmotic imbalance generated by a permeant solute. Thus the progressive loss of observed latency probably reflects a changing proportion of ruptured
lysosomes in the population. Size is not the only possible source of a graded response;
lysosomes from different cell types (hepatocytes, Kupffer cells) or of different intracellular
status (primary, secondary lysosomes) might well differ in permeability properties.
R. B. thanks the ScienceResearch Council for apostgraduate studentship.C. D. E. undertook
part of this work during the tenure of a Graduate Fellowship awarded by Rotary International,
to whom she expresses her thanks.
Arsenis, C., Gordon, J. S. & Touter, 0. (1970) J. Biol. Chem. 245,205-21 1
de Duve, C. (1965) Harvey Lrcf. 59,49-87
Lloyd, J. B. (1969) Biochem. J. 115,703-707
Lloyd, J. B. (1971) Biochem. J. 121,245-248
Lloyd, J. B. (1973) in Lysosomes andstorage Diseases (Hers, H. G . & Van Hoof, F., eds.), pp.
173-195, Academic Press, New York and London
Resistance of Urate Oxidase to Proteolytic Digestion
DENIS A. FITZPATRTCK and KEVIN F. McGEENEY
Department of Medicine and Therapeutics, University College, Dublin 4, Ireland
In a previous study the immunological properties of urate oxidase (urate-oxygen oxidoreductase, EC 1.7.3.3) from various sources was studied. It was shown that, whereas the
animalenzymeexhibited a high degree of structural homology (Fitzpatrick &McGeeney,
1975), antisera to urate oxidase from a mould, yeast and bacterium did not cross-react
with one another at all (Fitzpatrick, 1972). To delineate further the degree of heterogeneity of the microbial enzyme the stability of these three urate oxidases to proteolytic
digestion was studied.
Candidu utilis urate oxidase (grade 3; Toyobo Co., Osaka, Japan) was given by Dr. J.
Fukumoto, and Aspergillus flavus and Bacillus fastidiosus enzymes were gifts from
Professor M. Brunaud and Mr. Keld Pederson respectively. Proteolytic enzymes were
trypsin (EC 3.4.4.4) (Sigma type I ; 2.5mg/ml), a-chymotrypsin (EC 3.4.4.5) (Sigma type
V; 0.2mg/ml), di-isopropyl phosphorofluoridate-treated carboxypeptidase A (EC
3.4.2.1) (Sigma; 0.4mg/ml) and elastase (EC 3.4.4.7) (Sigma type III ; l.Omg/ml).
Urate oxidase (1.8-2.4 units; 0.5-0.8mg of protein) in 2.0ml of buffer was equilibrated
at 37"C, enzyme activity was measured and then 0.1 ml of proteinase added. At fixed
time-intervals samples were removed, diluted 10-fold with 0.1 M-Na2CO3and assayed
immediately in 0.02~-borate,pH9.0, containing 0.55mg of urate/ml. Assays were
carried out at 293 nm in a Unicam SP.800 spectrophotometer with thermostatically
controlled cell holders at 30°C. A unit of activity catalysed the conversion of I pmol of
substrate/min. The results are summarized in Fig. 1.
The three endopeptidases used caused a rapid loss of enzyme activity, whereas the
exopeptidases had a weaker effect. The three microbial urate oxidases did not exhibit
the same high degree of independence observed in the immunological study. This is no
doubt due to the lower specificity of the proteolytic digestion technique. Some differences, however, can be observed. The Aspargillus enzyme, in contrast with the other two
urate oxidases, was very resistant to elastase digestion (Fig. 1c), although it was digested
more quickly by the other two endopeptidases. The amino acid sequences of the enzymes
studied are not known, but the amino acid content of the Aspergillus enzyme has been
published (Labourer & Langlois, 1968) and it shows a high content of acidic and
basic amino acids. Elastase is specific for uncharged non-aromatic side chains (Shotton,
1970). The Bacillus and Candida enzymes were much more susceptible to chymotrypsin
than to trypsin.
Vol. 3