54P
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
Bacterial Carbohydrate Sulphamidase Induction and Heparin Degradation
By A. G. LLOYD, B. A. LAW, L. J. FOWLER and
G. EMBERY. (Department of Biochemistry, Medical
Biology Centre, Queen's University, BelfastBT7 1NN)
Payza & Korn (1956) and Korn & Payza (1956)
showed that the growth ofFlavobacteriumheparinum
in media containing heparin was accompanied by
the appearance of enzymes capable of degrading
this polysaccharide by depolymerization, 0desulphation and N-desulphation. Evidence for the
latter process was based on the appearance of free
hexosamine end groups, presumed to result from
the concerted action of the 'depolymerase' system
and a new enzyme tentatively designated as a
heparin sulphamidase. On the other hand, Linker &
Hovingh (1965, 1968), Yosizawa (1967) and
Dietrich (1968a,b) all demonstrated the production
of a range of monosaccharides and oligosaccharides
with intact sulphamate groupings obtained as a
result of the degradation of heparin, co-heparin and
heparin sulphates by enzyme extracts from F.
heparinum previously grown in media containing
heparin. These results apparently contradict the
original findings.
Lloyd, Embery, Wusteman & Dodgson (1966)
and Lloyd, Embery, Powell, Curtis & Dodgson
(1966) have already reported on the use of N-re[35S]sulphated heparin and authentic 2-deoxy2[35S]-sulphoamino-D-glucose in studies on sulphamidase systems of mammals in vivo and in vitro.
These compounds have now been employed in an
examination of sulphamidase induction in F.
heparinum N.C.I.B. 9290, liberation of inorganic
35SO42- ions being detected by the paper-electrophoresis method of Dodgson, Lloyd & Tudball
(1961).
The growth of F. heparinum in a glucose-free
tryptone-soy medium containing either N-re[35S]sulphated heparin (2.5mg./ml.) or 2-deoxy2[35S]-sulphoamino-D-glucose (2.5mg./ml.), at 25°
with aeration was accompanied in both instances
by the liberation of inorganic 35SO42- ions.
Degradation of the [35S]sulphamate linkages of
these compounds was also observed when washed
preparations of F. heparinum, previously grown in a
glucose-tryptone-soy broth, were 'adapted' in a
casein hydrolysate medium containing either the
35S-labelled polymer (2.5 mg./ml.) or the 35S-labelled
monomer (2.5mg./ml.) incubated at 250 with
aeration.
The capacity to liberate inorganic 35SO42- ions
was retained when freeze-dried whole-cell preparations of F. heparinum, previously grown on (or
adapted to) unlabelled heparin, were subsequently
suspended by homogenization in 0 025M-phosphate
buffer, pH 7-5, and incubated with either N-re[35S]sulphated heparin or 2-deoxy-2[35S]-sulphoamino-D-glucose at 250. Essentially similar results
were obtained with cells grown on (or adapted to)
the unlabelled monomer. Neither labelled derivative was degraded by suspensions of freeze-dried
F. heparinum previously grown in media devoid of
the sulphamate compounds.
It is also noteworthy that liberation of inorganic
35SO42- ions was not a feature of experiments
similar to those described above in which ProteuB
vulgari8 N.C.T.C. 4636 was substituted for F.
heparinum. This finding further substantiates the
view that these two micro-organisms are appreciably
different in their relative capacities to degrade
acidic glycosaminoglycans and related compounds
(cf. Yamagata, Saito, Habuchi & Suzuki, 1968).
B.A. L. and L.J.F. are indebted to the Medical Research
Council and the Science Research Council respectively for
the award of studentships. We gratefully acknowledge
financial support from the Nuffield Foundation.
Dietrich, C. P. (1968a). Fed. Proc. 27, 321.
Dietrich, C. P. (1968b). Biochem. J. 108, 647.
Dodgson, K. S., Lloyd, A. G. & Tudball, N. (1961). Biochem.
J. 79, 111.
Korn, E. D. & Payza, A. N. (1956). J. biol. Chem. 223, 859.
Linker, A. & Hovingh, P. (1965). J. biol. Chem. 240, 3724.
Linker, A. & Hovingh, P. (1968). Fed. Proc. 27, 529.
Lloyd, A. G., Embery, G., Powell, G. M., Curtis, C. G. &
Dodgson, K. S. (1966). Biochem. J. 98, 34P.
Lloyd, A. G., Embery, G., Wusteman, F. S. & Dodgson,
K. S. (1966). Biochem. J. 98, 33P.
Payza, A. N. & Korn, E. D. (1956). J. biol. Chem. 223, 853.
Yamagata, T., Saito, H., Habuchi, 0. & Suzuki, S. (1968).
J. biol. Chem. 243, 1523.
Yosizawa, Z. (1967). Biochim. biophys. Acta, 141, 600.
Degradation of 135S]Heparin by Mammalian
and Bacterial Sulphamidases
By A. G. LLOYD, L. J. FOWLER, G. EMBERY and
B. A. LAW. (Department of Biochemistry, Medical
Biology Centre, Queen's University, Belfast BT7
1NN)
Lloyd, Embery, Wusteman & Dodgson (1966)
showed that the urinary excretion of significant
quantities of inorganic 35SO42- ions was a consistent
feature of experiments involving the administration
of N-re-[35S]sulphated heparin to rats, but not after
the injection of authentic 2-deoxy-2[35S]-sulphoamino-D-glucose. When a wide range of mammalian
tissues was tested in systems in vitro, liberation of
inorganic 35SO42- ions from [35S]heparin was only
observed after prolonged incubation of the labelled
polymer with homogenates of rat spleen (Lloyd,
Embery, Powell, Curtis & Dodgson, 1966). Extracts
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
containing this system have been purified from the
supernatant obtained after centrifuging a 20% (w/v)
homogenate of fresh rat or pig spleen in 0 1 M-acetate
buffer, pH5-0, at l00000gav. at 2° for 45min. The
supernatant was heated to 60° for 5min., cooled
rapidly before centrifuging to remove precipitated
material, and then chromatographed on CMcellulose in O lM-acetate buffer, pH5.0.
The substrates used in the study of the purified
system included N-re-[35S]sulphated heparin,
authentic potassium 2-deoxy-2[35S]-sulphoaminoD-glucose (Lloyd, Wusteman, Tudball & Dodgson,
1964) and the following compounds isolated by
gel-filtration on Sephadex G-10 after the depolymerization of N-re-[35S]sulphated heparin with
extracts of Flavobacterium enzymes freed from
bacterial sulphamidase by treatment with protamine sulphate: 2 - deoxy - 2 [35S] - sulphoamino - D glucose 6-0-sulphate; 4,5-dehydroglucopyranosyluronic acid-2-deoxy-2[35S]-sulphoamino-D-glucose
0-sulphate (see Lloyd, Fowler, Law & Embery,
1968). Inorganic 35SO42- ion liberation was again
followed by the method of Dodgson, Lloyd &
Tudball (1961).
The properties of the mammalian enzyme were
compared with those of the sulphamidase induced in
F. heparinum (Lloyd, Law, Fowler & Embery, 1968),
partially purified by gel filtration on Sephadex G-200
but still containing trace amounts of the depolymerizing enzymes (Linker & Hovingh, 1965) plus a
sugar sulphate sulphohydrolase (EC 3.1.6.3) analogous in properties to the enzyme that can be induced in Trichoderma viride (Lloyd, Large, Davies,
Olavesen & Dodgson, 1968). Several differences in
the mode of action of the mammalian and bacterial
sulphamidases were revealed. Thus the mammalian
enzyme acted optimally at pH 5 0, whereas the optimum for the bacterial system was pH 7*2. Secondly,
the mammalian enzyme liberated inorganic 35SO42ions only from [35S]heparin (optimum substrate
concentration 50mg./ml.): on the other hand, the
bacterial enzyme preferentially degraded authentic
2 -deoxy-2[35S] -sulphoamino-D -glucose (optimum
substrate concentration 16mM; Km 5.26mM), also
cleaving the [35S]sulphamate groupings of 2-deoxy2[35S]-sulphoamino-D-glucose 6-0-sulphate and the
unsaturated uronic acid disaccharide [35S]sulphamate, as well as the intact 35S-labelled polymer
to a limited extent. The following substances (each
at 0 1 M) all inhibited the action of the mammalian
enzyme on [35S]heparin: inorganic S042- ions,
inorganic P043- ions, 2-deoxy-2-sulphoamino-Dglucose, L-serine N-sulphate (I), D-cycloserine Nsulphate (II) and phenyl sulphamate (III). Increasing concentrations of inorganic P043- and S042ions inhibited the action of the bacterial enzyme on
2-deoxy-2[35S]-sulphoamino-D-glucose but I, II
and III (at 0- 1 M) were without effect.
55P
L. J. F. and B. A. L. are indebted to the Science Research
Council and the Medical Research Council respectively for
the award of studentships. We gratefully acknowledge
financial support from the Nuffield Foundation.
Dodgson, K. S., Lloyd, A. G. & Tudball, N. (1961). Biochem.
J. 79, 111.
Linker, A. & Hovingh, P. (1965). J. biol. Chem. 223, 859.
Lloyd, A. G., Embery, G., Powell, G. M., Curtis, C. G. &
Dodgson, K. S. (1966b). Biochem. J. 98, 34P.
Lloyd, A. G., Embery, G., Wusteman, F. S. & Dodgson,
K. S. (1966a). Biochem. J. 98, 33P.
Lloyd, A. G., Fowler, L. J., Law, B. A. & Embery, G.
(1968). Biochim. biophy8. Acta, in the Press.
Lloyd, A. G., Large, P. J., Davies, M., Olavesen, A. H. &
Dodgson, K. S. (1968). Biochem. J. 108, 393.
Lloyd, A. G., Law, B. A., Fowler, L. J. & Embery, G.
(1968). Biochem. J., preceding communication.
Lloyd, A. G., Wusteman, F. S., Tudball, N. & Dodgson,
K. S. (1964). Biochem. J. 92, 68.
Lysine Control ofa-Aminoadipate and Penicillin Synthesis in Penicillium Chrysogenum
By S. A. GOULDEN and F. W. CHATTAWAY. (Glaxo
Laboratorie8 Ltd., Ulverston, Lancs., and Department
of BiocheMistry, University of Leeds)
It was observed by Demain (1957) that lysine was
a potent inhibitor of penicillin production and that
a-aminoadipate could reverse this inhibition. He
also showed that a-aminoadipate could stimulate
penicillin production in the absence of lysine. The
elucidation of the a-aminoadipate route for lysine
biosynthesis in yeast (Strassman & Ceci, 1965,
1966; Maragoudakis & Strassman, 1966) showed the
connexion between these two compounds, but their
relationship with penicillin biosynthesis remained
obscure.
Arnstein & Morris (1960) isolated a tripeptide, aaminoadipoylcysteinylvaline, and postulated that
it was an intermediate in penicillin biosynthesis.
The subsequent isolation of isopenicillin N by
Flynn, McCormick, Stamper, de Valeria & Godzeski
(1962), with an a-aminoadipoyl side chain, added
further circumstantial evidence for the involvement
of ao-aminoadipate in penicillin biosynthesis.
Demain (1966) suggested that the inhibition of
penicillin production by lysine is caused by
the end-product control exerted by lysine decreasing the availability of a-aminoadipate for pencillin
biosynthesis.
The present work has been concerned with lysinerequiring auxotrophs of Penicillium chrysogenum;
it has been found that a mutant blocked before
ac-aminoadipate can only make penicillin when
a-aminoadipate and lysine are added to the medium.
Mutants blocked after a-aminoadipate only require
the addition of lysine.