1760
BIOCHEMICAL SOCIETY TRANSACTIONS
reaction E and compound (VII) by reaction F. These results agreed with those previously
seen with the above Pseudomonas spp. However, the coupling of anaerobic steroid
degradation to nitrate reduction in the Pseudomonas spp. (Barnes et a/., 1975) was not
observed with this strain of E. coli. Menadione, not KN03, was necessary for steroid
degradation.
The natural hydrogen acceptor in the gut has not been identified, but vitamin K2is
present, synthesized mainly by Bacteroides fragilis (Gibbons & Engle, 1964). If the
reactions observed in vitro with E. colican occur in vivo then vitamin Kz could act as an
electron acceptor. In normal human subjects where the transit time of faecal material is
relatively short, nearly all bile acid secreted in the gut can be accounted for in the bile pool
and the faeces. However, in cases of bowel stagnation, such as blind-loop syndrome or
diverticulosis, significantly more bile acid is lost from the bile pool. In such areas of
stagnation, E. coli has been shown to be present up to 108cells/ml(Drasar & Hill, 1974).
Under such conditions high cell concentrations of E . coli could be in contact with bile
acids for a relatively long time.
The reactions observed with the above strain of E. coli support the hypothesis that
bile acid degradation in the gut could be implicated in the aetiology of colon cancer.
Barnes, P. J., Bilton, R. F., Fernandez, F., Hill, M. J. & Mason, A. N. (1975) Biochem. SOC.
Trans. 3,299-301
Barnes, P. J. Baty, J. D., Bilton, R. F. & Mason, A. N. (1976) Tetrahedron32,89-95
Cocucci, M. C. & Ferrari, A. (1965) Ann. Microbiof. Enzimof. 15,157-164
Drasar, B. S. &Hill, M. J. (1974)Human IntesrinalFlora, 1st edn., pp. 172-1 82, Academic Press,
London
Gibbons, R. J. & Engle, L. P. (1964) Science 146,1307-1309
Haslewood, G. A. D., Murphy, G . M. & Richardson, J. M. (1973) Clin. Sci. 44,95-98
Hayakawa, S . (1973) Adv. Lipid Res. 11,143-192
Hill, M. J. (1974) Cancer34,815-818
Kritchevsky, D., Martak, D. S. & Rothblat, G. H. (1963) Anal. Biochem. 5,388-392
Nagasawa, M., Watanabe, N., Hashiba, H., Tamura, G . & Arima, K. (1970)Agric. Biol. Chem.
34,798-800
Severina, L. O., Torgov, I. V., Skrjabin, G . K., Zaretskii, V. I., Wulfson, N. S. & Papernaja,
I. B. (1969) Tetrclhedron 25,5617-5622
Tenneson, M. E. (1977) Ph.D. Thesis, Council for National Academic Awards
Glucose Uptake of Pseudomonas aeruginosa and its Carbenicillin-Induced
L-Form
CHRISTOPHER J. BRANFORD WHITE, MICHAEL R. HORSMAN and
PETER S . ROWE
Department of Biology, Oxford Polytechnic, Headington, Oxford O X 3 QBP, U.K.
The metabolism of glucose in Pseudomonas aeruginosa has been described by Wang et al.
(1959). Furthermore it has been suggested that an inducible glucose-transport system
exists which may play a regulatory role in glucose metabolism (Ng & Dawes, 1973).
Recent studies indicate that glucose uptake occurs by either a high- or low-affinity
system and that the enzymes involved, glucose dehydrogenase and gluconate dehydrogenase, are orientated extracellularly (Roberts et al., 1973). These findings support the
view that glucose metabolism occurs by two distinct mechanisms which are associated
with extracellular and intracellular pathways. Ps.aeruginosa can adjust its metabolism
to either the extracellular or the intracellular compartment, depending on the glucose
concentration available in the external medium (Midgley & Dawes, 1973).
Studies on the native form and carbenicillin-induced L-form variant of Ps. aeruginosa
have clearly shown at least six major morphogical differences between them at the
cellular level (Hubert ef al., 1971). Moreover, an investigation on the passaged variant
1977
1761
571st MEETING, DUBLIN
2.0 L
0
L
-
I
20
L
-
I
40
.
i
2-2
-
60
80
Time (h)
Fig. I. Incorporation of ['4C]g/ucose into cell-wall hexosarnine
Identical amounts of ['4C]glucose was added to media of both passaged ( 0 ) and nonpassaged (A) forms of fs. aeruginosa. Each point is the mean of three analyses.
demonstrated radical changes in cell-wall composition and antibiotic-sensitivity
(Branford White et al., 1977). The present study was undertaken to compare the kinetics
of glucose uptake of both the native and homologous L-form and the possible relationship to changes in cell-wall structure.
A Ps. aeruginosa strain, serotype 6 (NP6) was obtained from a clinical source and
cultures grown in brain heart infusion both containing 3 % (w/v) NaCl and 10% (v/v)
horse serum (Spicer, 1976). L-forms (P6) were induced by continued passage of the NP6
form on increasing concentrations of carbenicillin up to 200,ug/ml and further
subcultures with this induced L-form were carried out in media supplemented with the
same concentration of carbenicillin. f14C]Glucose (specific radioactivity 1.72pCi/mg)
was added to both NP6 and P6 media and samples were removed after varying time
intervals up to 72h. Whole cells were then harvested by centrifugation and washed with
water osmotically stabilized with NaCl and horse serum. When the washings were free
from [14C]glucose, cell walls were isolated by the method previously described by
Wilkinson (1968). Cell-wall material was hydrolysed in constant-boiling HCI for 9 h at
110°C under N2, and hexosamines were isolated by ion-exchange chromatography on
Dowex 50W (X8; H+ form; BDH, Poole, Dorset, U.K.) by eluting a column with
2 M-HCI. Fractions were then assayed for total hexosamine content (Cessi & Piliego,
1960) and constituted 2.4% dry weight of the total cell wall for both bacterial species.
14C-labelledmaterial was measured in an Isocap 300 liquid-scintillation counter, with
Unisolve (Koch-Light Laboratories, Colnbrook, Bucks., U.K.) as the scintillant. The
amount of [14C]hexosaminedetected in the cell-wall fraction differed; that in the native
bacterium was 66 % of the amount found in the passaged variant. The uptake of ['*C]glucose into the hexosamine components is reported in Fig. 1. It appears that the rate of
[14C]gl~~ose
uptake into the amino sugar fraction of the cell wall occurs at a higher rate
in the carbenicillin-induced L-form. Moreover, the mean generation time of the variant
was 4 h compared with 2 h for the native bacterium at the exponential phase of growth.
Observations have shown that when the supply of glucose is restricted, fs. aeruginosa
adjusts its metabolism to an intracellular mechanism in order to facilitate the rapid
utilization of substrate (Midgely & Dawes, 1973). The above findings support the view
that the intracellular pathway used in preference to the extracellular system for the
L-form variant. Recent studies have shown that changes in fatty acid composition and
content do occur between the passaged and unpassaged forms. Furthermore analysis of
the lipopolysaccharide fraction revealed an increase in phosphate content in the
carbenicillin-induced variant (Branford White et al., 1977). It is postulated that changes
in the overall chemical composition of the cell wall could lower the efficiency of the
extracellular glucose-metabolizing route.
Vol. 5
1762
BIOCHEMICAL SOCIETY TRANSACTIONS
We are grateful to Mr. A. B. Spicer for his assistance.
Branford White, C. J., Rowe, P. S. Horsman, M. R. & Spicer, A. B. (1977)Biochem. SOC.Trans.
5, 1496-1498
Cessi, C. & Piliego, F. (1960) Biochem. J . 77,508-510
Hubert, E. G., Potter, C. S . , Hensley, T. J., Cohen, M., Kalmanson,G. M . & Cruze, L. B. (1 971)
Infect. Immun. 4, 60-72
Midgely, M. & Dawes, E. A. (1973) Biochem. J. 132,141-154
Ng, F. M. W. & Dawes, E. A. (1973) Biochem. J . 132, 129-140
Roberts, B. K., Midgely, M . & Dawes, E. A. (1973) J. Cen. Microbiol. 78,319-329
Spicer, A. B. (1976) J. Appl. BacrerioI. 40, 33-45
Wang, C. H., Stern, I. J. & Gilmour, C. M. (1959) Arch. Biophys. 81,489492
Wilkinson, S . G . (1968)J. Gen. Microbiol. 54,195-213
Release of Peptide-Bound Sialic Acid from Landschiitz Ascites-Tumour
Cells by Proteinase 1 of Aspergillus oryzae
RICHARD O’KENNEDY
Department of’Biochemistry, University College, Belfield, Dublin 4, Ireland
Brinase (proteinase 1 of Aspergillus oryzae) has been used in experimental therapy of
cancer, where it promotes fibrinolysis and also enhances the activity of the cellular
immune system (Thornes et d.,1972; Thornes, 1974). Exposure to brinase in vitro
causes release of sialopeptide from the surface of normal human lymphocytes and also,
but to a much lesser extent, from leukaemic cells (Smyth & O’Kennedy, 1977). The
effects of the enzyme on Landschutz ascites-tumour cells include altered membrane
permeability and growth enhancement (Smyth et al., 1971), loss of small amounts of
cholesterol (Smyth rt al., 1977), increased lysosomal permeability and unmasking of
membrane phospholipid from association with protein (Smyth et al., 1975). The present
communication reports the release of sialic acid in bound form from Landschutz ascites
tumour by brinase in vitro. The isolation and partial characterization of the sialopeptides
released are also described.
Tumour cells were (7-10 days old) were harvested in phosphate-buffered saline
(Hempling, 1958) from the peritoneal cavity of Schofield albino mice and washed three
times in phosphate-buffered saline to remove adherent ascitic-fluid components (Langley
& Ambrose, 1967). The cells (4 x 107/ml)were incubated at 37°C in a shaking-water bath
in phosphate-buffered saline alone or containing0.6pg of Brinase/ml (Astra A.B., Sodertalje, Sweden). Treatment was for 40min, since it was found that no further sialic acid
release occurred after this period. After centrifugation for 8min at lOOOg, the supernatants were deproteinized by precipitation with 5 % (w/v) trichloroacetic acid at 4°C.
The protein precipitates and samples of the protein-free supernatants were hydrolysed
for 1h at 80°C with 0.1 M-HCI. Sialic acid analyses were carried out on these and on
unhydrolysed samples of the protein-free supernatants. The method was that of Warren
(1959), the correction for deoxyribose being applied.
Control incubations in phosphate-buffered saline alone caused considerable loss of
sialic acid. Values for free, peptide- and protein-bound sialic acid (meansfs.E.M. for
seven experiments) were 12.5f2.0, 4.5k1.6 and 27.0+7.7nmol/109 cells respectively.
Innineexperiments incorporatingBrinase, no difference was found in the amounts of free
or protein-bound sialic acid released. However, the mean value for peptide-bound (nontrichloroacetic acid-precipitated) sialic acid was 22.0t 3.5nmol/109 cells, a significant
increase ( P < 0.01) over the relevant control. It is interesting to note that trypsin (6pg/ml)
had almost identical effects. Brinase differs from trypsin in being inactive against lysine
bonds (Bergvist, 1963).
Further examination of the sialopeptide released by Brinase was carried out by using
large-scale incubation mixtures (4 x 109-10 x 109cells). Supernatants from enzyme1977