Download Iron-Binding Compounds Formed by Micrococcus denitrificans

547th MEETING, LONDON
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Iron-Binding Compounds Formed by Micrococcus denitri9cans
G. H. TAIT
Department of Chemical Pathology, St. Mary's Hospital Medical School,
London W2 l P C , U.K.
2,3-Dihydroxybenzoic acid and a number of compounds containing it are excreted by
some micro-organisms grown in media deficient in iron (Neilands, 1972). In this report
it is shown that Micrococcus denitrificans grown in media deficient in iron excretes 2,3dihydroxybenzoic acid and two previously undescribed compounds containing 2,3dihydroxybenzoic acid and spermidine.
When M. denitri$caris is grown anaerobically in a defined nitrate medium deficient
in iron or containing high concentrations of calcium and copper, or aerobically in a defined ammonia medium deficient in iron (Tait, 1973), compounds which chelate iron
accumulate in the medium. Three compounds have been isolated which contain catechol
residues as shown by their reaction with Arnow's nitrite-molybdate reagent (cf. Corbin
& Bulen, 1969). Two of the compounds were extracted into ethyl acetate from medium
acidified to pH2. On extracting the ethyl acetate with 1 . 2 ~ - N a H C o one
~ , of them
(compound I) was extracted into the aqueous phase; the other (compound 111) remained
in the organic phase. These compounds were purified by chromatography on silicic acid.
Compound I was crystallized from hot water and compound 111was obtained as a white
powder by adding a concentrated solution in ethanol to water and freeze-drying the
suspension. The acidified medium from an iron-deficient aerobic culture, after extraction with ethyl acetate, was freeze-dried. The solid was extracted with ethanol, the
ethanolic solution was dried, and the residue was dissolved in water. Compound I1 was
purified by chromatography on Zeokarb 225 [H+]and then on CM-cellulose.
Compound I was found to be 2,3-dihydroxybenzoic acid. The melting point, infra-red
spectrum, ultra violet spectra in aqueous solutions of different pH values and in organic
solvents, and the chromatographic behaviour on paper in a number of solvents were
identical with those of an authentic sample.
Compound I1 contained 2,3-dihydroxybenzoic acid and spermidine in a molar ratio
of 2/1. It did not react with ninhydrin, showing that both primary amine groups of
spermidine were blocked. It reacted with fluorodinitrobenzene, and from the spectrum
of the product (Dubin, 1960) it was shown that the secondary amine group of spermidine
was free. It is concluded that compound I1 is N'N8-bis-(2,3-dihydroxybenzoyl)spermidine.
Compound I11 on acid hydrolysis yielded 2,3-dihydroxybenzoic acid, 2-hydroxybenzoicacid, L-threonineand spermidine in a molar ratio of 2 :1 : 1 : 1. Partial acid hydrolysis
yielded a dipeptide containing threonine and spermidine. The spectrum of the product
after reaction with fluorodinitrobenzene showed that the dipeptide did not have a free
secondary amine group, and thus it is probable that threonine is linked by its carboxyl
group to the secondary amine group of spermidine. On the basis of the enzymic experiments described below, and from the structure assigned to compound 11, it is probable
that compound I11 is 2-hydroxybenzoyl-N-~-threonyl-N~[N'N~-bis-(2,3-dihydroxybenzoy1)lspermidine. This compound forms a red equimolar complex with Fe3+.
A concentrated suspension of iron-deficient M . denitrgcans was disrupted by ultrasonication and centrifuged at 25000g for 30min. The clear supernatant, after dialysis,
was used as enzyme. The assay mixture contained Tris-HC1 buffer, pH8.8, MgCI2,
dithiothreitol, ATP, enzyme and, where added, L-threonine, spermidine, 2,3-dihydroxybenzoic acid and 2-hydroxybenzoic acid. [14C]Threonine or ['4C]spermidine was also
added. After incubation HCI and ethyl acetate were added. After thorough mixing the
ethyl acetate layer was removed. Portions of the ethyl acetate were taken for measurement of radioactivity and for paper chromatography.
On incubation of [14C]spermidine and 2,3-dihydroxybenzoic acid, with or without
threonine, a radioactive compound with the chromatographic properties of compound
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BIOCHEMICAL SOCIETY TRANSACTIONS
I1 was formed. No radioactivity was extracted into ethyl acetate in the absence of 2,3dihydroxybenzoic acid, or when 2-hydroxybenzoic acid was used in its place. A radioactive compound with the chromatographic properties of compound 111 was formed on
incubation of [14C]spermidine, threonine, 2,3-dihydroxybenzoic acid and 2-hydroxybenzoic acid.
On incubation of [‘4C]threonine, spermidine, 2,3-dihydroxybenzoic acid and 2hydroxybenzoic acid radioactive compound I11 was formed. In the presence of only one
of the aromatic acids only small amounts of compounds containing [14C]threoninewere
extracted into ethyl acetate.
When compound I1 was incubated with [14C]threonine and 2-hydroxybenzoic acid
it was converted into radioactive compound 111. Incubation of compound I1 with
[‘4C]threonine and 2,3-dihydroxybenzoic acid resulted in the formation of only a small
amount of a radioactive compound with the chromatographic properties of compound
111.
I thank Mrs. C. Eggleton for her excellent technical assistance and Dr. R. C. Davies for
helpful advice and discussions.
Corbin, J. L. & Bulen, W. A. (1969) Biochemistry 8,757-762
Dubin, D. T. (1960) J. Biol. Chem. 235 783-786
Neilands, J. B. (1972) Struct. Bonding 11, 145-170
Tait, G. H. (1973) Biochem. J. 131, 389-403
Effect of Growth Conditions and Dibntyryl Adenosine
3’:5’-Cyclic Monophosphate Treatment on L-Glutamate and L-Alanine
Transport in BMK28-CB3 Cells
DOUGLAS M. SCOTT and JOHN A. PATEMAN
Institute of Genetics, University of Glasgow, Glasgow GI 1 5JS, U.K.
Intracellular concentrations of naturally occurring amino acids and their analogues
have been shown to influence the inward transport of those molecules in bacterial, fungal
and certain mammalian cells either by transinhibition (Ring & Heinz, 1966; Belkhode
& Scholefield, 1969; Pall, 1971) or by control mechanisms effective on protein synthesis
(Pall, 1971 ;Phang et al., 1971). Treatment of tissue slices or cells in culture with various
hormones alters amino acid transport rates (Krawitt et al., 1970; Baril et al., 1969).
Receptors of hormones of the ‘primary messenger class’ (Robinson et al., 1971) are
closely related to and may be part of an adenyl cyclase system, and act to alter intracellular concentrations of the ‘secondary messenger’ cyclic AMP. Reports have claimed
that cyclic AMP or its dibutyryl derivative can alter the morphology of certain cell lines
(Hsie &Puck, 1971;Hsie et al., 1971; Sheppard, 1971), affect growth rates of certain cell
lines (Heidrick & Ryan, 1970; Blat et al., 1973), and alter the transport activities of
various metabolites e.g. nucleotides (Hauschka et al., 1972; Kram et al., 1973), sugars
(Kram & Tomkins, 1973) and phosphate (Blat et al., 1973).
The present paper describes the effect on L-alanine and L-glutamate uptake after the
following treatments (a), growth in the presence of certain amino acids (b), treatments
that are reported to alter intracellular cyclic AMP concentrations or (c), treatment of
the cells with dibutyryl cyclic AMP.
L-Glutamate and L-alanine transport were studied by determining the incorporation
of the appropriate labelled amino acid into cells grown in monolayers on coverslips or in
petri dishes. Before transport studies the cells were grown in Glasgow’s modified Eagles
medium plus 10% (v/v) or 0.5% (v/v) calf serum, with the addition in certain experiments of dibutyryl cyclic AMP (0.2-2m~)with or without theophylline (1 mM), or by
theophylline (1 mM) alone. In other experiments the medium was supplemented with
1974