Biochimica et Biophysics Acta, 753 (1983) 266-268 Elsevier

266

BBA Report

BBA 50051

ALKYLAMINES: NOVEL LIPID CONSTITUENTS IN DEZNOCOCCUS RADZOD URA NS

ROBERT ANDERSON

Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5CI (Canada)

(Received March lst, 1983)

(Revised manuscript received June 30th, 1983)

Key words: Alkylamine; Lipid composition; (Deinococcus radiodurans)

The major plasma membrane phosphoglycolipid of Deinococcus radioduruns contains a series of alkylamines as structural constituents. Gas chromatographic-mass spectrometric analysis of the N-acetylated alkylamines isolated from this lipid demonstrated that the major alkylamines are saturated C-15, C-16, C-17 and monounsaturated C-16, C-17 and C-18 species.

The reclassification of the formerly named Mi- crococcus radiodurans into the genus Deinococci (Greek, deinos = strange) was recently proposed on the basis of several distinguishing characteris-

tics, including complex cell wall structure, fatty

acid composition and radiation resistance [l]. Re-

cent studies have noted the presence of novel

membrane lipids in D. rudioduruns [2,3], the char-

acterization of which is of taxonomic interest as

well as of potential relevance to the understanding of the role of cell membranes in radiation protec-

tion. The major plasma membrane lipid compo-

nent [4] of D. rudioduruns is a phosphoglycolipid

(designated lipid 7 in Ref. 2). The present study

documents the characterization ,of a series of al- kylamines, hitherto unknown as lipid constituents,

derived from this major phosphoglycolipid.

Lipid extracts, prepared as previously described [2] from cultures of D. radiodurans, were subjected to thin-layer chromatography on silica gel H in chloroform/methanol/28% ammonia (65 : 35 : 5, v/v) for preparative isolation of lipid 7. The iso- lated lipid was hydrolyzed in aqueous methanolic- HCl [5] for 16 h at 70°C and, following evapora- tion of the solvent, the hydrolysis products were analyzed by thin-layer chromatography in chloro- form/methanol/water (65 : 25 : 4, v/v). In addi-

tion to fatty acid methyl esters and free fatty acids,

a ninhydrin-positive compound, ‘compound A’,

was observed, which had an R, of 0.27 as com-

pared with an R, of 0.23 for standard sphingosine. Infrared spectroscopy in KBr of compound A

showed major absorbancies at 3400-3500 cm-’

(broad; N-H stretching), 1580 cm-’ (N-H bending

of primary amine), 2860 and 2915 cm- ’ (C-H stretching), and 1400 and 1475 cm-’ (C-H bend-

ing). It was, accordingly, concluded that com-

pound A was an alkylamine or a mixture of al-

kylamines. In order to exclude the possibility that

compound A contained any hydroxyl groups,

aliquots of compound A were treated with acetic

anhydride in pyridine [6] (to acetylate any free

amino and hydroxyl groups) or with acetic

anhydride in methanol [5] (to acetylate only amino

groups). Either treatment yielded the same prod- uct (RF 0.85 in chloroform/methanol/28% am- monia (90: 10: 1, v/v)), demonstrating the ab- sence of hydroxyl groups on compound A.

Capillary gas liquid chromatography of the N- acetylated compound A (prepared with acetic anhydride in methanol) showed a series of compo- nents (Fig. 1). A standard, N-acetyl-l-hexade- cylamine (prepared by acetylation of authentic I-hexadecylamine (Aldrich)) had an identical re-

0005-2760/83/$03.00 0 1983 Elsevier Science Publishers B.V.

267

h

h

Retention Time

Fig. 1. Capillary gas liquid chromatogram of N-acetylated alkylamines (N-acetyl-compound A) from D. rudioduruns. A Hewlett-Packard

5880A gas chromatograph equipped with a capillary OV-1 column was used with programmed temperature gradient (40-190°C at

20”C/min, followed by a rise to 210°C at 2T/min).

Fig. 2. Mass spectral comparison of N-acetyl-1-hexadecylamine and the designated N-acetyl-16 : 0-alkylamine from D. radiodurans. Combined gas chromatography-mass spectrometry was performed using a Hewlett-Packard 5985 quadrupole mass spectrometer

coupled to a Hewlett-Packard 5840A gas chromatograph. A, Mass spectrum of N-acetyl-16 : 0-alkylamine (designated in Fig. 1) from

D. radioduruns; C, mass spectrum of standard N-acetyl-I-hexadecylamine; B, computed mass spectral difference comparison of A and

C.

tention time to that of one of the component peaks

in Fig. 1. This component peak was accordingly

designated 16 : 0 (Fig. 1). Comparative mass spec-

trometry of this component and the standard N-

acetyl-1-hexadecylamine showed virtual identity

(Fig. 2).

Mass spectra of the component peaks desig- nated 15 : 0, 16 : 0 and 17 :0 (Fig. 1) were con-

sistent with those expected for a normal N-acetyl

alkylamine series comprising 15, 16 and 17 carbon lengths, respectively; corresponding parent mass

peaks were detected at m/z 269, 283 and 297,

respectively. The mass spectra of the component peaks designated 16 : 1 and 17 : 1 were similar to those obtained for the corresponding saturated

species, except that all prominent signals at m/z 170 and above were shifted down 2 mass units. The two peaks designated 18 : 1 were so identified on the basis of their mass spectra, including a parent mass peak at m/z 309. Confirmation of the unsaturated nature of the peaks designated 16 : 1, 17 : 1 and 18 : 1 was obtained by the observation

that they disappeared after treatment of N-acetyl-

compound A with periodate-permanganate [7].

None of the saturated peaks, as designated in Fig.

1, was affected by the periodate-permanganate

procedure. Monounsaturated species, particularly

17 : 1 and 18 : 1, appeared to occur as two pre-

dominant components (Fig. l), presumably repre-

senting different locations of the double bond. A small proportion of the saturated 17 : 0 species

appeared to be a branched molecule (designated

17 : 0 br in Fig. 1) as judged by its short retention time, mass spectrum, including parent mass peak

at m/z 297, and its resistance to periodate-per-

manganate.

To the author’s knowledge, alkylamines of the

type reported here for D. radiodurans have not

been previously identified. Structurally, the closest analog to the alkylamines described here are the sphingosines, varieties of which have been dis- covered in both eukaryotic and prokaryotic organisms. Nevertheless, the occurrence of sphin- golipids in bacteria is rare. Among the best known

268

examples of bacterial sphingolipids occur in the Bacteriodes [8,9] although sphingolipids have also

been detected in Bdellovibrio [lo], Flavobacteria [l l] and Mycopfasma [12]. Virtually all sphingo-

sines characterized to date contain a 1,3-dihy-

droxy-2-amino-alkyl chain which may be linear,

branched or unsaturated. The alkylamines of D. rudioduruns are, thus, structurally distinct from

previously described sphingosines, primarily with

respect to their lack of hydroxyl groups. The major

phosphoglycolipid of D. radiodurans, from which

the alkylamines were isolated, shows infrared evi-

dence of an amide linkage (unpublished data).

This suggests that, as is the case with sphingosine,

the alkylamines of D. rudiodurans form more com-

plex lipid structures via acylation of the amino

group. Alkylamines apparently do not occur in the

‘free’ form in D. radiodurans, since analysis of

total lipid extracts [2] failed to detect any free

amino group-containing lipids. Further investiga-

tions will be necessary to determine whether such

alkylamines may be considered functional analogs

of sphingosine.

The work described here was supported by a

grant from the Medical Research Council of

Canada. I thank Dr. M.W. Khalil and the MRC

Group in Reproductive Biology for the use of the

capillary gas chromatograph. I am also grateful to

Mr. L. Marai for performing the coupled gas

chromatography-mass spectrometry.

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