Isolation, Purification and Characterization of Aurovertin B

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*Department of Biochemistry, Chelsea College, University of London, Manresa Road,. London S W3 6LX, U.K., and Biochemistry and Physiology Division, ...

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tubular intracristal spaces. Mitochondria under anaerobic conditions appeared similar to those in state 4. The rapidity of these changes on configuration has been investigated by rapid sampling. These changes are complete within lmin of bringing the mitochondria from state 4 to state 3 by ADP addition. Samples taken 50s after the change in state were identical with those withdrawn after 4min having distinct tubular cristae. These results show that, although different from beef heart mitochondria, turnip mitochondria do undergo configurational changes in a similar manner during changes in their metabolic states. Hackenbrock, C . R. (1966) J. Cell Biol.30,269-297

Isolation, Purification and Characterization of Aurovertin B M. D. OSSELTON,* H. BAUM* and R. B. BEECHEY *Department of Biochemistry, Chelsea College, University of London, Manresa Road, London S W3 6LX, U.K., and Biochemistry and Physiology Division, Woodstock Research Centre, Shell Research Ltd., Sittingbourne, Kent ME9 8AG, U.K. The aurovertins are synthesized by the fungus imperfectus Calcarisporium arbuscula (Preuss strain). The aurovertin described by Baldwin et al. (1964) is here called aurovertin D. It was shown by Lardy et al. (1964) to be a potent inhibitor of mitochondria1 ATP synthesis and ATPases (adenosine triphosphatases) (membrane-bound and soluble). R. B. Beechey (unpublished observations) and Bertina (1972) simultaneously and independently observed that C. arbuscula produces a number of substances possessing properties similar to those of aurovertin D. Thus :(a)ethanolic solutions have absorption maxima at 367-368nm, 355nm, 273-274nm and 267nm; (b) they fluoresce yellow-green when irradiated by light of wavelength 350-360nm; and (c)they react with 50 % (v/v) aq. H2S04to form purple-pink-coloured complexes after heating at 100°C for 5 min. The present communication describes the isolation, purification and recrystallization of aurovertin B. These are relatively easy procedures, and should enable individual workers to satisfy their own experimental requirements for aurovertin. Growth conditions C . arbuscula was obtained from the United States Department of Agriculture and stored on Czapek Dox agar slopes, pH 6.8, at 4°C. The culture medium F.14 (Baldwin et al., 1964) was employed for large-scale culture at pH 6.2,26"C and in darkness for 20 days. Preparation of crude extracts containing aurovertin

The extraction and purification procedures were performed under subdued light and at no stage was the temperature allowed to rise above 30°C. The mycelium was first separated from the growth medium. Extraction of thegrowth medium. The growth medium was filtered through glass-wool and then extracted with chloroform by continuous stirring at 4°C overnight (100ml of chloroform/250ml of growth medium). The chloroform extract was dried over anhydrous Na2S04,then after filtration the chloroform was removed in uacuo at 20°C to yield a deep yellow oil. Extraction of mycelium. The mycelium was chopped into pieces and washed twice with distilled water to remove slime, and was then extracted with acetone (1 litre/100g of tissue) by continuous stirring overnight at 4°C. The procedure described by Baldwin et al. (1964) was then followed to the stage of removal of the chloroform by distillation 1974

544th MEETING, LONDON

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Aurovertin A Aurovertin A, Aurovertin A2 Aurovertin B Aurovertin C Aurovertin D

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@ Origin W

Aurovertin E Aurovertin Aurovertin Aurovertin Aurovertin

F G H

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Fig. 1. T.I.c. of a mycelium extract of C. arbuscula The, silica-gel plate (layer thickness 0.2mm; Merck no. 5715) was developed with benzene-ethyl acetate (3 :7, v/v) at 20°C. Spots were located by spraying the plate with 50% (v/v) aq. HZSO4 followed by 5min heat at 100°C. Aurovertins appeared as pinkpurple spots, indicated by shading. Other materials on the plate stained either grey or yellow or brown. T.1.c. of aurovertins isolated from the growth medium exhibits the same pattern as the mycelium extract shown above. The nomenclature of the aurovertins was agreed with Dr. R. M. Bertha.

under reduced pressure. The resultant yellow oil was shown by t.1.c. to possess constituents similar to those in the medium extract (see Fig. 1). Isolation and purification of aurovertin B

Aurovertin B was purified from the combined crude extracts by either preparative t.1.c. or high-resolution liquid chromatography. Preparative t.1.c. was performed on 20cni x 20cm silica-gel 6OF254 plates (Merck no. 5715). Plates were developed in benzene-ethyl acetate (3 :7, v/v). Compounds were located by irradiating the developed plates with light of 350nm. Aurovertin B (RFapprox. 0.5) was the major component of the mixture and was eluted from the silica gel with methanol. The eluates were concentrated and rechromatographed by using the same solvent system. Aurovertin B was eluted from the plates, the solvent being evaporated before crystallization (see below). High-resolution liquid chromatography was performed on a system similar to that described by Dane et al. (1972) except that (a)warm solvent was not passed through the system and (b) the filter, saturator and thermostat were not included in our system. Two columns (1 m x 10mm) were used: (1) packed with Porasil A(60) equilibrated with dichloromethane [pressure at the top of the column 207kPa (301b/in2); initial flow rate 3ml/min). Constituents were eluted with a stepwise gradient, 0.2, 0.5, 0.7, 1.0, 1.3 and 1.5 % (v/v) methanol in dichloromethane. Fractions eluted by 0.7-1.5 % methanol were Vol. 2

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pooled, the solvents were evaporated under a stream of 02-free N3 and applied to the second column (silica gel H-elite 545) equilibrated with dichloromethane. The operating pressure at the column head was 1449-1484kPa (210-2151b/in2) and the initial flow rate 7.5ml/min. The constituents were eluted as described above. Aurovertin B was eluted from this column by 0.5 % methanol in dichloromethane. Fractions containing the aurovertin B peak (shown by t.1.c.) were pooled and concentrated as before. The aurovertin B obtained by either purification procedure was dissolved in a small volume of warm acetone and the solution was ultrafiltered. The solution was cooled and crystals of aurovertin B were obtained. Aurovertin B was recrystallized to m.p. 164166.5"C and stored at -20°C. Measurements of the accurate molecular weight of aurovertin B indicate a molecular formula of C2&208 (cf. C2sH3209 for aurovertin D; Beechey et al., 1967). The Lr., U.V. and proton-magnetic-resonance spectra of aurovertin B are qualitatively similar to those of aurovertin D. The ability of aurovertin B to inhibit ATP synthesis and the mitochondria1 ATPase appears to be the same as for aurovertin D. We acknowledge the help of Mr. D. T. Green, Milstead Laboratory, and Dr. F. Cottee, Burroughs Wellcome Research, Beckenham, Kent, for making mass-spectrum measurements, and Dr. L. J. Mulheirn, Milstead Laboratory, Shell Research Ltd., for his help in recrystallizing aurovertin B. M. D. 0. acknowledges financial support from the Science Research Council. Baldwin, C. L., Weaver, L. C.,Brooker, R. M., Jacobsen,T.N., Osborne, C.E., Jr. &Nash,H. A. (1964) Lloydia 27 (2), 88-95 Beechey, R. B., Williams, V., Holloway, C. T., Knight, I. G. & Roberton, A. M. (1967)Biochem. Biophys. Res. Commun.26, 339-341 Bertina, R. M. (1972) Ph.D. Thesis, University of Amsterdam, p. 18 Dane, J. N., Kennedy, G . J. & Knox, J. H. (1972) Nature (London) 237,77-81 Lardy, H. A., Connelly, J. L. &Johnson, D. (1964) Biochemistry 3,1961-1968

The Isolation and Properties of Venturicidin A P. LANGCAKE, R. B. BEECHEY, C. R. LINDOP, S. G. A. WICKINS, D. P. LEWORTHY, D. E. WIGGINS and J. M. BROUGHALL* Biochemistry and Physiology Division, Shell Research Ltd., Woodstock Agricultural Research Centre, Sittingbourne, Kent ME9 8AG, U.K. Venturicidin was originally isolated by Rhodes et al. (1961). Walter et al. (1967) showed that this antibiotic inhibited mitochondrial ATP synthesis. We shall present evidence that venturicidin A acts differently from oligomycin, another inhibitor of ATP synthesis. Because of potential interest to studies of oxidative phosphorylation, a convenient laboratory-scale method for the preparation of venturicidin A is described. Isolation The organism used for production of venturicidin was a strain of Streptomyces griseolus isolated as a laboratory contaminant which had been noted for its strong anti-fungal properties. Piricularia oryzae was the test organism in two types of bioassay used during the isolation: (a)a rapid semi-quantitative disc assay similar to that used by Stillwell et al. (1973); and (b) a quantitative bioassay involving measurement of the effects of test material on growth of the fungus in liquid medium, the dose-response curves from which enabled EDSovalues to be estimated. For the production of antibiotic, S. griseolus was grown for 48 h in shake culture in a 2 % (w/v) glucose-1 % (w/v) peptone salts medium at 28°C. After incubation, e l i t e (25g/l of culture) was added, mixed and the culture centrifuged (lOOOg, 20min, 5°C). Anti-fungal activity was extracted from the supernatant with ethyl acetate and from the mycelium-celite pellets

* CAPS student. 1974

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