The Effect of Alkylresorcinol on Lipid Metabolism in Azotobacter

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The Effect of Alkylresorcinol on Lipid Metabolism in Azotobacter chroococcum Joanna Rejmana and Arkadiusz Kozubekb,* a Institute of the Experimental Treatment of Cystic Fibrosis, H. S. Raffaele, 20132 Milano, It- aly b Institute of Biochemistry and Molecular Biology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland. E-mail: [email protected] * Author for correspondence and reprint requests Z. Naturforsch. 59c, 393Ð398 (2004); received November 12, 2003/March 16, 2004 We studied the effect of exogenous alkylresorcinols on the lipid metabolism of Azotobacter chroococcum. We observed that when 5-n-pentadecylresorcinol was present in the growth medium, the more endogenous alkylresorcinols were synthesized. Concurrently, a drop in the amount of phospholipids was observed. These changes were associated with increasing numbers of dormant cysts, while the number of vegetative cells diminished. The chemical nature of the alkylresorcinols synthesized by Azotobacter chroococcum was dependent on the duration of exposure of the bacteria to exogenous alkylresorcinols. When the exposure time was prolonged to four days, 5-n-nonadecylresorcinol (C 19:0) was substituted by 5-n- heneicosylresorcinol (C 21:0) and 5-n-tricosylresorcinol (C 23:0). Two fluorescent membrane probes, NBD-PE and TMA-DPH, further revealed that the presence of alkylresorcinols in the lipid bilayer restrains the phospholipid rotational motion. Key words: Phenolic Lipids, Alkylresorcinols, Azotobacter

Introduction 1971; Kumagai et al., 1971; Yamada et al., 1995). Alkylresorcinols are long-chain homologues of Remarkably, the alkyl chains of bacterial resorcin- orcinol (1,3-dihydroxy-5-methylbenzene). The nat- ols thus far isolated and identified were found to ural occurrence of these compounds or their deriv- be saturated. atives has been demonstrated in several plant fam- The precise physiological role of alkylresorci- ilies: Anacardiaceae, Ginkgoaceae, Gramineae, nols in bacteria remains to be explained. Never- Proteaceae, Myrsinaceae and Leguminosae (re- theless, their occurrence seems to be essential for viewed by Kozubek and Tyman, 1999). The pres- some bacteria. It has been speculated that their ence of alkylresorcinols has also been reported in amphiphilic nature allows them to be incorporated bacteria. The homologue 5-n-nonadecylresorcinol into the cell membrane, thus significantly changing its properties (Su et al., 1981). If vegetative cells (C19) was isolated for the first time from cysts of Azotobacter vinelandii, a gram negative, free-liv- of Azotobacter vinelandii undergo differentiation ing, nitrogen-fixing bacterium that lives in the soil to dormant cysts, their membrane phospholipids (Reusch and Sadoff, 1979, 1981, 1983; Segura et al., are substituted by 5-n-alkylresorcinols and 6-n-al- 2003). Another member of this family, Azotobac- kylpyrones (Reusch and Sadoff, 1981). Since these ter chroococcum, has been reported to be a rich resting cells are far more resistant to various source of alkylresorcinol homologues with side chemical and physical factors than vegetative cells, it is conceivable that they serve as endogenous chains varying from C13 to C27 (Batrakov et al., 1977; Kozubek et al., 1996). Interestingly, these anabiotic compounds (Bitkov et al., 1992; Nenas- compounds have also been isolated from the cul- hev et al., 1989, 1994). This hypothesis could be ture broth of Pseudomonas carboxydoflava (Osi- further supported by the observation that these pov et al., 1985) and Streptomyces (Aoyagi et al., compounds are also secreted into the growth media by several bacterial strains (Osipov et al., 1985; Tsuge et al., 1992). Abbreviations: NBD-PE, N-(7-nitrobenz-2-oxa-1,3-di- The purpose of our work was to examine the azol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoetha- nolamine; TMA-DPH, 1-(4-trimethylammoniumphe- effect of exogenous alkylresorcinols on the synthe- nyl)-6-phenyl-1,3,5-hexatriene-p-toluenesulfonate; MLV, sis of lipids in Azotobacter chroococcum. multilamellar vesicles.

0939Ð5075/2004/0500Ð0393 $ 06.00 ” 2004 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D 394 J. Rejman and A. Kozubek · Alkylresorcinols and Bacterial Lipid Metabolism

Materials and Methods Chromatography Microorganisms Resorcinolic lipids were separated from lipid ex- In our studies we utilized Azotobacter chroococ- tracts by column chromatography (1 ¥ 10 cm) on cum Az 12 strain. It was a kind gift from Stanislaw Silica Gel Si60 (Merck, Darmstadt, Germany) sus- Pietr from the Department of Agricultural Micro- pended in chloroform. The sample dissolved in biology, Agricultural University, Wroclaw. chloroform (1 mg/ml) was applied to the column. Fractions were eluted with chloroform/ethyl ace- Growth of bacteria tate (9:1, v/v). Thin-layer chromatography on Silica Gel Si60 The bacteria were cultivated on Burk’s nitrogen- RP-18 plastic sheets (Merck) was employed to an- free broth supplemented with glucose (1%). Er- alyze the composition of each fraction. To detect lenmeyer flasks (300 ml) containing 100-ml cul- alkylresorcinols, the plates developed in methanol/ tures were placed in a rotary shaker (150 rpm, am- water (95:5, v/v) were sprayed with 0.5% Fast Blue plitude 4 cm) and incubated at 30 ∞C. B ¥ BF4 solution (Chemapol, Prague, Czech Re- public) in 5% acetic acid. Extraction of lipids Bacteria were collected by centrifugation at Liposome preparation 4 ∞C, washed with 0.1 m MgSO4 and subsequently Liposomes were formed by mixing lipids [phos- freeze-dried. Lipids were extracted from the dry phatidylethanolamine and phosphatidylglycerol, material with a chloroform/methanol mixture (2:1, 4:1 (Avanti Polar Lipids, Alabaster, AL, USA)] in v/v). To separate solids from extracts, the suspen- a chloroform/methanol solution with methanol so- sion was centrifuged. The solvent was removed lutions of NBD-PE (Molecular Probes) or TMA- first in a vacuum rotary evaporator then in a vac- DPH (Molecular Probes) (0.5 mol%) and dried uum desiccator. Extracted lipids were dissolved in under a stream of nitrogen gas. The remnants of a chloroform/methanol solution (2:1, v/v) and solvents were removed by additional drying in stored under nitrogen at Ð20 ∞C. vacuo for 2 h. The lipid film was subsequently re- suspended in Tris (Tris-hydroxymethyl-amino- The effect of alkylresorcinols present in growth methane)-HCl buffer (5 ml, 10 mm, pH 7.4; Sigma, medium on lipids synthesis in Azotobacter Poznan, Poland) by intense vortexing, resulting in chroococcum Az 12 the formation of multilamellar vesicles (MLVs). Bacteria were cultivated in the presence of 5-n- This suspension was sonicated in a bath type soni- pentadecylresorcinol (0.5 mg/ml) for 1, 2, 3 and cator until clarity. 4 d. Subsequently, they were centrifuged and suspended in fresh medium without resorcinolic lip- Fluorescence measurements ids. This procedure was repeated five times to Each sample contained Tris-HCl buffer (2.7 ml, make sure that all unabsorbed resorcinols were re- 10 mm, pH 7.4), liposomes (containing 1 mg of moved from medium. The entire time of incuba- phospholipid) and a methanolic solution of all tion was 96 h thus bacteria were further cultivated studied resorcinols. Subsequently, fluorescence for an appropriate number of days (3, 2 or 1). Con- was measured with a spectrofluorimeter (SMF 25, trol bacteria were grown for 96 h in medium with- λ λ Kontron, Milan, Italy). NBD-PE ex 460 nm, em out alkylresorcinols. λ λ 534 nm; TMA-DPH ex 355 nm, em 450 nm. All other chemicals of the best available purity Quantitative determinations were from ALCHEM (Wroclaw, Poland). The amount of resorcinols was determined by the method described by Tluscik et al. (1981). To- Results tal phosphorus amount was determined by the The effect of exogenous alkylresorcinols on lipid method of Rouser et al. (1966). To determine the synthesis in Azotobacter chroococcum Az 12 amount of protein we employed the method of Lowry et al. (1955). To investigate the effect of exogenous alkylresorcinols on lipid synthesis in Azotobacter chroococcum Az 12 the bacteria were cultivated in me- J. Rejman and A. Kozubek · Alkylresorcinols and Bacterial Lipid Metabolism 395 dium containing 5-n-pentadecylresorcinol for 1, 2, creased with extending the time of 5-n-pentade- 3 and 4 d. Subsequently, the cultures were thor- cylresorcinol presence in the incubation broth. The oughly washed and re-suspended in fresh medium largest number of cysts (25%) was determined without resorcinol. when Azotobacter chroococcum cells were ex- The entire time of incubation was 96 h; there- posed to 5-n-pentadecylresorcinol for 4 d. fore the bacteria were further cultivated for an ap- In conclusion, the presence of alkylresorcinols propriate number of days (3, 2 or 1). Control bac- in growth medium significantly influences physio- teria were grown for 96 h in medium without logical activities of Azotobacter chroococcum. The alkylresorcinols. Table I presents the amount of degree of these changes correlates with the time phospholipids and alkylresorcinols in Azotobacter of exposure to these compounds. The drop in the chroococcum Az 12 cells following this treatment. amount of phospholipids is correlated with an The amount of phospholipids per mg of lyophi- increase in the amount of alkylresorcinols synthe- lized bacterial cells decreased with increasing ex- sized and the number of dormant cysts present in posure time to 5-n-pentadecylresorcinol in the culture. growth medium. When this compound was present in the medium for 4 d, the amount of phospholip- Chromatographic analysis of alkylresorcinols ids was reduced by almost 40%, relative to control extracted from Azotobacter chroococcum Az 12 cells. In contrast, the amount of alkylresorcinols increased during the course of the experiment. The chromatographic analysis of alkylresorci- Relative to control cells, the amount of resorcin- nols extracted from Azotobacter chroococcum olic lipids doubled already after 1 d incubation Az 12 is presented in Fig. 1. 5-n-Nonadecylresorci- with 5-n-pentadecylresorcinol present in medium. nol (C 19:0) was the major homologue isolated 4 d of incubation of Azotobacter chroococcum Az from Azotobacter chroococcum cells exposed to 12 in the presence of 5-n-pentadecylresorcinol re- exogenous alkylresorcinol (C 15:0) for 1 and 2 d sulted in a 15-fold increase of the amount of en- (lane 2 and 3). Extending the time of incubation dogenous resorcinols. Interestingly, the amount of to 3 d resulted in the occurrence of alkylresorci- alkylresorcinols increased more strongly than the nols with longer side chains (C 21:0 and C 23:0). amount of phospholipids decreased. However, the most prominent homologue was still The data presented in Table I indicate also that 5-n-nonadecylresorcinol (lane 4). The most pro- the longer the time of incubation with 5-n-penta- nounced change in the character of extracted ho- decylresorcinol, the lower the number of vegeta- mologues was observed while exogenous alkylre- tive bacterial cells in culture will be. When this sorcinols were present in growth medium for 4 d. compound was present in growth medium for 4 d, In these cells the amount of 5-n-nonadecylresorci- the amount of cells decreased by 25%. The nol was significantly reduced, while the presence number of vegetative cells which differentiated to of homologues with longer side chains (C 21:0 and metabolically dormant cysts progressively in- C 23:0) was greatly enhanced.

Table I. The effect of exogenous alkylresorcinols on the number of dormant and vegetative cells and lipid synthesis in Azotobacter chroococcum Az 12. The bacteria were first cultivated in medium containing 5-n-pentadecylresorcinol for 1, 2, 3 or 4 d. After intense washing the cells were re-suspended in fresh medium without resorcinols. The entire time of incubation was 96 h. Therefore the bacteria were further cultivated for an appropriate number of days (3, 2 or 1). Control bacteria were growing for 4 d in medium without alkylresorcinols. Three independent experiments were carried out in duplicate.

Number Percent Cell/cyst Phospholipids Alkylresorcinols Phospholipids/ of bacteria of cysts ratio [µg/mg of cellsa][µg/mg of cellsa] alkylresorcinols (in 1 µl after 96 h) (after 96 h) ratio

Control 600 ð 10 4 ð 1 150 80 ð 44ð 120 1 d 560 ð 15 5 ð 2 112 68 ð 310ð 1 6.8 2 d 500 ð 59ð 15562ð 511ð 2 5.6 4 d 450 ð 10 25 ð 31848ð 160ð 5 0.8 a Dry weight. 396 J. Rejman and A. Kozubek · Alkylresorcinols and Bacterial Lipid Metabolism

Fig. 2. The effect of alkylresorcinols on liposome membrane properties fluorescence intensity. Each sample contained Tris-HCl buffer (2.7 ml, 10 mm, pH 7.4), liposomes (containing 1 mg of phospholipid, preparation as Fig. 1. Chromatographic analysis of 5-n-alkylresorcinols described in Materials and Methods) and a methanolic extracted from Azotobacter chroococcum Az 12. The lip- solution of all studied resorcinols. Subsequently, fluores- ids were extracted from bacteria and separated by col- cence was measured with a spectrofluorimeter. umn chromatography as described in Materials and Methods. Thin-layer chromatography on Silica Gel Si60 RP-18 plastic sheets was employed to analyze the composition of each fraction. The plates were developed in the center of the lipid bilayer parallel to the lipid methanol/water (95:5, v/v) and sprayed with 0.5% Fast acyl chain axis. Its fluorescence polarization is high Blue B solution in 5% acetic acid. in the absence of rotational motion and is very (1) Alkylresorcinols standard mixture; (2) alkylresorcinols extracted from lyophilized Azotobacter chroococ- sensitive to reorientation of the long axis resulting cum after incubation with 5-n-pentadecylresorcinol for from interactions with surrounding lipids. On the 1 d; (3) alkylresorcinols extracted after incubation with other hand, the NBD-PE localizes in the head- 5-n-pentadecylresorcinol for 2 d; (4) alkylresorcinols ex- group region of the lipid bilayer, therefore it is tracted after incubation with 5-n-pentadecylresorcinol for 3 d; (5) alkylresorcinols extracted after incubation suitable to monitor changes in this region of the with 5-n-pentadecylresorcinol for 4 d. plasma membrane. To mimic the natural phos- The chromatographic pattern of alkylresorcinols ex- pholipid composition of prokaryotic organisms tracted from the lyophilized control bacteria was iden- (Reusch and Sadoff, 1979, 1981, 1983; Segura et al., tical to that presented for Azotobacter chroococcum incubated with 5-n-pentadecylresorcinol for 1 d (lane 2). 2003; Su et al., 1981), liposomes were formed from The intensity of the spots visualized with Fast Blue B a mixture of phosphatidylethanolamine and phos- (reacting with the ring structure) reflects the relative phatidylglycerol (4:1, v/v). Fig. 2 shows the amount of each homologue in the sample. changes in fluorescence intensity caused by the presence of alkylresorcinols in the lipid bilayer. The data indicate that these changes, for both ho- These results demonstrate that 5-n-pentadecyl- mologues, are most pronounced in the region resorcinol added to media promotes the synthesis monitored by TMA-DPH, in the center of the of endogenous alkylresorcinols. The alkyl chain plasma membrane. Although, 5-n-heneicosylresor- length of the newly synthesized homologues cinol had a stronger effect on lipid order in that increased with increasing time of incubation of the plasma membrane region. cells with 5-n-pentadecylresorcinol. These results are further supported by the results of fluorescence polarization of TMA-DPH The effect of alkylresorcinols on liposome and NBD-PE studies as a function of 5-n-heneico- membrane properties sylresorcinol concentration, indicating a decrease It has been suggested that alkylresorcinols may of phospholipids rotational motion in both bilayer incorporate into the plasma membrane and conse- regions (data not shown). quently change its properties (Bitkov et al., 1990, 1992; Kozubek et al., 1988, 1992). To examine Discussion this possibility, we employed fluorescent probes Our data reveal that exogenous resorcinols (NBD-PE and TMA-DPH) incorporated into li- strongly influence the lipid metabolism of Azoto- posomes containing alkylresorcinols (5-n-pentade- bacter chroococcum. Apparently, the exogenous cylresorcinol or 5-n-heneicosylresorcinol, 15 µm). alkylresorcinols cause metabolic changes in Azo- It is generally assumed that TMA-DPH resides in tobacter chroococcum cells, which cause the inhibi- J. Rejman and A. Kozubek · Alkylresorcinols and Bacterial Lipid Metabolism 397 tion of phospholipid synthesis. As a result, the bac- this process. Each additional -CH2- increases the teria stop dividing and switch to the synthesis of energy of interaction between neighbouring acyl longer alkylresorcinol homologues that subse- chains by approx. 2.1 kJ/mol. quently switch on the process of encystment. Re- On the other hand, gradual introduction of ex- cent data presented by Segura et al. (2003) indicat- ogenous alkylresorcinols into the plasma mem- ing that in Azotobacter vinelandii strains impaired brane causes changes in membrane fluidity that in poly-beta-hydroxybutyrate synthesis the pro- may restrict the functioning of membrane-associ- duction of alkylresorcinols is significally enhanced. ated proteins (Kozubek et al., 1992). This may in- As the common metabolic source for both beta- clude enzymes involved in the synthesis of bacte- hydroxybutyrate synthesis (one of the ketone bod- rial phospholipids, which are also associated with ies) and of alylresorcinols is the acetyl-CoA the the plasma membrane (Bell et al., 1971; Cronan data may suggest the feedback and the regulatory and Vagelos, 1972; Tsuge et al., 1992). This inacti- activities of 5-n-alkylresorcinols in the process of vation restrains the synthesis of phospholipids, re- encystment. For this purpose the cells may use two quired for generating new cells. They will stop di- alternative pathways. In the first one most of phos- viding and start to differentiate into metabolically pholipid’s and glyceride’s acyl chains are degraded dormant cysts. This process in bacteria from genus completely to acetyl-CoA from which, conse- Azotobacter is combined with excessive synthesis quently, both polyhydroxybutyrate and alkylresor- of alkylresorcinols. During the process of encyst- cinols are synthesised de novo. In an alternative ment, the length of the side chain of the synthe- pathway, already suggested earlier (Kozubek and sized alkylresorcinols increases, rendering the Tyman, 1999) and confirmed in the recent work of plasma membrane more rigid and resistant to vari- Suzuki et al. (2003), only a part of the acyl chains ous physical and chemical agents. Recently Segura is degraded and used for the synthesis of the 1,3- et al. (2003) demonstrated that synthesis of alkyl- dihydoxybenzene ring structure. The remaining resorcinols in Azotobacter vinelandii involves uti- chains are attached to these rings leading to the lisation of the same acetyl-CoA pool as those used formation of the 5-n-alkylresorcinol structure. Our for the synthesis of polyhydroxybutyrate (PHB). data further demonstrate that alkylresorcinols re- The inhibitory properties of other long-chain al- strain the rotational motion of the phospholipids kylresorcinol homologues against triglyceride syn- in the membrane bilayer that may result from the thesis has been also shown for animal adipocytes interaction between both types of the molecules, (Rejman and Kozubek, 2004). These data together as suggested previously for nonbacterial (liposo- with our observations suggest that alkylresorcinols mal) and bacterial membranes (Bitkov et al., 1992; may act as a trigger of the bacterial metabolism Kozubek et al., 1988). The introduction of resor- from the vegetative state to the formation of cysts. cinols with one saturated alkyl chain and a rela- tively large head group, able to form hydrogen Acknowledgement bonds, is an efficient way to reduce membrane fluidity. The synthesis of alkylresorcinols with longer The work was supported by the University of side chains (observed after 4 d) is the next step in Wroclaw research grants. 398 J. Rejman and A. Kozubek · Alkylresorcinols and Bacterial Lipid Metabolism

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