Presence of Cycloheximide Demonstrates Gluconeogenic - Journal of ...

JOURNAL OF BACTERIOLOGY, June 1983, p. 1472-1475

Vol. 154, No. 3

0021-9193/83/061472-04$02.00/0 Copyright C 1983, American Society for Microbiology

Incorporation of Radioactivity from Amino Acids in the Presence of Cycloheximide Demonstrates Gluconeogenic Activity in Mucor rouxii SURAIA SAID AND HtCTOR F. TERENZI* de Universidade Sdo Paulo, Faculdade de Medicina de Ribeirao Preto, Departamentos de Fisiologia e Bioquimica, Sdo Paulo, Brazil Received 27 September 1982/Accepted 10 March 1983

Mucor rouxii cells induced for gluconeogenesis incorporated radioactivity from [14C]glutamic acid into trichloroacetic acid-precipitable material in the presence of 200 ,ug of cycloheximide per ml. This metabolic capacity was repressed by hexoses and required amino acids for induction. These results suggest that the incorporation of amino acids in the presence of cycloheximide represents gluconeogenic activity with associated polysaccharide synthesis.

Previous work from this laboratory (10) demonstrated that cycloheximide increased severalfold the level of glycogen in Mucor rouxii mycelium incubated in a medium supplemented with a mixture of yeast extract and peptone as the source of carbon. This effect of cycloheximide required gluconeogenic activity and was not observed when the mycelium was grown under glucose-repressing conditions. Probably, the mechanism for this lies in the inhibition of protein synthesis by cycloheximide, thus increasing the endogenous amino acid pool, which was utilized for sugar synthesis by the gluconeogenic enzymes. In view of this observation, we suggested that kinetic measurements of polysaccharide synthesis in the presence of cycloheximide, utilizing gluconeogenic substrates, could give valuable information about gluconeogenic activity in the intact cell system. In this paper, we give measurements of the rate of incorporation of radioactivity from glutamic acid, in the presence of cycloheximide, into cold trichloroacetic acid (TCA)-precipitable material of M. rouxii germlings. The results suggest that this incorporation of label is evidence of gluconeogenic activity. (This paper is part of a thesis submitted by S.S. to the Department of Physiology of the School of Medicine of Ribeirao Preto, University of Sao Paulo, in partial fulfillment of the requirements for the Ph.D. degree.) M. rouxii NRRL 1894, a strain originally from C. W. Hesseltine (Northern Utilization Research and Development Division, Peoria, Ill.), was kindly provided by Roger Storck (Rice University, Houston, Tex.). Spores were produced and stored as described by Haidle and Storck (5). The basal saline solution described by Bartnicki-Garcia and Nickerson (1) was used

as the culture medium for all the experiments and was supplemented with Casamino Acids (Difco Laboratories, Detroit, Mich.) (0.2%), Casamino Acids and glucose (2%), or glutamic acid (5 mM) as the carbon source. A culture procedure, to be described in detail elsewhere, permitted us to obtain a homogeneous population of germlings growing exponentially. Briefly, this method consisted of two steps. (i) Spores were inoculated in liquid medium at a concentration of 107 spores per ml (when Casamino Acids was the sole carbon source) or 3 x 107 spores per ml (when glucose was also added). These cultures were incubated overnight at 30°C with mechanical agitation. Under these conditions, probably due to the high inoculum, development stopped at an early stage, and the cultures appeared to be a homogeneous population of cells exhibiting short (less than one diameter in size) germ tubes. (ii) Cells from cultures prepared as described above were har-

vested, rinsed, and resuspended in fresh medium, supplemented as indicated for each experiment, at a concentration of 5 x 106 cells per ml. These cells resumed growth immediately, and during the next 3 to 4 h, they grew at a constant exponential rate. All experiments were performed in this phase of development. After dilution into fresh medium, the germlings were incubated for 60 to 90 min. The exponentially growing culture was then harvested and suspended in an equal volume of saline without a carbon source containing 50 ,g of cycloheximide per ml. After 10 min, a solution containing 75 ,umol of L-[Ul4C]glutamic acid (specific activity, 0.0125 mCi/mmol) was added, and the cultures were maintained at 30°C with agitation. The final volume was 15 ml. Samples (1 ml each) were withdrawn at 10-min intervals

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FIG. 1. Effect of cycloheximide on the incorporation of radioactivity from glutamic acid in M. rouxii cells germinated in medium supplemented with 0.2% Casamino Acids (0) or 0.2% Casamino Acids and 2.0% glucose (0) as the carbon source. The rate of incorporation of radioactivity into cold TCA-precipitable material was determined as described in the text. The experimental points represent the percentage of the rate of incorporation of radioactivity in cycloheximide-treated cells as compared to that of cells without cycloheximide.

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during the next hour and precipitated in cold 10% TCA. These samples were collected on glass fiber filters (Whatman 934 AH), washed with cold 5% TCA containing an excess of unlabeled amino acid, dried, and counted in toluene-based scintillation fluid. The rate of incorporation of radioactive glutamic acid was expressed as micromoles per minute per milligram of protein. Protein was measured by the method of Lowry et al. (8) with bovine serum albumin as a standard. Cycloheximide was purchased from Sigma Chemical Co., St. Louis, Mo. L-U-14Camino acids were products of New England Nuclear Corp., Boston, Mass. All other chemicals were of the highest purity available. Cycloheximide is a potent inhibitor of incorporation of radioactivity from amino acids into TCA-precipitable material in M. rouxii (6) as well as in other cell systems. However, M. rouxii cells which had germinated in medium supplemented with amino acids as the sole source of carbon could incorporate radioactivity from glutamic acid into cold TCA-precipitable material even in the presence of an elevated concentration of cycloheximide (Fig. 1). The amount of label incorporated into the TCA precipitate in the presence of 200 p.g of antibiotic per ml was about 85% that of the control without cycloheximide. On the other hand, the incorporation of radioactivity was efficiently blocked with a low (10 ,ug/ml) dose of cycloheximide in cells which had been germinated in the presence of glucose. Similar results were obtained when other radioactive amino acids were utilized. For in-

TABLE 1. Distribution of radioactivity incorporated from [14C]glutamic acid in the presence or absence of cycloheximide into different cell fractionsa [14C]glutamic acid incorporated (,umol/mg of protein) Cycloheximide-treated cultures Fraction Control

Amino acid-grown

Glucose-grown cells

cells

Soluble in acetone-ethyl ether Fraction I

0.09 (3.9) 0.09 (4.2) 0.10 1.31 (56.7) 0.58 (26.8) 0.06 Fraction II 0.78 (33.8) 1.40 (64.8) 0.02 Final residue 0.13 (5.6) 0.09 (4.2) 0.02 a Cells (10 ml) previously germinated as indicated were labeled for 1 h with radioactive glutamic acid (5 mM) in the presence or absence of cycloheximide (50 ,ug/ml). After precipitation in cold 10%o TCA, the cell residue was extracted with 10 ml of acetone and twice with 10 ml of ethyl ether. The cell residue was then suspended in 4 ml of 1 N NaOH and heated in a boiling water bath for 10 min. The suspension was neutralized with 5 N HCl and centrifuged. The supernatant (fraction I) was saved, and the cell residue was processed as described by Becker (2) for the enzymatic hydrolysis of glycogen. After this treatment, the suspension was centrifuged, and the supernatant (fraction II) was saved. The sediment (final residue) was collected in a glass fiber filter, washed, and dried. The solvent extracts were pooled, evaporated in a water bath, redissolved in a toluene-based scintillation mixture, and counted. Radioactivity was also counted in all the other fractions. Figures represent the average of three independent experiments. Numbers in parentheses represent the percentage relative to total incorporation.

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stance, alanine, asparagine, and glutamine were incorporated in the presence of cycloheximide into the cold-TCA precipitate at a rate equivalent to 79, 160, and 42%, respectively, that of glutamic acid (unpublished observations). Other amino acids, such as leucine and glycine, were incorporated less efficiently, at a rate equivalent to 19 and 14%, respectively, that of glutamic acid (unpublished observations). In all cases, the radioactivity incorporated into the cold TCA precipitate was resistent to pronase digestion, indicating that it was not in protein. On the other hand, part of the label could be removed by extracting the TCA precipitate with acetone and ethyl ether, suggesting that it was in lipid fractions. The proportion of the label which could be removed with the organic solvents changed, depending on the radioactive amino acid used. Thus, about 25 to 30% of the label incorporated from alanine was solubilized by the acetoneether treatment (unpublished data), but less than 5% was removed when glutamic acid was used (Table 1). We examined the localization of the label incorporated from glutamic acid into different fractions of cells treated or not treated with cycloheximide (Table 1). More than 56% of the radioactivity incorporated in the absence of cycloheximide could be extracted by mild alkaline treatment. This extraction procedure solubilizes most of the cell protein (7) and the cell wall matrix material (3). The remainder of the label was found in glycogen (fraction 1I) and in the cell wall residue (final residue). On the other hand, the distribution of radioactivity in the cells treated with cycloheximide was quite different. In this case, the alkaline extraction solubilized only 26.8% of the radioactivity incorporated. It has been reported for fungal cells that cycloheximide inhibits the synthesis of glycoprotein as well as that of proteins but does not have shortterm effects on polysaccharide synthesis (3, 4, 9). Thus, the radioactivity present in the alkaline extract of cycloheximide-treated cells probably represented polysaccharides from the cell wall matrix (i.e., the mucoran fraction). The remainder of the label appeared in the glycogen fraction, in which it was increased onefold in comparison with the control, and in the cell wall residue (chitin-chitosan). These data confirm our previous results (10) showing that cycloheximide enhances the accumulation of glycogen in gluconeogenic cultures. For comparative purposes, the distribution of the label from glutamic acid in the TCA precipitate of glucose-grown cells is also shown in Table 1. The following experiments were designed to examine the effects of the carbon substrate of the growth medium on the capacity of the cells to incorporate radioactivity from glutamic acid

in the presence of cyclohFximide. When cells which had been germinated in the presence of glucose were transferred to a medium containing Casamino Acids as the sole source of carbon, they rapidly acquired the capacity to incorporate radioactivity from glutamic acid into cold TCAprecipitable material in the presence of cycloheximide (Fig. 2). Only 1.5 h of incubation of the glucose-grown cells in the amino acid medium was sufficient for full induction of this capacity. The induction process demanded protein synthesis and also required the existence of amino acids in the external medium. The elimination of exogenous glucose was not sufficient to promote the capacity to incorporate radioactivity from glutamic acid in the presence of cycloheximide in the glucose-grown cells.

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FIG. 2. Induction of the capacity to incorporate radioactivity from glutamic acid in the presence of cycloheximide. M. rouxii cells which had germinated in medium supplemented with 0.2% Casamino Acids and 2.0%o glucose (0) were transferred to medium supplemented with 0.2% Casamino Acids as the sole carbon source for 30 min (U), 60 min (A), 90 min (0), and 120 min (A) or to medium without a carbon source for 120 min (0). The cells were then transferred to medium containing radioactive glutamic acid (5 mM) and cycloheximide (50 F.g/ml), and the incorporation of radioactivity into cold TCA-precipitable material was determined as described in the text. The inset represents the incorporation rate of cells incubated in the presence of amino acids (0) or in the absence of a carbon source (0).

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VOL. 154, 1983

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FIG. 3. Effect of glucose on the capacity to incorporate radioactivity from glutamic acid in the presence of cycloheximide. M. rouxii cells germinated in medisupplemented with 0.2% Casamino Acids were transferred to medium supplemented with 0.2% Casamino Acids and 2.0o glucose and incubated for the time periods indicated. The cells were then transfeffed to a medium containing radioactive glutamic acid (5 mM) and cycloheximide (50 ,ug/ml), and the rate of incorporation of radioactivity into cold TCA-precipitable material was determined as described in the text. 0, Incorporation of radioactive glutamic acid; O, cell growth.

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In the experiments shown in Fig. 3, cells which germinated in medium supplemented with amino acids, and therefore able to incorporate radioactivity from amino acids in the presence of cycloheximide, were transferred to medium supplemented with amino acids and glucose. The capacity for incorporating radioactivity from glutamic acid decreased rapidly during the first 30 min and more slowly thereafter, suggesting that the enzymatic system responsible for the incorporation of the radioactive amino acid was partially inactivated and no longer synthesized in the presence of glucose. Other hexoses, such as mannose and fructose, were found to produce analogous effects. In summary, the ability to incorporate radioactivity from glutamic acid into TCA-precipitable material in the presence of cycloheximide is an adaptative property of M. rouxii, controlled by carbon catabolite repression and by amino

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acid induction. Our data strongly suggest that this incorporation of radioactivity represents gluconeogenic activity associated with polysaccharide synthesis. That is, the amino acid carbon was converted into sugar molecules, which in turn were used for the synthesis of glycogen and cell wall polysaccharides. Additional experiments with radioactive glucose (data not shown) proved that the rate of incorporation of this sugar into TCA-precipitable material in the presence of cycloheximide was the same for cells germinated under glycolytic or under gluconeogenic conditions. Therefore, it is possible that polysaccharide synthesis is not a limiting step in the chain of events intervening in the conversion of the amino acid carbon into polysaccharidic material. We believe that the rate of incorporation of the radioactive amino acid is a measurement of the in vivo activity of the gluconeogenic pathway. The experimental system described here may be of potential utility for studies on the regulatory properties of gluconeogenesis in intact Mucor cells and in similar microbial systems. This work was supported by grant B 39/79/245/00/00 from F/NEP. S.S. received fellowship 80-1115-0 from FAPESP. We thank Renato H. Migliorini for his continuous interest and support. LITERATURE CITED 1. Bartnicki-Garcia, S., and W. J. Nickerson. 1962. Nutrition, growth and morphogenesis of Mucor rouxii. J. Bacteriol. 84:841-858. 2. Becker, J. U. 1978. A method for glycogen determination in whole yeast cells. Anal. Biochem. 86:56-64. 3. Dow, J. M., and V. D. Villa. 1980. Oligoglucuronide production in Mucor rouxii: evidence for a role for endohydrolases in hyphal extension. J. Bacteriol. 142:939-944. 4. Elorza, M. V., and R. Santandreu. 1969. Effect of cycloheximide on yeast cell wall synthesis. Biochem. Biophys. Res. Commun. 36:741-747. 5. Haidle, C. W., and R. Storck. 1966. Control of dimorphism in Mucor rouxii. J. Bacteriol. 92:1236-1244. 6. Haidle, C. W., and R. Storck. 1966. Inhibition by cycloheximide of protein and RNA synthesis in Mucor rouxii. Biochem. Biophys. Res. Commun. 22:175-180. 7. Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3:447-454. 8. Lowry, 0. R., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 9. Sternlicht, E., D. Katz, and R. F. Rosenberger. 1973. Subapical wall synthesis and wall thickening induced by cycloheximide in hyphae of Aspergillus nidulans. J. Bacteriol. 114:819-823. 10. Terenzi, H. F., P. C. Mathias, E. Roselino, and R. H. Migliorini. 1982. Glycogen accumulation under gluconeogenic conditions in Mucor rouxii: effect of cycloheximide. Exp. Mycol. 6:180-185.