Planta (Berl.)128, 8 1 - 8 4 (1976)
Pl~Jl~__~ 9 by Springer-Verlag 1976
The Effect of Cyanide and some other Carbonyl Binding Reagents on Glycolate Excretion by Chlorella vulgaris Birgit Vennesland and Klaus Jetschmann Forschungsstelle Vennesland der Max-Planck-Gesellschaft, Harnackstr. 23, 1000 Berlin 33 (Dahlem)
Summary. The excretion of glycolate by illuminated Chlorella vulgaris cells at low CO2 tension can be stimulated about tenfold by substituting O2 for air, or by addition of cyanide, hydroxylamine, hydrazine or semicarbazide to the cells in air. For each reagent there is a concentration range giving a maximum effect. It is proposed, as a working hypothesis, that the H C N formed internally when the cells are illuminated in 02, may cause the glycolate excretion.
Introduction Cyanide has been identified as a component of an inactive form of nitrate reductase in Chlorella vulgaris (Lorimer et al., 1974). Pistorius et al. (1975) have presented evidence that, in these algae, H C N production is enhanced by high light intensity, high O2 tension, and low CO2 tension. These same conditions have previously been shown to cause a high rate of glycolate excretion by the same algal strain (Warburg and Krippahl, 1960). The question rises whether the intracellular generation of H C N might promote the excretion of glycolate. In the present paper, we describe how added HCN, hydroxylamine, hydrazine and semicarbazide all potentiate glycolate excretion by Chlorella.
Materials and Methods The strain of Chlorella vulgaris employed in these experiments is the same as that previously employed by Warburg and Krippahl (1960). The cells were grown on a nitrate-containing mineral salts medium in 5% CO2 in air under continuous illumination for two days, and harvested by centrifugation as previously described (Solomonson and Vennesland, 1972). They were washed twice in a nitratefree salt solution (5 g MgSO4.7H20, 2.5 g KH2PO4 and 2 g NaC1 per liter, adjusted to pH 6.8 with NaOH). The washed cells were suspended in the same salt solution to give about 50 gl packed cells per ml of suspension.
The experiments were conducted in conical Warburg flasks with a side arm and a central trough. One ml cell suspension, one ml 0.15 M Na-K phosphate buffer, pH 6.8 and one ml water were added to the main compartment of the flask with other additions as indicated. All incubations were conducted with shaking at 20 ~ C. In most of the experiments, the CO2 tension was held constant at 0. t % with a carbonate-bicarbonate buffer placed in the central trough (Warburg et al., 1961 ; Warburg and Krippahl, 1962). The evolution of Oz in light, and the consumption of 02 in the dark could thus be measured, as well as the eventual glycolate excretion. In the experiment with HCN, 150 ~tl cells were employed, the reagent was added as a gas (Gewitz and V61ker, 1960), and CO2 tension was not buffered. Unless otherwise specified, illumination was with white light, 2.5 cal per minute for 30 rain. The cells were then removed by centrifugation, and glycolate was determined on the supernatant as previously described (Warburg and Krippahl, 1960). Solutions of hydroxylamine hydrochloride, hydrazine sulfate, phenyl hydrazine hydrochloride and semicarbazide hydrochloride were adjusted to the pH of the suspending medium immediately before use. They were generally placed in the side arm of the Warburg flask, and mixed with the cells at the start of the equilibration period, about ten minutes before the commencement of illumination. None of these reagents interfered with the glycolate determination at the concentrations employed.
Results
Photosynthesis and Glycolate Excretion Chlorella cells grown in 5% C02, perform photosynthesis poorly in 0.1% CO2. Net rates of 02 evolution on illumination were in the range of 60 to 80 gl 02 per h per 50 ~tl cells in 0.1% CO2 in air. When 0.1% CO2 in O2 was substituted for 0.1% CO2 in air, the onset of illumination caused an initial development of positive pressure, which lasted only about ten minutes, after which the light did little more than compensate for the respiration. This inhibition of photosynthesis by O2 (Warburg effect) was accompanied by a large rise in glycolate excretion. There was no glycolate excreted in the dark, or in the light in 0.1% CO2 in argon. On illumination
B. Vennesland and K. Jetschmann : Glycolate Excretion by Chlorella vulgar&
82
P~
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2s
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' 016'
HCN, ~ moles
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]~
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o
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i
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o
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,
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i 150 SEMICARBAZIDE,~ moles
Fig. 1
'~o~o~o
>,
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1 2 3 Z, HYDROXYLAMINE,}amoles
,;
2'~
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HYDRAZINE,pmoles
Fig. 2
Fig. 1. (A) Effects o f H C N on glycolate excretion. Reaction mixtures contained 150 gl cells in 3 ml salt solution. For experimental details see Materials and Methods. (B) Effect of semicarbazide on glycolate excretion. Reaction mixtures contained 50 gl cells in 3 ml salt solution. For experimental details see Materials and Methods Fig. 2. (A) Effect of hydroxylamine on glycolate excretion. Procedure as in Fig. 1 B. (B) Effect of hydrazine on glycolate excretion. Procedure as in Fig. 1 B
in 0.1% CO2 in air, 0.2 to 0.3 gmol of glycolate were excreted by 50 gl cells in 30 min, whereas the values observed for glycolate excretion in 02 under otherwise identical conditions were about 1.5 to 2 gmol of glycolate.
Enhanced Glycolate Excretion in Air With cyanide, hydroxylamine, hydrazine and semicarbazide, it was possible to find a suitable concentration which brought the level of glycolate excretion in air about up to the level which was observed with the same cell suspension in oxygen. Figs. 1 and 2 show the amount of glycolate excreted with increasing concentrations of these reagents. The curves for cyanide, hydroxylamine and hydrazine show a well-defined and rather narrow maximum. In each case, the amount of reagent required to cause the greatest glycolate excretion was sufficient to cause a partial, but not a complete inhibition of photosynthesis. With phenyl hydrazine, no stimulation of glycolate excretion was observed at any concentration, up to and above those which inhibited photosynthesis completely. The amount of H C N which gave an optimal effect (Fig. 1A) was 0.1 gmol per 150 gl cells. In general, the effects of cyanide, hydroxylamine and hydrazine vary inversely with the quantity of cells employed as is expected for reagents which react with the cell constituents. Thus, with 50 gl cells, we would expect an optimal effect with about 0.03 to 0.04 gmol of added HCN. This is an approximation. It is difficult to locate the top of such a narrow peak. A maximum effect with hydroxylamine was ob-
served when about 0.5 gmol were added per 50 ~tl of cells (Fig. 2A); and for hydrazine, the maximum effect was observed when 1 to 1.5 ~tmol were added per 50 ~tl of cells (Fig. 2 B). With semicarbazide (Fig. 1B), the glycolate production was constant between 10 and 160 gmol reagent added. This was for an illumination time of 30 min. When the time of illumination was prolonged to 50 min, glycolate formation continued in the reaction mixtures with the smaller amounts of semicarbazide but not in the reaction mixtures with the larger amounts. The result was the development of a broad peak of glycolate production between 2 and 60 gmol of added semicarbazide. It is probable that a change of incubation time would give analogous shifts in the shapes of the other curves, but this has not been examined systematically. None of the reagents tested had any appreciable stimulatory effect in O.1% C02 in 02, when the rate of glycolate excretion was already high. Whether elicited by 02 or by carbonyl-binding reagents in air, glycolate excretion was inhibited by high C02 tension (5%).
Semicarbazide as a Trapping Agent for Glyoxylic Acid The semicarbazone of glyoxylic acid has an absorption band with a maximum at 252 nm and a millimolar extinction coefficient of 12.4 (Olson, 1959). Thus, small amounts of this substance can readily be detected. The absorbancy was measured against a blank containing semicarbazide. When 100 gl of cells were illuminated for 150 min with 90 gmol of semicarbazide in 0.1% CO2-air, 8 to 10gmol of glycolate and
B. Vennesland and K. Jetscbmann : Glycolate Excretion by Chlorella vulgaris
one ~tmol of glyoxylic semicarbazone were found in the suspending medium. Thus, the rate of glyoxylate formation appears to be slow. When the glycolate excretion was inhibited by raising the CO2 tension, the characteristic peak at 252 nm was replaced by a broad absorption at shorter wavelengths.
Discussion
The process of glycolate formation and metabolism is associated with the phenomenon of photorespiration in higher plants (Tolbert, 1971 ; Zelitch, 1971), which has evoked much recent interest because of its effect on the overall efficiency of photosynthesis (Tolbert, 1974; Zelitch, 1975). Glycolate metabolism in algae has many features in common with that of higher plants, though some of the details are different (Merrett and Lord, 1973). The excretion of glycolate, a phenomenon common in algae and first described by Tolbert and Zill (1956), is particularly intense at high 02 tension, low CO2 tension, and high light intensity (Warburg and Krippahl, 1960). A number of possible mechanisms for glycolate formation have been suggested. The route by way of ribulose diphosphate oxygenase is supported by good evidence (Andrews et aI., 1971; Bowes et al., 1971; Bowes and Ogren, 1972), and has the further attraction that it might account for the competitive effects of 02 and CO2 on glycolate formation. Glycolate is metabolized further by way of glyoxylate, glycine and serine to glycerate (Tolbert, 1974). The excretion of glycolate is thought to result when much glycolate is formed and its further metabolism is blocked. The present results show that a small amount of HCN, supplied externally, increases glycolate excretion of Chlorella vulgaris in air, to a level about equal to that observed in 100% 02. Hydroxylamine, hydrazine and semicarbazide behave similarly, except that progressively higher concentrations of these reagents are required. The effect of all these reagents on glycolate excretion is rather similar to that of isonicotinylhydrazide (INH), which has been studied extensively (Pritchard et al., 1962; Coombs and Whittingham, 1966; Marker and Whittingham, 1966; Gore et al., 1974). INH is thought to inhibit the mitochondrial conversion of glycine to serine, a pyridoxal phosphate mediated reaction (Gore et al., 1974; Coombs and Whittingham, 1966). All of these reagents employed in the present study should interfere with pyridoxal phosphate requiring reactions. It seems to be a reasonable working hypothesis that these reagents all affect glycolate excretion by a similar mechanism. HCN is a particularly effective inhibitor of pyridoxal phosphate mediated reactions.
83
HCN is also the most interesting of the various reagents tested because Chlorella vuIgaris can generate HCN (Gewitz et al., 1974; Lorimer et al., 1974). It seems more than a curious coincidence that the HCN production is enhanced by high 02 tension and high light intensity (Pistorius et al., 1975),just as is glycolate production. This suggests that it might be the internally generated HCN which blocks the metabolism of glycolate by cells at high 02 tension. Although we have no indication that the amounts of intracellularly generated cyanide ever reach a level as high as 60 nmol per 100 gl cells (the amount of added cyanide required to cause maximal glycolate excretion under the present conditions), it is probable that the cyanide generated internally is more effective biochemically than externally added cyanide. The oxidation of glycolate to glyoxylate is catalyzed by a so-called glycolic dehydrogenase instead of by the glycolic oxidase of higher plants (Nelson and Tolbert, 1970; Merrett and Lord, 1973). This glycolic dehydrogenase has been incompletely characterized as a cyanide-sensitive dehydrogenase with an unknown hydrogen acceptor. It is repressed by high CO2 tensions; and the capacity to excrete glycolate has been associated with the absence (or low concentration level) of this enzyme (Nelson and Tolbert, 1969; Cooksey, 1971). The possibility that internally generated HCN inhibits such an enzyme should also be considered.
References Andrews, T.J., Lorimer, G.H., Tolbert, N.E. : Incorporation of molecular oxygen into glycine and serine during photorespiration in spinach leaves. Biochemistry 10, 4777~4782 (1971) Bowes, G., Ogren, W.L., Hageman, R.H.: Phosphogtycolate production catalyzed by ribulose diphosphate carboxylase. Biochem. biophys. Res. Commun. 45, 716-722 (1971) Bowes, G., Ogren, W.L.: Oxygen inhibition and other properties of soybean ribulose-l,5-diphosphate carboxylase. J. biol. Chem. 247, 2171 2176 (1972) Cooksey, K.E. : Glycolate: Dichlorophenot indophenol oxidoreductase in Chlarnydomonas reinhardtii. Plant Physiol. 48, 267~69 (1971) Coombs, J., Whittingham, C.P. : The mechanism of inhibition of photosynthesis by high partial pressures of oxygen in Chlorella. Proc. roy. Soc. B, 164, 511-520 (1966) Gewitz, H.-S., Lorimer, G.H., Solomonson, L.P., Vennesland, B. : Presence of HCN in Chlorella vulgaris and its possible role in controlling the reduction of nitrate. Nature 249, 79-81 (1974) Gewitz, H.-S., V61ker, W. : Weiterentwicklung der manometrischen Methoden (Herstellung definierter Blausfiure-Konzentrationen). Z. Naturforsch. 15b, 625 (1960) Gore, G., HilI, H.M., Evans, R.B., Rogers, L.J.: Inhibition by isonicotinyl hydrazide of pigment formation in higher plants. Phytochemistry 13, 1657 1665 (1974) Lorimer, G.H., Gewitz, H.-S., V61ker, W., Solomonson, L.P., Vennesland, B. : The presence of bound cyanide in the naturally
84
B. Vennelsand and K. Jetschmann: Glycolate Excretion by Chlorella vulgaris
inactivated form of nitrate reductase of Chlorella vulgaris. J. biol. Chem. 249, 6074-6079 (1974) Marker, A.F.H., Whittingham, C.P. : The photoassimilation of glucose in Chlorella with reference to the role of glycollic acid. Proc. roy. Soc. B 165, 473--485 (1966) Merrett, M.J., Lord, J.M.: Glycollate formation and metabolism by algae. New Phytol. 72, 751 767 (1973) Nelson, E.B., Tolbert, N.E. : The regulation of glycolate metabolism in Chlamydomonas reinhardtii. Biochim. biophys. Acta (Amst.) 184, 263-270 (1969) Nelson, E.B., Tolbert, N.E. : Glycolate dehydrogenase in green algae. Arch. Biochem. Biophys. 141, 102-110 (1970) Olson, J.A. : Spectrophotometric measurement of a-keto acid semicarbazones. Arch. Biochem. Biophys. 85, 225-233 (1959) Pistorius, E.K., Gewitz, H.-S., Voss, H., Vennesland, B. : Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo. Planta (Berl.) 128, 73-80 (1976) Pritchard, G.G., Griffin, W.J., Whittingham, C.P. : The effect of carbon dioxide concentration, light intensity and isonicotinyl hydrazide on the photosynthetic production of glycollic acid by Chlorella. J. exp. Bot. 13, 176-184 (1962) Solomonson, L.P., Vennesland, B. : Nitrate reductase and chlorate
toxicity in Chlorella vulgaris Beijerinck. Plant Physiol. 50, 421 424 (1972) Tolbert, N.E.: Microbodies-peroxisomes and glyoxisomes. Ann. Rev. Plant Physiol. 22, 45-74 (1971) Tolbert, N.E. : Photorespiration. In : Algal Physiology and Biochemistry, Botanical Monographs. Vol. 10, pp. 474-504. Ed. : Stewart, W.D.P. Oxford-London-Edinburgh-Melbourue: Blackwell 1974 Tolbert, N.E., Zill, L.P. : Excretion of glycolic acid by algae during photosynthesis. J. biol. Chem. 222, 895-906 (1956) Warburg, O., Geissler, A.W., Lorenz, S. : CO2-Drucke fiber Bicarbonat-Carbonatgemischen. Z. Naturforsch. 16b, 283 (1961) Warburg, O., Krippahl, G. : Glykolsfiurebildung in Chlorella. Z. Naturforsch. 15b, 197-199 (1960) Warburg, O., Krippahl, G. : Weiterentwicklung der manometrischen 1-Gef'~iBmethoden. Z. Naturforsch. 17b, 631~632 (1962) Zelitch, I. : Photosynthesis, Photorespiration and Plant Productivity. New York: Academic Press 1971 Zelitch, I. : Improving the efficiency of photosynthesis. Science 188, 626-633 (I 975) Received 11 August; accepted 8 September 1975
Pl~Jl~__~ 9 by Springer-Verlag 1976
The Effect of Cyanide and some other Carbonyl Binding Reagents on Glycolate Excretion by Chlorella vulgaris Birgit Vennesland and Klaus Jetschmann Forschungsstelle Vennesland der Max-Planck-Gesellschaft, Harnackstr. 23, 1000 Berlin 33 (Dahlem)
Summary. The excretion of glycolate by illuminated Chlorella vulgaris cells at low CO2 tension can be stimulated about tenfold by substituting O2 for air, or by addition of cyanide, hydroxylamine, hydrazine or semicarbazide to the cells in air. For each reagent there is a concentration range giving a maximum effect. It is proposed, as a working hypothesis, that the H C N formed internally when the cells are illuminated in 02, may cause the glycolate excretion.
Introduction Cyanide has been identified as a component of an inactive form of nitrate reductase in Chlorella vulgaris (Lorimer et al., 1974). Pistorius et al. (1975) have presented evidence that, in these algae, H C N production is enhanced by high light intensity, high O2 tension, and low CO2 tension. These same conditions have previously been shown to cause a high rate of glycolate excretion by the same algal strain (Warburg and Krippahl, 1960). The question rises whether the intracellular generation of H C N might promote the excretion of glycolate. In the present paper, we describe how added HCN, hydroxylamine, hydrazine and semicarbazide all potentiate glycolate excretion by Chlorella.
Materials and Methods The strain of Chlorella vulgaris employed in these experiments is the same as that previously employed by Warburg and Krippahl (1960). The cells were grown on a nitrate-containing mineral salts medium in 5% CO2 in air under continuous illumination for two days, and harvested by centrifugation as previously described (Solomonson and Vennesland, 1972). They were washed twice in a nitratefree salt solution (5 g MgSO4.7H20, 2.5 g KH2PO4 and 2 g NaC1 per liter, adjusted to pH 6.8 with NaOH). The washed cells were suspended in the same salt solution to give about 50 gl packed cells per ml of suspension.
The experiments were conducted in conical Warburg flasks with a side arm and a central trough. One ml cell suspension, one ml 0.15 M Na-K phosphate buffer, pH 6.8 and one ml water were added to the main compartment of the flask with other additions as indicated. All incubations were conducted with shaking at 20 ~ C. In most of the experiments, the CO2 tension was held constant at 0. t % with a carbonate-bicarbonate buffer placed in the central trough (Warburg et al., 1961 ; Warburg and Krippahl, 1962). The evolution of Oz in light, and the consumption of 02 in the dark could thus be measured, as well as the eventual glycolate excretion. In the experiment with HCN, 150 ~tl cells were employed, the reagent was added as a gas (Gewitz and V61ker, 1960), and CO2 tension was not buffered. Unless otherwise specified, illumination was with white light, 2.5 cal per minute for 30 rain. The cells were then removed by centrifugation, and glycolate was determined on the supernatant as previously described (Warburg and Krippahl, 1960). Solutions of hydroxylamine hydrochloride, hydrazine sulfate, phenyl hydrazine hydrochloride and semicarbazide hydrochloride were adjusted to the pH of the suspending medium immediately before use. They were generally placed in the side arm of the Warburg flask, and mixed with the cells at the start of the equilibration period, about ten minutes before the commencement of illumination. None of these reagents interfered with the glycolate determination at the concentrations employed.
Results
Photosynthesis and Glycolate Excretion Chlorella cells grown in 5% C02, perform photosynthesis poorly in 0.1% CO2. Net rates of 02 evolution on illumination were in the range of 60 to 80 gl 02 per h per 50 ~tl cells in 0.1% CO2 in air. When 0.1% CO2 in O2 was substituted for 0.1% CO2 in air, the onset of illumination caused an initial development of positive pressure, which lasted only about ten minutes, after which the light did little more than compensate for the respiration. This inhibition of photosynthesis by O2 (Warburg effect) was accompanied by a large rise in glycolate excretion. There was no glycolate excreted in the dark, or in the light in 0.1% CO2 in argon. On illumination
B. Vennesland and K. Jetschmann : Glycolate Excretion by Chlorella vulgar&
82
P~
A A
2s
E3
E
, (D
' ' 0',3'
' 016'
HCN, ~ moles
'0;
]~
oo =L < Ld
o
8 >,
2,0 i
i
LIJ
o
1,0 d o
510
,
1~)0
i 150 SEMICARBAZIDE,~ moles
Fig. 1
'~o~o~o
>,
dco
{D
B
?
1 2 3 Z, HYDROXYLAMINE,}amoles
,;
2'~
3'2
HYDRAZINE,pmoles
Fig. 2
Fig. 1. (A) Effects o f H C N on glycolate excretion. Reaction mixtures contained 150 gl cells in 3 ml salt solution. For experimental details see Materials and Methods. (B) Effect of semicarbazide on glycolate excretion. Reaction mixtures contained 50 gl cells in 3 ml salt solution. For experimental details see Materials and Methods Fig. 2. (A) Effect of hydroxylamine on glycolate excretion. Procedure as in Fig. 1 B. (B) Effect of hydrazine on glycolate excretion. Procedure as in Fig. 1 B
in 0.1% CO2 in air, 0.2 to 0.3 gmol of glycolate were excreted by 50 gl cells in 30 min, whereas the values observed for glycolate excretion in 02 under otherwise identical conditions were about 1.5 to 2 gmol of glycolate.
Enhanced Glycolate Excretion in Air With cyanide, hydroxylamine, hydrazine and semicarbazide, it was possible to find a suitable concentration which brought the level of glycolate excretion in air about up to the level which was observed with the same cell suspension in oxygen. Figs. 1 and 2 show the amount of glycolate excreted with increasing concentrations of these reagents. The curves for cyanide, hydroxylamine and hydrazine show a well-defined and rather narrow maximum. In each case, the amount of reagent required to cause the greatest glycolate excretion was sufficient to cause a partial, but not a complete inhibition of photosynthesis. With phenyl hydrazine, no stimulation of glycolate excretion was observed at any concentration, up to and above those which inhibited photosynthesis completely. The amount of H C N which gave an optimal effect (Fig. 1A) was 0.1 gmol per 150 gl cells. In general, the effects of cyanide, hydroxylamine and hydrazine vary inversely with the quantity of cells employed as is expected for reagents which react with the cell constituents. Thus, with 50 gl cells, we would expect an optimal effect with about 0.03 to 0.04 gmol of added HCN. This is an approximation. It is difficult to locate the top of such a narrow peak. A maximum effect with hydroxylamine was ob-
served when about 0.5 gmol were added per 50 ~tl of cells (Fig. 2A); and for hydrazine, the maximum effect was observed when 1 to 1.5 ~tmol were added per 50 ~tl of cells (Fig. 2 B). With semicarbazide (Fig. 1B), the glycolate production was constant between 10 and 160 gmol reagent added. This was for an illumination time of 30 min. When the time of illumination was prolonged to 50 min, glycolate formation continued in the reaction mixtures with the smaller amounts of semicarbazide but not in the reaction mixtures with the larger amounts. The result was the development of a broad peak of glycolate production between 2 and 60 gmol of added semicarbazide. It is probable that a change of incubation time would give analogous shifts in the shapes of the other curves, but this has not been examined systematically. None of the reagents tested had any appreciable stimulatory effect in O.1% C02 in 02, when the rate of glycolate excretion was already high. Whether elicited by 02 or by carbonyl-binding reagents in air, glycolate excretion was inhibited by high C02 tension (5%).
Semicarbazide as a Trapping Agent for Glyoxylic Acid The semicarbazone of glyoxylic acid has an absorption band with a maximum at 252 nm and a millimolar extinction coefficient of 12.4 (Olson, 1959). Thus, small amounts of this substance can readily be detected. The absorbancy was measured against a blank containing semicarbazide. When 100 gl of cells were illuminated for 150 min with 90 gmol of semicarbazide in 0.1% CO2-air, 8 to 10gmol of glycolate and
B. Vennesland and K. Jetscbmann : Glycolate Excretion by Chlorella vulgaris
one ~tmol of glyoxylic semicarbazone were found in the suspending medium. Thus, the rate of glyoxylate formation appears to be slow. When the glycolate excretion was inhibited by raising the CO2 tension, the characteristic peak at 252 nm was replaced by a broad absorption at shorter wavelengths.
Discussion
The process of glycolate formation and metabolism is associated with the phenomenon of photorespiration in higher plants (Tolbert, 1971 ; Zelitch, 1971), which has evoked much recent interest because of its effect on the overall efficiency of photosynthesis (Tolbert, 1974; Zelitch, 1975). Glycolate metabolism in algae has many features in common with that of higher plants, though some of the details are different (Merrett and Lord, 1973). The excretion of glycolate, a phenomenon common in algae and first described by Tolbert and Zill (1956), is particularly intense at high 02 tension, low CO2 tension, and high light intensity (Warburg and Krippahl, 1960). A number of possible mechanisms for glycolate formation have been suggested. The route by way of ribulose diphosphate oxygenase is supported by good evidence (Andrews et aI., 1971; Bowes et al., 1971; Bowes and Ogren, 1972), and has the further attraction that it might account for the competitive effects of 02 and CO2 on glycolate formation. Glycolate is metabolized further by way of glyoxylate, glycine and serine to glycerate (Tolbert, 1974). The excretion of glycolate is thought to result when much glycolate is formed and its further metabolism is blocked. The present results show that a small amount of HCN, supplied externally, increases glycolate excretion of Chlorella vulgaris in air, to a level about equal to that observed in 100% 02. Hydroxylamine, hydrazine and semicarbazide behave similarly, except that progressively higher concentrations of these reagents are required. The effect of all these reagents on glycolate excretion is rather similar to that of isonicotinylhydrazide (INH), which has been studied extensively (Pritchard et al., 1962; Coombs and Whittingham, 1966; Marker and Whittingham, 1966; Gore et al., 1974). INH is thought to inhibit the mitochondrial conversion of glycine to serine, a pyridoxal phosphate mediated reaction (Gore et al., 1974; Coombs and Whittingham, 1966). All of these reagents employed in the present study should interfere with pyridoxal phosphate requiring reactions. It seems to be a reasonable working hypothesis that these reagents all affect glycolate excretion by a similar mechanism. HCN is a particularly effective inhibitor of pyridoxal phosphate mediated reactions.
83
HCN is also the most interesting of the various reagents tested because Chlorella vuIgaris can generate HCN (Gewitz et al., 1974; Lorimer et al., 1974). It seems more than a curious coincidence that the HCN production is enhanced by high 02 tension and high light intensity (Pistorius et al., 1975),just as is glycolate production. This suggests that it might be the internally generated HCN which blocks the metabolism of glycolate by cells at high 02 tension. Although we have no indication that the amounts of intracellularly generated cyanide ever reach a level as high as 60 nmol per 100 gl cells (the amount of added cyanide required to cause maximal glycolate excretion under the present conditions), it is probable that the cyanide generated internally is more effective biochemically than externally added cyanide. The oxidation of glycolate to glyoxylate is catalyzed by a so-called glycolic dehydrogenase instead of by the glycolic oxidase of higher plants (Nelson and Tolbert, 1970; Merrett and Lord, 1973). This glycolic dehydrogenase has been incompletely characterized as a cyanide-sensitive dehydrogenase with an unknown hydrogen acceptor. It is repressed by high CO2 tensions; and the capacity to excrete glycolate has been associated with the absence (or low concentration level) of this enzyme (Nelson and Tolbert, 1969; Cooksey, 1971). The possibility that internally generated HCN inhibits such an enzyme should also be considered.
References Andrews, T.J., Lorimer, G.H., Tolbert, N.E. : Incorporation of molecular oxygen into glycine and serine during photorespiration in spinach leaves. Biochemistry 10, 4777~4782 (1971) Bowes, G., Ogren, W.L., Hageman, R.H.: Phosphogtycolate production catalyzed by ribulose diphosphate carboxylase. Biochem. biophys. Res. Commun. 45, 716-722 (1971) Bowes, G., Ogren, W.L.: Oxygen inhibition and other properties of soybean ribulose-l,5-diphosphate carboxylase. J. biol. Chem. 247, 2171 2176 (1972) Cooksey, K.E. : Glycolate: Dichlorophenot indophenol oxidoreductase in Chlarnydomonas reinhardtii. Plant Physiol. 48, 267~69 (1971) Coombs, J., Whittingham, C.P. : The mechanism of inhibition of photosynthesis by high partial pressures of oxygen in Chlorella. Proc. roy. Soc. B, 164, 511-520 (1966) Gewitz, H.-S., Lorimer, G.H., Solomonson, L.P., Vennesland, B. : Presence of HCN in Chlorella vulgaris and its possible role in controlling the reduction of nitrate. Nature 249, 79-81 (1974) Gewitz, H.-S., V61ker, W. : Weiterentwicklung der manometrischen Methoden (Herstellung definierter Blausfiure-Konzentrationen). Z. Naturforsch. 15b, 625 (1960) Gore, G., HilI, H.M., Evans, R.B., Rogers, L.J.: Inhibition by isonicotinyl hydrazide of pigment formation in higher plants. Phytochemistry 13, 1657 1665 (1974) Lorimer, G.H., Gewitz, H.-S., V61ker, W., Solomonson, L.P., Vennesland, B. : The presence of bound cyanide in the naturally
84
B. Vennelsand and K. Jetschmann: Glycolate Excretion by Chlorella vulgaris
inactivated form of nitrate reductase of Chlorella vulgaris. J. biol. Chem. 249, 6074-6079 (1974) Marker, A.F.H., Whittingham, C.P. : The photoassimilation of glucose in Chlorella with reference to the role of glycollic acid. Proc. roy. Soc. B 165, 473--485 (1966) Merrett, M.J., Lord, J.M.: Glycollate formation and metabolism by algae. New Phytol. 72, 751 767 (1973) Nelson, E.B., Tolbert, N.E. : The regulation of glycolate metabolism in Chlamydomonas reinhardtii. Biochim. biophys. Acta (Amst.) 184, 263-270 (1969) Nelson, E.B., Tolbert, N.E. : Glycolate dehydrogenase in green algae. Arch. Biochem. Biophys. 141, 102-110 (1970) Olson, J.A. : Spectrophotometric measurement of a-keto acid semicarbazones. Arch. Biochem. Biophys. 85, 225-233 (1959) Pistorius, E.K., Gewitz, H.-S., Voss, H., Vennesland, B. : Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo. Planta (Berl.) 128, 73-80 (1976) Pritchard, G.G., Griffin, W.J., Whittingham, C.P. : The effect of carbon dioxide concentration, light intensity and isonicotinyl hydrazide on the photosynthetic production of glycollic acid by Chlorella. J. exp. Bot. 13, 176-184 (1962) Solomonson, L.P., Vennesland, B. : Nitrate reductase and chlorate
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