- Email: [email protected]
Conversion of oximes to mustard oil glucosides (Glucosinolates)
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
120
(1967)
Communications Conversion
of Oximes
Glucosides
to Mustard
hydroxylamine hydrochloride and sodium bicarbonate. The ether was removed with a stream of NZ and carrier phenylacetaldehyde was added. Phenylacetaldoxime-U-*4C separated out and was recrystallized twice from et,hyl ether/hexane. Both chemical and radiochemical yields were low; phenylacetonitrile and phenylacetamide were observed as by-products in the mother liquor. The phenylacetaldoxime was shown to be 98% radiochemically homogeneous upon thin-layer chromatography. Two experiments were carried out in which labelled compounds were administered in volumes less than 0.5 ml to the roots of plants of C. oficinalis previously grown to about 0.3 gm fresh weight in nutrient solution. In the first, or-ketoisovaleric acid oxime-U-l% or L-valine-U-l% (Radiochemical Centre, Amersham) was added to single plants, which were then exposed to continuous light for 21 hours. In the second experiment, isobutyraldoxime-UJ4C or L-valine-U-1% were administered to groups of three plants, which were enclosed in transparent containers (220 ml) to reduce evaporative losses of isobutyraldoxime-U1%. The plants were harvested after 48 hours including two 6.hour dark periods. In both cases, water was added to the plant roots as required. In the third experiment phenylalanine-U-l% or phenylacetaldoxime-UJ4C (first dissolved in 5 ~1 ethanol) were administered in 0.25 ml water to 30 six-day-old excised shoots of L. satioum. Water was added as required and the plants were exposed to continuous light for 48 hours. All plant tissue was extracted twice with boiling 80% aqueous ethanol and the radioactivity was determined. The combined ethanolic extracts were evaporated under reduced pressure and the residues were dissolved and suspended in small volumes of water for subsequent analysis. Glucosinolates were separated in two dimensions by thin-layer electrophoresis at pH 2.0 and chromatography with n-propanol:water:ethylacetate:acetic acid:pyridine (120:60 20:4:1) as mobile phase (9). Glucosinolates were detected with sprays of 0.05 M silver nitrate followed by 0.05 M potassium dichromate after drying at 100” or with bromocresol green indicator. Isopropylglucosinolate-‘4C was identified by chromatographic examination of isopropylthioureaJ4C formed from it (10).
Oil
(Glucosinolates)
Recently it has been shown that oximes may be intermediates in the biosynthesis of cyanogenic glycosides from amino acids. Tapper et al. (1) have shown t,hat isobutyraldoxime-U-l% is converted to linamarin-1% in Linum usitatissimum L. seedlings with high efficiency comparable to the corresponding conversion of L-valine-U-14C. In this communication we wish to report that isobutyraldoxime-U-‘*C is also incorporated into isopropylglucosinolate (glucoputranjivin) (I, R = isopropyl) in Cochlearia oficinalis L. plants and that phenylacetaldoxime-U-14C is incorporated into benzylglucosinolate (glucotropaeolin) (I, R = beneyl) in Lepidium sativum L. seedlings. NOSO,-
K +
NOH
R-g
R-:H
S-p
-D-Glucose
I
II Aldoximes
Glucosinolates SCHEME
1.
Incorporation experiments (Z-6) have shown that amino acids are precursors of several glucosinolate aglycones and also that a number of compounds other than amino acids or closely related compounds are ineffective as precursors. In particular phenylpyruvic acid oxime-14C was less effective than phenylalanine-14C as a precursor of bensylglucosinolate (4,7). We report here a similar finding with ol-ketoisovaleric acid oxime-U-‘*C as a precursor of isopropylglucosinolate. The or-ketoisovaleric acid oxime-U-K! (anti HO-COOH isomer) and isobutyraldoxime-U-14C were prepared as previously described (1). Phenylacet,aldehyde-U’% was obtained from L-phenylalanine-U-14C (1 mg, 15&, Radiochemical Centre, Amersham) by oxidation with N-bromosuccinimide (2 mg) for 15 minutes at 5” in aqueous solution (0.6 ml) in the presence of an excess of succinamide (50 mg) at pH 6.7 (8). The phenylacetaldehyde-U-14C was extracted into ethyl ether and added to an aqueous solution of an excess of 719
720
COMMUNICATIONS TABLE COMPARISON Expt.
Compound
Expt. 1
OF
PRECURSORS
OF
administered
I ISOPROPYLGLUCOSINOLATE Amount
n-Valine-U-W a-Ketoisovaleric acid oxime-U-i% L-Valine-U-i% n-Valine-U-l% Isobutyraldoxime-U-W
Expt. 2
sp. act. @C/mm&)
bmmole)
1.0
2490
1.2 4.0 1.0 3.8
1150 622 2490 390
% Converted to isopropylglucosinolate
0.87a <0.25* 0.70” 1.744 15.9
0 Corrected for assumed loss of i4COOH. TABLE COMPARISON
OF
L-PHENYLALANINE
AND
2
PHENYL.~CETALDOXIME
AS
PRECURSORS
OF
BENZYLGLUCOSINOLATE
Compound
administered
n-Phenylalanine-UJ’C Phenylacetaldoxime-U-14C
Amount
~mole)
1.1 1.2
sp. act. duC/mmole)
70 Converted benzylglucosinolate
440 23.1
4.3a 26
to
sp. act. of benzylthiourea WJmmole)
3.56 1.22
(1Corrected for assumed loss of i4COOH from n-phenylalanine-U-14C. Radioautography was used to locate compounds of interest on thin-layer chromatography plates. Incorporation of label into glucosinolates was determined by measuring radioactivity of compounds eluted after separation. The specific activity of the aglycone of benzylglucosinolate was measured by preparing benzylthiourea, by ammonia treatment of the benzyl isothiocyanate liberated by myrosinase. The amount of thiourea was determined by absorption at 244 rnp in ethanolic solution following purification by paper chromatography (10). All radioactivity was measured by liquid scintillation counting. Table I presents the results of the first and second experiments. The incorporation of n-valineW into isopropylglucosinolate, while not particularly high, is significant. Radioautographs of the separated metabolites showed that most radioactivity remained in the valine pool, with only small conversion to other compounds including leucine. No significant radioactivity was observed in other glucosinolates. Further, glucose separated by chromatography after myrosinase treatment of the purified isopropylglucosinolate-*4C contained negligible radioactivity. This suggests that n-valine is a specific precursor of the isopropylglucosinolate aglycone in C. oficinalis. Meakin (7) has reported a similar finding from a single preliminary experiment with Tropaeolum peregrinum which also contains isopropylglucosinolate. The incorporation from a-ketoisovaleric acid oxime-
W is low and confirms a previous finding that phenylpyruvic acid oxime was only weakly effective as a precursor of benzylglucosinolate (4, 7). Table I shows clearly that isobutyraldoxime is efficiently converted to isopropylglucosinolate. Radioautographs showed isopropylglucosinolate to be the most strongly labelled product from isobutyraldoxime-W, and only a trace of radioactivity was observed in other glucosinolates. Table II presents the result of the third experiment. Phenylalanine has previously been shown to be a precursor of benzylglucosinolate in Tropaeolum majus L. (2, 4). Our results confirm these findings in the unrelated L. sativum. Further, the high incorporation of radioactivity from phenylacetaldoxime-1% with comparatively low dilution suggests it to be a normal biosynthetic intermediate. The results reported in this communication, together with the finding of isobutyraldoxime as a precursor of the cyanogenic glucoside linamarin (l), and the unpublished findings of this laboratory that phenylacetaldoxime is a precursor of prunasin and prulaurasin in peach and cherry laurel shoots, respectively, all strongly suggest that aldoximes are normal intermediates in the formation of both cyanogenic glycosides and glucosinolates. Experiments with i6N-labelled amino acids have shown that the amino nitrogen is retained and is presumably oxidized during the biosynthesis of corresponding cyanogenic glycosides
COMMUNICATIONS (11, 12) and glucosinolates (4, 7). Therefore, any oxime intermediates cannot be formed by condensation of an aldehyde or ketone and any hypothetical bound or free form of hydroxylamine. This conclusion is supported by the finding that isobutyraldehyde was ineffective as a precursor of linamarin (1). The isolation of oximes from plant tissue and experiments with ‘SN-labelled compounds are required to confirm the suggested roles of oximes as intermediates. ACKNOWLEDGMENT The authors are indebted skillful technical assistance.
to W. D. Bennett
for
REFERENCES 1. TAPPER, B. A., CONN, E. E., AND BUTLER, G. W., Arch. Biochem. Biophys. 119, 593 (1967). 2. BENN, M. H., Chem. Znd. Lo@on 1907 (1962). M., PROCH~ZKA, Z., AND VERES, 3. KUT&EK, K . , Nature 194, 393 (1962). 4. UNDERHILL, E. W., AND CHISHOLM, M. D., Biochem. Biophys. Res. Commun. 14, 425 (1964). 5. SCHRAUDOLF, H., AND BERGMANN, F., Planta 67, 75 (1965). 6. CHISHOLM, M. D., AND WETTER, L. R., Can. J. Biochem. 44, 1625 (1966). of 7. MEAKIN, D., Ph.D. Thesis, Department Chemistry, University of Alberta at Calgary (1965). 8. GREENSTEIN, J. P., AND WINITZ, M., “Chemistry of the Amino Acids,” p. 1331. Wiley, New York (1961). 9. BIELESKI, R. L., AND TURNER, N. A., Anal. Biochem. 17,278 (1966). A., AND RUBINSTEIN, K., Acta Chem. 10. KjzR, Scand. 7,528 (1953.) 11. BUTLER, G. W., AND CONN, E. E., J. Biol. Chem. 239, 1674 (1964). 12. URIBE, E. G., AND CONN, E. E., J. Biol. Chem. 241, 92 (1966). B. A. TAPPER G. W. BUTLER Plant Chemistry Division, D.S.Z.R., and Department of Chemistry and Biochemistry Massey University Palmerston iVorth, New Zealand Received March 29, 1967
Failure
of Thrombin
to Affect
Platelet
Respiration Glycolysis has frequently been considered the primary pathway for ATP production in blood platelets (1, 2), partly because of evidence indi-
721
cating only a limited aerobic metabolism. The re port by Hussain and Newcomb (3) that addition of thrombin, which initiates the biochemical and morphological changes associated with clotting and clot retraction, caused an elevenfold increase in the rate of oxygen uptake by platelets was therefore significant. It indicated that platelets actually have a rather large capacity for aerobic metabolism under conditions of greatest energy requirement. In beginning a study of the role of respiration in platelet metabolism, I attempted to reproduce the experiments reported by Hussain and Newcomb. Through many attempts in which the procedures used were nearly identical to those reported or consisted of various modifications of those procedures, no evidence of any effect of thrombin on platelet respiration was observed. Other than a species difference, the only apparent explanation for the discrepancy wm my use of a Clark-type oxygen electrode (4), in which the stationary electrodes are immersed in a half-saturated KC1 solution that is separated from the test solution by a membrane permeable only to gases, whereas Hussain and Newcomb used a bare platinum electrode that dips directly into the test solution. An electrode similar (but not identical) to that used by Hussain and Newcomb was obtained and gave results similar to those reported. The evidence presented here indicates that the observation of a stimulation by thrombin of platelet respiration is an artifact of the electrode and that respiration is constant during thrombin-induced aggregation of platelets. Blood was collected from rats using siliconecoated glassware and 0.25 volume of ACD (acidcitrate-dextrose, NIH formula B) as anticoagulant, and was immediately cooled in an ice bath. Erythrocytes and white blood cells were removed by centrifugation at 1509 for 40 minutes. Platelets were sedimented from the supernatant fluid by centrifugation at 8oOg for 20 minutes and were washed twice in the platelet wash solution described by Hussain and Newcomb (3). Figure 1 is an example of a measurement of oxygen uptake using a Clark electrode. Addition of platelets initiated a decline in the concentration of oxygen. Thrombin had no effect on the rate of oxygen consumption but cyanide completely inhibited it. Figure 2 shows the result of experiments carried out under conditions identical to those in Fig. 1 except for the use of an exposed platinum electrode. Addition of platelets caused a steady decline in oxygen concentration (Fig. 2A), and thrombin apparently caused a sharp increase in the rate of oxygen consumption, which resulted in a trace very similar to those previously reported (3). However, when the experiment was repeated
OF
BIOCHEMISTRY
AND
BIOPHYSICS
120
(1967)
Communications Conversion
of Oximes
Glucosides
to Mustard
hydroxylamine hydrochloride and sodium bicarbonate. The ether was removed with a stream of NZ and carrier phenylacetaldehyde was added. Phenylacetaldoxime-U-*4C separated out and was recrystallized twice from et,hyl ether/hexane. Both chemical and radiochemical yields were low; phenylacetonitrile and phenylacetamide were observed as by-products in the mother liquor. The phenylacetaldoxime was shown to be 98% radiochemically homogeneous upon thin-layer chromatography. Two experiments were carried out in which labelled compounds were administered in volumes less than 0.5 ml to the roots of plants of C. oficinalis previously grown to about 0.3 gm fresh weight in nutrient solution. In the first, or-ketoisovaleric acid oxime-U-l% or L-valine-U-l% (Radiochemical Centre, Amersham) was added to single plants, which were then exposed to continuous light for 21 hours. In the second experiment, isobutyraldoxime-UJ4C or L-valine-U-1% were administered to groups of three plants, which were enclosed in transparent containers (220 ml) to reduce evaporative losses of isobutyraldoxime-U1%. The plants were harvested after 48 hours including two 6.hour dark periods. In both cases, water was added to the plant roots as required. In the third experiment phenylalanine-U-l% or phenylacetaldoxime-UJ4C (first dissolved in 5 ~1 ethanol) were administered in 0.25 ml water to 30 six-day-old excised shoots of L. satioum. Water was added as required and the plants were exposed to continuous light for 48 hours. All plant tissue was extracted twice with boiling 80% aqueous ethanol and the radioactivity was determined. The combined ethanolic extracts were evaporated under reduced pressure and the residues were dissolved and suspended in small volumes of water for subsequent analysis. Glucosinolates were separated in two dimensions by thin-layer electrophoresis at pH 2.0 and chromatography with n-propanol:water:ethylacetate:acetic acid:pyridine (120:60 20:4:1) as mobile phase (9). Glucosinolates were detected with sprays of 0.05 M silver nitrate followed by 0.05 M potassium dichromate after drying at 100” or with bromocresol green indicator. Isopropylglucosinolate-‘4C was identified by chromatographic examination of isopropylthioureaJ4C formed from it (10).
Oil
(Glucosinolates)
Recently it has been shown that oximes may be intermediates in the biosynthesis of cyanogenic glycosides from amino acids. Tapper et al. (1) have shown t,hat isobutyraldoxime-U-l% is converted to linamarin-1% in Linum usitatissimum L. seedlings with high efficiency comparable to the corresponding conversion of L-valine-U-14C. In this communication we wish to report that isobutyraldoxime-U-‘*C is also incorporated into isopropylglucosinolate (glucoputranjivin) (I, R = isopropyl) in Cochlearia oficinalis L. plants and that phenylacetaldoxime-U-14C is incorporated into benzylglucosinolate (glucotropaeolin) (I, R = beneyl) in Lepidium sativum L. seedlings. NOSO,-
K +
NOH
R-g
R-:H
S-p
-D-Glucose
I
II Aldoximes
Glucosinolates SCHEME
1.
Incorporation experiments (Z-6) have shown that amino acids are precursors of several glucosinolate aglycones and also that a number of compounds other than amino acids or closely related compounds are ineffective as precursors. In particular phenylpyruvic acid oxime-14C was less effective than phenylalanine-14C as a precursor of bensylglucosinolate (4,7). We report here a similar finding with ol-ketoisovaleric acid oxime-U-‘*C as a precursor of isopropylglucosinolate. The or-ketoisovaleric acid oxime-U-K! (anti HO-COOH isomer) and isobutyraldoxime-U-14C were prepared as previously described (1). Phenylacet,aldehyde-U’% was obtained from L-phenylalanine-U-14C (1 mg, 15&, Radiochemical Centre, Amersham) by oxidation with N-bromosuccinimide (2 mg) for 15 minutes at 5” in aqueous solution (0.6 ml) in the presence of an excess of succinamide (50 mg) at pH 6.7 (8). The phenylacetaldehyde-U-14C was extracted into ethyl ether and added to an aqueous solution of an excess of 719
720
COMMUNICATIONS TABLE COMPARISON Expt.
Compound
Expt. 1
OF
PRECURSORS
OF
administered
I ISOPROPYLGLUCOSINOLATE Amount
n-Valine-U-W a-Ketoisovaleric acid oxime-U-i% L-Valine-U-i% n-Valine-U-l% Isobutyraldoxime-U-W
Expt. 2
sp. act. @C/mm&)
bmmole)
1.0
2490
1.2 4.0 1.0 3.8
1150 622 2490 390
% Converted to isopropylglucosinolate
0.87a <0.25* 0.70” 1.744 15.9
0 Corrected for assumed loss of i4COOH. TABLE COMPARISON
OF
L-PHENYLALANINE
AND
2
PHENYL.~CETALDOXIME
AS
PRECURSORS
OF
BENZYLGLUCOSINOLATE
Compound
administered
n-Phenylalanine-UJ’C Phenylacetaldoxime-U-14C
Amount
~mole)
1.1 1.2
sp. act. duC/mmole)
70 Converted benzylglucosinolate
440 23.1
4.3a 26
to
sp. act. of benzylthiourea WJmmole)
3.56 1.22
(1Corrected for assumed loss of i4COOH from n-phenylalanine-U-14C. Radioautography was used to locate compounds of interest on thin-layer chromatography plates. Incorporation of label into glucosinolates was determined by measuring radioactivity of compounds eluted after separation. The specific activity of the aglycone of benzylglucosinolate was measured by preparing benzylthiourea, by ammonia treatment of the benzyl isothiocyanate liberated by myrosinase. The amount of thiourea was determined by absorption at 244 rnp in ethanolic solution following purification by paper chromatography (10). All radioactivity was measured by liquid scintillation counting. Table I presents the results of the first and second experiments. The incorporation of n-valineW into isopropylglucosinolate, while not particularly high, is significant. Radioautographs of the separated metabolites showed that most radioactivity remained in the valine pool, with only small conversion to other compounds including leucine. No significant radioactivity was observed in other glucosinolates. Further, glucose separated by chromatography after myrosinase treatment of the purified isopropylglucosinolate-*4C contained negligible radioactivity. This suggests that n-valine is a specific precursor of the isopropylglucosinolate aglycone in C. oficinalis. Meakin (7) has reported a similar finding from a single preliminary experiment with Tropaeolum peregrinum which also contains isopropylglucosinolate. The incorporation from a-ketoisovaleric acid oxime-
W is low and confirms a previous finding that phenylpyruvic acid oxime was only weakly effective as a precursor of benzylglucosinolate (4, 7). Table I shows clearly that isobutyraldoxime is efficiently converted to isopropylglucosinolate. Radioautographs showed isopropylglucosinolate to be the most strongly labelled product from isobutyraldoxime-W, and only a trace of radioactivity was observed in other glucosinolates. Table II presents the result of the third experiment. Phenylalanine has previously been shown to be a precursor of benzylglucosinolate in Tropaeolum majus L. (2, 4). Our results confirm these findings in the unrelated L. sativum. Further, the high incorporation of radioactivity from phenylacetaldoxime-1% with comparatively low dilution suggests it to be a normal biosynthetic intermediate. The results reported in this communication, together with the finding of isobutyraldoxime as a precursor of the cyanogenic glucoside linamarin (l), and the unpublished findings of this laboratory that phenylacetaldoxime is a precursor of prunasin and prulaurasin in peach and cherry laurel shoots, respectively, all strongly suggest that aldoximes are normal intermediates in the formation of both cyanogenic glycosides and glucosinolates. Experiments with i6N-labelled amino acids have shown that the amino nitrogen is retained and is presumably oxidized during the biosynthesis of corresponding cyanogenic glycosides
COMMUNICATIONS (11, 12) and glucosinolates (4, 7). Therefore, any oxime intermediates cannot be formed by condensation of an aldehyde or ketone and any hypothetical bound or free form of hydroxylamine. This conclusion is supported by the finding that isobutyraldehyde was ineffective as a precursor of linamarin (1). The isolation of oximes from plant tissue and experiments with ‘SN-labelled compounds are required to confirm the suggested roles of oximes as intermediates. ACKNOWLEDGMENT The authors are indebted skillful technical assistance.
to W. D. Bennett
for
REFERENCES 1. TAPPER, B. A., CONN, E. E., AND BUTLER, G. W., Arch. Biochem. Biophys. 119, 593 (1967). 2. BENN, M. H., Chem. Znd. Lo@on 1907 (1962). M., PROCH~ZKA, Z., AND VERES, 3. KUT&EK, K . , Nature 194, 393 (1962). 4. UNDERHILL, E. W., AND CHISHOLM, M. D., Biochem. Biophys. Res. Commun. 14, 425 (1964). 5. SCHRAUDOLF, H., AND BERGMANN, F., Planta 67, 75 (1965). 6. CHISHOLM, M. D., AND WETTER, L. R., Can. J. Biochem. 44, 1625 (1966). of 7. MEAKIN, D., Ph.D. Thesis, Department Chemistry, University of Alberta at Calgary (1965). 8. GREENSTEIN, J. P., AND WINITZ, M., “Chemistry of the Amino Acids,” p. 1331. Wiley, New York (1961). 9. BIELESKI, R. L., AND TURNER, N. A., Anal. Biochem. 17,278 (1966). A., AND RUBINSTEIN, K., Acta Chem. 10. KjzR, Scand. 7,528 (1953.) 11. BUTLER, G. W., AND CONN, E. E., J. Biol. Chem. 239, 1674 (1964). 12. URIBE, E. G., AND CONN, E. E., J. Biol. Chem. 241, 92 (1966). B. A. TAPPER G. W. BUTLER Plant Chemistry Division, D.S.Z.R., and Department of Chemistry and Biochemistry Massey University Palmerston iVorth, New Zealand Received March 29, 1967
Failure
of Thrombin
to Affect
Platelet
Respiration Glycolysis has frequently been considered the primary pathway for ATP production in blood platelets (1, 2), partly because of evidence indi-
721
cating only a limited aerobic metabolism. The re port by Hussain and Newcomb (3) that addition of thrombin, which initiates the biochemical and morphological changes associated with clotting and clot retraction, caused an elevenfold increase in the rate of oxygen uptake by platelets was therefore significant. It indicated that platelets actually have a rather large capacity for aerobic metabolism under conditions of greatest energy requirement. In beginning a study of the role of respiration in platelet metabolism, I attempted to reproduce the experiments reported by Hussain and Newcomb. Through many attempts in which the procedures used were nearly identical to those reported or consisted of various modifications of those procedures, no evidence of any effect of thrombin on platelet respiration was observed. Other than a species difference, the only apparent explanation for the discrepancy wm my use of a Clark-type oxygen electrode (4), in which the stationary electrodes are immersed in a half-saturated KC1 solution that is separated from the test solution by a membrane permeable only to gases, whereas Hussain and Newcomb used a bare platinum electrode that dips directly into the test solution. An electrode similar (but not identical) to that used by Hussain and Newcomb was obtained and gave results similar to those reported. The evidence presented here indicates that the observation of a stimulation by thrombin of platelet respiration is an artifact of the electrode and that respiration is constant during thrombin-induced aggregation of platelets. Blood was collected from rats using siliconecoated glassware and 0.25 volume of ACD (acidcitrate-dextrose, NIH formula B) as anticoagulant, and was immediately cooled in an ice bath. Erythrocytes and white blood cells were removed by centrifugation at 1509 for 40 minutes. Platelets were sedimented from the supernatant fluid by centrifugation at 8oOg for 20 minutes and were washed twice in the platelet wash solution described by Hussain and Newcomb (3). Figure 1 is an example of a measurement of oxygen uptake using a Clark electrode. Addition of platelets initiated a decline in the concentration of oxygen. Thrombin had no effect on the rate of oxygen consumption but cyanide completely inhibited it. Figure 2 shows the result of experiments carried out under conditions identical to those in Fig. 1 except for the use of an exposed platinum electrode. Addition of platelets caused a steady decline in oxygen concentration (Fig. 2A), and thrombin apparently caused a sharp increase in the rate of oxygen consumption, which resulted in a trace very similar to those previously reported (3). However, when the experiment was repeated