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Conversion of Methionine Hydroxy Analogue to Methionine in the Chick
TURKEY STEAKS Turkey on the table the year round. Human Nutrition Research Division and Animal Husbandry Research Division, Agricultural Research Service, and Poultry Division, Agricultural Marketing Service, Washington, D.C. p. IS. Klose, A. A., H. L. Hanson and H. Lineweaver, 1950. The freezing preservation of turkey meat steaks. Food Tech. 4 : 71-74. Smith, F., 1948. Turkey for Truman becomes tradition. Turkey World, 2 3 : 16-17.
673
Snedecor, G. W., 1956. Statistical Methods. Iowa State Univ. Press, Ames. pp. 10, 237. Taylor, M. H., 1963. The effect method of cooking has on quality and tenderness in machine knit turkey steaks. Unpublished data, Kansas State Univ., Manhattan, Kans. Wheeler, E. S., 1949. Factors affecting the storage life of frozen turkey steaks and filets. M.S. Thesis, Kansas State College Library, Manhattan, Kans.
R.
S.
GORDON
Monsanto Company, St. Louis, Mo. AND I.
W.
SIZER
Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. (Received for publication September 28, 1964)
Q I N C E the hydroxy analogue of me^ thionine (2-hydroxy, 4-methylthiobutyric acid) will support the growth of rats and chickens (Block and Jackson, 1932; Gordon and Sizer, 1954; Bird, 1952) the mechanism of its conversion to methionine was investigated. The utilization by the rat of alpha keto methionine and its conversion to methionine in the presence of suitable amino donors (Cahill and Rudolph, 1942; Meister and Tice, 1950; Meister et al., 1952 Meister, 1954) suggest that the keto acid is an intermediary between the hydroxy analogue and methionine. IN VIVO STUDIES
Synthetic S35-labelled methionine hydroxy analogue (Blake and Wineman, 1956) and labelled methionine (Abbott Laboratories, Chicago, Illinois) were administered intravenously or encapsulated, admixed on a standard chick ration ("LC2 and LC2M," Gordon and Sizer, 1955), to New Hampshire cockerels, 4-6 weeks old maintained in individual metabolism cages.
Methionine was isolated from liver protein of the sacrificed chicks by the method of Weiss et al. (1955), counted, combusted to sulfate and recounted. The specific activities (based on sulfur and corrected for background, self-absorption and counter efficiency) were found to check within 0.5%. As seen in Table 1, injected S35 derived from DL-methionine hydroxy analogue is incorporated as efficiently into liver protein methionine as is S35 derived from L-methionine, and more efficiently than that from DL-methionine. Further information on the intermediary metabolism of S35 methionine and the S35 hydroxy analogue of methionine was obtained by studying the accumulation of S35 in blood and excreta after administering an oral dose of labelled compound (see Table 2). Again, L-methionine and DL-methionine hydroxy analogue gave similar results, while DL-methionine did not accumulate in the blood to the same degree, but instead appeared in high concentration in the excreta [preliminary work
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Conversion of Methionine Hydroxy Analogue to Methionine in the Chick
674
R. S. GORDON AND I. W. SIZER
TABLE 1.—Incorporation into liver protein of S3* from 1 DL-methionine, L-melhionine and methionine hydroxy analogue*
70
IN VITRO STUDIES Chicken liver mitochondria were prepared by the procedure of Schneider and Hogeboom (1950) using 0.25M sucrose. The supernatant was recentrifuged for 20 minutes at 35,000 r.p.m. before use. The mitochondria were washed twice with 0.25M sucrose. Figure 1 shows the oxidation of the hydroxy analogue of methionine by a mitochondrial preparation of chicken liver. H 2 0 2 formation was demonstrated by the marked increase in oxygen consumption when both catalase and ethyl alcohol were added to the system (Keilin and Hartree, 1945). The corresponding keto acid (AKM), 2-keto-4-methylmercapto butyric acid was isolated at the end of the reaction TABLE 2.—Accumulation of 5 3 5 from labelled Lmethionine, vi.-methionine and VL-hydroxy methionine in plasma and in excreta* L-Methionine DL-Methionine Plasma after O . S h r . after4hrs.
3.31 mg.% S. 01 m g . %
Excreta after 24 hrs.
5.6%
1.85 m g . % 3.61 m g . % 19.3%
DL-Hydroxy Methionine 3.16 mg.% 5.02 mg.% 8.4%
* A dose of 10 microcuries of S35 was fed to each of four chicks. T h e data in the table represent the average of the four.
-/
60
/
+ Catalase t Alcohol p
50
"
/
'
/
40
/ /
+ Catalase
^AT
_
^
30
20
10
/" 20
40 60 Time (min's.)
80
100
FIG. 1. Oxidation of methionine hydroxy analogue by mitochondria. The Warburg vessels in both experiments contained 1 ml. washed mitochondria, 10 u.M phosphate buffer, pH 7.6, 250 units Worthington beef liver catalase in a final volume of 3 ml. The vessel labelled "catalase + alcohol" contained 0.1 ml. absolute ethyl alcohol. The center well of both vessels contained 0.2 ml. 10% KOH. Not shown on the figure is the very low oxygen consumption of mitochondria in the absence of added substrate.
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from the mixture after deproteinization (Awapara, 1953) by the procedure described by Meister (1953) as the sodium DL-Methionine salt (I). It was then converted into the 2, Hydroxy L-Methionine DL-Methionine 4-dinitrophenyl-hydrazone (II). Anal: I, Analogue C5H7OsSNa. Calculated: C, 35, 29; H, 4.5; 460 273 4S21 S, 18.84. Found: 35.21, H, 4.18; S, 18.80. 398 286 408 474 345 387 II, CnH 12 0 6 N 4 S. Calculated: C, 40.24; H, 381 304 509 3.69; N, 17.07; S, 9.77. Found: 40.32, H, 428 302 Average 439 3.68; N, 16.98; S, 9.83. The liver supernatant also oxidized hy* 10 microcuries/500 g. body weight were injected and samples taken two hours later. Four droxy methionine to keto methionine chicks were used per labelled compound. 1 The data are expressed as CPMX10~ 2 /mg. iso- (Baker, 1952) which was identified by lated methionine and are the average of duplicate chromatography. An aliquot of the heated determinations per individual liver. deproteinized reaction mixture was chrowith D-methionine yielded much more vari- matographed using butanol, acetic acid, able results and are reported elsewhere (Gordon, 1965)]. •t 1 • 1 1 1
675
CONVERSION OF METHIONINE HYDROXY ANALOGUE
TABLE 3.—Formation of methionine and hydroxy methionine by supernatants from rat and chicken liver* AKM 1 Found
Methionine Found
Hydroxy Acid Found
Additions Rat
Chick
Rat
Chick
Rat
0.0 7.1 7.3 2.6
0.2 1.0 8.1 0.8
0.1 1.7 1.6 6.8
0.0 1.2 0.5 1.4
M-M
M.M
None 10 M.M AKM 10 fi.M A K M + 1 0 M-M glutamine 10 M.M A K M + 1 0 ii.M leucine
0.0 7.7 1.2 8.0
Chick
0.0 1.4 1.0 0.7
water, 4:1:5. Sulfur-containing compounds were detected with iodoplatinate (Winegard et al., 1948). The composition of the reaction mixture could be measured with an error of less than 10% when compared to standard amounts of methionine, hydroxy methionine and keto methionine (Fowler, 1951; Mori, 1954). When keto methionine was used as substrate, the liver supernatant was shown by paper chromatography to form methionine as well as the hydroxy acid. For comparison, this same experiment was performed using the supernatant from rat liver prepared in exactly the same manner as the chicken liver supernatant. It is apparent from Table 3 that this liver fraction of rat or chick can convert alpha keto methionine to hydroxy methionine and methionine. As is well known, glutamine acts as an amino donor and causes methionine production in the rat. On the other hand, leucine, as well as valine and isoleucine, act as amino donors for methionine production in the chick, whereas glutamine is ineffective (Jenkins and Sizer, 1956). Table 4 illustrates, in contrast to the preceding observations, that the kidney principally converts the D-amino acid into the corresponding alpha-keto analogue. Presumably, the keto acid is then transported to the liver for participation in
well-known transamination reactions, provided blood levels of keto acid are below levels in the kidney tubule (Mason and Berg, 1952). DISCUSSION The conversion of the hydroxy analogue of methionine to methionine in the chicken involves the corresponding keto acid as an intermediate. This appears to be the general route for interconversion of the alphahydroxy acids in amino acids in other species (Meister et al., 1952; Baker, 1952; Holden et al., 1951). The formation of hydrogen peroxide in vitro suggests that a fiavoprotein is responsible for the enzymatic oxidation of the hydroxy acid to the TABLE 4.—Conversion of DL-alpha-hydroxy, alphaketo, L- and D-alpha-amino {methionine) thiolbutyric acids by chick kidney homogenates Composition of Reaction Mixture After 30 mins.* Substrate
alpha-Hydroxy alpha-Keto L-alpha-amino D-alpha-amino
alphaKeto
alphaAmino
%
%
Trace 99 10 99+
0 0 88 Trace
alphaHydroxy % 99+ 1 1 0
* The procedure of Bender and Krebs (1950) as modified by Baker (1952) was used. Reaction products were measured chromatographically (Mori, 1954; Fowler, 1951). In each case, the alpha-keto acids were isolated as the corresponding phenylhydrazones.
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* Reaction mixture contains 50 ju.M N a / K pohsphate buffer, pH 7.6, 1 ml. supernatant, equivalent to 0.25 ml. liver, total volume 3 ml. Incubation 1.5 hours at 37°. Sulfur-containing compounds were measured chromatographically using iodoplatinate. 1 AKM: alpha keto methionine.
676
R. S. GORDON AND I. W. SIZEE
ammonia formed are probably excreted quite easily (Berg, 1959). High blood levels of keto acid would work against re-absorption of keto acid formed in the process of converting D-amino acids to L-amino acids. Rapid reduction of keto acid in the liver leads to a "pool" of alpha-hydroxy acids and helps conserve essential carbon skeletons until adequate nitrogen is available and/or the need for protein synthesis becomes pressing (see proposed mechanism in Figure 2). Differences observed in uptake and excretion of S35 labelled DL- and L-methionine are quite similar to the results most recently reported by Edwards et al. (1964) who demonstrated that the conversion of the Disomer of methionine to utilizable L-isomer was slower than the uptake of L-methionine into protein in rapidly growing animals. The results reported here are also quite similar to those of Kinsell et al. (1948) who reported a high rate of D-methionine excretion but a negligible excretion of L-methionine after initial infusion of DLmethionine. These workers also reported a more rapid disappearance of the D-isomer from the plasma of the test subjects. Such experiments indicate that there is a relatively high threshold for the natural isomer of methionine insofar as a tubular re-absorption is concerned. Likewise, Camien et al. (1951) orally administered DL-methionine to human subjects and recovered approximately 25% of the D-isomer in urine. At the same time, these workers reported that only a small amount of L-isomer was excreted. In general, it seems reasonable to suppose that if the hydroxy analogue of methionine had not been made synthetically, it would have turned up later as one of a series of naturally occurring hydroxy acids important in methionine, valine, isoleucine and leucine anabolism in the chicken. It was brought to our attention that Lan-
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keto acid. This study of chicken liver confirms the study by Robinson et al. (1962). These workers isolated an MN "long chain L-alpha-hydroxy acid oxidase" which aerobically oxidizes L-hydroxy methionine to keto methionine plus hydrogen peroxide. Considering the earlier results of Mahler et al. (1953) and Tubbs and Greville (1959-1961), demonstrating that D-alpha hydroxy acids are oxidized by an enzyme or enzyme system different from L-alphahydroxy oxidase, the evidence reported here furthers the view, first put forth by Block and Jackson (1932), that both isomers of the hydroxy analogue of methionine are completely utilized. The ability of leucine, valine, isoleucine and methionine to share an amino group (via transamination) is remarkable and probably reflects an evolutionary pressure unique to avian species. Furthermore, we have completed several preliminary experiments (similar to those of Crampton and Smythe, 1953) which indicate that the hydroxy analogues of leucine and methionine (but not the keto analogues) are actively re-absorbed in the kidney against concentration gradients. In their work, Crampton and Smythe (1953) demonstrated that the D-isomers of alanine and methionine are only re-absorbed when the concentration of the amino acid in blood is below the level in the kidney tubule. This could explain the lowered uptake of DL-methionine observed. The fact that enzymes exist which will convert keto acids in the liver to hydroxy acids, as well as the fact that these hydroxy acids appear to be actively re-absorbed, seem to be part of the same evolutionary pattern. This is important because the amino acid oxidases (and particularly the D-amino acid oxidases) are present in so much higher concentration in kidney than in liver (Krebs, 1951) that keto acids and
CONVERSION OF M E T H I O N I N E HYDROXY ANALOGUE
COOH
677 - COOH
I
CHNK-
CHOH CH 0 CH 2 -*
CH.
-CH(CH3)2,-CH2CH(CH3)2,-eH(CH3)C2H5
FIG. 2. Summary of interconversion of alpha-hydroxy, alpha-keto, alpha-amino methyl thiobutyric acid (methionine) illustrating role of leucine, valine and isoleucine as amino donors.
ger (1965) has a paper in press confirming these results and demonstrating the interconversion of methionine and the keto and hydroxy analogues of methionine. SUMMARY 1. When young chicks are fed L-methionine—S35 or DL-alpha-hydroxy methionine analogue—S35, the label appears rapidly in the blood and is incorporated into protein methionine at similar rates. Under similar conditions, DL-methionine—S35 is taken up in protein in lesser amounts and accumulates in larger amounts in the excreta. 2. Chick liver homogenate fractions catalyze the oxidation of hydroxy methionine to keto methionine. 3. Chick or rat supernatant fractions of liver can convert keto methionine to either hydroxy methionine or to methionine. The production of methionine is much greater when leucine (chick) or glutamine (rat) is added to the system. Further, in transamination reactions in chicken (but not rat), methionine, leucine, isoleucine and valine serve as amino donors for the alpha keto
acids corresponding to each of these amino acids. REFERENCES Awapara, J., 1953. 2-Aminoethanesulfinic acid: an intermediate in the oxidation of cysteine in vivo. J. Biol. Chem. 203: 183-188. Baker, C. G., 1952. Enzymatic oxidation of Dand L-alpha-hydroxy acids. Arch. Biochem. Biophys. 4 1 : 325-332. Bender, A. E., and H. A. Krebs, 1950. The oxidation of various synthetic alpha-amino acids by mammalian D-amino acid oxidase, L-amino acid oxidase of cobra venom and the L- and D-amino acid oxidase of "Neurospora Crassa." Biochem. J. 46: 210-219. Berg, C. P., 1959. Protein and Amino Acid Nutrition, Academic Press, N.Y.: 83. Bird, F., 1952. A comparison of methionine and two of its analogues in the nutrition of the chick. Poultry Sci. 3 1 : 1095-1096. Blake, E. S., and R. J. Wineman, 1956. Poultry Feed, U. S. Patent 2,745,745. Block, R. J., and R. W. Jackson, 1932. Metabolism of cystine and methionine. J. Biol. Chem. 97: cvi.-cvii. Cahill, W. M., and G. G. Rudolph, 1942. The replaceability of DL-methionine in the diet of the rat with its alpha-keto acid analogue. J. Biol. Chem. 145: 201-205. Camien, M. N., R. B. Malin and M. S. Dunn, 1951. Urinary excretion of ingested t - and DL-
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 10, 2015
R*
678
R. S. GORDON AND I. W. SIZEE 208-221. Mason, M., and C. P. Berg, 1952. The metabolism of D- and L-tryptophan and D- and L-kynurenine by liver and kidney preparations. J. Biol. Chem. 195: 515-524. Meister, A., and S. V. Tice, 1950. Transamination from glutamine to alpha-keto acids. J. Biol. Chem. 187: 173-187. Meister, A., H. A. Sober, S. V. Tice and P. E. Fraser, 1952. Transamination and associated deamidation of asparagine and glutamine. J. Biol. Chem. 197: 319-330. Meister, A., 1954. Enzymatic transamination reactions involving arginine and ornithine. J. Biol. Chem. 206: 587-596. Meister, A., 1953. Sodium alpha-ketoisocaproate (CH 3 ) 2 CHCH 2 COCOONa-MoI. Wt. 152.1 (CeHoOsNa). Biochemical Preparations, 3 : 66. Mori, I., 1954. A new technique for quantitative paper chromatography. Science, 119: 653-654. Robinson, J. C , L. Keay, R. Molinari and I. W. Sizer, 1962. L-alpha-hydroxy acid oxidases of hog renal cortex. J. Biol. Chem. 237: 20012010. Schneider, W. C , and G. H. Hogeboom, 1950. Intracellular distribution of enzymes-V. Further studies on the distribution of cytochrome c in rat liver homogenates. J. Biol. Chem. 183: 123— 128. Tubbs, P. K., and G. D. Greville, 1961. The oxidation of D-alpha-hydroxy acids in animal tissues. Biochem. J. 8 1 : 104-114. Tubbs, P. K., and G. D. Greville, 1959. Dehydrogenation of D-lactate by a soluble enzyme from kidney mitochondria. Biochemica Biophysica Acta, 34: 290-291. Weiss, S., E. I. Anderson, P. T. Hsu and J. Stekol, 1955. An adaptation of the Floyd-Lavine procedure for the isolation of methionine to tracer work. J. Biol. Chem. 214: 239-244. Winegard, H. M., G. Toennies and R. J. Block, 1948. Detection of sulfur-containing amino acids on paper chromatograms. Science, 108: 506-507.
NEWS AND NOTES (Continued from page 658) (5) Technical Problems of Producing Wholesome Foods (6) Assessment of Food Quality (7) Modern Trends in the Academic Training of Food Scientists and Technologists
(8) Economic, Nutritional and Sociological Aspects of Food Processing, Manufacture and Consumption. Special invited papers as well as freely con-
(Continued on page 716)
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methionines measured microbiologically. Arch. Biochem. 30: 62-65. Crampton, R. F., and D. H. Smythe, 1953. The excretion of the enantiomorphs of amino acids, J. Physiol. 122: 1-10. Edwards, G. A., C. H. Edwards and E. L. Gadsden, 1964. On the mechanism of transport of methionine to body tissues. Fed. Proc. 23 : 136. Fowler, H. D., 1951. Quantitative paper chromatography. Nature, 168: 1123-1124. Gordon, R. S., 1965. Metabolism of other D- and L-hydroxy acids. Ann. New York Acad. Sci. In press. Gordon, R. S., and I. W. Sizer, 1954. Nutritional equivalence of methionine of hydroxy analogue (MHA) and methionine for growth. Federation Proc. 13: 58-59. Gordon, R. S., and I. W. Sizer, 1955. Ability of sodium sulfate to stimulate growth of the chicken. Science, 122: 1270. Holden, J. T., R. B. Wildman and E. E. Snell, 1951. Growth promotion by keto and hydroxy acids and its relation to vitamin B6. J. Biol. Chem. 191: 559-576. Jenkins, W. T., and I. W. Sizer, 1956. Transamination of certain aliphatic amino acids by an enzyme from chicken liver. Federation Proc. 15: 283. Keilin, D., and E. F. Hartree, 1945. Prop, of catalase catalysis of coupled oxidation of alcohol. Biochem. J. 39: 293-301. Kinsell, L. W., H. A. Harper, H. D. Barton, M. E. Hutchin, and J. R. Hess, 1948. Studies in methionine and sulfur metabolism, Part I. J. Clin. Invest. 27: 677-688. Krebs, H. A., 1951. The Enzymes. Academic Press, N.Y., Vol. 2, Part 1: 509. Langer, B. W., Jr., 1965. The biochemical conversion of 2-hydroxy-4-methylthiobutyric acid to methionine by the rat in vitro. Biochem. J. In press. Mahler, H. R., A. Tomisek and F. M. Huennekens, 1953. Studies on the cyclophorase system XXVI. The lactic oxidase. Exptl. Cell Research,
673
Snedecor, G. W., 1956. Statistical Methods. Iowa State Univ. Press, Ames. pp. 10, 237. Taylor, M. H., 1963. The effect method of cooking has on quality and tenderness in machine knit turkey steaks. Unpublished data, Kansas State Univ., Manhattan, Kans. Wheeler, E. S., 1949. Factors affecting the storage life of frozen turkey steaks and filets. M.S. Thesis, Kansas State College Library, Manhattan, Kans.
R.
S.
GORDON
Monsanto Company, St. Louis, Mo. AND I.
W.
SIZER
Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. (Received for publication September 28, 1964)
Q I N C E the hydroxy analogue of me^ thionine (2-hydroxy, 4-methylthiobutyric acid) will support the growth of rats and chickens (Block and Jackson, 1932; Gordon and Sizer, 1954; Bird, 1952) the mechanism of its conversion to methionine was investigated. The utilization by the rat of alpha keto methionine and its conversion to methionine in the presence of suitable amino donors (Cahill and Rudolph, 1942; Meister and Tice, 1950; Meister et al., 1952 Meister, 1954) suggest that the keto acid is an intermediary between the hydroxy analogue and methionine. IN VIVO STUDIES
Synthetic S35-labelled methionine hydroxy analogue (Blake and Wineman, 1956) and labelled methionine (Abbott Laboratories, Chicago, Illinois) were administered intravenously or encapsulated, admixed on a standard chick ration ("LC2 and LC2M," Gordon and Sizer, 1955), to New Hampshire cockerels, 4-6 weeks old maintained in individual metabolism cages.
Methionine was isolated from liver protein of the sacrificed chicks by the method of Weiss et al. (1955), counted, combusted to sulfate and recounted. The specific activities (based on sulfur and corrected for background, self-absorption and counter efficiency) were found to check within 0.5%. As seen in Table 1, injected S35 derived from DL-methionine hydroxy analogue is incorporated as efficiently into liver protein methionine as is S35 derived from L-methionine, and more efficiently than that from DL-methionine. Further information on the intermediary metabolism of S35 methionine and the S35 hydroxy analogue of methionine was obtained by studying the accumulation of S35 in blood and excreta after administering an oral dose of labelled compound (see Table 2). Again, L-methionine and DL-methionine hydroxy analogue gave similar results, while DL-methionine did not accumulate in the blood to the same degree, but instead appeared in high concentration in the excreta [preliminary work
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 10, 2015
Conversion of Methionine Hydroxy Analogue to Methionine in the Chick
674
R. S. GORDON AND I. W. SIZER
TABLE 1.—Incorporation into liver protein of S3* from 1 DL-methionine, L-melhionine and methionine hydroxy analogue*
70
IN VITRO STUDIES Chicken liver mitochondria were prepared by the procedure of Schneider and Hogeboom (1950) using 0.25M sucrose. The supernatant was recentrifuged for 20 minutes at 35,000 r.p.m. before use. The mitochondria were washed twice with 0.25M sucrose. Figure 1 shows the oxidation of the hydroxy analogue of methionine by a mitochondrial preparation of chicken liver. H 2 0 2 formation was demonstrated by the marked increase in oxygen consumption when both catalase and ethyl alcohol were added to the system (Keilin and Hartree, 1945). The corresponding keto acid (AKM), 2-keto-4-methylmercapto butyric acid was isolated at the end of the reaction TABLE 2.—Accumulation of 5 3 5 from labelled Lmethionine, vi.-methionine and VL-hydroxy methionine in plasma and in excreta* L-Methionine DL-Methionine Plasma after O . S h r . after4hrs.
3.31 mg.% S. 01 m g . %
Excreta after 24 hrs.
5.6%
1.85 m g . % 3.61 m g . % 19.3%
DL-Hydroxy Methionine 3.16 mg.% 5.02 mg.% 8.4%
* A dose of 10 microcuries of S35 was fed to each of four chicks. T h e data in the table represent the average of the four.
-/
60
/
+ Catalase t Alcohol p
50
"
/
'
/
40
/ /
+ Catalase
^AT
_
^
30
20
10
/" 20
40 60 Time (min's.)
80
100
FIG. 1. Oxidation of methionine hydroxy analogue by mitochondria. The Warburg vessels in both experiments contained 1 ml. washed mitochondria, 10 u.M phosphate buffer, pH 7.6, 250 units Worthington beef liver catalase in a final volume of 3 ml. The vessel labelled "catalase + alcohol" contained 0.1 ml. absolute ethyl alcohol. The center well of both vessels contained 0.2 ml. 10% KOH. Not shown on the figure is the very low oxygen consumption of mitochondria in the absence of added substrate.
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from the mixture after deproteinization (Awapara, 1953) by the procedure described by Meister (1953) as the sodium DL-Methionine salt (I). It was then converted into the 2, Hydroxy L-Methionine DL-Methionine 4-dinitrophenyl-hydrazone (II). Anal: I, Analogue C5H7OsSNa. Calculated: C, 35, 29; H, 4.5; 460 273 4S21 S, 18.84. Found: 35.21, H, 4.18; S, 18.80. 398 286 408 474 345 387 II, CnH 12 0 6 N 4 S. Calculated: C, 40.24; H, 381 304 509 3.69; N, 17.07; S, 9.77. Found: 40.32, H, 428 302 Average 439 3.68; N, 16.98; S, 9.83. The liver supernatant also oxidized hy* 10 microcuries/500 g. body weight were injected and samples taken two hours later. Four droxy methionine to keto methionine chicks were used per labelled compound. 1 The data are expressed as CPMX10~ 2 /mg. iso- (Baker, 1952) which was identified by lated methionine and are the average of duplicate chromatography. An aliquot of the heated determinations per individual liver. deproteinized reaction mixture was chrowith D-methionine yielded much more vari- matographed using butanol, acetic acid, able results and are reported elsewhere (Gordon, 1965)]. •t 1 • 1 1 1
675
CONVERSION OF METHIONINE HYDROXY ANALOGUE
TABLE 3.—Formation of methionine and hydroxy methionine by supernatants from rat and chicken liver* AKM 1 Found
Methionine Found
Hydroxy Acid Found
Additions Rat
Chick
Rat
Chick
Rat
0.0 7.1 7.3 2.6
0.2 1.0 8.1 0.8
0.1 1.7 1.6 6.8
0.0 1.2 0.5 1.4
M-M
M.M
None 10 M.M AKM 10 fi.M A K M + 1 0 M-M glutamine 10 M.M A K M + 1 0 ii.M leucine
0.0 7.7 1.2 8.0
Chick
0.0 1.4 1.0 0.7
water, 4:1:5. Sulfur-containing compounds were detected with iodoplatinate (Winegard et al., 1948). The composition of the reaction mixture could be measured with an error of less than 10% when compared to standard amounts of methionine, hydroxy methionine and keto methionine (Fowler, 1951; Mori, 1954). When keto methionine was used as substrate, the liver supernatant was shown by paper chromatography to form methionine as well as the hydroxy acid. For comparison, this same experiment was performed using the supernatant from rat liver prepared in exactly the same manner as the chicken liver supernatant. It is apparent from Table 3 that this liver fraction of rat or chick can convert alpha keto methionine to hydroxy methionine and methionine. As is well known, glutamine acts as an amino donor and causes methionine production in the rat. On the other hand, leucine, as well as valine and isoleucine, act as amino donors for methionine production in the chick, whereas glutamine is ineffective (Jenkins and Sizer, 1956). Table 4 illustrates, in contrast to the preceding observations, that the kidney principally converts the D-amino acid into the corresponding alpha-keto analogue. Presumably, the keto acid is then transported to the liver for participation in
well-known transamination reactions, provided blood levels of keto acid are below levels in the kidney tubule (Mason and Berg, 1952). DISCUSSION The conversion of the hydroxy analogue of methionine to methionine in the chicken involves the corresponding keto acid as an intermediate. This appears to be the general route for interconversion of the alphahydroxy acids in amino acids in other species (Meister et al., 1952; Baker, 1952; Holden et al., 1951). The formation of hydrogen peroxide in vitro suggests that a fiavoprotein is responsible for the enzymatic oxidation of the hydroxy acid to the TABLE 4.—Conversion of DL-alpha-hydroxy, alphaketo, L- and D-alpha-amino {methionine) thiolbutyric acids by chick kidney homogenates Composition of Reaction Mixture After 30 mins.* Substrate
alpha-Hydroxy alpha-Keto L-alpha-amino D-alpha-amino
alphaKeto
alphaAmino
%
%
Trace 99 10 99+
0 0 88 Trace
alphaHydroxy % 99+ 1 1 0
* The procedure of Bender and Krebs (1950) as modified by Baker (1952) was used. Reaction products were measured chromatographically (Mori, 1954; Fowler, 1951). In each case, the alpha-keto acids were isolated as the corresponding phenylhydrazones.
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 10, 2015
* Reaction mixture contains 50 ju.M N a / K pohsphate buffer, pH 7.6, 1 ml. supernatant, equivalent to 0.25 ml. liver, total volume 3 ml. Incubation 1.5 hours at 37°. Sulfur-containing compounds were measured chromatographically using iodoplatinate. 1 AKM: alpha keto methionine.
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R. S. GORDON AND I. W. SIZEE
ammonia formed are probably excreted quite easily (Berg, 1959). High blood levels of keto acid would work against re-absorption of keto acid formed in the process of converting D-amino acids to L-amino acids. Rapid reduction of keto acid in the liver leads to a "pool" of alpha-hydroxy acids and helps conserve essential carbon skeletons until adequate nitrogen is available and/or the need for protein synthesis becomes pressing (see proposed mechanism in Figure 2). Differences observed in uptake and excretion of S35 labelled DL- and L-methionine are quite similar to the results most recently reported by Edwards et al. (1964) who demonstrated that the conversion of the Disomer of methionine to utilizable L-isomer was slower than the uptake of L-methionine into protein in rapidly growing animals. The results reported here are also quite similar to those of Kinsell et al. (1948) who reported a high rate of D-methionine excretion but a negligible excretion of L-methionine after initial infusion of DLmethionine. These workers also reported a more rapid disappearance of the D-isomer from the plasma of the test subjects. Such experiments indicate that there is a relatively high threshold for the natural isomer of methionine insofar as a tubular re-absorption is concerned. Likewise, Camien et al. (1951) orally administered DL-methionine to human subjects and recovered approximately 25% of the D-isomer in urine. At the same time, these workers reported that only a small amount of L-isomer was excreted. In general, it seems reasonable to suppose that if the hydroxy analogue of methionine had not been made synthetically, it would have turned up later as one of a series of naturally occurring hydroxy acids important in methionine, valine, isoleucine and leucine anabolism in the chicken. It was brought to our attention that Lan-
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keto acid. This study of chicken liver confirms the study by Robinson et al. (1962). These workers isolated an MN "long chain L-alpha-hydroxy acid oxidase" which aerobically oxidizes L-hydroxy methionine to keto methionine plus hydrogen peroxide. Considering the earlier results of Mahler et al. (1953) and Tubbs and Greville (1959-1961), demonstrating that D-alpha hydroxy acids are oxidized by an enzyme or enzyme system different from L-alphahydroxy oxidase, the evidence reported here furthers the view, first put forth by Block and Jackson (1932), that both isomers of the hydroxy analogue of methionine are completely utilized. The ability of leucine, valine, isoleucine and methionine to share an amino group (via transamination) is remarkable and probably reflects an evolutionary pressure unique to avian species. Furthermore, we have completed several preliminary experiments (similar to those of Crampton and Smythe, 1953) which indicate that the hydroxy analogues of leucine and methionine (but not the keto analogues) are actively re-absorbed in the kidney against concentration gradients. In their work, Crampton and Smythe (1953) demonstrated that the D-isomers of alanine and methionine are only re-absorbed when the concentration of the amino acid in blood is below the level in the kidney tubule. This could explain the lowered uptake of DL-methionine observed. The fact that enzymes exist which will convert keto acids in the liver to hydroxy acids, as well as the fact that these hydroxy acids appear to be actively re-absorbed, seem to be part of the same evolutionary pattern. This is important because the amino acid oxidases (and particularly the D-amino acid oxidases) are present in so much higher concentration in kidney than in liver (Krebs, 1951) that keto acids and
CONVERSION OF M E T H I O N I N E HYDROXY ANALOGUE
COOH
677 - COOH
I
CHNK-
CHOH CH 0 CH 2 -*
CH.
-CH(CH3)2,-CH2CH(CH3)2,-eH(CH3)C2H5
FIG. 2. Summary of interconversion of alpha-hydroxy, alpha-keto, alpha-amino methyl thiobutyric acid (methionine) illustrating role of leucine, valine and isoleucine as amino donors.
ger (1965) has a paper in press confirming these results and demonstrating the interconversion of methionine and the keto and hydroxy analogues of methionine. SUMMARY 1. When young chicks are fed L-methionine—S35 or DL-alpha-hydroxy methionine analogue—S35, the label appears rapidly in the blood and is incorporated into protein methionine at similar rates. Under similar conditions, DL-methionine—S35 is taken up in protein in lesser amounts and accumulates in larger amounts in the excreta. 2. Chick liver homogenate fractions catalyze the oxidation of hydroxy methionine to keto methionine. 3. Chick or rat supernatant fractions of liver can convert keto methionine to either hydroxy methionine or to methionine. The production of methionine is much greater when leucine (chick) or glutamine (rat) is added to the system. Further, in transamination reactions in chicken (but not rat), methionine, leucine, isoleucine and valine serve as amino donors for the alpha keto
acids corresponding to each of these amino acids. REFERENCES Awapara, J., 1953. 2-Aminoethanesulfinic acid: an intermediate in the oxidation of cysteine in vivo. J. Biol. Chem. 203: 183-188. Baker, C. G., 1952. Enzymatic oxidation of Dand L-alpha-hydroxy acids. Arch. Biochem. Biophys. 4 1 : 325-332. Bender, A. E., and H. A. Krebs, 1950. The oxidation of various synthetic alpha-amino acids by mammalian D-amino acid oxidase, L-amino acid oxidase of cobra venom and the L- and D-amino acid oxidase of "Neurospora Crassa." Biochem. J. 46: 210-219. Berg, C. P., 1959. Protein and Amino Acid Nutrition, Academic Press, N.Y.: 83. Bird, F., 1952. A comparison of methionine and two of its analogues in the nutrition of the chick. Poultry Sci. 3 1 : 1095-1096. Blake, E. S., and R. J. Wineman, 1956. Poultry Feed, U. S. Patent 2,745,745. Block, R. J., and R. W. Jackson, 1932. Metabolism of cystine and methionine. J. Biol. Chem. 97: cvi.-cvii. Cahill, W. M., and G. G. Rudolph, 1942. The replaceability of DL-methionine in the diet of the rat with its alpha-keto acid analogue. J. Biol. Chem. 145: 201-205. Camien, M. N., R. B. Malin and M. S. Dunn, 1951. Urinary excretion of ingested t - and DL-
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R*
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R. S. GORDON AND I. W. SIZEE 208-221. Mason, M., and C. P. Berg, 1952. The metabolism of D- and L-tryptophan and D- and L-kynurenine by liver and kidney preparations. J. Biol. Chem. 195: 515-524. Meister, A., and S. V. Tice, 1950. Transamination from glutamine to alpha-keto acids. J. Biol. Chem. 187: 173-187. Meister, A., H. A. Sober, S. V. Tice and P. E. Fraser, 1952. Transamination and associated deamidation of asparagine and glutamine. J. Biol. Chem. 197: 319-330. Meister, A., 1954. Enzymatic transamination reactions involving arginine and ornithine. J. Biol. Chem. 206: 587-596. Meister, A., 1953. Sodium alpha-ketoisocaproate (CH 3 ) 2 CHCH 2 COCOONa-MoI. Wt. 152.1 (CeHoOsNa). Biochemical Preparations, 3 : 66. Mori, I., 1954. A new technique for quantitative paper chromatography. Science, 119: 653-654. Robinson, J. C , L. Keay, R. Molinari and I. W. Sizer, 1962. L-alpha-hydroxy acid oxidases of hog renal cortex. J. Biol. Chem. 237: 20012010. Schneider, W. C , and G. H. Hogeboom, 1950. Intracellular distribution of enzymes-V. Further studies on the distribution of cytochrome c in rat liver homogenates. J. Biol. Chem. 183: 123— 128. Tubbs, P. K., and G. D. Greville, 1961. The oxidation of D-alpha-hydroxy acids in animal tissues. Biochem. J. 8 1 : 104-114. Tubbs, P. K., and G. D. Greville, 1959. Dehydrogenation of D-lactate by a soluble enzyme from kidney mitochondria. Biochemica Biophysica Acta, 34: 290-291. Weiss, S., E. I. Anderson, P. T. Hsu and J. Stekol, 1955. An adaptation of the Floyd-Lavine procedure for the isolation of methionine to tracer work. J. Biol. Chem. 214: 239-244. Winegard, H. M., G. Toennies and R. J. Block, 1948. Detection of sulfur-containing amino acids on paper chromatograms. Science, 108: 506-507.
NEWS AND NOTES (Continued from page 658) (5) Technical Problems of Producing Wholesome Foods (6) Assessment of Food Quality (7) Modern Trends in the Academic Training of Food Scientists and Technologists
(8) Economic, Nutritional and Sociological Aspects of Food Processing, Manufacture and Consumption. Special invited papers as well as freely con-
(Continued on page 716)
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methionines measured microbiologically. Arch. Biochem. 30: 62-65. Crampton, R. F., and D. H. Smythe, 1953. The excretion of the enantiomorphs of amino acids, J. Physiol. 122: 1-10. Edwards, G. A., C. H. Edwards and E. L. Gadsden, 1964. On the mechanism of transport of methionine to body tissues. Fed. Proc. 23 : 136. Fowler, H. D., 1951. Quantitative paper chromatography. Nature, 168: 1123-1124. Gordon, R. S., 1965. Metabolism of other D- and L-hydroxy acids. Ann. New York Acad. Sci. In press. Gordon, R. S., and I. W. Sizer, 1954. Nutritional equivalence of methionine of hydroxy analogue (MHA) and methionine for growth. Federation Proc. 13: 58-59. Gordon, R. S., and I. W. Sizer, 1955. Ability of sodium sulfate to stimulate growth of the chicken. Science, 122: 1270. Holden, J. T., R. B. Wildman and E. E. Snell, 1951. Growth promotion by keto and hydroxy acids and its relation to vitamin B6. J. Biol. Chem. 191: 559-576. Jenkins, W. T., and I. W. Sizer, 1956. Transamination of certain aliphatic amino acids by an enzyme from chicken liver. Federation Proc. 15: 283. Keilin, D., and E. F. Hartree, 1945. Prop, of catalase catalysis of coupled oxidation of alcohol. Biochem. J. 39: 293-301. Kinsell, L. W., H. A. Harper, H. D. Barton, M. E. Hutchin, and J. R. Hess, 1948. Studies in methionine and sulfur metabolism, Part I. J. Clin. Invest. 27: 677-688. Krebs, H. A., 1951. The Enzymes. Academic Press, N.Y., Vol. 2, Part 1: 509. Langer, B. W., Jr., 1965. The biochemical conversion of 2-hydroxy-4-methylthiobutyric acid to methionine by the rat in vitro. Biochem. J. In press. Mahler, H. R., A. Tomisek and F. M. Huennekens, 1953. Studies on the cyclophorase system XXVI. The lactic oxidase. Exptl. Cell Research,