485

Biochem. J. (1975) 147, 485491 Printed in Great Britain

Purification and Properties of S-Alkyl-L-cysteine Lyase from Seedlings of Acaciafarnesiana Willd. By MENDEL MAZELIS and RICHARD K. CREVELING Department ofFood Science and Technology, University ofCalifornia, Davis, Calif. 95616, U.S.A. (Received 8 November 1974)

1. An S-alkyl-L-cysteine lyase (EC 4.4.1.6) was purified to apparent homogeneity from extracts of acetone-dried powders of the hypocotyls of etiolated 5-day-old seedlings of Acacia farnesiana Willd. 2. The enzyme catalyses a fl-elimination reaction and will utilize both the thioether and sulphoxide form ofthe substrate. 3. There is a broad specificity with regard to the alkyl substituent, but cystathionine is utilized very poorly. 4. The pH optimum is 7.8 and the Km value for the probable natural substrate L-djenkolate is 0.3 mM. 5. Both sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and ultracentrifugal analysis give a molecular weight of about 144000. 6. One mol of pyridoxal phosphate is bound/mol of enzyme. 7. The energy of activation with L-djenkolate as the substrate is 53.1 kJ/mol. 8. The enzyme has a partial specific volume of 0.56 and s20,w of 7.26S. A large number ofS-substituted cysteines have been identified among the non-protein nitrogen constituents of higher plants (Fowden, 1964). Both thioether and sulphoxide derivatives have been found. The enzyme alliin lyase (EC 4.4.1.4) has been described in a number of plant species (Stoll & Seebeck, 1951; Tsuno, 1958; Schwimmer et al., 1960; Kupiecki & Virtanen, 1960; Mazelis, 1963; Jacobsen et al., 1968). This enzyme utilizes only the sulphoxide derivatives as substrates according to the following overall reaction:

Experimental

0

2R-S-CH2-CH-C02 +H20 I NH3+

0

R-S-S-R+2CH3-C-C02

+

2NH4+

(1)

11 0

Another enzyme, alkylcysteine lyase (EC 4.4.1.6), has been found in several higher-plant species (Schwimmer & Kjaer, 1960; Mazelis & Fowden, 1973) and bacteria (Nomura et al., 1963; Murakami, 1960), which will cleave both the thioether and the sulphoxide derivative by fl-elimination. With the thioether as substrate the reaction proceeds as follows: R-S-CH2-CH-02- +H20 NH3+

RSH+ CH3-C--CO2- +NH4+

11

0 Vol. 147

Schwimmer & Kjaer (1960) obtained a very active alkylcysteine lyase preparation from the endosperm of seeds of Albizzia lophanta, but its purity was not known. We have reported the presence of a similar enzyme in the hypocotyl tissue of young etiolated seedlings of Acaciafarnesiana (Mazelis & Creveling, 1973). The present paper describes the purification of this enzyme essentially to homogeneity, and its kinetic and physical properties.

(2)

Enzyme assay The usual assay mixture consisted of 175mMTricine [N-tris(hydroxymethyl)methylglycine] buffer, pH7.8, 25,pM-pyridoxal phosphate, lOmM-L-djenkolate or 40mM-S-methyl-L-cysteine, and enzyme in a final volume of ml. After incubation in a water bath at 30°C for the desired time, Usually lOmin, the reaction was stopped by the addition of 3ml of 10% (w/v) trichloroacetic acid. Any precipitate that formed was removed by centrifugation and a portion of the reaction mixture analysed for pyruvate colorimetrically (Friedemann & Haugen, 1943) or for NH3. To determine NH3 0.5ml of 10% (w/v) trichloroacetic acid was added to a portion of the inactivated reaction mixture and the total volume made up to 3 ml with water, then I ml of Nessler's reagent was added and after 10min, the absorbance was measured at 435nm and compared with a standard curve obtained with (NH4)2SO4. One unit of enzyme activity produced 1 umol of product/min. Protein concentrations were determined by u.v.absorbance measurements at 280 and 260nm (Layne, 1957).

486 Germination of seedlings anid acetone-dried powder preparation The seeds of A. farnesiana were obtained from trees in the arboretum of the University of California, Davis. Seeds were gathered in every year from 1969 to 1972 and each crop was stored individually. Because of the toughness of the seed coat, the seeds were scarified in conc. H2SO4 for 90min with occasional stirring. The acid was decanted and the seeds were washed with large amounts of cold water. They were then aerated in a large beaker with cold water overnight and planted in trays of vermniculite the next day. After germination at 30°C for 5 days in the dark, the cotyledons were removed and discarded. The hypocotyls plus roots were washed in cold water, the excess of water was removed by blotting with paper towels, then the plant material was weighed and placed in a chilled Waring Blendor. Acetone (4-S5vol., v/w at -15°C) was added and the mixture blended at high speed for 30-60 s. The slurry was filtered and the precipitate washed with acetone while still on the filter paper by use of a filter flask. The precipitate was dried on the filter paper under suction and then spread out on paper towels at room temperature (21°C) overnight. The resulting product was ground in a mortar and pestle to a fine powder and stored in dark bottles at 100. This powder was the starting material for all enzyme isolations and purifications. A typical preparation would give about 30g of powder from 500g of hypocotyls plus roots.

Gel electrophoresis Discontinuous-gel electrophoresis was carried out by using 10 % (w/v) acrylamide gels containing 0.03 % N,N'-methylenebisacrylamide which stack at pH8.9 and run at pH9.5 as described in the Canalco catalogue (Disc Electrophoresis, 1974) or the 10% or 12.5 % discontinuous sodium dodecyl sulphatepolyacrylamide-gel systems of Laemrni (1970) as modified by Traut et al. (1973). After electrophoresis the gels were stained with 0.25 % Coomassie Blue in 5% (v/v) methanol and 7.5% (v/v) acetic acid. Gel densitometry was done with a Gilford spectrophotometer and a model 2410 linear transport accessory scanning at SSOnm for Coomassie Blue.

Amino acid analysis Protein samples were hydrolysed in 6M-HCI in sealed tubes at 100°C for 24 and 72h. The samples were dried under vacuum in a desiccator, and redissolved in water to give a concentration equivalent to O.1mg of protein/mi. Portions of the hydrolysed sample were analysed on a Beckman 121 amino acid analyser with an expanded scale. Tryptophan was determined on an unhydrolysed protein sample by the spectrophotometric method of Edelhoch (1967).

M. MAZELIS AND R. K. CREVELING

Ultracentrifugation analysis The sedimentation coefficient s was determined by boundary sedimentation in a Beckman model E analytical ultracentrifuge equipped with a photoelectric scanner, The rotor velocity was 60000rev./ min at 200C, A double-sector cell was used and the cell was scanned at 4min intervals. Chemicals L-Djenkolate sulphoxide was a gift from Dr. L. Fowden, Dept. of Botany, University College, London, U,K.; S-methyl-L-cysteine sulphoxide was prepared by oxidation of S-methyl-L-cysteine with acid H202 (Stoll & Seebeck, 1951). The product was recrystallized from ethanol-water solutions. SPropyl-L-cysteine was a gift from Dr. J. F. Carson, Western Regional Research Laboratory, Albany, Calif., U.S.A.; L-cysteine S-sulphate was synthesized by the method of Segel & Johnson (1963). /-Cyano-Lalanine was a gift from Dr. E. E. Conn, Department of Biochemistry, University of California, Davis, Calif., U.S.A. All other chemicals were purchased from commercial sources. The NH3 colour reagent was purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. and used directly in nesslerization assays. Results Purification procedure All operations were done in the cold (4-100C) unless otherwise stated. Acetone-dried powder (5g) was suspended in 500ml of 0.05M-potassium phosphate, pH7.2, and stirred for 30min. The suspension was strained through cheesecloth and the filtrate centrifuged for 30min at 23000g. The supernatant solution was decanted and the volume measured. Foreach lOOml, lOml of 1 % (w/v) protamine sulphate was added slowly and the mixture stirred for 15min, followed by centrifugation at 25000g for 15min. The supernatant solution was decanted and 17.6g of solid (NH4)2SO4/lOOml added slowly with stirring to give a final saturation of 30%. The precipitate that formed was removed by centrifugation and discarded then 16.2g of (NH4)2SO4/lOOmn was added to give a saturation of 55%. The precipitate that appeared was collected by centrifugation and dissolved in 0.01 m-potassium phosphate, pH 7.2. This solution was placed on a column (2.5 cm x 75 cm) of Sephadex G-200, eluted with 0.01 M-potassium phosphate, pH 7.2, and 9.5 ml fractions were collected. The fractions containing enzyme having at least twice the specific activity of the starting material on the column were combined and the entire volume was placed on a column (2cm x 24cm) of DEAE-cellulose. Elution of the enzyme was carried out with a 0.05MTris-HCI buffer, pH 8.0, and a gradient of increasing NaCl concentration which was prepared by connect1975

487

S-ALKYL-L-CYSTEINE LYASE Table 1. Summary ofpurification procedure

S-Methyl-L-Cysteine was used as the substrate for determining activity. The assay was done as described in the Experimental section. Recovery Total vol. Total protein Total activity Specific activity (%) (units/mg) Purification (units) (mg) (nil) Fraction 100 1.0 1.1 2617 2379 390 1. Acetone-dried powder extract 2. Protamine treatment; supernatant solution 3. 30-55Y/-satd. (NH4)2SO4 precipitate 4. Sephadex G-200 combined filtrate 5. DEAE-cellulose eluate Tube 32 Tube 33

406

1705

2046

1.2

1.1

78

16

238

1904

8.0

7.3

72

63

88

1637

20

18

62

14 16

560 533

40 33

36

21 20

6.5 6.5

30

q .4

I *w

a

0

Tube no.

Fig, 1. Comparison between the absorbance at 280nm (--) and enzyme activity (-- --) of the eluate from the DEAEcellulose column For details see the text. ing a reservoir solution of 0,05M-Tris-HCl, pH18.0, containing 1 .0M-NaCl to a mixing chamber holding 20Qml of 0.05M-Tris-HCI, pH18.0. The elution rate was adjusted to about 32ml/h and 6.5m1 fractions were collected, The test tubes having the highest activity were located and kept individually at -15°C, andwerethawedasneededtoremoveenzyme samples. The enzyme was very stable under these conditions, retaining complete activity for over 5 months even with repeated thawing and refreezing. Table 1 summarizes a typical purification. For convenience the substrate used for enzyme assay in this case was S-methyl-L-cysteine. In other cases L-djenkolate was used with the same results. Enzyme purity The elution profile of the enzyme obtained from the DEAE-cellulose column is sbown in Fig. 1. Vol. 147

Front (+)

Fig. 2. Densitometer tracing of a polyacrylamide disc gel with 40kg of enzyme protein stained with Coomassie Blue The most active fraction from the DEAE-cellulose column was used.

The fractions with the highest specific activity were examined by disc-gel electrophoresis. Fig. 2 shows a densitometric recording of a gel on which 40pg of protein was electrophoresed, There is one symmetrical sharp band of protein, and the enzyme by this criterion is essentially homogeneous. In other preparations there was sometimes a small band immediately behind the main band, but only 5-10% the size of the main peak. pH optimum Activity with L-djenkolate as the substrate was measured at various pH values with several different

M. MAZELIS AND R. K. CREVELING

488 buffers such as phosphate, Hepes [2-(N-2-hydroxyethylpiperazin-N'-yl)ethanesulphonic acid], Tris and Tricine. The optimum pH appeared to be in the region 7.7-7.9 in every case. However, the absolute value of the activity varied with the buffer used. Tricine gave the highest values and was used consistently in the assay procedure. Substrate specificity Table 2 summarizes the results obtained in determining the specificity of the enzyme with regard to the substituent replacing the thiol proton. There is a wide variety of structures that allow cleavage to proceed at a significant rate. Conversion of the thioester into the sulphoxide does not imnpair the ability of the enzyme to utilize the substrate. In fact, with L-djenkolate, which might be considered the natural substrate, conversion into the sulphoxide appears to enhance the rate of utilization markedly. It is ofconsiderable interest that L-cystathionine, which can be considered a substituted homologue of L-djenkolate, is a relatively poor substrate for this enzyme. fl-Cyano-L-alanine was tested as an inhibitor of the lyase activity. It was extremely effective, giving an inhibition of over 70% at 1 mm concentration. The K1 value for /J-cyano-L-alanine was determined by the graphical method of Dixon (1953). The fit of the data to a straight line was by use of a computer program for a linear regression curve. The correlation coefficient for the data from one substrate concentration was 0.97 and for the second substrate concentration 0.99. The Kt obtained was 304uM. Products of the reaction With L-djenkolate and S-methyl-L-cysteine as substrates the amount of pyruvate produced under Table 2. Substrate specificity The utilization of each substrate was measured at the same concentration and under the same conditions as the reference compound L-djenkolate. Relative activity Compound 100 L-Djenkolate 144 L-Djenkolate sulphoxide 13 L-Cystathionine DL-Allocystathionine

5

L-Cysteic acid

5

S-Methyl-L-cysteine

51

S-Methyl-L-cysteine sulphoxide

38 36 36 90 81 52 3 46

S-Methyl-L-cysteine

S-Propyl-L-cysteine L-Cysteine-S-sulphate

L-Cystine S-Carbamyl-L-Cysteine S-Carboxymethyl-L-cysteine

S-Benzyl-L-cysteine

Table 3. Effect ofcommon inhibitors ofpyridoxalphosphate reactions The components of the reaction mixture were as described in the Experimental section but including the inhibitor where listed. Enzyme (4,ug of protein) with a specific activity of 36units/mg was used in each case. (Concn. (mM) Relative activity Inhibitor None

NH20H KCN

Isonicotinic acid hydrazide Amino-oxyacetic acid

0.1 1.0 1.0 3.0 1.0 3.0 0.1 1.0

100 70 7 50 25 96 87 26 5

the usual assay conditions was compared with the amount of NH3. In both cases the amounts were equivalent. Since the colorimetric 2,4-dinitrophenylhydrazine assay is not specific for pyruvate, a largescale preparation was made to recover sufficient product to characterize chemically. S-Methyl-Lcysteine (1 mmol) was dissolved in 5ml of water and the solution adjusted to pH7.8, 125nmol of pyridoxal phosphate and 8units of step-5 enzyme were added and incubated at 30°C. At 60min another 8 units of enzyme were added. Samples ofthe reaction mixture were withdrawn at intervals and assayed for pyruvate by the usual method. After 200min the reaction had gone to completion. Then 200ml of 0.2% 2,4-dinitrophenylhydrazine in 2M-HCI was added to the reaction mixture and the entire solution placed in the cold. After 4 days the mixture was filtered and the precipitate washed with water and dried. The yield was 211mg, a yield of 80% of the theoretical value. The product was recrystallized from ethanol and water. A known sample of pyruvate 2,4-dinitrophenylhydrazone was prepared in a similar manner. Themelting point ofthe product from the enzymic reaction and the known pyruvate derivative was 216°C, which conforms with the literature value (Handbook of Biochemistry, 1968). The i.r. spectra of the known pyruvate derivative and the enzymic product were measured and found to be identical.

Pyridoxal phosphate requirement In the absence of exogenous pyridoxal phosphate the activity is decreased by 10-25 %. The effect of a number of inhibitors of reactions involving pyridoxal phosphate are shown in Table 3. Hydroxylamine and CN- were both effective at 1 mM. Amino-oxyacetic acid was the most effective inhibitor tested. The spectrum of the enzyme at pH7.2 showed a small absorbance peak at 498 nm and a larger one at 1975

489

S-ALKYL-L-CYSTEINE LYASE 420nm, in addition to the usual strong protein absorbance at 280nm. The ratio E280/E420 is 11. The absorption spectrum did not show any significant shifts on the addition of L-cysteine. Samples of an enzyme preparation having a specific activity of 33 units/mg of protein were assayed for bound pyridoxal phosphate by the colorimetric procedure of Wada & Snell (1961): 6nmol of pyridoxal phosphate/ mg of protein was found. On the basis of a molecular weight of approx. 145000 (the evidence for which is presented below) this gives a value of 0.9mol of cofactor/mol of protein. Kinetic constants The Michaelis constants for both L-djenkolate and S-methyl-L-cysteine used as substrates were determined. The data were examined by use of a computer program for a linear regression curve by using a straight-line form of the Michaelis-Menten equation. The correlation coefficient in each case was greater than 0.99. The K, for L-djenkolate was 0.3 mM

and Vmax. of 76units/mg. With S-methyl-L-cysteine as the substrate a Km of 5.3 mM and Vn,ax. of 62 units/mg were obtained. The activation energy for the lyase reaction with L-djenkolate was determined by means of the Arrhenius relationship between velocity and temperature. From the slope of the straight line obtained by plotting the inverse of the reaction rate against the inverse of the absolute temperature (Fig. 3), the activation energy calculated is 53.1 kJ/mol. The slope was obtained by use of the same program for a linear regression curve above, and the correlation coefficient was greater than 0.99.

60

11-

40 4,

bo 201

0

x e0

8 _1

20 30

31

32

33

34

35

104/T Fig. 3. Determination of the activation energy for the enzymic cleavage of L-djenkolate by using the Arrhenius equation The reaction components were as described in the Experimental section, with 4,ug of enzyme having a specific activity of 30units/mg. Incubation time was 5min. V is umol of pyruvate produced. Vol. 147

I.C

0.6 0

v~

0.2

v

t

Origin

(-)

Origin

(+)

Fig. 4. Densitometer tracing of sodium dodecyl sulphatetreated lyase on sodium dodecyl sulphate-12.5% (w/v) polyacrylamide disc gel after staining with Coomassie Blue For details see the text.

Physical properties The partial specific volume of the protein was obtained by means of a digital precision densitometer by using the procedure of Kratky et al. (1969). A value of 0.56 was calculated for the lyase. The 520,w was determined from the experimental s value by using the partial specific volume as determined experimentally and is 7.26S. This would correspond to an estimated molecular weight of 142000. A sample of the purified lyase was examined by sodium dodecyl sulphate-polyacrylamide-disc-gel electrophoresis. A densitometer tracing of the gel is depicted in Fig. 4. The results show that the lyase consists of two dissimilar subunits of different molecular weights. By using proteins of known molecular weight and sodium dodecyl sulphatepolyacrylamide-gel electrophoresis a linear relationship was obtained between molecular weight and mobility. The marker proteins used and their molecular weights were conalbumin (75000), bovine serum albumin (68000), ovalbumin (48000), carboxypeptidase A (34000) and trypsin (23300). The larger subunit of the lyase has a molecular weight of 96000 and the smaller 48 000. The molecular weight of the oligomer, 144000, agrees closely with the molecular weight calculated from the ultracentrifuge data. Amino acid composition The results of the amino acid analysis of a sample of

the enzyme protein that had been hydrolysed in

490

M. MAZELIS AND R. K. CREVELING Table 4. Aminio acid compositin ofpuirified lyase The hydrolysis of the protein and analysis of the constituent amino acids are described in the text. Content

(pmol/mg) Lysine Histidine Arginine Tryptophan Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cysteine Valine Methionine Isoloucine Leucine Tyrosine Phenylalanine

(24h) 0.43 0.12 0.17

(Umol/mg)

(Corrected or averaged)

(72h)

(,mol/mg)

0.42 0.11 0.16

0.43 0.12 0.17 0.12* 0.66 0.42t 0.59t 0.47 0.34 0.41 0.46 0.016 0.35 0.11 0.34 0.52 0.23t 0.18

0.66 0.41 0.57 0.45 0.32 0.41 0.47 0.008 0.33 0.12 0.32 0.56 0.21 0.19

0.66 0.39 0.53 0.49 0.36 0.40 0.45 0.005 0.37 0.10 0.36 0.54 0.16 0.17 * See the Experimental section for method of determination. t Extrapolated to zero time of hydrolysis, by assuming first-order kinetics.

6M-HCI are given in Table 4. The number of residues of each amino acid was calculated from the ,umol of amino acid/sample weight and on the basis of an enzyme molecular weight of 144000. Discussion The enzyme described in the present report appears to be very similar to that found in Albizzia lophanta by Schwimmer & Kjaer (1960). The enzyme is speific for the -S-CHH2-CH(NI2)CO2H moiety, but a wide range of substituents can replace the thiol bydrogen, and the S atom can be oxidized to the sulphoxide without loss of activity as a substrate. Just as in the Albizzia enzyme, the presence of terminal carboxyl group in the alkyl substituent usually decreases the rate of cleavage of the substrate. This is not always the case, however, since the enzyme does cleave L-djenkolate and L-cystine, both possessw ing terminal carboxyl groups and also acting as excellent substrates. It is very interesting that s.-cystathionine is such a poor substrate for both this and the Albizzia enzyme. L-Djenkolate can be considered perhaps as a S-substituted homologue of cystathionine and L-cystine as a S-substituted analogue. Giovanelli & Mudd (1971 have purified a cystathionase from spinach leaves which has a much greater specificity for cystathionine, but this enzyme also cleaves djenkolic acid at a faster rate. AlCyano-L-alanine has been shown by Pfeffer & a

(mol of amino acid/ 144000g of protein) 62 17 24 18 95

60 85 68 49

59

66 2 50 16 49 75 33 26

Ressler (1967) to be a potent inhibitor of liver cystathionase, and it also inhibits the spinach leaf enzyme (Giovanelli & Mudd, 1971). We have shown that it is a very effective inhibitor of this Acacia enzyme. In fact the K, for the spinach leaf enzyme of 32pM is almost identical with our value of 30AM for the A. farnesiana enzyme. Therefore it appears that even if cystathionine is not a good substrate for the latter enzyme, similar binding sites and reaction mechanisms may be involved. The use of the term 'good' in describing a substrate is only relative, since our most purified fraction is 50 times more active with cystathionine than that reported for the same fraction of the spinach leaf preparation (Giovanelli & Mudd, 1971). For most of the enzymes involved in the metabolism ofamino acids in higher plants, the evidence to date for the involvement of pyridoxal phosphate has been of an indirect nature. The presence of pyridoxal phosphate in the Acacia enzyme was definitely established. The nature of the binding of the cofactor, whether to the large subunit or to the small, and the location and structure of the active site are future areas

for study.

A reasonable question is what physiological function does the enzyme perform. The fact that there is such a high concentration of enzyme in the hypocotyl and root, and the strong mercaptan-like odour which is most prominent at the juncture of the shoot and root, fits in well with the suggestion made by

1975

S-ALKYL-L-CYSTEINE LYASE Mazelis & Fowden (1973) that during the early germination period while the cotyledon, hypocotyl and radicle are in the soil they are very susceptible to invasion by soil pathogens. Volatile sulphur compounds such as mercaptans and sulphides are good inhibitors of growth of many fungi. The release of such compounds would certainly give the seedlings a better chance of establishing themselves in the presence of potential pathogenic organisms. This work was supported by Contract 2-6633 from the National Aeronautics and Space Administration. We thank Dr. D. W. Gruenwedel for use of the digital densitometer and analytical ultracentrifuge and Mr. Joseph Fu and Mrs. Susan Brown for their technical assistance. We also thank Mrs. E. Beckman for doing the amino acid analysis and quantification of the results.

References Disc Electrophoresis (1974) Catalog of CANALCO Chemical Co., pp. 23-25, Rockville, Md. Dixon, M. (1953) Biochem. J. 55, 170-171 Edelhoch, H. (1967) Biochemistry 6, 1948-1954 Fowden, L. (1964) Annu. Rev. Biochem. 33,173-204 Friedemann, T. E. & Haugen, P. E. (1943) J. Biol. Chem. 147,415-442 Giovanelli, J. & Mudd, S. H. (1971) Biochim. Biophys. Acta 227, 654-670 Handbook of Biochemistry (1968) Selected Data for MolecularBiology(Sober, H. A., ed.), p. B-26, Chemical Rubber Co., Cleveland, Ohio

Vol. 147

491 Jacobsen, J. V., Yamaguchi, Y., Mann, L. K., Howard, F. D. & Bernhard, R. A. (1968) Phytochemistry 7, 1099-1108 Kratky, O., Leopold, H. & Stabinger, H. (1969) Z. Angew. Phys. 4, 273-277 Kupiecki, F. P. &Virtanen, A. 1. (1960) Acta Chem. Scand. 14, 1913-1918 Laemmli, U. K. (1970) Nature (London) 227, 680-685 Layne, E. (1957) Methods Enzymol. 3, 451-454 Mazelis, M. (1963) Phytochemistry 2, 15-22 Mazelis, M. & Creveling, R. K. (1973) Abstr. Pacific Slope Biochem. Conf. p. 18 Mazelis, M. & Fowden, L. (1973) Phytochemistry 12, 1287-1289 Murakami, F. (1960) Bitamin 20, 126-131 Nomura, J., Nishiguka, Y. & Hayaishi, 0. (1963) J. Biol. Chem. 238, 1441-1446 Pfeffer, M. & Ressler, C. (1967) Biocheni. Pharmacol. 16, 2299-2308 Schwimmer, S. & Kjaer, A. (1960) Biochim. Biophys. Acta 42, 316-324 Schwimmer, S., Carson, J. F., Makower, R. V., Mazelis, M. & Wong, F. F. (1960) Experientia 16, 449-450 Segel, I. H. & Johnson, M. J. (1963) Anal. Biochem. 5, 330-337 Stoll, A. & Seebeck, E. (1951) Advan. Enzymol. 11, 377-400 Traut, R. R., Bollen, A., Sun, T. T., Hershey, J. W. B., Sundberg, J. & Pierce, L. R. (1973) Biochemistry 12, 3266-3273 Tsuno, S. (1958) Bitamin 14, 659-664 Wada, H. & Snell, E. E. (1961) J. Biol. Chem. 236, 20892095