multiple molecular forms of l-amino acid oxidase

MULTIPLE MOLECULAR FORMS OF L-AMINO ACID OXIDASE*

Daniel Wellnert and Melvin B. Hayes$ Department of Biochemistry, Tufts University,

School of Medicine, Boston, Mass.

When L-amino acid oxidase is purified and crystallized from the venom of the Eastern Diamondback rattlesnake (Crotalus adamanteus), it usually exhib- its three components on electrophoresis, although a single symmetrical peak is obtained on ultracentrifugation.’ On the other hand, the venom of the Cotton- mouth moccasin (Ancistrodon p . pisciwrus) appears to contain only one form of the enzyme.’ The three rattlesnake isozymes and the moccasin enzyme are similar in many respects: they have a molecular weight of about 130,000, they contain two moles of flavin adenine dinucleotide (FAD) per mole, and they have indistinguishable absorption spectra. They are also similar with respect to enzymatic activity, specificity, kinetics, and heat stability. I t appears, therefore, tha t the “active sites” of these enzymes are very similar or identical.

In addition to the multiplicity of molecular forms arising from the exis- teme of isozymes, 1.-amino acid oxidase can also exist in multiple forms differing from one another in catalytic properties. Thus, it was found by Singer and Kearney that the L-amino acid oxidase of A . p. pisciwrus as well as tha t of a number of other snakes‘ is capable of existing in an active and a n inactive form, and that these two forms are interconvertible. The same phenomenon was observed with the crystalline enzyme from C. adamanteus.”‘ The active form “u,” and the inactive form, “8,” have similar electrophoretic mobilities, sedimentation coefficients, and solubilities.”’ Although they also appear identical when examined by immunochemical techniques, they differ significantly in their absorption spectra and optical rotatory dispersions.” The data have been interpreted to indicate tha t the interconversion of a and 0 involves a conformational change restricted to a portion of the molecule which includes the “active site.”

Recently, in the course of studies on the interaction of p-chloromercuribenzoate (PCMB) with L-amino acid oxidase, we have observed the formation of another form of the enzyme, “7.” The y form is enzymatically inactive, but differs from 8 in spectral and other properties.$

‘This work was supported in part by grant AM-08462 from the National Institutes of Health, U. S. Public Health Service.

t Recipient of a Lederle Medical Faculty Award. Present address: Department of Biochemistry, Cornell University Medical College, New York, y. Y.

$Part of this work has been presented in a preliminary report. §Present address: Department of Biochemistry, Cornell University Medical College,

New York, N . Y. 118

3

Wellner & Hayes : L-Amino Acid Oxidase

MATERIAIS A N D M E r t i o ~ ) ~

119

Snake venoms were obtained from Ross Allen's Reptile Institute, Silver Springs, Fla. 1.-Amino acid oxidase was purified and crystallized as described previously.' Except where noted otherwise, all the experiments were carried out with twice-crystallized enzyme, consisting of a mixture of the three isozymes. Pure A and C isozymes were obtained by sampling a Tiselius cell after electrophoretic separation. PCMB was obtained as the sodium salt from Cal- biochem. PCMB-carboxyl-"C was obtained from Tracerlab. I ts radiochem- ical purity was ascertained by showing that no change in specific radioactivity occurred on repeated recrystallization after mixing with unlabeled compound. Radioactivity was measured in a thin-window, gas-flow counter. All radioac- tive samples were dissolved in the same buffer and the same volume was plated. Since the small amount of protein present did not affect the counting efficiency significantly, no corrections for self-absorption were necessary. PCMB concentrations were determined spectrophotometrically, using the ex- tinction coefficients given by Boyer.' Enzyme concentrations were calculated from a value of 1.79 for the absorbency at 275 mp of a solution containing 1 mg/ml. Enzymatic activity was measured as described previously,'' using L-phenylalanine as substrate. The specific activity of the a form ranged from nine to 12.2 units/pg.

The conversion of a to p was carried out by incubating the enzyme in 0.1 M sodium phosphate buffer, pH 7.5, for one hr a t 37" C. The conversion of @ t o (Y was accomplished by adjusting the pH of the solution to 5 with dilute acetic acid, and then incubating the enzyme for one hr a t 37" C.

Separation of enzyme from unbound PCMB was accomplished on a 15 x 1 cm column of Sephadex G-25 a t 4" C. Since it was found tha t a small amount of PCMB is strongly adsorbed to Sephadex, the column was equilibrated with a solution of 6.5 x 10 ' M PCMB of the same specific radioactivity as that used in the experiments, in 0.1 M sodium phosphate, pH 7.5. The enzyme was eluted from the column in the same buffer, and the constant, low background of radioactivity was subtracted from the values reported here.

1

R~suLrs

FIGURE 1 shows the electrophoretic pattern obtained with the crystalline L-amino acid oxidase isolated from the fresh venom of a single rattlesnake. Three isozymes are present in this preparation. In another preparation from a single snake, component C was missing, and the concentration of A was greater than that of B.' When the enzyme is crystallized from the pooled venom of a large number of snakes, isozyme A is usually the most abundant. Thus, considerable individual variation in the occurrence of the three isozymes exists in this species.

Although a complete amino acid analysis of each of the three isozymes is not yet available, a comparison of the amino acid composition of pure A with

120 Annals New Y ork Academy of Sciences

FIGlJRE 1. Descending electrophoretic pattern of crystalline L-amino acid from the venom of a single snake. Photograph was taken after 154 min. Migration was from right (cathode) to left. The three components are referred to in the text as A, B, and C, in order of decreasing negative charge. Buffer: 0.1 M 2-amino-2-hydroxymethyi-l,3-propanediol- HCI, pH 7.2.

that of a mixture of the three isozymes may be made from the data of TABLE 1. I t may be seen that, whereas the value for a number of amino acids (histidine, aspartic acid, valine, methionine, leucine, tyrosine, and phenylalanine) are very similar in the two preparations, significant differences are found with lysine, arginine, and half-cystine. I t is not yet possible to determine whether the amino acid composition of A accounts for its greater negative electrophoretic mobility, however, since the amide nitrogen content has not yet been accurately determined. The results suggest tha t the three isozymes differ in amino acid composition and sequence. It is possible, however, that some parts of the molecules are identical. This may be true for the “active site,” as dis- cussed above, and probably also for the antibody-binding sites. Thus, i t was shown that isozymes A and C are serologically indistinguishable and tha t they react with the same antibody m~lecu le s .~”” I t should be pointed out that , as the half-cystine values given in TABLE 1 have been estimated only as cystine in the protein hydrolysate, they must be considered as tentative until more accurate determinations, such a s cysteic acid analyses of the oxidized protein, are carried out. A study of the amino acid composition of the individual isozymes is in progress.

L-Amino oxidase is also capable of existing in an active form, a , and an in: active form, @, which appear to differ in the conformation of the “active site.”” In order to investigate the possibility that sulfhydryl groups might be in- volved in the a to @ interconversion, the effect of P C M B on this reaction was investigated. Asshown in FIGURE 2, after treating 0 with P C M B a t 0’ C, pH 5.0, much less activity returned under reactivating conditions than in the con- trol in which PCMB was omitted. Since, as shown below, P C M B does not inhibit the enzyme, we postulated that P C M B had reacted to yield a new form

Wellner & Hayes: I,-Amino Acid Oxidase 121

TABLE 1 AMINO ACID COMPOSITION OF L-AMINO ACID OXIDASE'

Amino Acid Residue

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-Cystine Valine Methionine Jsoleucine Leucine Tyrosine Phenylalanine

Isozyme A (moles Residue per 130,000 g)

74.5 29.1 62.4 115.8 69.4 73.2 118.0 45.6 77.7 84.0 20.2 66.6 18.3 68.1 78.4 58.4 55.2

Mixture of Isozymes A, B, and C

(moles Residue per 130,000 g)

81.6 30.2 78.2 116.4 67.2 70.3 116.1 41.2 73.4 83.2 11.5 67.4 18.6 69.8 78.2 58.1 55.4

*The proteins were denatured by heating for 5 min at 1OO"C, washed with distilled water, and hydrolyzed with 6 N HC1 at 110°C. The values from a 20-hr and a 70-hr hydrolysis were averaged, except for those of threonine, serine, and cystine, which were extrapolated to 0 time, and for d i n e and iso- leucine, for which the 70-hr value was used. Tryptophan and amide nitrogen have not been determined

of the enzyme, y, which could not be converted to (Y under our standard reactivating conditions. The formation of y appeared to be a slow reaction, since after two hr at Oo C it was only about haIf compIete (FIGURE 2). The kinetics of this reaction were therefore investigated, as shown in FIGURE 3. I t may be seen that, while all the activity was recovered initially, very little activity was recovered after six or seven hr of reaction with PCMB a t pH 5 and 0" C. At pH 7.5, on the other hand, j3 appeared to be quite stable in the presence of PCMB, since it could be completely converted to a even after 24 hr. When a solution of a was kept at 0" C in the presence of PCMB, either a t pH 5 or pH 7.5, no significant loss of activity was detected after 48 hr or longer.

Several questions were raised by these results: Is y the PCMB derivative of 8, or are other changes also taking place in the enzyme molecule? Does PCMB react with fl at pH 7.5 or only at pH 5? Does PCMB react with a? What is the stoichiometry of the reaction between PCMB and the enzyme?

In order to answer these questions, it was necessary to measure the binding

122

100 c J 6 k 80 - z LL

6 0 lR Y

4 0 - > I- W

- 2 0

0

Annals New Y ork Academy of Sciences

I I I I I

- I I I I 0 30 60 90 I20

TIME A T 37" (MIN.)

FIGURE 2. Effect of PCMB on the conversion of 0 to a. A solution o,f the (3 form of the enzyme was preincubated at 0" C, pH 5, in the presence of 2 x 10- M PCMB for about two hr. It was then warmed to 37" C and aliquots were removed at various times for activity measurements (lower curve). The upper curve represents a similar experiment, except that PCMB was omitted.

of PCMB to the enzyme directly. This was accomplished by the use of radio- active PCMB combined with gel filtration on Sephadex. This technique has been used successfully with other enzyme systems.

FIGURE 4 shows that PCMB binds to the /3 form of the enzyme a t pH 7.5. I t could be calculated that, in this experiment, 2.3 moles of PCMB were bound per mole of enzyme. Thus, it appears tha t the enzyme contains two groups, presumably sulfhydryl groups, which react with PCMB under these conditions. Similar results were obtained with /3 at pH 5, a at pH 5, and (Y a t pH 7.5. The values for the amount of PCMB bound ranged from 2.3 to 2.7 moles per mole of enzyme. In order to find out whether the same or different groups were reactive in the a and p forms, the u form was allowed to react with PCMR, converted to the /3 form, and again allowed to react with PCMB. The enzyme, now in the y form, was applied to a Sephadex G-25 column. The results are shown in FIGURE 5. I t was found in this experiment that 2.4 moles of PCMB were bound per mole of enzyme. I t appears, therefore, that the same groups were reacting in the (Y and /3 enzymes, since otherwise one would have expected to find a total of four moles of bound PCMB per mole of enzyme.

I 1 13

Wellner & Hayes: L-Amino Acid Oxidase 123

100

80

60

40

20

TIME AT oo (HOURS)

I I I I Ih

0

FIGURE 3. Kinetics of conversion of p to y a t 0' C in the presence of PCMB. A splu- tion of the M PCMB at pH 5 (solid circles) or a t pH 7.5 (circles). After various times, aliquots were removed, warmed to 3 7 O C for one hr (after adjusting the pH to 5 when necessary), and the activity was measured.

form of the enzyme was incubated a t Oo C in the presence of 2 x 10


1.2 -

6 '.O -

3 Y

0.8 - b N

k

> V Z w

0.6 -

0.4 - 0 v) m

0.2 -

48

40

n + Y

32 i I

24 ?\ 0

16 1 ti 3

x

8

0 0

10 20 30 40 50

EFFLUENT VOLUME (ML)

FIGURE 4. Binding of PCMB-14C to 8-L-amino acid o$dase. The f l f o p of the enzyme was allowed to react with labeled PCMB (2 x 10 . M, 2.76 x 10 cpmlpmole) at pH 7.5 for 72 hr at 0" C. The solution was then applied to a column of Sephadex G-25 as described in the text.

As shown in FIGURE 6, the reaction between PCMB and the enzyme is not instantaneous. Thus, after five min, only about 0.5 mole of PCMB was bound per mole of enzyme.

When the PCMB derivative of 8, isolated by gel filtration as described in FIGURE 4, was incubated for one hr a t 37O C immediately after lowering the pH to 5, it was converted to a, as evidenced by return of the enzymatic activity. However, as shown above (FIGURE 3), little activity was recovered when the enzyme was allowed to stand for some time in the cold at p H 5 before being warmed to 37" C. Thus, i t may be concluded tha t y is not simply a P C M B derivative of 0, but that its formation involves other changes in the protein molecule. As shown below, this reaction is accompanied by marked changes in the visible and ultraviolet spectrum of the enzyme.

The experiment described in FIGURE 7 demonstrates tha t the P C M B de-


U T 3 E 0.8 n b N

5 0.6

> u z w 0.4 OD a 0 v) s 0.2

0

5 10 15 20 25 30

FRACTION NUMBER

25

- 2o t

Y

15

?' 0

10 * r' 6 u

5

0

FIGURE 5. Binding of PCMB-14C to y-L-amino acid ofidase. The cx-fop of the enzyme was allowed to react with labeled PCMB (2 x 10 M, 2.76 x 10 cpmlpmole) at pH 7.5 for 36 hr at 0" C. The enzyye was then converted to the fl form and allowed to react with labeled PCMB (2 x 10- M) at pH 5 for 48 hr at 0" C. The solution was then applied to a column of Sephadex G-25 as described in the text.

rivative of a is fully active and may be converted to the PCMB derivative of 0 a t pH 7.5 and 37O C. As indicated by the small return of activity (FIGURE 7, right side), the enzyme was largely converted to the y form a t pH 5 and 0' C, although no free PCMB was present in the solution. The formation of y could be prevented by treating the PCMB derivative with mercaptoethanol or dithiothreitol before acidifying the solution.

The spectra of the three enzyme forms are shown in FIGURES 8 and 9. I t may be seen that the visible spectrum of the y enzyme is markedly different from that of either a or (3. I t is shifted toward shorter wavelengths and one of the peaks is considerably decreased in intensity. The maxima are a t 378 and 456 mp, as compared to 390 and 462 mp for the a enzyme, and 387 and 458 mp for the @ enzyme. (Combination of the a or (3 enzyme with PCMB did not affect their visible absorption spectra significantly.) A characteristic feature of the y spectrum is the pronounced shoulder on the long wavelength side of

126

W g 0.2 0 cn m 6 -

0

Annals New York Academy of Sciences

I I I I 1

Annals New York Academy of Sciences

I I I I 1

10 20 30 40 50

EFFLUENT VOLUME (ML.)

FIGURE 6. Binding of PCMB-I4 C to a-L-amino acid oxidqy after five F in . The a form of l.-amino acid oxidase was allowed to react with PCMB- C (2 x 10 M, 2.76 x lo6 cpm/pmole) for five min at pH 7.5 and 0" C, and immediately applied to a Sephadex C-25 column as described in the text.

the 456 mp peak. Such a shoulder has been observed in the spectra of a number of flavoproteins, but it is present only to a small extent in the a and b forms of L-amino acid oxidase.

Although the y form of L-amino acid oxidase is inactive and is not reac- tivated, like the @ form, by incubation at pH 5 and 37" C, it is not an irrever- sibly denatured form of the enzyme. Thus, unlike heat-denatured enzyme, i t is still soluble and retains its bound FAD. I n addition, on standing a t 0" C a t pH 7.5, it is slowly converted to a form, presumably (?, which can in turn he converted to active enzyme. This is demonstrated by the experiment described in FIGURE 10.

DISCUSSION

The results reported here show that the L-amino acid oxidase of Crotalus adamanteus may occur, even in a single snake, in three forms differing in electrophoretic mobility and amino acid composition, and also may undergo tran- sitions between three molecular forms differing in enzymatic and spectral properties. The latter have been designated as a, 8, and y, and their intercon-


I I I I II I I I 1 -.

0 20 40 60" 0 20 40 60

TIME A T 37" (MIN.)

FIGURE 7. Conversiosn of a to y in the absence of free PCMB. A solution of a L-amino acid oxidase (7.7 x 10- M) was reacted with PCMB (3.3 x 10 ' M) for 48 hr a t pH 5 and 0' C. It was then freed of unreacted PCMB by passage through a Sephadex (2-25 column equilibrated with 0.1 M sodium phosphate, pH 7.5. The enzyme was then warmed to 37' C for one hr, and during this time, aliquots were removed for activity measurements (left side of Figure). After adjusting the pH to 5 with dilute acetic acid, the solution was kept a t 0" C for 24 hr. The enzyme was then warmed to 37' C for one hr, and, during this time, aliquots were removed for activity measurements (right side of Figure).

version is summarized in FIGURE 11. T h e y form is obtained from t h e P C M B derivative of t h e 0 form at pH 5 a n d 0" C. Since, in t h e absence of P C M B , the 0 enzyme is stable a t pH 5, it appears t h a t P C M B affects primarily the stability of t h e 0 conformation. I t may be concluded from t h e data t h a t sulfhydryl groups are not essential for enzymatic activity, since P C M B does not inacti- vate the enzyme even af ter combining with it. Other sulfhydryl reagents, such as N-ethylmaleimide, iodoacetate, or iodoacetamide, d o not inhibit t h e enzyme either, and, under t h e same conditions as used here for t h e P C M B reaction, d o not appear t o favor t h e formation of t h e y form (D. Wellner and M. B. Hayes, unpublished results). I t is not yet known, however, whether they react with the enzyme under these conditions.

128 Annals New York Academy of Sciences

t I I I I I I I

350 380 410 440 470 500 530

WAVELENGTH ( m p )

FIGURE 8. Visible spectra of a, 8, and y forms of L-amino acid oxidase (PCMB derivatives). Conditions: 1 mg/ml enzyme in 0.1 M sodium phosphate buffer, pH 7.5; 0; C; 1 cm light path. The PCMB derivative of a was obtained by reacting a with 2 x 10- M PCMB in 0.1 M sodium phosphate buffer, pH 7.5, for 24 hr at 0' C. This solution was incubated at 37" C for one hr to obtain the PCMB derivative of 8. The pH was then adjusted to 5 with dilute acetic acid and the solution was kept at 0' C for five days. The PCMB derivative of y thus obtained was transferred by gel filtration to 0.1 M sodium phosphate, pH 7.5, immediately before the spectrum was taken.

One curious aspect of the reactions shown in FIGURE 10 is that , whereas a t 0" C the PCMB derivative of p is converted to y a t pH 5, a t 37" C, it is converted to a. It is possible tha t this behavior may be explained by a differ- ence in the energy of activation of the two reactions. I t has been shown by Singer and Kearney" tha t the energy of activation of the a,p interconversion is extremely high (42,500 cal/mole). The rate of this reaction is therefore highly temperature dependent. I t takes place quite well a t 37" C but does not proceed at an appreciable rate, in either direction, below 10' C. If the PSr transition has a low energy of activation, i t may be expected tha t its rate at 37" C would not be very different from tha t at Oo C. Thus, under conditions where the PCMB derivative of is unstable and may undergo a transition either to a or to 7 , viz., a t pH 5, the direction of the transition may differ at 0' C and 37" C because of the effect of temperature on the relative velocity of the two reactions.

The visible absorption spectra of the a, 8, and y forms of the enzyme differ from one another and from the absorption spectrum of free FAD. It may


WAVELENGTH (my)

FIGURE 9. Ultraviolet spectra of a, 8, and y forms of L-amino acid oxidase (PCMB derivatives). Conditions as in FIGURE 8.

therefore be concluded that the interaction between FAD and the protein is different in the three cases. These results, together with the optical rotatory dispersion and immunochemical studies reported previously,’ suggest that these three forms of the enzyme differ in the conformation of their “active sites.”

Another example of an enzyme whose stability is altered by reaction with PCMB is mitochondria1 DPNH-dehydrogenase. This enzyme has been reported by Cremona and Kearney14 to react with sulfhydryl reagents a t 0’ C without loss of activity. However, when the enzyme was subsequently warmed, its activity with ferricyanide as electron acceptor decreased. This loss of activity was attributed to an irreversible conformational change in the enzyme. A possible interpretation of these results is that interaction between sulfhydryl groups and other groups on the protein play an important role in maintaining the stability of a particular conformation. This may also be true of L-amino acid oxidase. Of interest in this connection is the evidence recently obtained by Dunnill and Green’j that sulfhydryl groups in @-lactoglobulin, which react very slowly with PCMB, become exposed following a pH-dependent conformational change in the protein.


10

8

6

4

2

0-s -- 0 - - 0

-rs 0 --- -

m

FIGURE 10. Recovery of enzymatic activity from y L-amino acid oxidase. The activity of the PCMB derivative of y. obtained as described in FIGURE 8, was measured after various times of storage a t pH 7.5 and Oo C (triangles). The activity of the PCMB derivatives of a (circles) and b (squares) are included for comparison. Open symbols: di- rect activity measurements; solid symbols: activity after incubation at pH 5 for one hr a t 37" C.


FIGURE 11. Scheme for the interconversion of a, (3, and y forms of L-amino acid oxidase.

SUMMARY

L-Amino acid oxidase of Crotalus adamenteus venom occurs a s three elec- trophoretically separable isozymes. These are designated as A, B, and C in order of decreasing negative charge. The three isozymes are indistinguishable by spectral, ultracentrifugal, or enzymatic measurements. All three are glyco- proteins of molecular weight 130,000. They contain two moles of FAD per mole. An amino acid analysis of component A gave results significantly different from tha t of a preparation containing all three isozymes, suggesting tha t the three proteins differ in amino acid composition and sequence.

Each of the isozymes described above may, in turn, exist in multiple molecular forms. These are designated as a, @, and y, and they differ in their enzymatic and spectral properties. Whereas a is enzymatically active, @ and y are not. The a form may be converted to the b form by incubation a t pH 7.5 for one hr at 37" C and may be regenerated by incubating /3 at pH 5 for one hr at 37" C. This reversible inactivation was discovered and studied by Singer and Kearney.' A comparison of the optical rotatory dispersion and other properties of a and @ have suggested that the interconversion of these two forms of the enzyme involves a conformational change at the active site.5 The third form, 7 , differs markedly from either a or @ in spectral properties. I t cannot be converted to a by conditions under which B is converted to a. I t is obtained by treating the p-chloromercuribenzoate (PCMB) derivative of @ for several hr a t pH 5 and a t 0" C. Whea either the a or p form of the enzyme is treated with "C-PCMB and the unreacted PCMB is removed by gel filtration on Sephadex G-25, it is found that about two moles of PCMB are bound per mole of enzyme. Reaction with PCMB results in no impairment of the enzymatic activity or of the ability to undergo the a=@ interconversion. The PCMB derivative of /3 is converted to y a t pH 5 and 0" C, to a a t pH 5 and 37" C, and is stable at pH 7.5. Neither @ itself nor the PCMB derivative of a is converted to y a t pH 5. These three forms of the enzyme are thought to differ in the conformation of the active site.

132 Annals New York Academy of Sciences

ACKNOWLEDGMENT

W e are indebted to Dr. Maurice Liss for his help with the amino acid analyses.

REFERENCES

1. WELLNER, D .&A. MEISTER. 1960. J. Biol. Chem. 235: 2013. 2. SINGER,T . P. & E. B. KEARNEY. 1950. Arch. Biochem. 29 190. 3. SINGER, T. P. & E. B. KEARNEY. 1951. Arch. Biochem. 33: 377. 4. SINGER, T. P. & E. B. KEARNEY. 1951. Arch. Biochem. 33: 397. 5. WELLNER, D. 1966. Biochemistry 5 1585. 6. DUKE, F. R. & E. A. WEISKOPF. 1966. Ann. N. Y. Acad. &i. 136 123. 7. SINGER, T. P. & E. B. KEARNEY. 1951. Arch. Biochem. 33: 414. 8. WELLNER, D. & M. B. HAYES. 1966. Abstracts of papers, 152nd Meeting, Am.

9. ROYER, P. D. 1954. J. Am. Chem. Soc. 76 4331. 10. WELLNER, D. 1966. In Flavins and Flavoproteins. E. C. Slater, Ed. Elsevier.

11. HUMMEL, J. P. & W. J. DREYER. 1962. Biochim. Biophys. Acta 63: 530. 12. FAIRCLOUGH, C . F. & J.S. FRUTON. 1966. Biochemistry 5: 673. 13. WELLNER. V. P. &A. MEISTER. 1966. Biochemistry 5 872 (1966). 14. CREMONA, T. & E. B. KEARNEY. 1965. J. Biol. Chem. 240: 3645. 15. DUNNILL, P.& D. W.GREEN. 1965. J. Molec. Biol. 15: 147.

Chem. Soc. New York, N.Y.

Amsterdam, Netherlands. 223.

multiple molecular forms of l-amino acid oxidase

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