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Purification and properties of riboflavin-binding protein from the egg white of the duck (Anas platyrhynckos)
339
Biochimica et Biophysica Acta, 623 (1980) 339--347 © Elsevier/North-Holland Biomedical Press
BBA 38422
PURIFICATION AND PROPERTIES OF RIBOFLAVIN-BINDING PROTEIN FROM THE EGG WHITE OF THE DUCK (ANAS PLATYRHYNCKOS)
K. MUNIYAPPA and P. R A D H A K A N T H A ADIGA
Department o f Biochemistry, Indian Institute of Science, Bangalore 560 012 (India)
(Received October 1st, 1979) Key words: Riboflavin-binding protein; Molecular weight estimation; Amino acid composition; (Anas platyrhynckos)
Summary Riboflavin-binding protein was purified from the egg white of domestic duck and some of its properties were investigated. The protein was homogeneous by the criteria of gel filtration on Sephadex G-100 and electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, had molecular weight of 36 000 + 1000 and, unlike the chicken egg white protein (Mr 32 000 + 2000), was devoid of covalently-bound carbohydrate. It was similar to the chicken riboflavin-binding protein in its behavior on ion-exchange celluloses and affinity to interact with the flavin and its coenzymes, but differed significantly in amino acid composition in that it completely lacked proline and contained less of methionine and arginine. The protein partially cross-reacted with the specific antiserum to chicken riboflavin-binding protein with a spur during immunodiffusion analysis.
Introduction
The avian egg contains adequate amounts of all the necessary nutrients, including vitamins and other micronutrients for the proper development of the prospective embryo. In the case of chicken, the deposition of at least some of the vitamins in the egg involves the obligatory participation of carrier proteins specific for each of these vital nutrients [1--5]. As early as 1959, Rhodes et al. [5 ], while exploring the use of CM-cellulose for the fractionation of chicken egg white, discovered the presence of a unique fiavoprotein which bound riboflavin more avidly than its c0enzymes, but was devoid of any enzymatic activity. A similar flavin-binding protein was subsequently isolated from the egg yolk [6,7]. The biological importance of this protein as a vitamin-carrier, indispensable for embryonic development and survival, was elucidated much later
340 [8]. The egg flavoproteins are phosphoglycoproteins with molecular weight of 32 000 (white) and 36 000 (yolk), and appear to be products of a single structural gene [9]. While the egg white proteins originate from the oviduct, the yolk proteins are secreted by the avian liver, and transported through the circulation; to be deposited in the developing oocyte [10]. Because of several attractive features associated with this flavoprotein, in recent years considerable attention has been focused on its purification, physicochemical properties and quantification [ 11 ], the structural features involved in protein-ligand interaction [12--15] and hormonal induction and regulation of its biosynthesis [16--18]. While the existence of similar flavoproteins in the eggs of several avian species has been indicated earlier [19], no systematic studies on purification and molecular characteristics of these proteins from other oviparous species appear to have been reported so far. In this paper, we describe the isolation of the riboflavin-binding protein from the egg white of domestic duck (Anas platyrhynckos) and compare some of its properties with those of the chicken flavoprotein. Materials Fresh duck eggs were obtained from the local market. Potato acid phosphatase was procured from Sigma Chemical Co. (St. Louis, MO). The sources of other biochemicals, reagents and gels have been referred to earlier [11 ]. Methods
Isolation and purification of riboflavin-binding protein. Unless otherwise mentioned, all the steps were carried out at 4°C. The m e t h o d employed for isolation was essentially similar to that described by Murthy and Adiga [11 ] for chicken egg white protein with minor modifications. Briefly, the egg white, physically separated from the yolk, was sonicated for 2 min at 4 mA using a Bronson sonifier (Model $75) and the clarified protein was dialysed against 0.1 M acetate buffer, pH 4.3. After loading the protein on a DEAE-cellulose column (2.0 cm X 75 cm) pre-equilibrated with the above buffer and washing off the unadsorbed proteins, the absorbed proteins were eluted with 0.5 M NaC1 in 0.1 M acetate buffer at pH 4.3. Fractions having absorption both at 280 nm (protein) and 445 n m (riboflavin) were pooled, dialyzed against water and fractionated on a CM-ceUulose column (1.5 cm X 60 cm) using NaC1 gradient (0 -* 0.5 M NaC1 in 0.025 M sodium acetate buffer, pH 3.6). Fractions having a high absorption at 280 nm were pooled, dialyzed against water, lyophilized and tested for the capacity to bind riboflavin [12]. Analytical polyacrylamide gel disc electrophoresis. Polyacrylamide disc gel electrophoresis was carried out with 7.5% gels at pH 8.3 according to the m e t h o d of Davies [20] and gels were stained and destained as described elsewhere [11]. Neutral sugars were estimated according to Dubois et al. [21]. Staining for the glycoprotein was carried out as described by Zacharius et al. [22]. Molecular weight determination. Molecular weight of the purified protein was determined by gel filtration chromatography on a Sephadex G-100 column
341 [23] and b y electrophoresis using 7.5% SDS-polyacrylamide gels [24]. The following proteins of k n o w n molecular weight served as markers in both the procedures: c y t o c h r o m e c ( 1 3 7 0 0 ) , chymotrypsinogen ( 2 5 0 0 0 ) , ovalbumin (44 000), bovine serum albumin (68 000, monomer), phosphorylase A (95 000) and yeast alcohol dehydrogenase (125 000). Spectral measurements. Absorption spectral changes were recorded with a Cary 14 or Unicam (Model SP 500) spectrophotometer. The concentrations of solutions of the apo-riboflavin-binding protein and riboflavin were routinely determined spectrophotometrically using the calculated values of e2s0 = 4.75 • 104 M -1 • cm -l and e44s = 1.25 • 104 M -1 • cm -~ [25] respectively. Fluorimetric titrations were carried o u t with a Perkin-Elmer spectrofluorimeter (Model 203). During titration of the apo-protein with riboflavin, protein fluorescence was measured at 340 nm following excitation at 280 nm. Similarly, flavin fluorescence was excited at 370 nm and emission measured at 520 nm.
Treatment of the apo-riboflavin-binding protein with potato acid phosphatase. The apo-protein (15 mg) in 1.5 ml of 0.1 M sodium acetate buffer (pH 5.3) was treated with 0.1 ml of the acid phosphatase (4590 units) and dialysed against 30 ml of buffer at 37°C in a water bath. 1 ml aliquots of the dialysate were removed at specifified intervals and analysed for inorganic phosphate [26]. The total phosphate analysis was carried o u t concurrently on the dephosphorylated and untreated apo-protein. Immunological techniques. Antiserum to the chicken riboflavin-binding prorein was raised in albino rabbits as described b y Murthy et al. [12]. Double immunodiffusion analysis on agar was performed as described b y Ouchterlony [27]. Amino acid analysis. The protein (5 mg) was hydrolyzed in vacuo in 6 M HC1 at l l 0 ° C for 24 h and the amino acid composition determined in an automatic amino acid analyzer (Electroselenium Ltd., U.K.). The details of analysis and calculations were according to the standard procedures [28]. Tryptophan content was determined chemically b y the procedure of Spies and Chambers [29]. Protein determination. Protein concentrations were measured as described b y L o w r y et al. [30] using bovine serum albumin as the standard. Results and Discussion
Purification of riboflavin-binding protein When the processed crude egg white protein was applied on the DEAE-cellulose column and the ion-exchanger was extensively washed with the equilibration buffer (0.1 M sodium acetate buffer, pH 4.3) until the effluent was free of material absorbing at 280 nm, the vast majority of the protein was excluded from the column as monitored spectrophotometrically. The absorbed protein was then eluted with 0.5 M NaC1/0.1 M sodium acetate buffer (pH 4.3). Fractions with absorption at b o t h 445 nm (riboflavin) and 280 nm ( p r o t e i n ) w e r e pooled and concentrated b y lyophilization. Polyacrylamide gel disc electrophoresis o f this preparation revealed t w o closely spaced protein bands. Further purification of the riboflavin-binding protein was achieved b y chromatography on a CM-cellulose column using a linear salt gradient (0 -~ 0.5 M
342
NaC1 in 0.025 M sodium acetate buffer, pH 3.6) as the eluent. The protein was found to resolve as two distinct peaks. The protein species which emerged first as a relatively small peak appeared to be ovomucoid (since it gave a precipitin band during immunodouble diffusion on agar, against a specific antiserum to purified chicken ovomucoid) and did not interact with riboflavin. The larger protein peak, emerging after the first, possessed flavin binding capacity apart from partially cross-reacting with the mono-specific antiserum to purified chicken riboflavin-binding protein [12]. The pooled fractions corresponding to the second protein peak were dialyzed and concentrated. From a dozen duck eggs, a yield of 36 mg of the purified protein was realized.
Criteria o f purity The purified protein exhibited a single stainable band on electrophoresis using 7.5% polyacrylamide gels {data not shown). A duplicate gel, when stained for glycoprotein [22], did not take up the stain, indicating the absence of bound carbohydrate residues. In conformity with this, quantitative methods for total bound carbohydrate [21] also gave negative results. This is in marked contrast to the chicken egg flavo-proteins, both of which are glycoproteins [11]. The physiological relevance of the lack of covalently bound oligosaccharide in the duck protein is not understood at present. Nevertheless, these results confirm an earlier observation with the chicken ovoflavoprotein that bound carbohydrate residues are dispensable in terms of the protein-flavin interaction [ 11 ]. It is pertinent to mention here that the duck yolk riboflavin-binding protein isolated by a procedure recently developed to purify chicken yolk protein [7] is in fact a glycoprotein (Muniyappa, K. and Adiga, P.R., unpublished data) which is in conformity with the postulate [2,31] that the carbohydrate moieties of the yolk glycoproteins may serve as recognition sites at specific receptor loci on the outer surface of the plasma membranes of the developing oocyte for facilitating preferential uptake and deposition in the yolk [ 32 ]. Molecular weight The molecular weight of the purified protein was determined by gel filtration chromatography on Sephadex G-100 as well as by SDS-polyacrylamide gel electrophoresis. By both these procedures, a single molecular species with an apparent molecular weight of 36 000 + 1000 was observed. Thus, in terms of molecular weight the duck flavoprotein, though devoid of covalently bound carbohydrate, is slightly larger than chicken egg white protein (32 000 + 2000) [11] and more akin to the chicken yolk flavoprotein (36000 + 2000) [7]. It could be easily separated from the chicken egg white flavoprotein during coelectrophoresis on SDS-polyacrylamide gel electrophoresis (unpublished data). Immunological cross reactivity On immunodouble diffusion analysis on agar against the antiserum to purified chicken egg white riboflavin-binding protein [12] the duck protein exhibited a single precipitin line which, however, was not completely confluent with that obtained with chicken egg white riboflavin-binding protein (Fig. 1). The unequivocal detection of a spur in the precipitin band suggests that the duck ovoflavoprotein, while possessing some common antigenic determinants
343
Fig. 1. D o u b l e - i m m u n o d i f f u s i o n analysis o f r i b o f l a v i n - b i n d i n g p r o t e i n . T h e c e n t r e well c o n t a i n e d a n t i s e r u m to c h i c k e n r i b o f l a v i n - b i n d i n g p r o t e i n . T h e p e r i p h e r a l wells, 1 a n d 2, c o n t a i n r i b o f l a v i n - b i n d i n g p r o t e i n f r o m c h i c k e n egg w h i t e ; 3 a n d 4 c o n t a i n r i b o f l a v i n - b i n d i n g p r o t e i n f r o m d u c k ' s egg w h i t e . 20/~g of e a c h w a s a p p l i e d .
(and hence partial sequence homology) with the chicken riboflavin:binding protein, is distinguishable from the latter in finer details of molecular architecture. Similar observations have been made regarding the immunological features of other egg white proteins such as lysozyme, ovomucoid and conalbumin from different avian species [19]. Amino acid analysis Further evidence for the above premise stems from an examination of the amino acid composition of the protein (Table I). It may be seen that there are
TABLE I C O M P A R I S O N OF AMINO ACID COMPOSITIONS OF T H E DUCK AND T H E C H I C K E N E G G W H I T E RIBOFLAVIN-BINDING PROTEINS Amino acid
A s p a r t i c acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine Haif-cysteine Valine Methionine Isoleucine Tyrosine Phenylalanine Lysine Histidine Axginine *** Tryptophan Phosphorus
Residues/mol OvotlavoProtein Duck *
Chicken * *
14.0 11.0 26.0 39.2 nil 7.8 16.0 12.6 10.0 3.6 24.0 8.2 11.0 18.4 16.0 1.2 *** 9.0 7.4
20.0 7.7 28.8 36.4 9.7 8.3 13.9 15.9 5.7 8.2 14.8 9.5 7.0 17.2 9.2 5.6 9.3 7.0
* N o r m a l i z e d to M r 36 0 0 0 o f t h e n a t i v e p r o t e i n . ** T h e v a l u e s are t a k e n f I o m R e f . [ 3 3 ] . *** N o r m a l i z e d to o n e a r g i n i n e r e s i d u e .
344 clear cut quantitative and qualitative differences between the duck and chicken egg white proteins, which is reminiscent of observations regarding the pronounced variations in amino acid composition (besides other characteristics) among homologous egg proteins from different avian species [19]. For example, proline is completely absent in the duck flavoprotein in contrast to nearly 10 residues per mol in the chicken flavoprotein [33]. Further, the duck protein appears to be significantly richer in hydrophobic amino acids (valine + leucine) and histidine, though the reverse seems to be the case with respect to the contents of methionine and arginine. Of special significance is the presence of comparable number of residues of tryptophan, half-cysteine and acidic amino acids in both the proteins. The fact that these functional moities are conserved assumes importance in the context of our earlier findings with the chicken ovoflavoprotein [12] that these residues are critically involved in protein-ligand interaction.
Dephosphorylation and flavin binding The finding that the purified duck riboflavin-binding protein harbours the same number of protein b o u n d phosphate residues (Table I) as the chicken protein, prompted an investigation on their functional significance. Treatment with potato acid phosphatase resulted in a rapid dephosphorylation of both the apoand holoproteins. More than 80% of the protein b o u n d phosphate was released when incubated with the enzyme at 37°C for 3--5 h. If more enzyme was added after 3 h of incubation, up to 95% of the total b o u n d phosphate could be liberated. However, the dephosphorylated apo-riboflavin-binding protein could bind riboflavin with an efficiency comparable to that of untreated protein (unpublished data), showing that the covalently b o u n d phosphate residues are dispensable in terms of protein-ligand interaction. Similar conclusion has been arrived at in the case of the chicken ovoflavoprotein [ 5 ].
Spectral changes on flavin-protein interaction In Fig. 2 the changes in visible absorption spectrum of riboflavin on interaction with the purified duck apo-riboflavin-binding protein are depicted. The spectral changes are characterised b y a remarkable hypochromism of both the 370 nm and 450 nm bands without a shift of band positions. Based on a comparison of absorption spectra of flavins in solvents of various polarity [34,35], this phenomenon of hypochromicity at 370 nm has been attributed to the involvement of hydrophilic or polar interactions [36]. In this respect, the duck protein appears to be similar to the chicken egg white flavin-binding protein. Furthermore, the magnitude of hypochromicity was found to be proportional to the extent of flavin-apo-protein interaction. However, no significant spectral shifts in the absorption band at 450 nm were concomittantly observed in contrast to the situation obtained with the chicken ovoflavoprotein [37]. In the latter case, besides hypochromicity, a red shift of 450 nm band accompanied b y appearance of shoulders, has been recorded and this has been interpreted to represent the isoalloxazine ring of the flavin getting deeply buried in the hydrophobic environment of the apo-protein, following protein-ligand interaction. Based on this interpretation, it appears likely that the loci where the flavin interacts with the duck apo-flavoprotein are relatively less hydrophobic than those of the chicken protein.
345
0.~
(A) O~
O.g
~0.4
0.~
\
0.1 1
I
i
i
i
0.6
O.S
0.4
O.2 0.1 i
I:
(c)
0.~ 0.4 0..4
0J
0
350
45o
ssO
WIVIEL E NGTH (nm)
Fig. 2. Visible ab so rp tion spectra of the flavin-apo-protein complexes. (A) A bs orpt i on spectra of riboflavin and its co mp lex with the apo-protein. I, Riboflavin (30 /~M), II, I plus the apo-protein (30 /~M). (B) Absorp tion spectra of FMN and its c o m p l e x with the apo-protein I, FMN (30 #M). n0 I plus apo-protein (30 #M) (C) A b s o r p t i o n spectra of FAD and its c o m p l e x w i t h the apo-protein. I, FAD (30/~M), II, I plus apo-protein (30 pM).
Since the magnitude of hypochromicity at 370 nm, and 450 nm bands appears to be a, function of the extent of protein-ligand interaction, it is clear from the data depicted in Fig. 2 that FAD and FMN have relatively less affinity than the free flavin to interact with the apo-flavoprotein at equimolar
346 concentrations, which is in agreement with the findings with the chicken aporiboflavin-binding protein [ 5 ].
Quenching of flavin fluorescence The titration curve for flavin-apo-protein interaction showed a sharp inflection point at equivalence point due to complete quenching of flavin fluorescence (Fig. 3). From these data, the molar extinction co-efficient of the apoprotein at 280 nm was calculated to be 4.75 • 104 M -1 • cm -1 assuming 1 : 1 binding stoichiometry of protein-flavin interaction. The quenching of flavin fluorescence is in accordance with the findings of Rhodes et al. '[5] with the chicken flavo-protein and is reminiscent of the observation with D-amino acid oxidase-bound FAD complexing with benzoate [38,39]. The binding of flavin also resulted in quenching of protein fluorescence and had an association constant Ks of 4.0 • 10 s M -1 at pH 7.0, a value comparable to t h a t calculated for the chicken egg white flavoprotein [40]. The above data thus demonstrate that the homologous flavin-binding pro-
II/
100
(A)
~. 50
5
10 RIBOFLAVIN x 10-I'M
15
(B)
100
o u,. 50
0
I 0.2
I I I 0.4 0.6 0.B RIBOFLAVIN(~M)
I 1.0
F i g . 3. F l u o r i m e t r i c t i t r a t i o n o f t h e d u c k a p o - r i b o f l a v i n - b i n d i n g p r o t e i n w i t h r i b o f l a v i n . ( A ) T i t r a t i o n utilizing flavin fluorescence. Fluorescence of riboflavin excited at 370 nm was measured at 520 nm. The a m o u n t s o f r i b o f l a v i n i n d i c a t e d 410 #1 a l i q u o t s ) w e r e a d d e d t o 3 . 0 m l o f a n a p o - p r o t e i n s o l u t i o n ( a b s o r b ance at 280 nm, 0.026) (I) or to 0.1 M sodium phosphate buffer, pH 7.0 (II). The extinction coefficient o f t h e p r o t e i n a t 2 8 0 n m w a s c a l c u l a t e d t o b e 4 . 7 5 • 1 0 4 M - 1 • c m -1 a s s u m i n g a 1 : 1 ( M / M ) b i n d i n g r a t i o . B. T i t r a t i o n u t i l i z i n g p r o t e i n f l u o r e s c e n c e , F l u o r e s c e n c e o f t h e a p o - p r o t e i n e x c i t e d a t 2 8 0 n m w a s measured at 350 nm. The amounts of riboflavin indicated were added to 3.0 ml of an apo-protein solution (440 nM).
347
teins from the egg whites of the two avian species of different taxonomic groups exhibit close physico-chemical and functional kinships, such as behavior on ion~xchange cellulose, preferential interaction with the free vitamin, rather than its coenzyme forms, with attendent quenching of flavin fluorescence and changes in visible absorption spectrum, etc. However, it is also clear that beneath these apparent similarities, they also display discrete differences in finer details o f molecular characteristics. The findings that these subtle changes do not significantly influence the functional characteristics of the protein testifies to the importance of the protein for avian embryonic development and may be related to the fact that flavin binding is directly to the protein and not through a prosthetic group or through other non-amino acid residues. References 1 Sonneborn, D.W. and Hensen, H.J. (1970) Science 168, 591--592 2 Muniyappa, K. and Adiga, P.R. (1979) Biochem. J. 177, 887--894 3 Fraser, D.R. and Emtage, J.S. (1976) Biochem. J. 160, 671--682 4 Meslaz, H.W., Camper, S.A. and White, H.B. (1978) J. Biol. Chem. 253, 6 9 7 9 - - 6 9 8 2 5 Rhodes, M.B., Bennet, N. and Feeney, R.E. (1959) J. Biol. Chem. 234, 2 0 5 4 - - 2 0 6 0 6 0 s t r o w s k i , W., Skahzynski, B. and Zak, Z. (1962) Biochim. Biophys. Acta 59, 515--517 7 Murthy, U.S., Sreekrishna, K. and Adiga, P.R. (1979) Anal. Biochem. 92, 345--350 8 Winter, W.P., Buss, E.G., Clagett, C.O. and Boucher, R.V. (1967) Comp. Biochem. Physiol. 22, 892 --897 9 Winter, W.P., Buss, E.G., Clagett, C.O. and Boucher, R.V. (1967) Comp. Biochem. Physiol. 22, 897--906 10 Heald, P.J. and McLachlan, P.M. (1965) Biochem. J. 94, 32--39 11 Murthy, U.S. and Adiga, P.R. (1977) Indian J. Biochem. Biophys. 14, 118--124 12 Murthy, U.S., Podder, S.K. and Adiga, P.R. (1976) Biochim. Biophys. Acta 434, 69--81 13 Blankenhorn, G. (1978) Eur. J. Biochem. 82, 155--160 14 FarreH, H.M., Mallette, M.E., Buss, E.G. and Clagett, C.O. (1969) Biochim. Biophys. Acta 194, 433--442 15 Lubas, B., Soltysik, M., Steczko, J. and Ostxowski, W. (1977) FEBS Lett. 7 9 , 1 7 9 - - 1 8 2 16 Murthy, U.S. and Adiga, P.R. (1978) Biochim. Biophys. Acta 538, 364--378 17 Murthy, U.S. and Adiga, P.R. (1977) Biochem. J. 166, 6 47--650 18 Murthy, U.S. and Adiga, P.R, (1978) Biochem. J. 170, 331--335 19 Feeney, R.E. and Allison, R.G. (1969) Evolutionary Biochemistry of Proteins, pp. 58--91, Wiley Interscience, New Y o r k 20 Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404---427 21 Dubois, M., Gilles, K.A., Hamilton, J. and Rebers, P.A. (1956) Anal. Chem. 28, 350--356 22 Zacharius, R.M., Zell, T.E., Merrison, J.H. and Woodlock, J.J. (1969) Anal. Biochem. 30, 148--152 23 Andrews, P. (1964) Biochem. J. 91, 22--33 24 Weber, K. an d Osborne, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 25 Whitby, L.G. (1953) Biochem. J. 54, 4 3 7 - - 4 4 2 26 Fiske, C.H. and Subba Rao, Y. (1925) J. Biol. Chem. 66, 375--400 27 Ou chterlo ny, O. (1967) in H a n d b o o k of E x p e r i m e n t a l I m m u n o l o g y (Weir, D.M., ed.), pp. 655--706, Blackweli Publication, Oxford 28 Spackman, D.H., Stein, M. and Moore, S. (1950) Anal. Chem. 30, 1190---1206 29 Spies, J.R. and Chambers, D.C. (1949) Anal. Chem. 21, 1 2 4 9 - - 1 2 6 6 30 Lowry , O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 31 Cutting, J.A. and Roth, T.F. (1973) Biochim. Biophys. Acta 298, 951--955 32 Yusko, S.G. an d R o t h , T.F. (1976) J. Supramol. Struct. 4, 8 9 - 9 7 33 Phillips, J. (1969) Ph.D. Thesis, Pennsylvania State University, U.S.A. 34 Massey, V., Curti, B. and Ganther, H. (1966) J. Biol. Chem. 241, 2347--2357 35 Harbury, H.A., LaNone, K.F., Loach, P.A. and Amick, R.M. (1959) Proc. Natl. Acad. Sei. U.S.A. 45, 1708--1717 36 Kotaki, A., Naoi, N., Okuda, J. and Yagi, K. (1967) J. Biochem. 6 1 , 4 0 4 - - 4 0 6 37 Nishikimi, M. and Yagi, K. (1969) J. Biochem. 6 6 , 4 2 7 - - 4 2 9 38 Yagi, K. and Ozanoa (1962) Biochim. Biophys. Aeta 5 6 , 4 1 3 - - 4 1 9 39 Kotaki, A., Naoi, M. and Yagi, K. (1966) J. Biochem. 59, 625--628 40 Nishikimi, M. and Kyogoku, Y. (1973) J. Biochem. 73, 1 2 3 3 - - 1 2 4 2
Biochimica et Biophysica Acta, 623 (1980) 339--347 © Elsevier/North-Holland Biomedical Press
BBA 38422
PURIFICATION AND PROPERTIES OF RIBOFLAVIN-BINDING PROTEIN FROM THE EGG WHITE OF THE DUCK (ANAS PLATYRHYNCKOS)
K. MUNIYAPPA and P. R A D H A K A N T H A ADIGA
Department o f Biochemistry, Indian Institute of Science, Bangalore 560 012 (India)
(Received October 1st, 1979) Key words: Riboflavin-binding protein; Molecular weight estimation; Amino acid composition; (Anas platyrhynckos)
Summary Riboflavin-binding protein was purified from the egg white of domestic duck and some of its properties were investigated. The protein was homogeneous by the criteria of gel filtration on Sephadex G-100 and electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, had molecular weight of 36 000 + 1000 and, unlike the chicken egg white protein (Mr 32 000 + 2000), was devoid of covalently-bound carbohydrate. It was similar to the chicken riboflavin-binding protein in its behavior on ion-exchange celluloses and affinity to interact with the flavin and its coenzymes, but differed significantly in amino acid composition in that it completely lacked proline and contained less of methionine and arginine. The protein partially cross-reacted with the specific antiserum to chicken riboflavin-binding protein with a spur during immunodiffusion analysis.
Introduction
The avian egg contains adequate amounts of all the necessary nutrients, including vitamins and other micronutrients for the proper development of the prospective embryo. In the case of chicken, the deposition of at least some of the vitamins in the egg involves the obligatory participation of carrier proteins specific for each of these vital nutrients [1--5]. As early as 1959, Rhodes et al. [5 ], while exploring the use of CM-cellulose for the fractionation of chicken egg white, discovered the presence of a unique fiavoprotein which bound riboflavin more avidly than its c0enzymes, but was devoid of any enzymatic activity. A similar flavin-binding protein was subsequently isolated from the egg yolk [6,7]. The biological importance of this protein as a vitamin-carrier, indispensable for embryonic development and survival, was elucidated much later
340 [8]. The egg flavoproteins are phosphoglycoproteins with molecular weight of 32 000 (white) and 36 000 (yolk), and appear to be products of a single structural gene [9]. While the egg white proteins originate from the oviduct, the yolk proteins are secreted by the avian liver, and transported through the circulation; to be deposited in the developing oocyte [10]. Because of several attractive features associated with this flavoprotein, in recent years considerable attention has been focused on its purification, physicochemical properties and quantification [ 11 ], the structural features involved in protein-ligand interaction [12--15] and hormonal induction and regulation of its biosynthesis [16--18]. While the existence of similar flavoproteins in the eggs of several avian species has been indicated earlier [19], no systematic studies on purification and molecular characteristics of these proteins from other oviparous species appear to have been reported so far. In this paper, we describe the isolation of the riboflavin-binding protein from the egg white of domestic duck (Anas platyrhynckos) and compare some of its properties with those of the chicken flavoprotein. Materials Fresh duck eggs were obtained from the local market. Potato acid phosphatase was procured from Sigma Chemical Co. (St. Louis, MO). The sources of other biochemicals, reagents and gels have been referred to earlier [11 ]. Methods
Isolation and purification of riboflavin-binding protein. Unless otherwise mentioned, all the steps were carried out at 4°C. The m e t h o d employed for isolation was essentially similar to that described by Murthy and Adiga [11 ] for chicken egg white protein with minor modifications. Briefly, the egg white, physically separated from the yolk, was sonicated for 2 min at 4 mA using a Bronson sonifier (Model $75) and the clarified protein was dialysed against 0.1 M acetate buffer, pH 4.3. After loading the protein on a DEAE-cellulose column (2.0 cm X 75 cm) pre-equilibrated with the above buffer and washing off the unadsorbed proteins, the absorbed proteins were eluted with 0.5 M NaC1 in 0.1 M acetate buffer at pH 4.3. Fractions having absorption both at 280 nm (protein) and 445 n m (riboflavin) were pooled, dialyzed against water and fractionated on a CM-ceUulose column (1.5 cm X 60 cm) using NaC1 gradient (0 -* 0.5 M NaC1 in 0.025 M sodium acetate buffer, pH 3.6). Fractions having a high absorption at 280 nm were pooled, dialyzed against water, lyophilized and tested for the capacity to bind riboflavin [12]. Analytical polyacrylamide gel disc electrophoresis. Polyacrylamide disc gel electrophoresis was carried out with 7.5% gels at pH 8.3 according to the m e t h o d of Davies [20] and gels were stained and destained as described elsewhere [11]. Neutral sugars were estimated according to Dubois et al. [21]. Staining for the glycoprotein was carried out as described by Zacharius et al. [22]. Molecular weight determination. Molecular weight of the purified protein was determined by gel filtration chromatography on a Sephadex G-100 column
341 [23] and b y electrophoresis using 7.5% SDS-polyacrylamide gels [24]. The following proteins of k n o w n molecular weight served as markers in both the procedures: c y t o c h r o m e c ( 1 3 7 0 0 ) , chymotrypsinogen ( 2 5 0 0 0 ) , ovalbumin (44 000), bovine serum albumin (68 000, monomer), phosphorylase A (95 000) and yeast alcohol dehydrogenase (125 000). Spectral measurements. Absorption spectral changes were recorded with a Cary 14 or Unicam (Model SP 500) spectrophotometer. The concentrations of solutions of the apo-riboflavin-binding protein and riboflavin were routinely determined spectrophotometrically using the calculated values of e2s0 = 4.75 • 104 M -1 • cm -l and e44s = 1.25 • 104 M -1 • cm -~ [25] respectively. Fluorimetric titrations were carried o u t with a Perkin-Elmer spectrofluorimeter (Model 203). During titration of the apo-protein with riboflavin, protein fluorescence was measured at 340 nm following excitation at 280 nm. Similarly, flavin fluorescence was excited at 370 nm and emission measured at 520 nm.
Treatment of the apo-riboflavin-binding protein with potato acid phosphatase. The apo-protein (15 mg) in 1.5 ml of 0.1 M sodium acetate buffer (pH 5.3) was treated with 0.1 ml of the acid phosphatase (4590 units) and dialysed against 30 ml of buffer at 37°C in a water bath. 1 ml aliquots of the dialysate were removed at specifified intervals and analysed for inorganic phosphate [26]. The total phosphate analysis was carried o u t concurrently on the dephosphorylated and untreated apo-protein. Immunological techniques. Antiserum to the chicken riboflavin-binding prorein was raised in albino rabbits as described b y Murthy et al. [12]. Double immunodiffusion analysis on agar was performed as described b y Ouchterlony [27]. Amino acid analysis. The protein (5 mg) was hydrolyzed in vacuo in 6 M HC1 at l l 0 ° C for 24 h and the amino acid composition determined in an automatic amino acid analyzer (Electroselenium Ltd., U.K.). The details of analysis and calculations were according to the standard procedures [28]. Tryptophan content was determined chemically b y the procedure of Spies and Chambers [29]. Protein determination. Protein concentrations were measured as described b y L o w r y et al. [30] using bovine serum albumin as the standard. Results and Discussion
Purification of riboflavin-binding protein When the processed crude egg white protein was applied on the DEAE-cellulose column and the ion-exchanger was extensively washed with the equilibration buffer (0.1 M sodium acetate buffer, pH 4.3) until the effluent was free of material absorbing at 280 nm, the vast majority of the protein was excluded from the column as monitored spectrophotometrically. The absorbed protein was then eluted with 0.5 M NaC1/0.1 M sodium acetate buffer (pH 4.3). Fractions with absorption at b o t h 445 nm (riboflavin) and 280 nm ( p r o t e i n ) w e r e pooled and concentrated b y lyophilization. Polyacrylamide gel disc electrophoresis o f this preparation revealed t w o closely spaced protein bands. Further purification of the riboflavin-binding protein was achieved b y chromatography on a CM-cellulose column using a linear salt gradient (0 -~ 0.5 M
342
NaC1 in 0.025 M sodium acetate buffer, pH 3.6) as the eluent. The protein was found to resolve as two distinct peaks. The protein species which emerged first as a relatively small peak appeared to be ovomucoid (since it gave a precipitin band during immunodouble diffusion on agar, against a specific antiserum to purified chicken ovomucoid) and did not interact with riboflavin. The larger protein peak, emerging after the first, possessed flavin binding capacity apart from partially cross-reacting with the mono-specific antiserum to purified chicken riboflavin-binding protein [12]. The pooled fractions corresponding to the second protein peak were dialyzed and concentrated. From a dozen duck eggs, a yield of 36 mg of the purified protein was realized.
Criteria o f purity The purified protein exhibited a single stainable band on electrophoresis using 7.5% polyacrylamide gels {data not shown). A duplicate gel, when stained for glycoprotein [22], did not take up the stain, indicating the absence of bound carbohydrate residues. In conformity with this, quantitative methods for total bound carbohydrate [21] also gave negative results. This is in marked contrast to the chicken egg flavo-proteins, both of which are glycoproteins [11]. The physiological relevance of the lack of covalently bound oligosaccharide in the duck protein is not understood at present. Nevertheless, these results confirm an earlier observation with the chicken ovoflavoprotein that bound carbohydrate residues are dispensable in terms of the protein-flavin interaction [ 11 ]. It is pertinent to mention here that the duck yolk riboflavin-binding protein isolated by a procedure recently developed to purify chicken yolk protein [7] is in fact a glycoprotein (Muniyappa, K. and Adiga, P.R., unpublished data) which is in conformity with the postulate [2,31] that the carbohydrate moieties of the yolk glycoproteins may serve as recognition sites at specific receptor loci on the outer surface of the plasma membranes of the developing oocyte for facilitating preferential uptake and deposition in the yolk [ 32 ]. Molecular weight The molecular weight of the purified protein was determined by gel filtration chromatography on Sephadex G-100 as well as by SDS-polyacrylamide gel electrophoresis. By both these procedures, a single molecular species with an apparent molecular weight of 36 000 + 1000 was observed. Thus, in terms of molecular weight the duck flavoprotein, though devoid of covalently bound carbohydrate, is slightly larger than chicken egg white protein (32 000 + 2000) [11] and more akin to the chicken yolk flavoprotein (36000 + 2000) [7]. It could be easily separated from the chicken egg white flavoprotein during coelectrophoresis on SDS-polyacrylamide gel electrophoresis (unpublished data). Immunological cross reactivity On immunodouble diffusion analysis on agar against the antiserum to purified chicken egg white riboflavin-binding protein [12] the duck protein exhibited a single precipitin line which, however, was not completely confluent with that obtained with chicken egg white riboflavin-binding protein (Fig. 1). The unequivocal detection of a spur in the precipitin band suggests that the duck ovoflavoprotein, while possessing some common antigenic determinants
343
Fig. 1. D o u b l e - i m m u n o d i f f u s i o n analysis o f r i b o f l a v i n - b i n d i n g p r o t e i n . T h e c e n t r e well c o n t a i n e d a n t i s e r u m to c h i c k e n r i b o f l a v i n - b i n d i n g p r o t e i n . T h e p e r i p h e r a l wells, 1 a n d 2, c o n t a i n r i b o f l a v i n - b i n d i n g p r o t e i n f r o m c h i c k e n egg w h i t e ; 3 a n d 4 c o n t a i n r i b o f l a v i n - b i n d i n g p r o t e i n f r o m d u c k ' s egg w h i t e . 20/~g of e a c h w a s a p p l i e d .
(and hence partial sequence homology) with the chicken riboflavin:binding protein, is distinguishable from the latter in finer details of molecular architecture. Similar observations have been made regarding the immunological features of other egg white proteins such as lysozyme, ovomucoid and conalbumin from different avian species [19]. Amino acid analysis Further evidence for the above premise stems from an examination of the amino acid composition of the protein (Table I). It may be seen that there are
TABLE I C O M P A R I S O N OF AMINO ACID COMPOSITIONS OF T H E DUCK AND T H E C H I C K E N E G G W H I T E RIBOFLAVIN-BINDING PROTEINS Amino acid
A s p a r t i c acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine Haif-cysteine Valine Methionine Isoleucine Tyrosine Phenylalanine Lysine Histidine Axginine *** Tryptophan Phosphorus
Residues/mol OvotlavoProtein Duck *
Chicken * *
14.0 11.0 26.0 39.2 nil 7.8 16.0 12.6 10.0 3.6 24.0 8.2 11.0 18.4 16.0 1.2 *** 9.0 7.4
20.0 7.7 28.8 36.4 9.7 8.3 13.9 15.9 5.7 8.2 14.8 9.5 7.0 17.2 9.2 5.6 9.3 7.0
* N o r m a l i z e d to M r 36 0 0 0 o f t h e n a t i v e p r o t e i n . ** T h e v a l u e s are t a k e n f I o m R e f . [ 3 3 ] . *** N o r m a l i z e d to o n e a r g i n i n e r e s i d u e .
344 clear cut quantitative and qualitative differences between the duck and chicken egg white proteins, which is reminiscent of observations regarding the pronounced variations in amino acid composition (besides other characteristics) among homologous egg proteins from different avian species [19]. For example, proline is completely absent in the duck flavoprotein in contrast to nearly 10 residues per mol in the chicken flavoprotein [33]. Further, the duck protein appears to be significantly richer in hydrophobic amino acids (valine + leucine) and histidine, though the reverse seems to be the case with respect to the contents of methionine and arginine. Of special significance is the presence of comparable number of residues of tryptophan, half-cysteine and acidic amino acids in both the proteins. The fact that these functional moities are conserved assumes importance in the context of our earlier findings with the chicken ovoflavoprotein [12] that these residues are critically involved in protein-ligand interaction.
Dephosphorylation and flavin binding The finding that the purified duck riboflavin-binding protein harbours the same number of protein b o u n d phosphate residues (Table I) as the chicken protein, prompted an investigation on their functional significance. Treatment with potato acid phosphatase resulted in a rapid dephosphorylation of both the apoand holoproteins. More than 80% of the protein b o u n d phosphate was released when incubated with the enzyme at 37°C for 3--5 h. If more enzyme was added after 3 h of incubation, up to 95% of the total b o u n d phosphate could be liberated. However, the dephosphorylated apo-riboflavin-binding protein could bind riboflavin with an efficiency comparable to that of untreated protein (unpublished data), showing that the covalently b o u n d phosphate residues are dispensable in terms of protein-ligand interaction. Similar conclusion has been arrived at in the case of the chicken ovoflavoprotein [ 5 ].
Spectral changes on flavin-protein interaction In Fig. 2 the changes in visible absorption spectrum of riboflavin on interaction with the purified duck apo-riboflavin-binding protein are depicted. The spectral changes are characterised b y a remarkable hypochromism of both the 370 nm and 450 nm bands without a shift of band positions. Based on a comparison of absorption spectra of flavins in solvents of various polarity [34,35], this phenomenon of hypochromicity at 370 nm has been attributed to the involvement of hydrophilic or polar interactions [36]. In this respect, the duck protein appears to be similar to the chicken egg white flavin-binding protein. Furthermore, the magnitude of hypochromicity was found to be proportional to the extent of flavin-apo-protein interaction. However, no significant spectral shifts in the absorption band at 450 nm were concomittantly observed in contrast to the situation obtained with the chicken ovoflavoprotein [37]. In the latter case, besides hypochromicity, a red shift of 450 nm band accompanied b y appearance of shoulders, has been recorded and this has been interpreted to represent the isoalloxazine ring of the flavin getting deeply buried in the hydrophobic environment of the apo-protein, following protein-ligand interaction. Based on this interpretation, it appears likely that the loci where the flavin interacts with the duck apo-flavoprotein are relatively less hydrophobic than those of the chicken protein.
345
0.~
(A) O~
O.g
~0.4
0.~
\
0.1 1
I
i
i
i
0.6
O.S
0.4
O.2 0.1 i
I:
(c)
0.~ 0.4 0..4
0J
0
350
45o
ssO
WIVIEL E NGTH (nm)
Fig. 2. Visible ab so rp tion spectra of the flavin-apo-protein complexes. (A) A bs orpt i on spectra of riboflavin and its co mp lex with the apo-protein. I, Riboflavin (30 /~M), II, I plus the apo-protein (30 /~M). (B) Absorp tion spectra of FMN and its c o m p l e x with the apo-protein I, FMN (30 #M). n0 I plus apo-protein (30 #M) (C) A b s o r p t i o n spectra of FAD and its c o m p l e x w i t h the apo-protein. I, FAD (30/~M), II, I plus apo-protein (30 pM).
Since the magnitude of hypochromicity at 370 nm, and 450 nm bands appears to be a, function of the extent of protein-ligand interaction, it is clear from the data depicted in Fig. 2 that FAD and FMN have relatively less affinity than the free flavin to interact with the apo-flavoprotein at equimolar
346 concentrations, which is in agreement with the findings with the chicken aporiboflavin-binding protein [ 5 ].
Quenching of flavin fluorescence The titration curve for flavin-apo-protein interaction showed a sharp inflection point at equivalence point due to complete quenching of flavin fluorescence (Fig. 3). From these data, the molar extinction co-efficient of the apoprotein at 280 nm was calculated to be 4.75 • 104 M -1 • cm -1 assuming 1 : 1 binding stoichiometry of protein-flavin interaction. The quenching of flavin fluorescence is in accordance with the findings of Rhodes et al. '[5] with the chicken flavo-protein and is reminiscent of the observation with D-amino acid oxidase-bound FAD complexing with benzoate [38,39]. The binding of flavin also resulted in quenching of protein fluorescence and had an association constant Ks of 4.0 • 10 s M -1 at pH 7.0, a value comparable to t h a t calculated for the chicken egg white flavoprotein [40]. The above data thus demonstrate that the homologous flavin-binding pro-
II/
100
(A)
~. 50
5
10 RIBOFLAVIN x 10-I'M
15
(B)
100
o u,. 50
0
I 0.2
I I I 0.4 0.6 0.B RIBOFLAVIN(~M)
I 1.0
F i g . 3. F l u o r i m e t r i c t i t r a t i o n o f t h e d u c k a p o - r i b o f l a v i n - b i n d i n g p r o t e i n w i t h r i b o f l a v i n . ( A ) T i t r a t i o n utilizing flavin fluorescence. Fluorescence of riboflavin excited at 370 nm was measured at 520 nm. The a m o u n t s o f r i b o f l a v i n i n d i c a t e d 410 #1 a l i q u o t s ) w e r e a d d e d t o 3 . 0 m l o f a n a p o - p r o t e i n s o l u t i o n ( a b s o r b ance at 280 nm, 0.026) (I) or to 0.1 M sodium phosphate buffer, pH 7.0 (II). The extinction coefficient o f t h e p r o t e i n a t 2 8 0 n m w a s c a l c u l a t e d t o b e 4 . 7 5 • 1 0 4 M - 1 • c m -1 a s s u m i n g a 1 : 1 ( M / M ) b i n d i n g r a t i o . B. T i t r a t i o n u t i l i z i n g p r o t e i n f l u o r e s c e n c e , F l u o r e s c e n c e o f t h e a p o - p r o t e i n e x c i t e d a t 2 8 0 n m w a s measured at 350 nm. The amounts of riboflavin indicated were added to 3.0 ml of an apo-protein solution (440 nM).
347
teins from the egg whites of the two avian species of different taxonomic groups exhibit close physico-chemical and functional kinships, such as behavior on ion~xchange cellulose, preferential interaction with the free vitamin, rather than its coenzyme forms, with attendent quenching of flavin fluorescence and changes in visible absorption spectrum, etc. However, it is also clear that beneath these apparent similarities, they also display discrete differences in finer details o f molecular characteristics. The findings that these subtle changes do not significantly influence the functional characteristics of the protein testifies to the importance of the protein for avian embryonic development and may be related to the fact that flavin binding is directly to the protein and not through a prosthetic group or through other non-amino acid residues. References 1 Sonneborn, D.W. and Hensen, H.J. (1970) Science 168, 591--592 2 Muniyappa, K. and Adiga, P.R. (1979) Biochem. J. 177, 887--894 3 Fraser, D.R. and Emtage, J.S. (1976) Biochem. J. 160, 671--682 4 Meslaz, H.W., Camper, S.A. and White, H.B. (1978) J. Biol. Chem. 253, 6 9 7 9 - - 6 9 8 2 5 Rhodes, M.B., Bennet, N. and Feeney, R.E. (1959) J. Biol. Chem. 234, 2 0 5 4 - - 2 0 6 0 6 0 s t r o w s k i , W., Skahzynski, B. and Zak, Z. (1962) Biochim. Biophys. Acta 59, 515--517 7 Murthy, U.S., Sreekrishna, K. and Adiga, P.R. (1979) Anal. Biochem. 92, 345--350 8 Winter, W.P., Buss, E.G., Clagett, C.O. and Boucher, R.V. (1967) Comp. Biochem. Physiol. 22, 892 --897 9 Winter, W.P., Buss, E.G., Clagett, C.O. and Boucher, R.V. (1967) Comp. Biochem. Physiol. 22, 897--906 10 Heald, P.J. and McLachlan, P.M. (1965) Biochem. J. 94, 32--39 11 Murthy, U.S. and Adiga, P.R. (1977) Indian J. Biochem. Biophys. 14, 118--124 12 Murthy, U.S., Podder, S.K. and Adiga, P.R. (1976) Biochim. Biophys. Acta 434, 69--81 13 Blankenhorn, G. (1978) Eur. J. Biochem. 82, 155--160 14 FarreH, H.M., Mallette, M.E., Buss, E.G. and Clagett, C.O. (1969) Biochim. Biophys. Acta 194, 433--442 15 Lubas, B., Soltysik, M., Steczko, J. and Ostxowski, W. (1977) FEBS Lett. 7 9 , 1 7 9 - - 1 8 2 16 Murthy, U.S. and Adiga, P.R. (1978) Biochim. Biophys. Acta 538, 364--378 17 Murthy, U.S. and Adiga, P.R. (1977) Biochem. J. 166, 6 47--650 18 Murthy, U.S. and Adiga, P.R, (1978) Biochem. J. 170, 331--335 19 Feeney, R.E. and Allison, R.G. (1969) Evolutionary Biochemistry of Proteins, pp. 58--91, Wiley Interscience, New Y o r k 20 Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404---427 21 Dubois, M., Gilles, K.A., Hamilton, J. and Rebers, P.A. (1956) Anal. Chem. 28, 350--356 22 Zacharius, R.M., Zell, T.E., Merrison, J.H. and Woodlock, J.J. (1969) Anal. Biochem. 30, 148--152 23 Andrews, P. (1964) Biochem. J. 91, 22--33 24 Weber, K. an d Osborne, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 25 Whitby, L.G. (1953) Biochem. J. 54, 4 3 7 - - 4 4 2 26 Fiske, C.H. and Subba Rao, Y. (1925) J. Biol. Chem. 66, 375--400 27 Ou chterlo ny, O. (1967) in H a n d b o o k of E x p e r i m e n t a l I m m u n o l o g y (Weir, D.M., ed.), pp. 655--706, Blackweli Publication, Oxford 28 Spackman, D.H., Stein, M. and Moore, S. (1950) Anal. Chem. 30, 1190---1206 29 Spies, J.R. and Chambers, D.C. (1949) Anal. Chem. 21, 1 2 4 9 - - 1 2 6 6 30 Lowry , O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 31 Cutting, J.A. and Roth, T.F. (1973) Biochim. Biophys. Acta 298, 951--955 32 Yusko, S.G. an d R o t h , T.F. (1976) J. Supramol. Struct. 4, 8 9 - 9 7 33 Phillips, J. (1969) Ph.D. Thesis, Pennsylvania State University, U.S.A. 34 Massey, V., Curti, B. and Ganther, H. (1966) J. Biol. Chem. 241, 2347--2357 35 Harbury, H.A., LaNone, K.F., Loach, P.A. and Amick, R.M. (1959) Proc. Natl. Acad. Sei. U.S.A. 45, 1708--1717 36 Kotaki, A., Naoi, N., Okuda, J. and Yagi, K. (1967) J. Biochem. 6 1 , 4 0 4 - - 4 0 6 37 Nishikimi, M. and Yagi, K. (1969) J. Biochem. 6 6 , 4 2 7 - - 4 2 9 38 Yagi, K. and Ozanoa (1962) Biochim. Biophys. Aeta 5 6 , 4 1 3 - - 4 1 9 39 Kotaki, A., Naoi, M. and Yagi, K. (1966) J. Biochem. 59, 625--628 40 Nishikimi, M. and Kyogoku, Y. (1973) J. Biochem. 73, 1 2 3 3 - - 1 2 4 2