Structural studies of blood-group substance A from hog-stomach linings

52

BIOCHIMICA ET BIOPHYSICA ACTA BBA 85005

STRUCTURAL STUDIES OF BLOOD-GROUP

SUBSTANCE A

FROM HOG-STOMACH LININGS N. K. KOCHETKOV, V. A. DEREVITSKAYA, S. G. KARA-MURZA AND V. (~. ZHAROV Institute for Chemistry of Natural Products, U.S.S.R. Academy of Sciences, Moscow (U.S.S.R.)

(Received August 9th, 1963) SUMMARY Blood-group substance A from pooled hog-stomach linings was subjected to hydrolysis by a protease from asian influenza virus and to hydroxylaminolysis. Half of the polymer appeared in the dialyzable fraction after proteolysis. The non-dialyzable polysaccharide fragment retained only aspartic acid. Hydroxylaminolysis results in 30 % yield of dialyzable products. The non-dialyzable fragment contained no aspartic acid after this cleavage. Mono- and oligosaccharides, peptides and short-chain glycopeptides were detected among the dialyzable products of hydroxylaminolysis. On the basis of the results obtained, a general plan of the structure of blood-group substance A is proposed. INTRODUCTION Despite the numerous investigations into the immunology and immunochemistry of blood-group substances a-3, there is almost no information concerning their chemical structure. The most recent works of KABAT4,5 and of MORGAN6,7 give some idea of the possible terminal carbohydrate residues which are the immunological determinants, but it is difficult to form a concept of the general structure of these biopolymers on the basis of available experimental data, including those of hydrazinolysis s,9. There is also no evidence as to the nature of the bond between the carbohydrate and peptide moieties of the molecule; the assumptions of the existence of N-glycoside 1° and 0-glycoside n bonds still remain conjectural. No sufficient proof has been forthcoming either of the report that a fragment containing ether-linked sugar and amino acid residues 1~ has been isolated from these substances. The outstanding biological role of the blood-group substances makes highly desirable a knowledge of their chemical structure. A possible approach to this could be development of fragmentation methods for these molecules and determination of the resultant partial structures. We have accordingly carried out the proteolysis and hydroxylaminolysis of blood-group A substance which has resulted in certain conclusions about its general structure and which gives promise of future results. EXPERIMENTAL Paper chromatography and electrophoresis

Paper chromatography and electrophoresis were carried out on type "M" paper of the Leningrad No. 2 factory, using the following solvent systems: (A) n - b u t a n o l Biochim. Biophys. Acta, 83 (t964) 52 0o

STRUCTURAL STUDIES OF BLOOD GROUP A SUBSTANCE

53

acetic acid-water (4: I : I, V/V), (B) pyridine-n-butanol-benzene-water (3 : 5 : I : 3, v/v), (C) n-butanol-pyridine-water (3: 2:1.5, v/v), and (D) isoamyl alcohol saturated with 1% acetic acid. Electrophoresis was carried out in the horizontal apparatus EFA-I in pyridineacetate buffer (2 ml pyridine, 4 ml acetic acid, water up to I 1; pH 4.2-4.4), 25 V/cm, 3 h; and in 0.025 M veronal buffer (pH 8.6), 20 V/cm. Two-dimensional electrophoresis-chromatography was carried out as follows: the substances were subjected to paper electrophoresis in pyridine-acetate buffer; the electropherogram was dried and then chromatographed in the second dimension in System A. Sugar-containing substances were detected by means of aniline phthalate, silver nitrate and periodate reagent; amino acid-containing substances were detected by means of ninhydrin.

Thin-layer chromatography Thin-layer chromatography was carried out on a fixed silica-gel layer in the systems (E) phenol-water (75:25, v/v), (G) n-propanol-ammonia (70:30, v/v), (H) n-butanol- acetic acid- water (60: 20 : 20, v/v), (I) ethanol- ammonia (77 : 23, v/v). The substances were detected by means of periodate reagent, ninhydrin and sulphuric acid.

Blood-group substance A Blood-group substance A was isolated from hog-stomach lining by KABAT'Sla method with the difference that, in our case, the substance was again dissolved in water and reprecipitated with three volumes of alcohol. The polymer was then triturated with alcohol, dried, fractionated from phenolic solutions 14 and dialyzed against distilled water. Because, for the solution of the problem posed here, it was desirable to have an average sample of the biopolymer, the stomach linings of 50-200 hogs were used for the isolation*. The polymer** obtained was electrophoretically homogeneous, as ascertained by electrophoresis on glass powder 15 in borate buffer (pH 7.5), 460 V, I ~A, and, on paper in acetate buffer (pH 4). The serological activity, as determined by the haemagglutination-inhibition test, was found to be equal to 0.4-0. 5 ~g. The monomer composition of the biopolymer was determined by the following methods : combined galactose and fucose content by the anthrone technique 18, amino sugars by a modified ELSON-MORGAN1~ method, contents of galactose, fucose, glucosamine and galactosamine separately by the aniline phthalate method 18. The peptide content of the polymer was determined according to LOWRY et al. 19.

Protease.from asian influenza virus A new proteolytic enzyme was isolated from the asian influenza virus, Strain A-2 "Krasnodar" lOl-59"**. * The s u b s t a n c e A w h e n isolated from pooled hog-stomach linings exhibited some slight H (O) activity, b u t it seems unlikely t h a t this c o n t a m i n a t i o n could in a n y w a y h a v e affected the results of the p r e s e n t first a p p r o x i m a t i o n to the mucopolysaccharide structure. ** The monosaccharide and amino acid composition of the isolated p r e p a r a t i o n was in full agreement w i t h reported d a t a on blood-group substance A. Details of this p a r t of the w o r k will be published elsewhere. *** According to our new d a t a the source of protease requires additional investigation.

Biochim. Biophys. Acta, 83 (1964) 52-60

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N . K . KOCHETKOV et al.

The isolation was achieved from tile allantoic fluid prepared by the inoculation of Io-days-old chick embryos. The liquid was centrifuged (2ooo rev./min, 3o min) to free it from erythrocytes and other insoluble particles. The virus-containing supernatant was decanted and the virus particles were removed by centrifugation (45 ooo rev./min, 2 h). The precipitate was twice resuspended in o.oo5 M phosphate buffer (pH 7.o) and removed by centrifugation. The resuspended precipitate was then centrifuged at 7oo-8oo rev./min for 3o rain to remove possible aggregated particles and twice-recrystallized trypsin was added to the supernatant in amounts of I m g per 2o-3o ml of allantoic fluid. After incubating at 37 ° for I 6 - I 8 h, the preparation was dialyzed for 72-96 h against 2oo vol. of distilled water at 4 % This was followed by centrifugation of the aqueous solution (45ooo rev./min, 2 h) to remove any viral debris. The enzyme was precipitated from aqueous solution by means of acetone. The fraction which precipitated from the solution, when acetone was added up to a concentration of 2o-35 %, was removed by centrifugation and dissoh;ed in the minimum amount of water. The undissolved residue was removed bv centrifugation and the solution was subjected to freeze-drying. The enzyme was found to be of high proteolytic activity (proteolytic factor Cx = I23, with casein as substrate) and of broad substrate specificity (tests on albumin, casein, gelatin and mucin). It had neither glycosidase (test on maltose, cellobiose, lactose and sucrose) nor esterase (test on O-aminoacyl derivatives of glucose and phenylalanine esters) activity. No special enzymic studies of the enzyme were carried out except to establish that its maximum activity at 36-38o occurs at pH 6.35 -7.I.

Proteolysis of blood-group substa~ce d The substance was incubated for 48 h at 37 ° in phosphate buffer of pH 7.o, with I - 5 % of its weight of enzyme. The products were then dialyzed or fractionated on Sephadex.

HydroxyIaminolysis of blood-group substance A Hydroxylamine base was prepared from the hydrochloride by adding the theoretical amount of sodium ethylate in absolute ethanol. A 4 % solution of the biopolymer was incubated for 3 4 h at room temperature in 5 M aqueous solution of hydroxylamine. The solution was then freeze-dried until hydroxylamine had been removed completely and the residue, after being taken up in water, was dialyzed against water. The diffusate was evaporated under vacuum at 3o ° until a small volume remained; it was then freeze-dried.

Preparative fractionation on Sephadex The fractionation was carried out on Sephadex G-5o "Medium" using a column of 38o mm ~ 18 ram. The composition of the fractions (each of which was 5 ml in volume) was followed by standard techniques.

Preparative electrophoresis Electrophoresis was carried out in pyridine-acetate buffer for 3 h. The electropherogram was dried and a narrow strip cut out, which was used to detect the location of the ninhydrin-staining substances. The main portion of the electropherogram was then cut into zones which were eluted with 25 % ethanol. Biochim. Biophyr. Acia, 83 (1904) 5 ' 0o

STRUCTURAL STUDIES OF BLOOD GROUP A SUBSTANCE

55

RESULTS AND DISCUSSION

It has already been said that in order to obtain data leading to the first general conceptions of the structure of blood-group substance A it was subjected to proteolysis and hydroxylaminolysis. The substance isolated by us from the hog-stomach lining had high specific activity and was free of extraneous protein or polysaccharide matter as was shown by its electrophoretic homogeneity. Fractional precipitation by ethanol 2° from aqueous copper acetate solutions of the polymer afforded two fractions. These were completely identical in carbohydrate composition, so that their difference could be only with respect to molecular weights. The lack of homogeneity in the molecular weight of the polymer (and also certain minor structural differences in the blood-group substances, which to all appearances are due to individual differences in the animals and are subjected to fluctuations arising from seasonal, nutritional and other external factors) were of no consequence to the problem which we had set before us.

Proteolysis of blood-group substance A Very little study has been devoted to the proteolysis of blood-group substances. It has been shown 21,22 that ficin and papain cause them to lose much of their ability to inhibit haemagglutination, despite relatively little change in their composition. Only insignificant amounts of amino acids and peptides are eliminated, but considerable fall in viscosity of the polymer solutions is observed. Trypsin, pepsin and chymotrypsin do not act on these substances. For cleavage of the peptide moiety of bloodgroup substance A as stated above we isolated a protease from the asian influenza virus, which proved highly efficient in this respect. To obtain an idea of the products formed in the proteolytic reaction, the mixture resulting from incubation of the polymer with the protease was subjected to dialysis against water and the diffusate was analysed by thin-layer chromatography (with Systems E, G, H and I) and paper chromatography (with System B). A large variety of amino acids was detected, resulting from breakdown of the peptide chains and autolysis of the enzyme. Free monosaccharides were absent; however, after acid hydrolysis of the diffusate (0.5 N HC1, 8 h) fucose and galactose were found chromatographically (System B). It thus follows that, besides amino acids, proteolysis led to the liberation of small oligosaccharides or fragments containing both sugars and amino acids, which yielded reducing sugars only after hydrolysis. Isolation of the high-molecular-weight proteolysis product (Fragment I) by dialysis proved tedious and, in consequence, this was carried out on Sephadex. The separation of the mixture obtained on incubation with protease is shown in Fig. I. One can see from Fig. I that Fragment I appeared as a single, clearly defined peak, indicating its homogeneity. Following discharge of the high-molecular-weight fraction which amounted to about 50 % of the initial biopolymer, the low-molecular-weight proteolysis products came through the column. Molecular weight determinations of Fragment I have not yet been made, but the specific viscosity of its aqueous solutions compared with that of the initial biopolymer (o.145 compared with 0.286 for 0.39 % at 34 °) bears evidence of a rather high molecular weight. Hydrolysis of Fragment I (0.5 N HC1, 8 h) gave a mixture of monosaccharides consisting of fucose, galactose and amino sugars. The fucose and galactose content Biochim. Biophys. Acta, 83 (1964) 52-60

56

N.K. KOCHETKOVgt al.

was close to that of the initial biopolymer, whereas the amino sugar content was much greater (71.2 compared with 3o.8 %). Amino sugars were practically absent from tile diffusate and hence were liberated only in insignificant amounts during proteolysis, so that they must be predominantly in Fragment I. On the contrary, galactose and fucose are partially split off during proteolysis and should therefore be present in appreciable quantities also in other parts of the molecule. 0.5

0.4

0.,3 c o L o

0.2

L

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i

°o

10

~o

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40

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Fig. 1. F r a c t i o n a t i o n of proteolytic p r o d u c t s on S e p h a d e x G-5 o.

Practically only one amino acid, aspartic acid, was revealed on hydrolysis of Fragment I under peptide-cleavage conditions (6 N HC1, IOO-IO5°, 16-18 h). It was identified by means of thin-layer chromatography (Systems E, G, H, I), electrophoresis (pyridine-acetate buffer) and also electrophoresis followed by paper chromatography (System A). On spotting with very high concentrations of hydrolysate, the chromatogram showed traces of other acids, which was present in amounts which were negligible in comparison with that of aspartic acid, so that it could not be taken into account in forming a general idea of the biopolymer structure. These findings clearly indicate that aspartic acid is directly attached to the polysaccharide moiety of the blood-group substance A and is apparently the binding unit between the polysaccharide and peptide parts of the molecule. To confirm this, Fragment I was subjected to dinitrophenylation followed by acid hydrolysis (6 N HC1, lOO-lO5 °, 16-18 h). Only DNP-aspartic acid and traces of aspartic acid (evidently due to incomplete dinitrophenylation) were detected in the hydrolysate by electrophoresis (pyridine-acetate buffer) and paper chromatography (System D). Hence the entire aspartic acid contained in Fragment I must be bound directly to the polysaccharide chain, and is not combined in peptide chains. Further proof of this lies in the fact that no amino acids are liberated as such on the action of aminopeptidase upon Fragment I. Aspartic acid must therefore form the bridge between the polysaccharide and peptide moieties of blood-group substance A. Moreover, comparison between the aspartic acid contents of Fragment I (o.5 %) and of the initial biopolymer (o.95 %) showed an appreciable amount of this acid to be involved in such bridging Biochim. Biophys..4 cla, 83 (t 904) 5z -6o

STRUCTURAL STUDIES OF BLOOD GROUP

A

SUBSTANCE

57

function. Finally, if one takes into account the high molecular weight of Fragment I and that its molecules therefore contain several aspartic acid residues, which are all at the sites of linkage between the carbohydrate and peptide moieties, one must conclude that the biopolymer has a branched structure.

Hydroxylaminolysis of blood-group substance A Although hydroxylaminolysis has been frequently applied to the study of ester and O-peptide bonds in proteins, peptides .3, 34 and more complex biopolymers (see, for example, ref. 25) it has not been utilized hitherto in the case of the blood-group substances. Hydroxylaminolysis is a well known means for readily splitting the ester bond (see, for example, refs. 26 and 27). This reaction was studied in our laboratory 28 using specially synthesized model compounds 2°. Since under drastic conditions, it can also affect other linkages ~°-33 we made a special study of the stability of glycoside and amide bonds towards hydroxylamine. Under the conditions of hydroxylaminolysis which we selected (see experimental part), simple glycosides, di-, and oligosaccharides and also N-aminoacyl derivatives of aminosugars were found not to undergo fission. But, even if a small number of bonds other than ester bonds does break during the action of hydroxylamine upon such a complex molecule as the blood-group substance, this could have no significant effect on the conclusions drawn. Hydroxylaminolysis of blood-group substance A gave a complex mixture of products, which, as in the case of proteolysis, could be divided into a high-molecularweight fragment (Fragment II) and low-molecular-weight fragments. In this case dialysis against water was found to be the most convenient way for their separation. The diffusate, containing the low-molecular-weight fragments (about 30 % of the initial weight of the biopolymer), was subjected to electrophoresis and paper chromatography. The best separation was achieved by two-dimensional paper electrophoresis-chromatography (see experimental part). Hydroxylaminolysis yields a large number of low-molecular-weight fragments, 20-22 spots being clearly discerned on the electropherochromatogram*. The spots can be divided into three groups, namely: those due to the periodate reagent and silver nitrate and therefore to the oximes of sugars or small oligosaccharides; those due to the ninhydrin reaction, corresponding to lower peptides and those giving both the ninhydrin and periodate reactions and therefore due to substances containing both monosaccharides and amino acids, most likely glycopeptides. In order to obtain further information about the nature of the low-molecularweight fragments of hydroxylaminolysis, the diffusate was subjected to preparatory electrophoresis on paper (see experimental part). Analysis by paper chromatography (System B) of the neutral products eluted from the electropherograms revealed galactose, fucose and N-acetylhexosamine oximes, the densest spot being given by fucose oxime. That it was just these monosaccharides which were present in the lowmolecular-weight fractions was confirmed by the fact that on hydrolysis (0.5 N HC1, 8 h, ioo °) of the mixture obtained on evaporation of the diffusate, fucose, galactose and small amounts of glucosamine and galactosamine were revealed by chromatography. A substance of unknown nature with Rgalaetose = 2 was also detected. * A l t h o u g h the n u m b e r and relative positions of the spots varied slightly for various bloodgroup substance specimens, the general picture shown in Fig. 2 r e m a i n e d the same during repeated runs.

Biochim. Biophys. Acta, 83 (I964) 52-60

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N.K. KOCHETKOV6t a[,.

Some of the substances in the mobile zones from preparative electrophoresis were also investigated. Acid hydrolysis of these substances showed the presence of neutral monosaccharides --galactose and fucose. Because the electrophoretic mobility of these substances could be due only to amino acids, this was further proof that tile lowmolecular-weight products of hydroxylaminolysis, besides mono- and oligosaccharides also contain small glycopeptides. The non-dialyzing, high-molecular-weight fraction of the hydroxylaminolysis products (Fragment II), emerges in the form of a well expressed peak on passing the products through a Sephadex column. Fragment II is a glycopeptide, considerably reduced in its content of amino acids in comparison with the initial biopolymer (9 %, instead of 16 % for the latter). The monosaccharide composition of the fragment exhibited no qualitative difference from that of the biopolymer despite the fact that a study of the low-molecular-weight fragments formed by hydroxylaminolysis showed that the sugars eliminated were predominately galactose and fucose. Very characteristic is the distribution of some amino acids especially aspartic acid among the hydroxylaminolysis products. The hydrolysate of Fragment II (following hydrolysis by 6 N HC1, 24 h, ioo ° and evaporation under vacuum to remove the HC1) contained only traces of aspartic acid, as shown by electrophoresis and paper chromatography. Hence it is almost completely removed from the polymer by hydroxylaminolysis. From this it follows that aspartic acid which, as seen from the results of proteolysis, links the polysaccharide part of the biopolymer with the peptide moiety could be ester-bonded via one of its carboxyl groups. However, not all the peptide chains of the biopolymer are eliminated by hydroxylaminolysis. This shows that aspartic acid is not the only bonding unit between the polysaccharide and peptide moieties of the biopolymer. The peptide chains which are not eliminated by the action of hydroxylamine, bnt undergo rupture by the protease of the influenza virus, are apparently also present in the blo(~d-group substances.

Electrophoresis Q Start 4

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Fig. 2. Separation of low-molecular-weight Fig. 3. General scheme of the structure of a hydroxylaminolytic products of b l o o d - g r o u p blood-group substance molecule. substance A by electrophoresis in combination with chromatography. O, spots due to periodate reagent; 0, spots due to ninhydrin: O, spots due to both ninhydrin and periodate reagent. l~iochim. Biophv~..h:ta, S3 (I,~4) 52 I~o

STRUCTURAL STUDIES OF BLOOD GROUP A SUBSTANCE

59

Also worthy of note is that glutamic acid, which could also have formed the links between the polymer moieties, differs very little in its content in the highmolecular-weight Fraction II from that in the initial substance.

General structure of blood-group substance A Analysing the data we obtained in the proteolysis and hydroxylaminolysis of blood-group substance A, a general outline of the structure of this important biopolymer can be postulated. Schematically it is represented in Fig. 3. The backbone of the biopolymer is apparently a polysaccharide chain. This backbone was liberated almost as such from the biopolymer on proteolysis and together with a large number of pendant peptide chains on hydroxylaminolysis. This central part of the biopolymer molecule contains the major portion of the N-acetylhexosamine residues together with lesser amounts of galactose and fucose, which is in complete agreement with data on the periodate oxidation of blood-group substances 34. The branching of the polysaccharide backbone can be established only after detailed study of the Fragment I by the standard methods of carbohydrate chemistry, which is at present under progress. The results of proteolysis and hydroxylaminolysis indicate that several, or even many, peptide chains are joined to the backbone and that therefore the polymer must be of a rather highly branched structure. Some of these peptide chains are linked to the polysaccharide via a s p a l i c acid and the almost complete removal of the latter on hydroxylaminolysis shows that this link must be of the ester type. The liberation of numerous mucopolysaccharides, in particular of fucose and galactose, on proteolysis and hydroxylaminolysis, indicates that a certain amount of the monosaccharide residues must be on the periphery of the molecule and apparently linked with peptide chains. Evidence directly supporting this view is to be found in the liberation of small-sized glycopeptides by hydroxylaminolysis. The nature of the bond between these peripheral carbohydrate residues and the peptides may be at least partially elucidated from a study of the fragments obtained by proteolysis and hydroxylaminolysis. Work in this direction is also in progress. CONCLUSIONS

The general structure of blood-group substance A outlined here is in harmony with a number of data regarding their immunological determinants. Thus MORGAN and KABAT have shown that the terminal oligosaccharides determine the group specificity of these biopolymers 35-39 and by specific cleavage of the end carbohydrate residues one can change the specificity of the polymer; for example, pass over from substance A to substance H, etc. A comparison of data reported in the literature with those obtained in the present study leads to the inference that the specific substances of various blood groups, that are of the same origin, possess similar polysaccharide backbones and differ in their peripheral framework. REFERENCES 1 E. A. KABAT, Blood Group Substances, Academic Press, New York, 1956. 2 W. T. J. MORGAN, Bull. Soc. Chim. Biol., 42 (1961) 1591. 3 M. STACEY AND S. A. BARKER, Carbohydrates of Living Tissues, D. v a n Nostrand, London, 1962. 4 G. SCHIFFMAN, E. A. KABAT AND S. LESKOWITZ, J. Am. Chem. Soc., 84 (1962) 73.

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KOCHETKOV gt

al.

5 W . M. WATKINS, M. L. ZARNITZ AND E. A. tXABAT, Nature, 195 (1962) 12o 5. 8 T. I. PAINTER, W . M. WATKINS AND W . T. J. MORGAN, Nature, 193 (19621 lO42. 7 W ~. T. J. MORGAN, Bull. Soc. Chim. Biol., 42 (1961) 159. 8 Z. YOSIZAWA, Biochim. Biophys. Acta, 52 (19611 588. 9 Z. YOSIZAWA AND T. SATO, Biochim. Biophys../tcla, 52 (1961) 591. 10 H . MASAMUNE AND S. HFKOMORI, Tohoku J. Exptl. Med., 04 (I956) 281. 11 I. WERNER, ,4cta Soc. Med. Upsalien., 58 (1953) 1. 12 H. MASAMUNE, Proc. Intern. Congr. Biochem., 3rd, Brussels, I955, A c a d e m i c P r e s s , N e w Y o r k , I95(), p. 72. 13 E . i . KABAT, Blood Group Substances, A c a d e m i c P r e s s , N e w Y o r k , 1956, p. 135. 14 W . T. J. MORGAN AND H. K. KING, Biochem. J., 37 (19431 64015 B. J. HOCEVAR AND D. H. NORTHCOTE, Nature, i 7 9 (I957) 488. 16 G. N. SAITSEVA AND T. N. AFANASEVA, Biohhimiya, 22 (1957) lO35. 17 C. J. RONDEL AND W. T. J. MORGAN, Biocherrl. J., 61 (19.55) 586. 18 S. B. MISRA AND V. Ix. MOHAN, J. Sci. Ind. Res. India, 19 (I96O) 173. 19 (). H . LOWRY, N. J. ]{OSEBROUGH, A. L. FARR AND t{. J. t~ANDALL, J. Biol. Chem., 193 (1951) 265 . 2o A. J. ERSKINE AND J. K. N. J o N E s , Can. J. Chem., 34 (1950 ) 8 2 L 21 A. PUSZTAI AND W. T. J. MORGAN, Nature, 182 (1958) 1"148. 22 A. PUSZTAI AND W. T. J. MORGAN, Biochem. J., 8 I (I9()l) (139, 048. 23 p. M. GALLOP, S. SEIFTER AND E. MEILMAN, Nature, 183 (I959) I659. 24 S. SEIFTER, P. H. GALLOP AND E. MEILMAN, J. Biol. Chem., 235 (I96O) 2613. 25 M. CHAPUT, G. MlCHEt. AND E . LEDERER, Biochim. Biophys. Acla, 63 ([902) 31o. 26 H. (;. ZACHAU, Chem. Per., 93 (196o) 1822. 27 H . G. ZACHAU AND W . ['~ARAU, Chem. Bet., 93 (196o) 183o28 V. A. DEREVITSKAYA, L. M. LIKHOSHERSTOV AND N. Ix. NOCHETKOV, ]giN. ,lkad. Nauk SSSR Old. Khim. Nauk, i n t h e p r e s s . 29 N. K. tX~OCHETKOV, V. ,\. DEREVYFSKAYA, L. M. LIKHOSHERSTOV, N. V. MOLOTSOV AND S. (~. [{ARA-MURZA, Tetrahedron, 18 (I962) 273. 30 p. M. GALLOP, S. SEIFTER AND E. MEILMAN, Nature, 183 (1959) I659. 31 W. [. WILLIAMS, 1. I.ITWIN AND ('. B. THORNE, J. Biol. Chem., 212 (t955) 43732 C. BRAUNITZER, Bioehim. Biophys. ,4cta, 19 (I956) 57433 M. L1EFLANDER AND (). WACKER, Z. Physiol. Chem., 327 (t962) L95. 34 G. SCHIFFMAN, E. i . KABAT AND M. THOMPSON, J. Am. Chem. Soc., 84 (19(i21 403 . a5 A. POSZTAI AND V(. T. J. MORGAN, Nature, [82 (1958) (148. 3 6 M. g. ZERNITZ AND E. A. KABAT, J. Asq/L Chem. Soc., 82 (190o) 3953a7 W. M. WATKINS AND W. T. J. MORGAN, Nature, 18o 0 9 5 7 ) 1o36as W . T. J. MORGAN, Bull. Soc. Chim. Biol., 42 (190l) 1591. 39 W . M. WATKINS, Bull. Sot. Chim. Biol., 42 (I96o) 1590-

Biochim. Biophys. :lcta, 83 (19~14) 52 ()o