/. Biochem. 86, 1573-1585 (1979)
Enzymatic Properties of Neuraminidases from Arthrobacter ureafaciens Yoshihiro UCHIDA, Yoji TSUKADA, and Tsunetake SUGIMORI Kyoto Research Laboratories, Marulrin Shoyu Co., Ltd., Uji, Kyoto 611 Received for publication, June 25, 1979
Neuraminidase I and neuraminidase II from Arthrobacter ureafaciens were characterized. As determined by gel filtration on Ultrogel AcA 44, the molecular weights of neuraminidases I and II were 51,000 and 39,000, respectively. Neuraminidases I and II were similar to each other in their enzymatic properties except for the substrate specificities towards gangliosides and erythrocyte stroma. Their optimal pHs were between 5.0 and 5.5 with TV-acetylneuraminosyl-lactose or bovine submaxillary mucin as substrates, but with colominic acid as a substrate, the pH optimum was between 4.3 and 4.5. They were most active around 53°C, were stable between pH 6.0 and 9.0, and were thermostable up to 50°C. They did not require CaI+ for activity and were not inhibited by EDTA. They were inhibited only slightly or not at all by /?-chloromercuribenzoic acid or Hg t+ . Both neuraminidases I and II were able to hydrolyze the a-ketosidic linkage of iV-glycolylneuraminic acid as well as that of iV-acetylneuraminic acid, and were able to liberate substantially all of the sialic acid from various kinds of substrates. However, they cleaved only about 50% of the sialic acid from bovine submaxillary mucin. The saponification of bovine submaxillary mucin by mild alkali treatment, on the other hand, resulted in an increased susceptibility to the neuraminidases and brought about the complete liberation of sialic acid. Remarkable differences were observed between neuraminidases I and II as regards substrate specificities on gangliosides; the initial rate of hydrolysis by neuraminidase I was 74 times, and its maximum velocity constant was 91 times those of neuraminidase II. The addition of sodium cholate markedly stimulated the enzymatic hydrolysis of gangliosides, and increased the maximum velocity constant of neuraminidase I twofold and that of neuraminidase II 143-fold. Although neuraminidases I and II were able to hydrolyze (a,2-3), (a,2-6), and (a,2-8) linkages, the initial rate of hydrolysis of 7V-acetylneuraminosyI-a,2-6-lactose was greater than that of the o,2-3-isomer.
Sialic acid (JV-acylated neuraminic acid) commonly occurs as the terminal residue of carbohydrate chains in many glycoproteins or glycolipids. Since sialic acid has many biological functions (7Vol. 86, No. 5, 1979
75), neuraminidase [sialidase, EC 3.2 1.18] can markedly influence the biological behavior of macromolecules and cells. Neuraminidase preparations free from contaminating enzymes have long
1573
1574
Y. UCHIDA, Y. TSUKADA, and T. SUGIMORI
been sought, since such preparations would be particularly useful in investigating the roles of sialic acid in glycoconjugates in living bodies. In the previous paper (14), we reported the distribution of neuraminidase in Arthrobacter, and its purification by a novel affinity chromatography procedure using colominic acid as a selective adsorbent. The purified neuraminidase from Arthrobacter ureafaciens has been shown to be free from activities of protease, Ar-acetylneuraminic acid aldolase, phospholipase C, and glycosidases. By gel filtration on Ultrogel AcA 44, the purified neuraminidase was divided into two active peaks which were tentatively designated as neuraminidase I and neuraminidase n . The present paper describes the enzymatic characteristics of the two neuraminidases from Arthrobacter ureafaciens. A preliminary report has already appeared (IS). MATERIALS AND METHODS Preparation of Neuraminidase—Neuraminidase from Arthrobacter ureafaciens was prepared and purified as described previously (14). By gel filtration on Ultrogel AcA 44, the purified neuraminidase was divided into two peaks which were tentatively designated as neuraminidase I and neuraminidase II (14). These two neuraminidases were used in the present experiments. Assay of Neuraminidase Activity—The assay system contained the following components in a total volume of 200[t\: 50fil of substrate solution, 50 pil of 200 mM buffer solution, and 100 ft\ of enzyme preparation. The reaction was conducted at 37CC for lOmin, and the amount of A^acetylneuraminic acid liberated was compared with that of a control containing the same components except for the enzyme. A/-Acetylneuraminic acid was determined by a modification of the thiobarbituric acid procedure of Aminoff (14). One unit of neuraminidase activity was defined as the amount that releases 1 (imo\ of N-acetylneuraminic acid per min under the standard reaction conditions using colominic acid as a substrate (14). Molecular Weight Determination—The molecular weight of the enzyme was estimated by gel filtration based on the method of Andrews (16). A column of Ultrogel AcA 44 (2.6 x 100 cm) was
equilibrated with lOOmM phosphate buffer (pH 6.8) and run with the same buffer at a flow rate of 25 ml per h. The following standard proteins were used for calibration; cytochrome c, myoglobin, chymotrypsinogen A, ovalbumin, and albumin. The partition coefficient, Kav, was calculated using the equation of Laurent and Killander (17). Thin-Layer Chromatography—Products of neuraminidase digestion were applied to a thin layer Silica Gel 60 chromatographic plate (Merck) pretreated with 0.2 M sodium dihydrogen phosphate (18), and the plate was developed three times with rt-propanol-1 N NH4OH-water ( 6 : 2 : 1 , v/v) as a solvent (19-21). Sialic acid was detected by spraying with the diphenylamine-aniline reagent (22). Chemicals—Colominic acid (Na salt) and iV-acetylneuraminic acid were the products of our company (24). iV-Acetylneuraminosyl-Iactose, bovine submaxillary mucin, bovine brain gangliosides, fetuin, transferrin, and trypsin inhibitor were obtained from Sigma Chemical Company. Human erythrocyte stroma was from Miles Laboratories, Inc. The following compounds were generous gifts: 7/-acetylneuraminosyl-a,2-3-lactose (23) and JV-acetylneuraminosyl-a,2-6-lactose, Dr. M. Koseki, Fukushima Medical College; iV-glycolylneuraminic acid and glycoproteins from the eggs of sea urchins (Hemicentrotus pulcherrimus and Anthocidaris crassispina) (20), Dr. K. Hotta, Kitasato University. Ultrogel AcA 44 was obtained from LKB, Bromma, Sweden. The calibration proteins were obtained from both Boehringer Mannheim and Mann Research Laboratories. Other chemicals were from Nakarai Chemicals Ltd., Kyoto. RESULTS AND DISCUSSION pH Optimum and Stability—The effects of pH on the rates of hydrolysis of A'-acetylneuraminosyllactose, bovine submaxillary mucin, and colominic acid are illustrated in Fig. 1. There was little difference in pH optima between neuraminidases I and II. However, the pH optima varied with the substrate; with TV-acetylneuraminosyl-lactose and bovine submaxillary mucin, the optima lay between 5.0 and 5.5, while with colominic acid, / . Biochem.
PROPERTIES OF A. urcafaciens NEURAMINIDASE
3
4
5
6
7
8
3
pH
4
1575
5
6
8
8
pH
pH
Fig. 1. Effect of pH on the reaction rate. 7^-Acetylneuraminosyl-lactose (A), bovine submaxillary mucin (B), or colominic acid (C) was employed as a substrate. Either acetate buffer (pH 3.5-5.9) or phosphate buffer (pH 5.4-8.0) was used. The other reaction conditions were as described in " MATERIALS AND METHODS." Data are shown for neuraminidase I ( # ) and neuraminidase
n(o).
10
pH
pH
Fig. 2. Effect of pH on the stability of neuraminidase. Neuraminidase I ( • ) and neuraminidase II (O) were incubated at 37°C for 24 h in 20 HIM buffers of pH 2.2 to 10.4; HCl-CH,COONa and CH,COOH-CH,COONa buffer for pH 2.2 to 5.9, and NaH,PO4-K,HPO4 and K,HPO4-NaOH buffer for pH 5.9 to 10.4. The remaining neuraminidase activities were determined under the standard reaction conditions. the optima were between 4.3 and 4.5. In order to remove the sialic acid residues from animal cells or tissues, neuraminidase is often allowed to react in physiological phosphate-saline Vol. 86, No. 5, 1979
solution, a typical buffer solution of neutral pH. As shown in Fig. 1, the pH activity profiles of neuraminidases I and n with bovine submaxillary mucin as a substrate gave broad optima ranging
1576
Y. UCHIDA, Y. TSUKADA, and T. SUGIMORI
from 4.5 to 6.0. Since no marked depression of the activities was observed around neutral pH, neuraminidases from Arthrobacter ureafaciens are considered to be suitable for the treatment of substrates in a physiological pH environment. As shown in Fig. 2, neuraminidases I and II are both stable at 37°C for 24 h in buffers from pH 6.0 to 9.0. This result indicates that the neuraminidases can remain active during prolonged incubation when the reaction is conducted at around neutral pH. Thermal Reactivity and Stability—The standard reaction mixture containing 50 fil of substrate (colominic acid, Na salt, 4.0 mg/ml), 50 p\ of 200 HIM acetate buffer (pH 4.5), and 100 ft\ of enzyme preparation was allowed to react at various temperatures for 10 min, and the amount of iV-acetylneuraminic acid released was compared with that of a control containing the same components except for the enzyme. The results are illustrated in Fig. 3. Neuraminidases I and II both had optima at around 53°C.
a
The neuraminidases were then incubated at pH 6.8 in a water bath maintained at various temperatures for 10 min, followed by chilling in an ice bath. An aliquot of the treated enzymes was assayed for remaining neuraminidase activity by the standard procedure and compared with a control kept at 5°C. Neuraminidases I and II were both found to be thermostable up to 50°C, as shown in Fig. 4A. The time courses of thermal inactivation of the enzymes at 60°C were followed as shown in Fig. 4B. Incubation for 60 min resulted in 75 % inactivation of neuraminidases I and II. Calcium ions had no protective effect. Effects of Metal Ions on the Enzyme Activity— The effects of metal ions at either 1 ITM or 10 ITIM on the activities of neuraminidases I and II were investigated. The experimental results are sum-
io •
i
20
30
40
Minutes a t 60*C
40
SO
60
70
Temperature (*C)
Fig. 3. Effect of temperature on the reaction rate. The reaction conditions (with the exception of reaction temperature) were as described in " MATERIALS AND METHODS." Data are shown for neuraminidase I ( # ) and neuraminidase II (O)-
Fig. 4. Thermal stabilities of neuraminidases I and IJ. Neuraminidase I ( • ) and neuraminidase II (O) were heated in 20 mM phosphate buffer (pH 6.8). The remaining neuraminidase activities were determined under the standard reaction conditions described in the text. (A) Thermal inactivation of neuraminidase incubated at various temperatures for 10 min. (B) Time course of the thermal inactivation of neuraminidase incubated at 60°C. / . Biochem.
PROPERTIES OF A. ureafaciens NEURAMINIDASE TABLE I. Effects of metals on neuraminidases I and II from Arthrobacter ureafaciens. Neuraminidase I or II (250 fil) was preincubated with 250 fi\ of sample solution (40 mM or 4 mM) at 37°C for 10 min, then 100 fi\ aliquots were taken and the activities were assayed as described in the text.
1577
Effects of Various Compounds on the Enzyme Activity—Various types of compounds were examined for inhibitory or stimulatory action on the enzyme. As shown in Table II, no marked differences were observed between neuraminidases I and II. Chelating agents such as a.a'-dipyridyl, 8-hydroxyquinoline, o-phenanthroline, sodium diRelative activity (%) Final ethyldithiocarbamate, thiourea, sodium pyrophosCompound cone. Neuraminidase Neuraminidase phate, and EDTA, SH-inhibitors such as mono(mM) I II iodoacetic acid, sodium arsenite, sodium arsenate, jj-chloromercuribenzoic acid, and mercuric chloride None 100 100 (Table I), SH-compounds (S-S dissociating agents) LiCl 10 100 100 such as L-ascorbic acid, 2-mercaptoethanol, gluNaCl 10 100 99 tathione, L-cysteine, sodium thioglycollate, and KC1 10 98 96 dithiothreitol, and carbonyl group inhibitors such MgCl, 1 94 92 as sodium bisulfite, hydroxylamine, semicarbazide, 1 92 CaCl, 93 thiosemicarbazide, and hydrazine sulfate were neither inhibitors nor activators of the enzyme. 1 BaCl, 94 93 Oxidizing agents such as N-bromosuccinimide and CuCl, 1 93 95 iodine, however, were shown to be highly inhibi1 94 ZnSO. 95 tory. CdCl, 1 71 78 At present, neuraminidases from V. cholerae HgCl, 1 91 91 and Cl. perfringens have been prepared in a purified 92 1 91 Mna, state and are widely used for research purposes. FeCl, 1 91 88 It has been reported that neuraminidase from CoCl, 1 89 91 Cl. perfringens is inhibited by p-chloromercuribenzoic acid and Hg l+ (35). Neuraminidases from NiCl, 1 96 98 A. ureafaciens were only slightly or not at all Pb(CH,COO) . 1 66 67 inhibited by p-chloromercuribenzoic acid or Hg I+ , in striking contrast to the Cl. perfringens neumarized in Table I. Neuraminidases I and II raminidase; they did not require Ca1+ for activity were both inhibited to some extent by Cd1+ and and were not inhibited by EDTA, which is in Pb1+, but were not much affected by other metal striking contrast to the V. cholerae neuraminidase. ions. The purified neuraminidase preparation from Neuraminidases can usually be classified into A. ureafaciens was shown to be free from contwo groups according to the requirement for Ca t+ ; taminating enzymes (14). The indifference of this one group requires Ca t+ for activity and is inhibited enzyme to calcium would be of particular value in by EDTA, and the other does not require Ca1+ studies either on the relation of membrane-bound and is not inhibited by EDTA. Neuraminidases calcium to membrane sialoglycan structure (40-43) belonging to the former are produced by Vibrio or on the exchangeability and permeability of Ca1+ cholerae (25-28), Corynebacterium diphtheria (28- in the cells (44-46); the sialic acid accounts for a 30), and Streptococcus group K (31, 32), and those significant component of cation binding on the belonging to the latter have been obtained from cell surface, with particular affinity for calcium, Clostridium perfringens (33-36), Diplococcus pneu- as demonstrated with red blood cell membrane moniae (37), Pasteurella multocida (38), Strepto- (43), and might also be of importance in the myces albus (39), and Streptomyes spp. (35). regulation of the Ca*+ permeability in cultured Neuraminidases from Arthrobacter ureafaciens heart cells (45). belong to the latter group; they do not require Substrate Specificity—The substrate specifiCa1+ for activity and are not inhibited by EDTA cities of neuraminidases I and II were studied with (Table II). various substrates. As reported by Cassidy et al.
Vol. 86, No. 5, 1979
1578
Y. UCHIDA, Y. TSUKADA, and T. SUGIMORI
TABLE II. Effects of various compounds on neuraminidases I and II from Arthrobacter weafaciens. Enzyme activities were assayed after preincubation as described in Table L Compound
Final cone. (mM)
None
Relative activity (%) Nauraminidase I
Neuraminidase 11
100
100
EDTA
1
99
98
a, a'-Dipyridyl
1
101
102
8-HydroxyquinoIine
1
100
97
o-Phenanthroline
1
90
94
Sodium diethyldithiocarbamate
1
88
95
Thiourea
1
96
100
Sodium pyrophosphate
1
89
94
Monoiodoacetic acid
1
92
97
Sodium arsenite
1
86
92
Sodium arsenate
1
88
98
/>-Chloromercuribenzoic acid
1
94
93
L-Ascorbic acid
1
91
90
2-Mercaptoethanol
1
94
94
Glutathione
1
92
94
L-Cysteine HQ
1
95
95
Sodium thioglycollate
1
93
92
Dithiothreitol
1
95
94
Sodium bisulfate
1
89
88
Hydroxylamine HC1
1
96
93
Semicarbazide HC1
1
91
97
Thiosemicarbazide HC1
1
95
99
Hydrazine sulfate
1
90
95
Sodium fluoride
1
91
93
Sodium azide
1
94
96
100
102
2
1
0.01
38
22
0.001
90
91
0.5
4
4
0.05
21
33
0.005
90
92
10
68
69
1
95
95
Phenylmethylsulfonyl fluoride
1
A'-Bromosuccinimide
0.1
Iodine
Hydrogen peroxide
/ . Biochem.
1579
PROPERTIES OF A. ureafaciens NEURAMINIDASE
TABLE EU. Substrate specificity studies with neuraminidases I and II from Arthrobacter ureafaciens. Each substrate, containing 1.5 /jmol of bound form sialic acid per ml of incubation mixture, was hydrolyzed with 0.015 unit of neuraminidase at 37°C for 10 min in 50 mM acetate buffer, pH 4.5 (colominic acid) or pH 5.0 (other substrates). The released sialic acid was determined by the method described in the text. Substrate
JV-Acetylneuraminosyl-ar, 2-3-lactose iV-Acetylneuraminosyl-ar, 2-6-lactose Colominic acid Bovine submaxillary mucin Bovine brain gangliosides Fetuin (calf serum) Transferrin (human) Sea urchin egg glycoprotein HP b Sea urchin egg glycoprotein A O
Sialic acid released (/imol/mg enzyme/min)
Linkage of ciolir flCld
Neuraminidase I
Neuraminidase n
76.4 120.0 47.8 43.1 58.8 45.0 56.6 37.9 35.2
114.9 180.2 79.1 36.2
a, 2-3 Jelly coat glycoprotein of eggs of the sea urchin, Hemicentrotus pulcherrimus {20). e Jelly coat glycoprotein of eggs of the sea urchin, Anlhocidaris crassispina (20). TABLE IV. Hydrolysis of various substrates by neuraminidases I and II from Arthrobacter ureafaciens. Each substrate, containing 1.5 [imo\ of bound sialic acid per ml of incubation mixture, was hydrolyzed with 0.15 unit of neuraminidase at 37°C in 50 mM acetate buffer, pH 4.5 (colominic acid) or pH 5.0 (other substrates). At appropriate intervals, aliquots were withdrawn and the released sialic acid was determined by the method described in the text. Sialic acid released (%) Neuraminidase I
Substrate
A'-Acetylneuraminosyl-a, 2-3-lactose JV-Acetylneuraminosyl-ar, 2-6-lactose Colominic acid Bovine submaxillary mucin Bovine submaxillary mucin (saponified)* Bovine brain gangliosides Fetuin (calf serum) Transferrin (human) Trypsin inhibitor (ovomucoid) Erythrocyte stroma (human) Sea urchin egg glycoprotein HP b Sea urchin egg glycoprotein AC e
Neuraminidase II
10 min
90 min
10 h
10 min
90 min
10 h
48
95
100
48
96
100
62
99
100
61
99
100
43
90
99
43
91
100
38
52
52
29
48
50
66
99
100
61
97
100
44
51
53
trace
5
25
45
82
95
23
73
94
46
87
100
42
85
99
40
65
70
39
66
70
39
72
97
3
21
66
32
56
62
18
49
61
26
64
74
16
51
71
» The saponification of bovine submaxillary mucin was performed according to the method of Reid et al. (54). Jelly coat gJycoprotein of the sea urchin, Hemicentrotus pulcherrimus (20). e Jelly coat glycoprotein of eggs of the sea urchin, Anthocidaris crassispina (20). b
Vol. 86, No. 5, 1979
1580 (34), two parameters of the hydrolytic reactions were compared in the cases of neuraminidases I and II; the initial rate and the final extent of hydrolysis. In order to determine the initial rate of hydrolysis, a sufficient amount of substrate was allowed to react with a limited amount of enzyme, as shown in Table HL Under these conditions, the release of sialic acid was linear with time for at least lOmin, and hence the reaction rate obtained here was regarded as the initial rate of hydrolysis. Table HI shows the results of substrate specificity studies based on the initial rates of hydrolysis. In order to determine the final extent of hydrolysis, on the other hand, reactions were performed with a sufficient amount of enzyme as shown in Table IV. Aliquots were removed from the reaction mixtures at various times and analysed for free sialic acid. Table IV shows the time course of release of sialic acid. These results demonstrate that the structure of the substrate can influence the initial rate and final extent of hydrolysis, and that neuraminidase I is distinct from neuraminidase II as regards substrate specificities. In the present work, neuraminidases from A. ureafaciens were produced in a culture medium containing colominic acid as a sole source of carbon. The excreted neuraminidases attacked a variety of substrates as well as colominic acid. The glycoproteins isolated from the jelly coat of the eggs of sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina, were reported to be rich in iV-glycolylneuraminic acid (20). As shown in Tables III and IV, sialic acid was released from the sea urchin glycoproteins by both neuraminidases I and II. Thin-layer chromatography was applied for the separation and detection of Nacetylneuraminic acid and iV-glycolylneuraminic acid. As shown in Fig. 5, only iV-glycolylneuraminic acid was detected in the hydrolysate of the sea urchin glycoprotein, only iV-acetylneuraminic acid in that of colominic acid, and a mixture of TV-acetylneuraminic acid and lactose in that of JV-acetylneuraminosyl-lactose. These results demonstrate that neuraminidases I and II are both able to hydrolyze the a-ketosidic linkage of TV-glycolylneuraminic acid as well as that of .N-acetylneuraminic acid, and also demonstrate that both neuraminidases are able to split off sialic acid not only from colominic acid, in which sialic acids are a,2-8 linked (47, 48), but also from
Y. UCHIDA, Y. TSUKADA, and T. SUGIMORI
Fig. 5. Thin-layer chromatography of neuraminidase reaction products. Reaction products obtained by neuraminidase digestion as shown in Table IV were used for analysis. Experimental details are given in "MATERIALS AND METHODS." (A) Af-Acetylneuraminic acid; (B) N-glycolylneuraminic acid; (C) lactose; (D), (E), and (F) are the reaction products formed by neuraminidase I from W-acetylneuraminosyllactose, colominic acid, and from jelly coat glycoprotein of eggs of the sea urchin, Anthocidaris crassispina, respectively. The reaction products formed by neuraminidase II gave identical chromatograms.
N-acetylneuraminosyl-lactose, bovine submaxillary mucin, fetuin, transferrin, trypsin inhibitor, and bovine brain gangliosides, in which sialic acid residues are a,2-3 or a,2-6 linked. Therefore, it is concluded that both neuraminidases can hydrolyze (a,2-3), (ar,2-6), and (or.2-8) linkages, as has been reported for other neuraminidases of bacterial origin (34, 49, 50). The initial rate of hydrolysis, however, varied with the substrate examined and was particularly outstanding with the two isomers of N-acetylneuraminosyl-lactose (Table III); the initial rate of hydrolysis of Ar-acetylneuraminosyl-o,2-6-Iactose J. Biochem.
PROPERTIES OF A. ureafaciens NEURAMINIDASE was greater than that of the or,2-3-isomer, indicating that the linkage of sialic acid influences the rate of hydrolysis. The substrate specificities of A. ureafadens neuraminidases on JV-acetylneuraminosyl-lactoses were different from that of Cl. perfringens neuraminidase; the latter enzyme has been reported to attack TV-acetylneuraminosyla,2-3-lactose at approximately twice the rate observed with the a,2-6-isomer (34). Bovine submaxillary mucin was not completely hydrolyzed by A. ureafadens neuraminidases; about 50% of the sialic acids in bovine submaxillary mucin remained uncleaved even after prolonged incubation, as shown in Table IV. The similar tendency was observed with Cl. perfringens neuraminidase (34). The sialic acids of native bovine submaxillary mucin are composed of Nacetylneuraminic acid, iV-glycolylneuraminic acid, 7V-acetyl-7-O-acetylneuraminic acid, iV-acetyl-8-Oacetylneuraminic acid, iV-acetyl-7,8-di-O-acetylneuraminic acid, and ./V-acetyl-tri-O-acetylneuraminic acid (51-53). As shown in Table IV, removal of the 0-acetyl groups of bovine submaxillary mucin by treatment with 0.1 N potassium hydroxide for 30 min at room temperature (54) brought about the complete hydrolysis of sialic acids. It has been reported that the release of these 0-acetylated
1581 neuraminic acids by V. cholerae and Cl. perfringens neuraminidase is retarded by the presence of bound iV-aceryl- and TV-glycolylneuraminic acid (52). The results presented here show that some O-acetylated neuraminic acids are resistant to A. ureafadens neuraminidases, but further studies on this point are necessary. When bovine brain gangliosides were used as a substrate, marked differences in substrate specificity were observed between neuraminidases I and II; neuraminidase I can hydrolyze the gangliosides rather easily, while neuraminidase II liberates Nacetylneuraminic acid very slowly (Tables III and IV). Recently Sugano et al. reported that A. ureafadens neuraminidase can hydrolyze monosialoganglioside GMi (55), which was believed to be resistant to various neuraminidases of viral, bacteria], and mammalian origin (56-59), and they also reported that the addition of detergents, especially bile salts, resulted in a marked increase in the enzymatic hydrolysis of G M t (55). The bovine brain gangliosides are a mixture of monosialoganglioside, disialoganglioside, and trisialoganglioside (60). The effect of detergent on the hydrolysis of bovine brain gangliosides was studied here. As shown in Table V, neuraminidase I
TABLE V. Hydrolysis of gangliosides by neuraminidases I and n in the presence or absence of sodium cholate. Bovine brain gangliosides (Sigma, type III, 1.5 fimol per ml incubation mixture as sialic acid content) were hydrolyzed with 0.08 unit of neuraminidase at 37°C in the presence or absence of sodium cholate (3 fimol per ml of incubation mixture). Aliquots were withdrawn and the released sialic acid was determined by the method described in the text.
Neuraminidase I
None Sodium cholate None+Sodium cholate* None+Neuraminidase I* None+Neuraminidase n»
Neuraminidase II
None Sodium cholate None+Sodium cholate* None+Neuraminidase I* None+Neuraminidase II*
» Addition was made after 10 h. Vol. 86, No. 5, 1979
Sialic acid released (%)
Addition
Enzyme
10 min
90 min
10 h
24 h
48h
34.9 55.8
50.1 74.4
56.4 100
34.9 34.9 34.9
50.1 50.1 50.1
54.1 93.0 54.1 54.1 54.1
97.7 56.9 56.4
57.4 100 100 57.8 56.4
Enzymatic Properties of Neuraminidases from Arthrobacter ureafaciens Yoshihiro UCHIDA, Yoji TSUKADA, and Tsunetake SUGIMORI Kyoto Research Laboratories, Marulrin Shoyu Co., Ltd., Uji, Kyoto 611 Received for publication, June 25, 1979
Neuraminidase I and neuraminidase II from Arthrobacter ureafaciens were characterized. As determined by gel filtration on Ultrogel AcA 44, the molecular weights of neuraminidases I and II were 51,000 and 39,000, respectively. Neuraminidases I and II were similar to each other in their enzymatic properties except for the substrate specificities towards gangliosides and erythrocyte stroma. Their optimal pHs were between 5.0 and 5.5 with TV-acetylneuraminosyl-lactose or bovine submaxillary mucin as substrates, but with colominic acid as a substrate, the pH optimum was between 4.3 and 4.5. They were most active around 53°C, were stable between pH 6.0 and 9.0, and were thermostable up to 50°C. They did not require CaI+ for activity and were not inhibited by EDTA. They were inhibited only slightly or not at all by /?-chloromercuribenzoic acid or Hg t+ . Both neuraminidases I and II were able to hydrolyze the a-ketosidic linkage of iV-glycolylneuraminic acid as well as that of iV-acetylneuraminic acid, and were able to liberate substantially all of the sialic acid from various kinds of substrates. However, they cleaved only about 50% of the sialic acid from bovine submaxillary mucin. The saponification of bovine submaxillary mucin by mild alkali treatment, on the other hand, resulted in an increased susceptibility to the neuraminidases and brought about the complete liberation of sialic acid. Remarkable differences were observed between neuraminidases I and II as regards substrate specificities on gangliosides; the initial rate of hydrolysis by neuraminidase I was 74 times, and its maximum velocity constant was 91 times those of neuraminidase II. The addition of sodium cholate markedly stimulated the enzymatic hydrolysis of gangliosides, and increased the maximum velocity constant of neuraminidase I twofold and that of neuraminidase II 143-fold. Although neuraminidases I and II were able to hydrolyze (a,2-3), (a,2-6), and (a,2-8) linkages, the initial rate of hydrolysis of 7V-acetylneuraminosyI-a,2-6-lactose was greater than that of the o,2-3-isomer.
Sialic acid (JV-acylated neuraminic acid) commonly occurs as the terminal residue of carbohydrate chains in many glycoproteins or glycolipids. Since sialic acid has many biological functions (7Vol. 86, No. 5, 1979
75), neuraminidase [sialidase, EC 3.2 1.18] can markedly influence the biological behavior of macromolecules and cells. Neuraminidase preparations free from contaminating enzymes have long
1573
1574
Y. UCHIDA, Y. TSUKADA, and T. SUGIMORI
been sought, since such preparations would be particularly useful in investigating the roles of sialic acid in glycoconjugates in living bodies. In the previous paper (14), we reported the distribution of neuraminidase in Arthrobacter, and its purification by a novel affinity chromatography procedure using colominic acid as a selective adsorbent. The purified neuraminidase from Arthrobacter ureafaciens has been shown to be free from activities of protease, Ar-acetylneuraminic acid aldolase, phospholipase C, and glycosidases. By gel filtration on Ultrogel AcA 44, the purified neuraminidase was divided into two active peaks which were tentatively designated as neuraminidase I and neuraminidase n . The present paper describes the enzymatic characteristics of the two neuraminidases from Arthrobacter ureafaciens. A preliminary report has already appeared (IS). MATERIALS AND METHODS Preparation of Neuraminidase—Neuraminidase from Arthrobacter ureafaciens was prepared and purified as described previously (14). By gel filtration on Ultrogel AcA 44, the purified neuraminidase was divided into two peaks which were tentatively designated as neuraminidase I and neuraminidase II (14). These two neuraminidases were used in the present experiments. Assay of Neuraminidase Activity—The assay system contained the following components in a total volume of 200[t\: 50fil of substrate solution, 50 pil of 200 mM buffer solution, and 100 ft\ of enzyme preparation. The reaction was conducted at 37CC for lOmin, and the amount of A^acetylneuraminic acid liberated was compared with that of a control containing the same components except for the enzyme. A/-Acetylneuraminic acid was determined by a modification of the thiobarbituric acid procedure of Aminoff (14). One unit of neuraminidase activity was defined as the amount that releases 1 (imo\ of N-acetylneuraminic acid per min under the standard reaction conditions using colominic acid as a substrate (14). Molecular Weight Determination—The molecular weight of the enzyme was estimated by gel filtration based on the method of Andrews (16). A column of Ultrogel AcA 44 (2.6 x 100 cm) was
equilibrated with lOOmM phosphate buffer (pH 6.8) and run with the same buffer at a flow rate of 25 ml per h. The following standard proteins were used for calibration; cytochrome c, myoglobin, chymotrypsinogen A, ovalbumin, and albumin. The partition coefficient, Kav, was calculated using the equation of Laurent and Killander (17). Thin-Layer Chromatography—Products of neuraminidase digestion were applied to a thin layer Silica Gel 60 chromatographic plate (Merck) pretreated with 0.2 M sodium dihydrogen phosphate (18), and the plate was developed three times with rt-propanol-1 N NH4OH-water ( 6 : 2 : 1 , v/v) as a solvent (19-21). Sialic acid was detected by spraying with the diphenylamine-aniline reagent (22). Chemicals—Colominic acid (Na salt) and iV-acetylneuraminic acid were the products of our company (24). iV-Acetylneuraminosyl-Iactose, bovine submaxillary mucin, bovine brain gangliosides, fetuin, transferrin, and trypsin inhibitor were obtained from Sigma Chemical Company. Human erythrocyte stroma was from Miles Laboratories, Inc. The following compounds were generous gifts: 7/-acetylneuraminosyl-a,2-3-lactose (23) and JV-acetylneuraminosyl-a,2-6-lactose, Dr. M. Koseki, Fukushima Medical College; iV-glycolylneuraminic acid and glycoproteins from the eggs of sea urchins (Hemicentrotus pulcherrimus and Anthocidaris crassispina) (20), Dr. K. Hotta, Kitasato University. Ultrogel AcA 44 was obtained from LKB, Bromma, Sweden. The calibration proteins were obtained from both Boehringer Mannheim and Mann Research Laboratories. Other chemicals were from Nakarai Chemicals Ltd., Kyoto. RESULTS AND DISCUSSION pH Optimum and Stability—The effects of pH on the rates of hydrolysis of A'-acetylneuraminosyllactose, bovine submaxillary mucin, and colominic acid are illustrated in Fig. 1. There was little difference in pH optima between neuraminidases I and II. However, the pH optima varied with the substrate; with TV-acetylneuraminosyl-lactose and bovine submaxillary mucin, the optima lay between 5.0 and 5.5, while with colominic acid, / . Biochem.
PROPERTIES OF A. urcafaciens NEURAMINIDASE
3
4
5
6
7
8
3
pH
4
1575
5
6
8
8
pH
pH
Fig. 1. Effect of pH on the reaction rate. 7^-Acetylneuraminosyl-lactose (A), bovine submaxillary mucin (B), or colominic acid (C) was employed as a substrate. Either acetate buffer (pH 3.5-5.9) or phosphate buffer (pH 5.4-8.0) was used. The other reaction conditions were as described in " MATERIALS AND METHODS." Data are shown for neuraminidase I ( # ) and neuraminidase
n(o).
10
pH
pH
Fig. 2. Effect of pH on the stability of neuraminidase. Neuraminidase I ( • ) and neuraminidase II (O) were incubated at 37°C for 24 h in 20 HIM buffers of pH 2.2 to 10.4; HCl-CH,COONa and CH,COOH-CH,COONa buffer for pH 2.2 to 5.9, and NaH,PO4-K,HPO4 and K,HPO4-NaOH buffer for pH 5.9 to 10.4. The remaining neuraminidase activities were determined under the standard reaction conditions. the optima were between 4.3 and 4.5. In order to remove the sialic acid residues from animal cells or tissues, neuraminidase is often allowed to react in physiological phosphate-saline Vol. 86, No. 5, 1979
solution, a typical buffer solution of neutral pH. As shown in Fig. 1, the pH activity profiles of neuraminidases I and n with bovine submaxillary mucin as a substrate gave broad optima ranging
1576
Y. UCHIDA, Y. TSUKADA, and T. SUGIMORI
from 4.5 to 6.0. Since no marked depression of the activities was observed around neutral pH, neuraminidases from Arthrobacter ureafaciens are considered to be suitable for the treatment of substrates in a physiological pH environment. As shown in Fig. 2, neuraminidases I and II are both stable at 37°C for 24 h in buffers from pH 6.0 to 9.0. This result indicates that the neuraminidases can remain active during prolonged incubation when the reaction is conducted at around neutral pH. Thermal Reactivity and Stability—The standard reaction mixture containing 50 fil of substrate (colominic acid, Na salt, 4.0 mg/ml), 50 p\ of 200 HIM acetate buffer (pH 4.5), and 100 ft\ of enzyme preparation was allowed to react at various temperatures for 10 min, and the amount of iV-acetylneuraminic acid released was compared with that of a control containing the same components except for the enzyme. The results are illustrated in Fig. 3. Neuraminidases I and II both had optima at around 53°C.
a
The neuraminidases were then incubated at pH 6.8 in a water bath maintained at various temperatures for 10 min, followed by chilling in an ice bath. An aliquot of the treated enzymes was assayed for remaining neuraminidase activity by the standard procedure and compared with a control kept at 5°C. Neuraminidases I and II were both found to be thermostable up to 50°C, as shown in Fig. 4A. The time courses of thermal inactivation of the enzymes at 60°C were followed as shown in Fig. 4B. Incubation for 60 min resulted in 75 % inactivation of neuraminidases I and II. Calcium ions had no protective effect. Effects of Metal Ions on the Enzyme Activity— The effects of metal ions at either 1 ITM or 10 ITIM on the activities of neuraminidases I and II were investigated. The experimental results are sum-
io •
i
20
30
40
Minutes a t 60*C
40
SO
60
70
Temperature (*C)
Fig. 3. Effect of temperature on the reaction rate. The reaction conditions (with the exception of reaction temperature) were as described in " MATERIALS AND METHODS." Data are shown for neuraminidase I ( # ) and neuraminidase II (O)-
Fig. 4. Thermal stabilities of neuraminidases I and IJ. Neuraminidase I ( • ) and neuraminidase II (O) were heated in 20 mM phosphate buffer (pH 6.8). The remaining neuraminidase activities were determined under the standard reaction conditions described in the text. (A) Thermal inactivation of neuraminidase incubated at various temperatures for 10 min. (B) Time course of the thermal inactivation of neuraminidase incubated at 60°C. / . Biochem.
PROPERTIES OF A. ureafaciens NEURAMINIDASE TABLE I. Effects of metals on neuraminidases I and II from Arthrobacter ureafaciens. Neuraminidase I or II (250 fil) was preincubated with 250 fi\ of sample solution (40 mM or 4 mM) at 37°C for 10 min, then 100 fi\ aliquots were taken and the activities were assayed as described in the text.
1577
Effects of Various Compounds on the Enzyme Activity—Various types of compounds were examined for inhibitory or stimulatory action on the enzyme. As shown in Table II, no marked differences were observed between neuraminidases I and II. Chelating agents such as a.a'-dipyridyl, 8-hydroxyquinoline, o-phenanthroline, sodium diRelative activity (%) Final ethyldithiocarbamate, thiourea, sodium pyrophosCompound cone. Neuraminidase Neuraminidase phate, and EDTA, SH-inhibitors such as mono(mM) I II iodoacetic acid, sodium arsenite, sodium arsenate, jj-chloromercuribenzoic acid, and mercuric chloride None 100 100 (Table I), SH-compounds (S-S dissociating agents) LiCl 10 100 100 such as L-ascorbic acid, 2-mercaptoethanol, gluNaCl 10 100 99 tathione, L-cysteine, sodium thioglycollate, and KC1 10 98 96 dithiothreitol, and carbonyl group inhibitors such MgCl, 1 94 92 as sodium bisulfite, hydroxylamine, semicarbazide, 1 92 CaCl, 93 thiosemicarbazide, and hydrazine sulfate were neither inhibitors nor activators of the enzyme. 1 BaCl, 94 93 Oxidizing agents such as N-bromosuccinimide and CuCl, 1 93 95 iodine, however, were shown to be highly inhibi1 94 ZnSO. 95 tory. CdCl, 1 71 78 At present, neuraminidases from V. cholerae HgCl, 1 91 91 and Cl. perfringens have been prepared in a purified 92 1 91 Mna, state and are widely used for research purposes. FeCl, 1 91 88 It has been reported that neuraminidase from CoCl, 1 89 91 Cl. perfringens is inhibited by p-chloromercuribenzoic acid and Hg l+ (35). Neuraminidases from NiCl, 1 96 98 A. ureafaciens were only slightly or not at all Pb(CH,COO) . 1 66 67 inhibited by p-chloromercuribenzoic acid or Hg I+ , in striking contrast to the Cl. perfringens neumarized in Table I. Neuraminidases I and II raminidase; they did not require Ca1+ for activity were both inhibited to some extent by Cd1+ and and were not inhibited by EDTA, which is in Pb1+, but were not much affected by other metal striking contrast to the V. cholerae neuraminidase. ions. The purified neuraminidase preparation from Neuraminidases can usually be classified into A. ureafaciens was shown to be free from contwo groups according to the requirement for Ca t+ ; taminating enzymes (14). The indifference of this one group requires Ca t+ for activity and is inhibited enzyme to calcium would be of particular value in by EDTA, and the other does not require Ca1+ studies either on the relation of membrane-bound and is not inhibited by EDTA. Neuraminidases calcium to membrane sialoglycan structure (40-43) belonging to the former are produced by Vibrio or on the exchangeability and permeability of Ca1+ cholerae (25-28), Corynebacterium diphtheria (28- in the cells (44-46); the sialic acid accounts for a 30), and Streptococcus group K (31, 32), and those significant component of cation binding on the belonging to the latter have been obtained from cell surface, with particular affinity for calcium, Clostridium perfringens (33-36), Diplococcus pneu- as demonstrated with red blood cell membrane moniae (37), Pasteurella multocida (38), Strepto- (43), and might also be of importance in the myces albus (39), and Streptomyes spp. (35). regulation of the Ca*+ permeability in cultured Neuraminidases from Arthrobacter ureafaciens heart cells (45). belong to the latter group; they do not require Substrate Specificity—The substrate specifiCa1+ for activity and are not inhibited by EDTA cities of neuraminidases I and II were studied with (Table II). various substrates. As reported by Cassidy et al.
Vol. 86, No. 5, 1979
1578
Y. UCHIDA, Y. TSUKADA, and T. SUGIMORI
TABLE II. Effects of various compounds on neuraminidases I and II from Arthrobacter weafaciens. Enzyme activities were assayed after preincubation as described in Table L Compound
Final cone. (mM)
None
Relative activity (%) Nauraminidase I
Neuraminidase 11
100
100
EDTA
1
99
98
a, a'-Dipyridyl
1
101
102
8-HydroxyquinoIine
1
100
97
o-Phenanthroline
1
90
94
Sodium diethyldithiocarbamate
1
88
95
Thiourea
1
96
100
Sodium pyrophosphate
1
89
94
Monoiodoacetic acid
1
92
97
Sodium arsenite
1
86
92
Sodium arsenate
1
88
98
/>-Chloromercuribenzoic acid
1
94
93
L-Ascorbic acid
1
91
90
2-Mercaptoethanol
1
94
94
Glutathione
1
92
94
L-Cysteine HQ
1
95
95
Sodium thioglycollate
1
93
92
Dithiothreitol
1
95
94
Sodium bisulfate
1
89
88
Hydroxylamine HC1
1
96
93
Semicarbazide HC1
1
91
97
Thiosemicarbazide HC1
1
95
99
Hydrazine sulfate
1
90
95
Sodium fluoride
1
91
93
Sodium azide
1
94
96
100
102
2
1
0.01
38
22
0.001
90
91
0.5
4
4
0.05
21
33
0.005
90
92
10
68
69
1
95
95
Phenylmethylsulfonyl fluoride
1
A'-Bromosuccinimide
0.1
Iodine
Hydrogen peroxide
/ . Biochem.
1579
PROPERTIES OF A. ureafaciens NEURAMINIDASE
TABLE EU. Substrate specificity studies with neuraminidases I and II from Arthrobacter ureafaciens. Each substrate, containing 1.5 /jmol of bound form sialic acid per ml of incubation mixture, was hydrolyzed with 0.015 unit of neuraminidase at 37°C for 10 min in 50 mM acetate buffer, pH 4.5 (colominic acid) or pH 5.0 (other substrates). The released sialic acid was determined by the method described in the text. Substrate
JV-Acetylneuraminosyl-ar, 2-3-lactose iV-Acetylneuraminosyl-ar, 2-6-lactose Colominic acid Bovine submaxillary mucin Bovine brain gangliosides Fetuin (calf serum) Transferrin (human) Sea urchin egg glycoprotein HP b Sea urchin egg glycoprotein A O
Sialic acid released (/imol/mg enzyme/min)
Linkage of ciolir flCld
Neuraminidase I
Neuraminidase n
76.4 120.0 47.8 43.1 58.8 45.0 56.6 37.9 35.2
114.9 180.2 79.1 36.2
a, 2-3 Jelly coat glycoprotein of eggs of the sea urchin, Hemicentrotus pulcherrimus {20). e Jelly coat glycoprotein of eggs of the sea urchin, Anlhocidaris crassispina (20). TABLE IV. Hydrolysis of various substrates by neuraminidases I and II from Arthrobacter ureafaciens. Each substrate, containing 1.5 [imo\ of bound sialic acid per ml of incubation mixture, was hydrolyzed with 0.15 unit of neuraminidase at 37°C in 50 mM acetate buffer, pH 4.5 (colominic acid) or pH 5.0 (other substrates). At appropriate intervals, aliquots were withdrawn and the released sialic acid was determined by the method described in the text. Sialic acid released (%) Neuraminidase I
Substrate
A'-Acetylneuraminosyl-a, 2-3-lactose JV-Acetylneuraminosyl-ar, 2-6-lactose Colominic acid Bovine submaxillary mucin Bovine submaxillary mucin (saponified)* Bovine brain gangliosides Fetuin (calf serum) Transferrin (human) Trypsin inhibitor (ovomucoid) Erythrocyte stroma (human) Sea urchin egg glycoprotein HP b Sea urchin egg glycoprotein AC e
Neuraminidase II
10 min
90 min
10 h
10 min
90 min
10 h
48
95
100
48
96
100
62
99
100
61
99
100
43
90
99
43
91
100
38
52
52
29
48
50
66
99
100
61
97
100
44
51
53
trace
5
25
45
82
95
23
73
94
46
87
100
42
85
99
40
65
70
39
66
70
39
72
97
3
21
66
32
56
62
18
49
61
26
64
74
16
51
71
» The saponification of bovine submaxillary mucin was performed according to the method of Reid et al. (54). Jelly coat gJycoprotein of the sea urchin, Hemicentrotus pulcherrimus (20). e Jelly coat glycoprotein of eggs of the sea urchin, Anthocidaris crassispina (20). b
Vol. 86, No. 5, 1979
1580 (34), two parameters of the hydrolytic reactions were compared in the cases of neuraminidases I and II; the initial rate and the final extent of hydrolysis. In order to determine the initial rate of hydrolysis, a sufficient amount of substrate was allowed to react with a limited amount of enzyme, as shown in Table HL Under these conditions, the release of sialic acid was linear with time for at least lOmin, and hence the reaction rate obtained here was regarded as the initial rate of hydrolysis. Table HI shows the results of substrate specificity studies based on the initial rates of hydrolysis. In order to determine the final extent of hydrolysis, on the other hand, reactions were performed with a sufficient amount of enzyme as shown in Table IV. Aliquots were removed from the reaction mixtures at various times and analysed for free sialic acid. Table IV shows the time course of release of sialic acid. These results demonstrate that the structure of the substrate can influence the initial rate and final extent of hydrolysis, and that neuraminidase I is distinct from neuraminidase II as regards substrate specificities. In the present work, neuraminidases from A. ureafaciens were produced in a culture medium containing colominic acid as a sole source of carbon. The excreted neuraminidases attacked a variety of substrates as well as colominic acid. The glycoproteins isolated from the jelly coat of the eggs of sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina, were reported to be rich in iV-glycolylneuraminic acid (20). As shown in Tables III and IV, sialic acid was released from the sea urchin glycoproteins by both neuraminidases I and II. Thin-layer chromatography was applied for the separation and detection of Nacetylneuraminic acid and iV-glycolylneuraminic acid. As shown in Fig. 5, only iV-glycolylneuraminic acid was detected in the hydrolysate of the sea urchin glycoprotein, only iV-acetylneuraminic acid in that of colominic acid, and a mixture of TV-acetylneuraminic acid and lactose in that of JV-acetylneuraminosyl-lactose. These results demonstrate that neuraminidases I and II are both able to hydrolyze the a-ketosidic linkage of TV-glycolylneuraminic acid as well as that of .N-acetylneuraminic acid, and also demonstrate that both neuraminidases are able to split off sialic acid not only from colominic acid, in which sialic acids are a,2-8 linked (47, 48), but also from
Y. UCHIDA, Y. TSUKADA, and T. SUGIMORI
Fig. 5. Thin-layer chromatography of neuraminidase reaction products. Reaction products obtained by neuraminidase digestion as shown in Table IV were used for analysis. Experimental details are given in "MATERIALS AND METHODS." (A) Af-Acetylneuraminic acid; (B) N-glycolylneuraminic acid; (C) lactose; (D), (E), and (F) are the reaction products formed by neuraminidase I from W-acetylneuraminosyllactose, colominic acid, and from jelly coat glycoprotein of eggs of the sea urchin, Anthocidaris crassispina, respectively. The reaction products formed by neuraminidase II gave identical chromatograms.
N-acetylneuraminosyl-lactose, bovine submaxillary mucin, fetuin, transferrin, trypsin inhibitor, and bovine brain gangliosides, in which sialic acid residues are a,2-3 or a,2-6 linked. Therefore, it is concluded that both neuraminidases can hydrolyze (a,2-3), (ar,2-6), and (or.2-8) linkages, as has been reported for other neuraminidases of bacterial origin (34, 49, 50). The initial rate of hydrolysis, however, varied with the substrate examined and was particularly outstanding with the two isomers of N-acetylneuraminosyl-lactose (Table III); the initial rate of hydrolysis of Ar-acetylneuraminosyl-o,2-6-Iactose J. Biochem.
PROPERTIES OF A. ureafaciens NEURAMINIDASE was greater than that of the or,2-3-isomer, indicating that the linkage of sialic acid influences the rate of hydrolysis. The substrate specificities of A. ureafadens neuraminidases on JV-acetylneuraminosyl-lactoses were different from that of Cl. perfringens neuraminidase; the latter enzyme has been reported to attack TV-acetylneuraminosyla,2-3-lactose at approximately twice the rate observed with the a,2-6-isomer (34). Bovine submaxillary mucin was not completely hydrolyzed by A. ureafadens neuraminidases; about 50% of the sialic acids in bovine submaxillary mucin remained uncleaved even after prolonged incubation, as shown in Table IV. The similar tendency was observed with Cl. perfringens neuraminidase (34). The sialic acids of native bovine submaxillary mucin are composed of Nacetylneuraminic acid, iV-glycolylneuraminic acid, 7V-acetyl-7-O-acetylneuraminic acid, iV-acetyl-8-Oacetylneuraminic acid, iV-acetyl-7,8-di-O-acetylneuraminic acid, and ./V-acetyl-tri-O-acetylneuraminic acid (51-53). As shown in Table IV, removal of the 0-acetyl groups of bovine submaxillary mucin by treatment with 0.1 N potassium hydroxide for 30 min at room temperature (54) brought about the complete hydrolysis of sialic acids. It has been reported that the release of these 0-acetylated
1581 neuraminic acids by V. cholerae and Cl. perfringens neuraminidase is retarded by the presence of bound iV-aceryl- and TV-glycolylneuraminic acid (52). The results presented here show that some O-acetylated neuraminic acids are resistant to A. ureafadens neuraminidases, but further studies on this point are necessary. When bovine brain gangliosides were used as a substrate, marked differences in substrate specificity were observed between neuraminidases I and II; neuraminidase I can hydrolyze the gangliosides rather easily, while neuraminidase II liberates Nacetylneuraminic acid very slowly (Tables III and IV). Recently Sugano et al. reported that A. ureafadens neuraminidase can hydrolyze monosialoganglioside GMi (55), which was believed to be resistant to various neuraminidases of viral, bacteria], and mammalian origin (56-59), and they also reported that the addition of detergents, especially bile salts, resulted in a marked increase in the enzymatic hydrolysis of G M t (55). The bovine brain gangliosides are a mixture of monosialoganglioside, disialoganglioside, and trisialoganglioside (60). The effect of detergent on the hydrolysis of bovine brain gangliosides was studied here. As shown in Table V, neuraminidase I
TABLE V. Hydrolysis of gangliosides by neuraminidases I and n in the presence or absence of sodium cholate. Bovine brain gangliosides (Sigma, type III, 1.5 fimol per ml incubation mixture as sialic acid content) were hydrolyzed with 0.08 unit of neuraminidase at 37°C in the presence or absence of sodium cholate (3 fimol per ml of incubation mixture). Aliquots were withdrawn and the released sialic acid was determined by the method described in the text.
Neuraminidase I
None Sodium cholate None+Sodium cholate* None+Neuraminidase I* None+Neuraminidase n»
Neuraminidase II
None Sodium cholate None+Sodium cholate* None+Neuraminidase I* None+Neuraminidase II*
» Addition was made after 10 h. Vol. 86, No. 5, 1979
Sialic acid released (%)
Addition
Enzyme
10 min
90 min
10 h
24 h
48h
34.9 55.8
50.1 74.4
56.4 100
34.9 34.9 34.9
50.1 50.1 50.1
54.1 93.0 54.1 54.1 54.1
97.7 56.9 56.4
57.4 100 100 57.8 56.4
