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The molecular size of neuraminidase from Vibrio cholerae (strain 4Z)
177
SHORT COMMUNICATIONS
These studies afford no evidence for S-Mg as a competitive inhibitor. Experiments at higher pH, where the fraction of S-Mg would be increased considerably, are not feasible because of the solubility product of Mg(OH), (1.2. lO-11 at I8 °, see ref. 5). However, these results do indicate that considerable corrections for substrate complexing are necessary in studies of the reaction rate as a function of substrate or Mg~+ concentration, particularly at higher pH values. It is presumed that the conclusions drawn here with respect to Mg~+ and aspartate apply also to the substrate L-threo-3-methylaspartate and to the other divalent metal cations, namely Mn 2+, CoS+ and Ni 2+, which were found to be activators of the enzyme s. Financial support of this project by the National Institutes of Health, Unite States Public Health Service (GM-IIO4O-OI) is gratefully acknowledged. Financial assistance to R. S. was provided by the Medical Student Research Training Program, University of Pennsylvania.
Department of Biochemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pa. (U.S.A.)
HAROLD J. BRIGHT ROBERT SILVERMAN
H. A. BARKER, R. D. SMYTH, R. M. WILSONANDH. WEISBACH,J. Biol. Chem., 234 (1959) 320. L. MICHAELISANDiV[.L. MENTEN,Biochem, Z., 35 (1911) 386. a R. F. LOMBAND A. E. MARTELL,J. Phys. Chem., 57 (1953) 690. 4 H. LINEWEAVERAND D. BORK, J. Am. Chem. Soc., 56 (1934) 658, Handbook of Chemistry and Physics, 35th Ed., Chemical Rubber Publishing Co., Cleveland, 1958, p. 1641. * H. J. BRIGHTAND L. L. INGRAHAM,Biochim. Biophys. Acta, 44 (196o) 586. z
Received October I8th, 1963
Biochim. Biophys. Acta, 81 (1964) 175-177
sc 11091
The molecular size of neuraminidase from Vibrio cholerae (strain 4Z) The enzyme neuraminidase (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) from Vibrio cholerae has been purified and crystallized independently by two groups. One preparation, isolated from an unspecified strain (MOHR AND SCI-IRAMM1) had sedimentation coefficients of 1.35 S at pH 8.5 and 2.35 S at pH 5.5. The other preparation (ADA, FRENCH AND LIND 2) from the strain 4 Z of V. cholerae was grown in the presence of sialyl lactose as inducer and appeared to be considerably larger, having a sedimentation coefficient of 5.5 S at pH 6. 7 and 5.3 S at pH 8. 5 (see ref. 3). The diffusion constant of this latter preparation was also determined and from these values a molecular weight of about 90 ooo was calculated 3. The molecular size of neuraminidase from the 4 Z strain of V. cholerae has now been further investigated. N-terminal amino acid analysis of the 4Z-strain enzyme was carried out using Abbreviation: PTH, phenylthiohydantoin.
Biochim. Biophys. Acta, 81 (1964) 177-18o
178
SHORT COMMUNICATIONS
a recently developed micromodification (LAVER4) of the EDMAN5 method. The enzyme was coupled with [35S]phenylisothiocyanate of high specific activity and the N-terminal amino acid(s) converted to the [asSIPTH derivatives. These were mixed with authentic samples of appropriate unlabelled phenylthiohydantoins and chromatographed in Solvents D and F of EDMAN AND SJOQUIST6. After drying, the positions of the unlabelled phenylthiohydantoins were marked out under ultraviolet light and the chromatograms were scanned for radioactivity. In Solvent F, the major radioactive peak coincided with PTH-alanine (Fig. Ia). PTH-alanine and P T H - t r y p t o p h a n are poorly separated in Solvent F but well separated in Solvent D. The main radioactive spot on the Solvent-F chromatogram was therefore eluted with glacial acetic acid and rechromatographed in Solvent D. Apart from a trace at the origin, all of the radioactivity was associated with PTH-alanine (Fig. Ib). The same result was obtained with three different preparations of neuraminidase, each from the 4 Z strain. Radioactive peaks corresponding to other PTH-amino acids were always seen but none was consistently obtained so that these probably came from contaminating proteins or peptides. The recovery of PTH-alanine was in the proportion of I mole per 9 ° ooo g of protein, when corrected for losses. These were not known exactly but the corrections made were based on the yield of PTH-alanine (87%), obtained by analysing in parallel rabbit y-globulin known to have I mole of N-terminal alanine per 18o ooo g of proteinT, s. It should be pointed out that if the recovery of PTH-alanine from the neuraminidase was in fact less than 87%, our estimate for the molecular weight would be unduly high. Ultracentrifugation studies of purified preparations and of filtrates of V. cholerae also yielded estimates of the size of this enzyme. Moving partition (YPHANTIS AND WAUGH) and fixed partition cells were used in a Spinco Model E ultracentrifuge. Sedimentation coefficients were estimated by the moving-boundary method (measurement of Schlieren patterns) when purified preparations were examined and b y the transport method (measurement of enzyme activity recovered from above the partition) for both preparations, using Eqn. 28b of SCItACHMAN9. Similar S values were obtained by the transport method for purified and crude preparations of neuraminidase and were about lO% less than S values calculated from the moving boundary method for purified preparations (see above). In some control experiments when the sedimentation behaviour of bovine plasma albumin (Armour laboratories, Fraction V, o.25%, w/v in phosphate buffered saline, p H 6.8) was studied, similar differences between S values obtained by the two methods were observed. Thus, there was no evidence that the enzyme activity in crude or purified preparations of this enzyme sedimented at a slower rate than the boundary observed optically when purified preparations were examined. Further evidence that crude filtrates of cultures of V. cholerae did not contain neuraminidase activity associated with a low-molecular-weight fraction was provided b y gel-filtration studies. A culture filtrate was mixed with bovine plasma albumin (mol. wt. 65 ooo) and with pancreatic ribonuclease (mol. wt. 17 0oo) and the mixture chromatographed on a column of the dextran gel, Sephadex G 75 (Fig. 2). Neuraminidase and bovine plasma albumin were eluted together from the column and before the appearance of ribonuclease in the effluent. The evidence presented agrees with the earlier finding that the neuraminidase produced by the 4 Z strain of V. cholerae has a molecular weight of about 9 ° ooo Biochim. Biophys. Acta, 81 (1964) I77 18o
179
SHORT COMMUNICATIONS
(a) 12000 10000 8000 6000 E ~4000 u
J Origin
Asp
Thr
Olyc Position on chromatogrem
Alo
Val
Solvept front
(b) 14000
12000 10000 8000 E ~c6000
4000
3 :_~
2000
Origin
Tryp
Alcl Position on chrornatogrern
Solvent frQnt
Fig. I. (a) D i s t r i b u t i o n of radioactivity on a c h r o m a t o g r a m (Solvent F) of the 3sS-labelled hit e r m i n a l a m i n o acid P T H s f r o m i . i m g of purified n e u r a m i n i d a s e (V. cholerae, strain 4Z). T h e positions of unlabelled m a r k e r p h e n y l t h i o h y d a n t o i n s added to the reaction m i x t u r e are s h o w n ; t h e relative positions of other a m i n o acid P T H s in Solvent F are given b y EDMAN AND S J O Q U I S T 6. A total of 4 ° 970 c o u n t s / m i n (counted on the p a p e r strip) w a s in the P T H - a l a n i n e spot. The [35S]phenylisothiocyanate used in this e x p e r i m e n t h a d 3.83" lO 6 c o u n t s / m i n per /2mole w h e n c o u n t e d as its sym-diphenylthiourea derivative u n d e r the same conditions as used for the exp e r i m e n t a l strip. (b) D i s t r i b u t i o n of radioactivity on a c h r o m a t o g r a m (Solvent D) showing s e p a r a t i o n of P T H - a l a n i n e a n d P T H - t r y p t o p h a n . The material applied was eluted f r o m the s p o t corresponding to P T H - a l a n i n e on a c h r o m a t o g r a m r u n in Solvent F.
Bioahim. Biophys. Acta, 8I (I964) I 7 7 - I 8 o
180
SHORT COMMUNICATIONS q-
i
P o.2
"7
i
O-I
..Q <
15
~O Tube
25
30
35
number
Fig. 2. C h r o m a t o g r a p h y of crude neuraminidase, bovine p l a s m a a l b u m i n and purified pancreatic ribonuclease on a column of the d e x t r a n gel, Sephadex G 75 (Pharmacia, Sweden). The column, 1.3 X 17. 5 cm, was equilibrated with a solution containing o.I M NaC1 dnd o.oi M Tris-maleic acid buffer (pH 5.7). io/~g ribonuclease and 5 ° mg a l b u m i n were dissolved in 0. 5 ml of crude neuraminidase (containing a b o u t 0. 3/*g of neuraminidase) and the m i x t u r e t h e n c h r o m a t o g r a p h e d on t h e Sephadex column, the developing buffer being t h a t described above, the flow rate 1.5 m l / h and the fraction size, o. 4 ml. [2]--[~, albumin, estimated b y m e a s u r i n g the absorba n c y (28o m/2) of the effluent; O - - - O, neuraminidase, estimated as described previously; & - - ~ , ribonuclease, estimated b y SHORTMAN'S10 method.
and it is probable that the molecule consists of a single polypeptide chain. Thus, it is unlikely that this enzyme is simply a polymer of the enzyme reported by MOHR AND SCHRAMM.
This work was supported in part by a grant from the National Health and Medical Research Council, Australia.
John Curtin School of Medical Research, Australian National University, Canberra (Australia) Walter and Eliza Hall Institute, Royal Melbourne Hospital Post O]fice, Melbourne (Australia)
W . G. LAVER
J. PYE G. L. ADA
1 E. 1V~OHRAND G. SCHi~AMM, Z. Naturforsch., i 5 b (196o) 568. z G. L. ADA, E. L. FRENCH AND P. E. LIND, J. Gen. Microbiol., 24 (1961) 409. 3 j . PYE AND C. C. CURTAIN, J. Gen. Microbiol., 24 (1961) 423 . 4 W. G. LAVER, Biochim. Biophys. Acta, 53 (1961) 469. P. EDMAN, Ann. N . Y . •cad. Sci., 88 (196o) 6o2. e p. EDMAN AND J. SJoQUiST, Acta Chem. Scand., io (1956) 15o 7. 7 R. R. PORTER, Biochem. J., 46 (195 o) 473. 8 M. L. McFADDEN AND E. L. SMITH, J. Biol. Chem., 214 (1955) 185. 9 H. K. SCHACHMAN, Ultracentrifugation in Biochemistry, Academic Press, N e w York, 1959, p. 86. 10 K. SHORTMAN, Biochim. Biophys. Acta, 5I (I96I) 37-
Received August 8th, 1963 Biochim. Biophys. Acta, 81 (1964) 177-18o
SHORT COMMUNICATIONS
These studies afford no evidence for S-Mg as a competitive inhibitor. Experiments at higher pH, where the fraction of S-Mg would be increased considerably, are not feasible because of the solubility product of Mg(OH), (1.2. lO-11 at I8 °, see ref. 5). However, these results do indicate that considerable corrections for substrate complexing are necessary in studies of the reaction rate as a function of substrate or Mg~+ concentration, particularly at higher pH values. It is presumed that the conclusions drawn here with respect to Mg~+ and aspartate apply also to the substrate L-threo-3-methylaspartate and to the other divalent metal cations, namely Mn 2+, CoS+ and Ni 2+, which were found to be activators of the enzyme s. Financial support of this project by the National Institutes of Health, Unite States Public Health Service (GM-IIO4O-OI) is gratefully acknowledged. Financial assistance to R. S. was provided by the Medical Student Research Training Program, University of Pennsylvania.
Department of Biochemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pa. (U.S.A.)
HAROLD J. BRIGHT ROBERT SILVERMAN
H. A. BARKER, R. D. SMYTH, R. M. WILSONANDH. WEISBACH,J. Biol. Chem., 234 (1959) 320. L. MICHAELISANDiV[.L. MENTEN,Biochem, Z., 35 (1911) 386. a R. F. LOMBAND A. E. MARTELL,J. Phys. Chem., 57 (1953) 690. 4 H. LINEWEAVERAND D. BORK, J. Am. Chem. Soc., 56 (1934) 658, Handbook of Chemistry and Physics, 35th Ed., Chemical Rubber Publishing Co., Cleveland, 1958, p. 1641. * H. J. BRIGHTAND L. L. INGRAHAM,Biochim. Biophys. Acta, 44 (196o) 586. z
Received October I8th, 1963
Biochim. Biophys. Acta, 81 (1964) 175-177
sc 11091
The molecular size of neuraminidase from Vibrio cholerae (strain 4Z) The enzyme neuraminidase (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) from Vibrio cholerae has been purified and crystallized independently by two groups. One preparation, isolated from an unspecified strain (MOHR AND SCI-IRAMM1) had sedimentation coefficients of 1.35 S at pH 8.5 and 2.35 S at pH 5.5. The other preparation (ADA, FRENCH AND LIND 2) from the strain 4 Z of V. cholerae was grown in the presence of sialyl lactose as inducer and appeared to be considerably larger, having a sedimentation coefficient of 5.5 S at pH 6. 7 and 5.3 S at pH 8. 5 (see ref. 3). The diffusion constant of this latter preparation was also determined and from these values a molecular weight of about 90 ooo was calculated 3. The molecular size of neuraminidase from the 4 Z strain of V. cholerae has now been further investigated. N-terminal amino acid analysis of the 4Z-strain enzyme was carried out using Abbreviation: PTH, phenylthiohydantoin.
Biochim. Biophys. Acta, 81 (1964) 177-18o
178
SHORT COMMUNICATIONS
a recently developed micromodification (LAVER4) of the EDMAN5 method. The enzyme was coupled with [35S]phenylisothiocyanate of high specific activity and the N-terminal amino acid(s) converted to the [asSIPTH derivatives. These were mixed with authentic samples of appropriate unlabelled phenylthiohydantoins and chromatographed in Solvents D and F of EDMAN AND SJOQUIST6. After drying, the positions of the unlabelled phenylthiohydantoins were marked out under ultraviolet light and the chromatograms were scanned for radioactivity. In Solvent F, the major radioactive peak coincided with PTH-alanine (Fig. Ia). PTH-alanine and P T H - t r y p t o p h a n are poorly separated in Solvent F but well separated in Solvent D. The main radioactive spot on the Solvent-F chromatogram was therefore eluted with glacial acetic acid and rechromatographed in Solvent D. Apart from a trace at the origin, all of the radioactivity was associated with PTH-alanine (Fig. Ib). The same result was obtained with three different preparations of neuraminidase, each from the 4 Z strain. Radioactive peaks corresponding to other PTH-amino acids were always seen but none was consistently obtained so that these probably came from contaminating proteins or peptides. The recovery of PTH-alanine was in the proportion of I mole per 9 ° ooo g of protein, when corrected for losses. These were not known exactly but the corrections made were based on the yield of PTH-alanine (87%), obtained by analysing in parallel rabbit y-globulin known to have I mole of N-terminal alanine per 18o ooo g of proteinT, s. It should be pointed out that if the recovery of PTH-alanine from the neuraminidase was in fact less than 87%, our estimate for the molecular weight would be unduly high. Ultracentrifugation studies of purified preparations and of filtrates of V. cholerae also yielded estimates of the size of this enzyme. Moving partition (YPHANTIS AND WAUGH) and fixed partition cells were used in a Spinco Model E ultracentrifuge. Sedimentation coefficients were estimated by the moving-boundary method (measurement of Schlieren patterns) when purified preparations were examined and b y the transport method (measurement of enzyme activity recovered from above the partition) for both preparations, using Eqn. 28b of SCItACHMAN9. Similar S values were obtained by the transport method for purified and crude preparations of neuraminidase and were about lO% less than S values calculated from the moving boundary method for purified preparations (see above). In some control experiments when the sedimentation behaviour of bovine plasma albumin (Armour laboratories, Fraction V, o.25%, w/v in phosphate buffered saline, p H 6.8) was studied, similar differences between S values obtained by the two methods were observed. Thus, there was no evidence that the enzyme activity in crude or purified preparations of this enzyme sedimented at a slower rate than the boundary observed optically when purified preparations were examined. Further evidence that crude filtrates of cultures of V. cholerae did not contain neuraminidase activity associated with a low-molecular-weight fraction was provided b y gel-filtration studies. A culture filtrate was mixed with bovine plasma albumin (mol. wt. 65 ooo) and with pancreatic ribonuclease (mol. wt. 17 0oo) and the mixture chromatographed on a column of the dextran gel, Sephadex G 75 (Fig. 2). Neuraminidase and bovine plasma albumin were eluted together from the column and before the appearance of ribonuclease in the effluent. The evidence presented agrees with the earlier finding that the neuraminidase produced by the 4 Z strain of V. cholerae has a molecular weight of about 9 ° ooo Biochim. Biophys. Acta, 81 (1964) I77 18o
179
SHORT COMMUNICATIONS
(a) 12000 10000 8000 6000 E ~4000 u
J Origin
Asp
Thr
Olyc Position on chromatogrem
Alo
Val
Solvept front
(b) 14000
12000 10000 8000 E ~c6000
4000
3 :_~
2000
Origin
Tryp
Alcl Position on chrornatogrern
Solvent frQnt
Fig. I. (a) D i s t r i b u t i o n of radioactivity on a c h r o m a t o g r a m (Solvent F) of the 3sS-labelled hit e r m i n a l a m i n o acid P T H s f r o m i . i m g of purified n e u r a m i n i d a s e (V. cholerae, strain 4Z). T h e positions of unlabelled m a r k e r p h e n y l t h i o h y d a n t o i n s added to the reaction m i x t u r e are s h o w n ; t h e relative positions of other a m i n o acid P T H s in Solvent F are given b y EDMAN AND S J O Q U I S T 6. A total of 4 ° 970 c o u n t s / m i n (counted on the p a p e r strip) w a s in the P T H - a l a n i n e spot. The [35S]phenylisothiocyanate used in this e x p e r i m e n t h a d 3.83" lO 6 c o u n t s / m i n per /2mole w h e n c o u n t e d as its sym-diphenylthiourea derivative u n d e r the same conditions as used for the exp e r i m e n t a l strip. (b) D i s t r i b u t i o n of radioactivity on a c h r o m a t o g r a m (Solvent D) showing s e p a r a t i o n of P T H - a l a n i n e a n d P T H - t r y p t o p h a n . The material applied was eluted f r o m the s p o t corresponding to P T H - a l a n i n e on a c h r o m a t o g r a m r u n in Solvent F.
Bioahim. Biophys. Acta, 8I (I964) I 7 7 - I 8 o
180
SHORT COMMUNICATIONS q-
i
P o.2
"7
i
O-I
..Q <
15
~O Tube
25
30
35
number
Fig. 2. C h r o m a t o g r a p h y of crude neuraminidase, bovine p l a s m a a l b u m i n and purified pancreatic ribonuclease on a column of the d e x t r a n gel, Sephadex G 75 (Pharmacia, Sweden). The column, 1.3 X 17. 5 cm, was equilibrated with a solution containing o.I M NaC1 dnd o.oi M Tris-maleic acid buffer (pH 5.7). io/~g ribonuclease and 5 ° mg a l b u m i n were dissolved in 0. 5 ml of crude neuraminidase (containing a b o u t 0. 3/*g of neuraminidase) and the m i x t u r e t h e n c h r o m a t o g r a p h e d on t h e Sephadex column, the developing buffer being t h a t described above, the flow rate 1.5 m l / h and the fraction size, o. 4 ml. [2]--[~, albumin, estimated b y m e a s u r i n g the absorba n c y (28o m/2) of the effluent; O - - - O, neuraminidase, estimated as described previously; & - - ~ , ribonuclease, estimated b y SHORTMAN'S10 method.
and it is probable that the molecule consists of a single polypeptide chain. Thus, it is unlikely that this enzyme is simply a polymer of the enzyme reported by MOHR AND SCHRAMM.
This work was supported in part by a grant from the National Health and Medical Research Council, Australia.
John Curtin School of Medical Research, Australian National University, Canberra (Australia) Walter and Eliza Hall Institute, Royal Melbourne Hospital Post O]fice, Melbourne (Australia)
W . G. LAVER
J. PYE G. L. ADA
1 E. 1V~OHRAND G. SCHi~AMM, Z. Naturforsch., i 5 b (196o) 568. z G. L. ADA, E. L. FRENCH AND P. E. LIND, J. Gen. Microbiol., 24 (1961) 409. 3 j . PYE AND C. C. CURTAIN, J. Gen. Microbiol., 24 (1961) 423 . 4 W. G. LAVER, Biochim. Biophys. Acta, 53 (1961) 469. P. EDMAN, Ann. N . Y . •cad. Sci., 88 (196o) 6o2. e p. EDMAN AND J. SJoQUiST, Acta Chem. Scand., io (1956) 15o 7. 7 R. R. PORTER, Biochem. J., 46 (195 o) 473. 8 M. L. McFADDEN AND E. L. SMITH, J. Biol. Chem., 214 (1955) 185. 9 H. K. SCHACHMAN, Ultracentrifugation in Biochemistry, Academic Press, N e w York, 1959, p. 86. 10 K. SHORTMAN, Biochim. Biophys. Acta, 5I (I96I) 37-
Received August 8th, 1963 Biochim. Biophys. Acta, 81 (1964) 177-18o