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Some molecular properties of pneumoccal neuraminidase isoenzymes
82 4
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35768 SOME MOLECULAR P R O P E R T I E S OF PNEUMOCCAL NEURAMINIDASE ISOENZYMES
S. W. TANENBAUM AND S.-C. SUN
Department of Microb,ology, College of Physicians and Surgeons, Columbm Umversity, New York, N.Y. 10032 (U.S.A.) (Received October I6th, 197 o)
SUMMARY
An estimated molecular weight of 7 ° 6oo ~: IOOO was determined for each of four pneumococcal neuraminidase iscenzyme groups by gel filtration chromatography. The identical value was found for the most homogeneous of these, the CM-II fraction, following its treatment with 8 M urea-o.I M ]5-mercaptoethanol and chromatography on a molecular sieve equilibrated with the denaturant. The resultant protein, though still coincident with residual enzymatic activity according to disc gel electropherograms, now displayed a migration towards the anode. An indistinguishable, carbamylated enzyme species was obtained upon reaction of the undenatured CM-II with o.I M KCNO. The gel-filtered CM-I and DEAE-II isoenzyme populations were individually resolved into two, well-separated isoenzyme bands upon electrophoresis. These results indicate a lack of subunit structure for the isoneuraminidases, and suggest that the appearance of the numerous multiple forms is a consequence of charge differences.
The finding that pneumococcal neuraminidase can exist in a multiplicity of forms has been recorded earlierk Among the possible explanations then offered to account for the appearance of these isoenzymes were: aggregation-disaggregation, subunit dissociation and recombination, differences in conformational states, heterogeneity in small ligand binding, or variations in primary sequence. This communication now presents evidence that the major neuraminidase isoenzyme groups have identical molecular weights approximating 7 ° 6o0 -+- ILOO, and that they do not appear to possess a subunit structure. These data, therefore, restrict the choice of hypotheses to account for the manifestation of isoneuraminidases to those involving charge difference. Following overnight growth of Diplococcus pneumoniae (Strain 70) and separation of the cells from the broth, the growth filtrate was brought to 0.70 saturation with (NH4)~SO 4 at 5 °. The separated protein was dialyzed, and the (NH4)~SO4 concentration was adjusted to 0.40 saturation. Precipitated protein was removed, Bioch,m. B~ophys. Acta, 229 (1971) 824-828
PNEUMOCOCCAL NEURAMINIDASE ISOENZYMES
70
I C
I I IT A OHIN
825
I G
6O
50
-~
4O
> 30
2O
lO
t ........ 10 4
I ........ 10 5
I 10 6
Log Molecular Weight Fig. 1. Gel f i l t r a t i o n of p u r i f i e d p n e u m o c o c c a l n e u r a m i n i d a s e s (N) on S e p h a d e x G - i o o (0--0) a n d on Biogel P - I o o ( 0 - - - 0 ) c o l u m n s of 1.8 c m × 32 c m p a c k e d d i m e n s i o n . M a r k e r p r o t e i n s : C, c y t o c h r o m e c, 2.o rag; A, a - c h y m o t r y p s i n o g e n , 2.o m g ; O, o v a l b u m i n , 2.o m g ; H, h e m o g l o b i n , 2.5 m g ; G, v G - i m m u n o g l o b u l i n , 1. 5 mg. N e u r a m i n i d a s e f r a c t i o n s (2- 7 mg) a n d t h e a p p r o p r i a t e m a r k e r s were e l u t e d w i t h 0.025 M c i t r a t e - p h o s p h a t e buffer (pH 6.o)--o.i M KCI s o l u t i o n a t 5 °. M e a s u r e d f r a c t i o n s w i t h i n t h e 1.1-1. 3 ml r a n g e were collected. Solid r e c t a n g l e on B i oge l line i n d i c a t e s t h e d i s t r i b u t i o n r a n g e f o u n d for t h e v a r i o u s i s o e n z y m e p r e p a r a t i o n s w h i c h a re l i s t e d in T a b l e I.
and the supernatant was then raised to 0.65 (NH4)2SO, saturation. After dialysis against o.oi M citrate-phosphate buffer (pH 6.0) the enzyme fraction was concentrated by ultrafiltration (Diaflo XM-5o membrane). The major isoenzyme populations TABLE I ESTIMATED MOLECULAR WEIGHTS OF PNEUMOCOCCAL ISOENZYMES AND THEIR MODIFICATIONS FOLLOWING GEL FILTRATION ON SEPHADEX AND POLYACRYLAMIDE Unless n o t e d otherwise, p r o t e i n s were c h r o m a t o g r a p h e d i n a c c o r d a n c e w i t h t h e d e t a i l s g i v e n in t h e l e g e n d to Fig. I.
Gel support
Isoenzyme fraction
Number of determznations
Molecular weight
Sephadex G-loo
CM-II
I
74 IOO
Biogel P-lOO
CM-I CM-I + o.I M K C N O CM-II CM-II + 8 M u r e a o.i M f l - m e r c a p t o e t h a n o l C M - I I + o.~ M K C N O DEAE-I DEAE-II
2 I 2
7 ° 5 oo, 75 7 ° 0 7 ° 3oo" 7 ° 8o0, 73 3 ° 0
2 i 2 2
7° 7° 71 7°
80o, 70 6oo** 5oo * ooo, 72 400 600, 7 ° 500
* C o l u m n e q u i l i b r a t e d w i t h o . i M KCNO. ** C o l u m n e q u i l i b r a t e d w i t h 8 M u r e a - o , i M f l - m e r c a p t o e t h a n o l .
Biochim. Biophys. Acta, 229 (I971) 824-828
826
s.w.
T A N E N B A U M , S.-C. SUN
were then resolved into their DEAE-I, D E A E - I I , CM-I, and CM-II groups by chromatography on ion-exchange celluloses 1. Initial determination of the molecular weight of the CM-II isoenzyme was carried out by gel filtration on Sephadex G-Ioo by the method of ANDREWS~. This provided a value of approx. 74 ooo (Fig. i). Because of known anomalies concerning elution retardation a and Sephadex inhibition 4 of lysozyme, this estimation for neuraminidase was checked using Biogel P-Ioo as the chromatographic sieve 5. Two such determinations gave molecular weights which averaged 72 ooo. Similar evaluation of the molecular weights of the other neuraminidase isoenzymes and of certain of their chemical modifications gave almost identical data (Table I). Polyacrylamide gel electrophoretic analysis of peak tubes from symmetrical enzyme activity curves which were obtained from these gel filtrations showed that the CM-II isoenzyme contained only one protein-staining band. This band coincided with the enzymatic region which was detected in a replicate zymogram (Fig. 2A). The specific activity of this apparently homogeneous CM-II isoenzyme was 600, several-fold- higher than the crystalline neuraminidase isoenzyme mixture previously described 1. After pooling and concentrating the protein under the Biogel sieved CM-II peak area, this isoenzyme was incubated at 25 ° for 2 h with 8 M urea-o.I M fl-mercaptoethanol (pH 6.6). An aliquot of this denatured enzyme was diluted 2o-fold with phosphate-citrate buffer for assay. Following such treatment, it was found with Collocalia mucoid as substrate, that the recoverable enzymatic activity of CM-II preparations ranged from 25 to 40 %. The bulk of the denatured protein was subjected to gel filtration on Biogel P-ioo, as outlined under Fig. I. Alternatively, acrylamide molecular sieving was carried out on a Biogel column which had been equilibrated with 8 M urea and o.i M/5-mercaptoethanol a. The molecular weight as determined by either chronlatographic technic remained essentially unchanged (Table I). Electro-
A
N
D
10
10
B
E
10
10
C
F
10
10
Fig. 2. Disc gel electrophoretic patterns and their parallel electropherograms for Biogel P-iooresolved neuramimdase isoenzymes. Electrophoresis was performed on 7.5% polyacrylamide gel (pH 8.5) at 4 mA per tube. Protein stain, Coomassie blue; enzyme assays were carried out on thirty 1.5-mm slices taken from frozen replicate gels1. Abscissa, relative enzyme activity (nmoles of N-acetylneuraminic acid released) with Collocalia mucold. (A) CM-II, (B) CM-II treated with urea-fl-mereaptoethanol denaturant, (C) CM-II treated with o.I M KCNO, (D) CM-I, (E) DEAE-II, (F) DEAE-I. Biochim. B,ophys. Acta, 229 (1971) 824-828
PNEUMOCOCCAL NEURAMINIDASE ISOENZYMES
827
phoretic examination of the denatured CM-II revealed that a total conversion to an anodal-migrating protein species, still identical with the residual enzymatic activity, had taken place (Fig. 2B). Although control tubes of the denaturant reaction mixture failed to evince a positive WERNER6 test for cyanate ion, it was considered probable that carbamylation 7 of the enzyme had occurred. When the native CM-II fraction was exposed to o.I M KCNO under comparable reaction conditions, the anticipated conversion (Fig. 2C) resulted. Disc gel electrophoresis of the molecular sieve-resolved CM-I enzyme group revealed considerable heterogeneity with regards to inactive protein. Furthermore, this fraction was seen to contain two isoenzyme subsets (Fig. 2D). When this latter heterogeneous fraction was carbamylated with o.I M KCNO and was subsequently filtered through Biogel P-ioo equilibrated with cyanate, a molecular weight for the enzymatic components of approx. 70 ooo was again obtained (Table I). Electropherograrns (Fig. 2E) of the DEAE-II isoenzyme population displayed one major and one minor protein band, each exhibiting enzymatic activity. The A~80 mu/A~6o m,u ratio of this highly purified isoenzyme mixture was 1.75. The DEAE-I isoenzyme was shown to consist of a considerable quantity of an inactive protein band, together with a smaller proportion of neuranfinidase (Fig. 2F). Whether this cathodal-migrating enzymatic component of the DEAE-I fraction is identical with the CM-II isoenzyme is as yet unclear; although preliminary evidence based on disc gel coelectrophoresis indicates that such is not the case. However, with the exception of the possibly cationic DEAE-I, the other isoenzyme populations migrated electrophoretically in rough accord with their net charge as earlier indicated by order of appearance from the two cellulose ion-exchangers 1 during increased salt gradients. The results of these experiments demonstrate that the major pneumococcal isoenzyme groups, as well as their subsets, are each comprised of enzymes of molecular weight around 7 ° 600. Their properties, therefore, do not resemble the associateddissociated pneumococcal dihydrofolate reductase 8, but may rather parallel the twelve uterine cathepsin D isoenzymes, which appeared at identical elution volumes upon Sephadex G-ioo chromatography 9. It should be mentioned parenthetically, that the molecular weight of Clostridium perfringens neuraminidase has been recently estimated by gel filtration to be 56 ooo (ref. io). The finding that the CM-II isoenzyme was not changed in its point of emergence on gel filtration after denaturation with urea in the presence of fl-mercaptoethanol, taken together with the fact that disc gel electrophoresis of the denatured protein did not reveal additional bands, strongly suggests that the pneumococcal neuraminidases lack subunit structure. The complete conversion of this isoenzyme into the carbamylated derivative on treatment with "cyanate-free" urea has revealed the presence on these proteins of a functional group or groups with extreme reactivity for this ion. Furthermore, the homogeneity of this derivative on disc gel electrophoresis also affirmed the apparent purity of the unsubstituted CM-II species. The most likely explanation to account for these results is that pneumococcal neuraminidase isoenzymes consist of differently charged species, which in turn are due either to translational or post-translational structural differences, or to different conformational states. Such putative sequential heterogeneity could also reflect partial peptidase cleavage of a uniform parental enzyme, as has been shown for Escherichia coli alkaline phosphatase mutants n. This alternative is deemed unBioch,m. B,ophys. Acta, 229 (1971) 824-828
828
S . W . TANENBAUM, S.-C. SUN
likely, though not excluded, on the basis of experiments which were carried out with the initial 0.70 saturated (NH~)2SO 4 precipitate obtained from the growth medium. This fraction, which contains almost all the filtrate protein including neuraminidase, was protease-negative in casein hydrolysis 12 and azocoll lv14 tests and also failed to produce additional color in a sensitive ninhydrin assay which used denatured casein as substrate. Therefore, barring multiple conformational transitions, the present evidence points to the biosynthesis of the several neuraminidase isoenzymes as a consequence of population heterogeneity of the pneumococcus strain, or ,in part, to the presence of allelic genes. ACKNOWLEDGEMENT
This research was supported by a grant (NSF GB-5316 ) from the National Science Foundation. REFERENCES I S . W . TANENBAUM, J. GULBINSKY, M. KATZ AND S.-C. SUN, Bioch*m. Biophys. Acta, 198 (197 o) 242. 2 P. ANDREWS, in D. SLICK, Methods of Biochemical Analysis, Vol. 18, Interscience, N e w York, 197o, p. I. 3 P. F. DAVlSON, Science, 161 (1968) 906. 4 J. R. WHITAKER, Anal. Chem., 35 (1963) 195o. 5 A. IV[. DEL C. BATTLE, J. Chromatog., 28 (1967) 82. 6 E. A. WERNER, J. Chem. Soc., 123 (1923) 2577. 7 G. R. STARK, W. H. STEIN AND S. MOORE, J. Biol. Chem., 235 (196o) 3177 . 8 F. M. SIROTNAK AND W. A. WILLIAMS, Arch. Biochem. Biophys., 136 (197 o) 580. 9 A. I. SAPOLSKY AND J. F. WOESSNER, Federation Proc., 29 (197 o) 924. IO E. BALKE AND R. DRZENIEK, Z. Naturforsch., 24b (I97 o) 599. I I S. NATORI AND A. GAREN, J. Mol. Biol., 49 (I97 o) 577. 12 A. R. SUBRAMANIAN AND G. KALNITSKY, Biochemistry, 3 (1964) 1861. 13 C. L. OAKLEY, G. H. WARRACK AND W. E. VAN HEYNINGEN, J. Pathol. Bacteriol., 58 (1946) 229. 14 L. M. SREERNY, J. MEYER AND E. BACHEM, Lab. Invest., 4 (1955) 27.
Biochim. Biophys. Acta, 229 (1971) 824-828
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35768 SOME MOLECULAR P R O P E R T I E S OF PNEUMOCCAL NEURAMINIDASE ISOENZYMES
S. W. TANENBAUM AND S.-C. SUN
Department of Microb,ology, College of Physicians and Surgeons, Columbm Umversity, New York, N.Y. 10032 (U.S.A.) (Received October I6th, 197 o)
SUMMARY
An estimated molecular weight of 7 ° 6oo ~: IOOO was determined for each of four pneumococcal neuraminidase iscenzyme groups by gel filtration chromatography. The identical value was found for the most homogeneous of these, the CM-II fraction, following its treatment with 8 M urea-o.I M ]5-mercaptoethanol and chromatography on a molecular sieve equilibrated with the denaturant. The resultant protein, though still coincident with residual enzymatic activity according to disc gel electropherograms, now displayed a migration towards the anode. An indistinguishable, carbamylated enzyme species was obtained upon reaction of the undenatured CM-II with o.I M KCNO. The gel-filtered CM-I and DEAE-II isoenzyme populations were individually resolved into two, well-separated isoenzyme bands upon electrophoresis. These results indicate a lack of subunit structure for the isoneuraminidases, and suggest that the appearance of the numerous multiple forms is a consequence of charge differences.
The finding that pneumococcal neuraminidase can exist in a multiplicity of forms has been recorded earlierk Among the possible explanations then offered to account for the appearance of these isoenzymes were: aggregation-disaggregation, subunit dissociation and recombination, differences in conformational states, heterogeneity in small ligand binding, or variations in primary sequence. This communication now presents evidence that the major neuraminidase isoenzyme groups have identical molecular weights approximating 7 ° 6o0 -+- ILOO, and that they do not appear to possess a subunit structure. These data, therefore, restrict the choice of hypotheses to account for the manifestation of isoneuraminidases to those involving charge difference. Following overnight growth of Diplococcus pneumoniae (Strain 70) and separation of the cells from the broth, the growth filtrate was brought to 0.70 saturation with (NH4)~SO 4 at 5 °. The separated protein was dialyzed, and the (NH4)~SO4 concentration was adjusted to 0.40 saturation. Precipitated protein was removed, Bioch,m. B~ophys. Acta, 229 (1971) 824-828
PNEUMOCOCCAL NEURAMINIDASE ISOENZYMES
70
I C
I I IT A OHIN
825
I G
6O
50
-~
4O
> 30
2O
lO
t ........ 10 4
I ........ 10 5
I 10 6
Log Molecular Weight Fig. 1. Gel f i l t r a t i o n of p u r i f i e d p n e u m o c o c c a l n e u r a m i n i d a s e s (N) on S e p h a d e x G - i o o (0--0) a n d on Biogel P - I o o ( 0 - - - 0 ) c o l u m n s of 1.8 c m × 32 c m p a c k e d d i m e n s i o n . M a r k e r p r o t e i n s : C, c y t o c h r o m e c, 2.o rag; A, a - c h y m o t r y p s i n o g e n , 2.o m g ; O, o v a l b u m i n , 2.o m g ; H, h e m o g l o b i n , 2.5 m g ; G, v G - i m m u n o g l o b u l i n , 1. 5 mg. N e u r a m i n i d a s e f r a c t i o n s (2- 7 mg) a n d t h e a p p r o p r i a t e m a r k e r s were e l u t e d w i t h 0.025 M c i t r a t e - p h o s p h a t e buffer (pH 6.o)--o.i M KCI s o l u t i o n a t 5 °. M e a s u r e d f r a c t i o n s w i t h i n t h e 1.1-1. 3 ml r a n g e were collected. Solid r e c t a n g l e on B i oge l line i n d i c a t e s t h e d i s t r i b u t i o n r a n g e f o u n d for t h e v a r i o u s i s o e n z y m e p r e p a r a t i o n s w h i c h a re l i s t e d in T a b l e I.
and the supernatant was then raised to 0.65 (NH4)2SO, saturation. After dialysis against o.oi M citrate-phosphate buffer (pH 6.0) the enzyme fraction was concentrated by ultrafiltration (Diaflo XM-5o membrane). The major isoenzyme populations TABLE I ESTIMATED MOLECULAR WEIGHTS OF PNEUMOCOCCAL ISOENZYMES AND THEIR MODIFICATIONS FOLLOWING GEL FILTRATION ON SEPHADEX AND POLYACRYLAMIDE Unless n o t e d otherwise, p r o t e i n s were c h r o m a t o g r a p h e d i n a c c o r d a n c e w i t h t h e d e t a i l s g i v e n in t h e l e g e n d to Fig. I.
Gel support
Isoenzyme fraction
Number of determznations
Molecular weight
Sephadex G-loo
CM-II
I
74 IOO
Biogel P-lOO
CM-I CM-I + o.I M K C N O CM-II CM-II + 8 M u r e a o.i M f l - m e r c a p t o e t h a n o l C M - I I + o.~ M K C N O DEAE-I DEAE-II
2 I 2
7 ° 5 oo, 75 7 ° 0 7 ° 3oo" 7 ° 8o0, 73 3 ° 0
2 i 2 2
7° 7° 71 7°
80o, 70 6oo** 5oo * ooo, 72 400 600, 7 ° 500
* C o l u m n e q u i l i b r a t e d w i t h o . i M KCNO. ** C o l u m n e q u i l i b r a t e d w i t h 8 M u r e a - o , i M f l - m e r c a p t o e t h a n o l .
Biochim. Biophys. Acta, 229 (I971) 824-828
826
s.w.
T A N E N B A U M , S.-C. SUN
were then resolved into their DEAE-I, D E A E - I I , CM-I, and CM-II groups by chromatography on ion-exchange celluloses 1. Initial determination of the molecular weight of the CM-II isoenzyme was carried out by gel filtration on Sephadex G-Ioo by the method of ANDREWS~. This provided a value of approx. 74 ooo (Fig. i). Because of known anomalies concerning elution retardation a and Sephadex inhibition 4 of lysozyme, this estimation for neuraminidase was checked using Biogel P-Ioo as the chromatographic sieve 5. Two such determinations gave molecular weights which averaged 72 ooo. Similar evaluation of the molecular weights of the other neuraminidase isoenzymes and of certain of their chemical modifications gave almost identical data (Table I). Polyacrylamide gel electrophoretic analysis of peak tubes from symmetrical enzyme activity curves which were obtained from these gel filtrations showed that the CM-II isoenzyme contained only one protein-staining band. This band coincided with the enzymatic region which was detected in a replicate zymogram (Fig. 2A). The specific activity of this apparently homogeneous CM-II isoenzyme was 600, several-fold- higher than the crystalline neuraminidase isoenzyme mixture previously described 1. After pooling and concentrating the protein under the Biogel sieved CM-II peak area, this isoenzyme was incubated at 25 ° for 2 h with 8 M urea-o.I M fl-mercaptoethanol (pH 6.6). An aliquot of this denatured enzyme was diluted 2o-fold with phosphate-citrate buffer for assay. Following such treatment, it was found with Collocalia mucoid as substrate, that the recoverable enzymatic activity of CM-II preparations ranged from 25 to 40 %. The bulk of the denatured protein was subjected to gel filtration on Biogel P-ioo, as outlined under Fig. I. Alternatively, acrylamide molecular sieving was carried out on a Biogel column which had been equilibrated with 8 M urea and o.i M/5-mercaptoethanol a. The molecular weight as determined by either chronlatographic technic remained essentially unchanged (Table I). Electro-
A
N
D
10
10
B
E
10
10
C
F
10
10
Fig. 2. Disc gel electrophoretic patterns and their parallel electropherograms for Biogel P-iooresolved neuramimdase isoenzymes. Electrophoresis was performed on 7.5% polyacrylamide gel (pH 8.5) at 4 mA per tube. Protein stain, Coomassie blue; enzyme assays were carried out on thirty 1.5-mm slices taken from frozen replicate gels1. Abscissa, relative enzyme activity (nmoles of N-acetylneuraminic acid released) with Collocalia mucold. (A) CM-II, (B) CM-II treated with urea-fl-mereaptoethanol denaturant, (C) CM-II treated with o.I M KCNO, (D) CM-I, (E) DEAE-II, (F) DEAE-I. Biochim. B,ophys. Acta, 229 (1971) 824-828
PNEUMOCOCCAL NEURAMINIDASE ISOENZYMES
827
phoretic examination of the denatured CM-II revealed that a total conversion to an anodal-migrating protein species, still identical with the residual enzymatic activity, had taken place (Fig. 2B). Although control tubes of the denaturant reaction mixture failed to evince a positive WERNER6 test for cyanate ion, it was considered probable that carbamylation 7 of the enzyme had occurred. When the native CM-II fraction was exposed to o.I M KCNO under comparable reaction conditions, the anticipated conversion (Fig. 2C) resulted. Disc gel electrophoresis of the molecular sieve-resolved CM-I enzyme group revealed considerable heterogeneity with regards to inactive protein. Furthermore, this fraction was seen to contain two isoenzyme subsets (Fig. 2D). When this latter heterogeneous fraction was carbamylated with o.I M KCNO and was subsequently filtered through Biogel P-ioo equilibrated with cyanate, a molecular weight for the enzymatic components of approx. 70 ooo was again obtained (Table I). Electropherograrns (Fig. 2E) of the DEAE-II isoenzyme population displayed one major and one minor protein band, each exhibiting enzymatic activity. The A~80 mu/A~6o m,u ratio of this highly purified isoenzyme mixture was 1.75. The DEAE-I isoenzyme was shown to consist of a considerable quantity of an inactive protein band, together with a smaller proportion of neuranfinidase (Fig. 2F). Whether this cathodal-migrating enzymatic component of the DEAE-I fraction is identical with the CM-II isoenzyme is as yet unclear; although preliminary evidence based on disc gel coelectrophoresis indicates that such is not the case. However, with the exception of the possibly cationic DEAE-I, the other isoenzyme populations migrated electrophoretically in rough accord with their net charge as earlier indicated by order of appearance from the two cellulose ion-exchangers 1 during increased salt gradients. The results of these experiments demonstrate that the major pneumococcal isoenzyme groups, as well as their subsets, are each comprised of enzymes of molecular weight around 7 ° 600. Their properties, therefore, do not resemble the associateddissociated pneumococcal dihydrofolate reductase 8, but may rather parallel the twelve uterine cathepsin D isoenzymes, which appeared at identical elution volumes upon Sephadex G-ioo chromatography 9. It should be mentioned parenthetically, that the molecular weight of Clostridium perfringens neuraminidase has been recently estimated by gel filtration to be 56 ooo (ref. io). The finding that the CM-II isoenzyme was not changed in its point of emergence on gel filtration after denaturation with urea in the presence of fl-mercaptoethanol, taken together with the fact that disc gel electrophoresis of the denatured protein did not reveal additional bands, strongly suggests that the pneumococcal neuraminidases lack subunit structure. The complete conversion of this isoenzyme into the carbamylated derivative on treatment with "cyanate-free" urea has revealed the presence on these proteins of a functional group or groups with extreme reactivity for this ion. Furthermore, the homogeneity of this derivative on disc gel electrophoresis also affirmed the apparent purity of the unsubstituted CM-II species. The most likely explanation to account for these results is that pneumococcal neuraminidase isoenzymes consist of differently charged species, which in turn are due either to translational or post-translational structural differences, or to different conformational states. Such putative sequential heterogeneity could also reflect partial peptidase cleavage of a uniform parental enzyme, as has been shown for Escherichia coli alkaline phosphatase mutants n. This alternative is deemed unBioch,m. B,ophys. Acta, 229 (1971) 824-828
828
S . W . TANENBAUM, S.-C. SUN
likely, though not excluded, on the basis of experiments which were carried out with the initial 0.70 saturated (NH~)2SO 4 precipitate obtained from the growth medium. This fraction, which contains almost all the filtrate protein including neuraminidase, was protease-negative in casein hydrolysis 12 and azocoll lv14 tests and also failed to produce additional color in a sensitive ninhydrin assay which used denatured casein as substrate. Therefore, barring multiple conformational transitions, the present evidence points to the biosynthesis of the several neuraminidase isoenzymes as a consequence of population heterogeneity of the pneumococcus strain, or ,in part, to the presence of allelic genes. ACKNOWLEDGEMENT
This research was supported by a grant (NSF GB-5316 ) from the National Science Foundation. REFERENCES I S . W . TANENBAUM, J. GULBINSKY, M. KATZ AND S.-C. SUN, Bioch*m. Biophys. Acta, 198 (197 o) 242. 2 P. ANDREWS, in D. SLICK, Methods of Biochemical Analysis, Vol. 18, Interscience, N e w York, 197o, p. I. 3 P. F. DAVlSON, Science, 161 (1968) 906. 4 J. R. WHITAKER, Anal. Chem., 35 (1963) 195o. 5 A. IV[. DEL C. BATTLE, J. Chromatog., 28 (1967) 82. 6 E. A. WERNER, J. Chem. Soc., 123 (1923) 2577. 7 G. R. STARK, W. H. STEIN AND S. MOORE, J. Biol. Chem., 235 (196o) 3177 . 8 F. M. SIROTNAK AND W. A. WILLIAMS, Arch. Biochem. Biophys., 136 (197 o) 580. 9 A. I. SAPOLSKY AND J. F. WOESSNER, Federation Proc., 29 (197 o) 924. IO E. BALKE AND R. DRZENIEK, Z. Naturforsch., 24b (I97 o) 599. I I S. NATORI AND A. GAREN, J. Mol. Biol., 49 (I97 o) 577. 12 A. R. SUBRAMANIAN AND G. KALNITSKY, Biochemistry, 3 (1964) 1861. 13 C. L. OAKLEY, G. H. WARRACK AND W. E. VAN HEYNINGEN, J. Pathol. Bacteriol., 58 (1946) 229. 14 L. M. SREERNY, J. MEYER AND E. BACHEM, Lab. Invest., 4 (1955) 27.
Biochim. Biophys. Acta, 229 (1971) 824-828