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Arylaminopeptidase activities in human cariogenic and non-cariogenic oral bacteria
Arch oral Bid. Vol. 16, pp. 675680,
1971. Pergamon Press. Printed in Great Britain.
SHORT COMMUNICATION ARYLAMINOPEPTIDASE ACTIVITIES IN HUMAN CARIOGENIC AND NON-CARIOGENIC ORAL BACTERIA H. OYA*, T. NAGATSU*,Y. KOBAYASHI~ and M. TAKEI? *Department of Biochemistry and tDepartment of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan
M~~KINEN (1966) reported the presence of arylaminopeptidase activity in human whole saliva and in human dental plaque. The presence of various arylaminopeptidases in human duct saliva and in human whole saliva, which hydrolyse amino acid-fi-naphthylamides, has also been reported (OYA, YAMAMOTO and NAGATSU,1968). The hydrolysis of glycyl-prolyl+naphthylamide was found to be relatively higher than those of other amino acid+-naphthylamides (NAGATSU,NAGATSUand YAMAMOTO,1968). The enzyme responsible for the hydrolysis of glycyl-prolyl-naphthylamide, i.e., glycyl-prolyl-naphthylamidase, in human submaxillary gland has been proved to be an enzyme distinct from other arylaminopeptidases (OYA and NAGATSU, 1969). Glycyl-prolyl-naphthylamidase was tist discovered in rat liver by HOPSU-HAVUand GLENNER(1966). Since the amino acid sequence of Gly-Pro-X is present in the collagen molecule (it has been found in hydrolysates from achilles tendon of cattle by SCHROHENLOHER, OGLE and LOGAN 1959), glycyl-prolyl-naphthylamidase might be expected to have an important role in the degradation of collagen in the oral cavity. The high activity of arylaminopeptidases in human whole saliva was attributed to oral bacteria (MKKINEN,1966; OYA et al., 1968). If bacteria in carious plaque contain arylaminopeptidases such as glycyl-prolylnaphthylamidase, destruction of dentine organic matrix by proteolysis might be possible. The presence of arylaminopeptidase activity in human carious dentine was demonstrated histochemically (LARMAS,M~~KINENand SCHEININ,1968). They proposed the hypothesis that decalcification and proteolysis might occur simultaneously, and in the same region. TAKEIet al. (1968) and KOBAYASHI et al. (1969) showed that the predominant species of bacteria in the carious plaque changed from streptococci to lactobacilli during the course of the development of a carious cavity in dentine. Therefore, it seemed important to determine those bacterial species which possess high proteolytic enzyme activities. In order to examine the relations of bacterial arylaminopeptidases to the development of caries, we have examined the enzyme activities in several cariogenic and noncariogenic oral bacteria. 675
676
H. OYA, T. NAGATSU,Y.
KOBAYAWI
AND
M. TAKEI
Test organisms were Streptococcus mitis ATCC 9811, Streptococcus salivarius ATCC 9222, Streptococcus faecalis IID 10541, cariogenic streptococci (strains PK-1, HS-6, CG-5, LM-7, E-49), Lactobacillus acidophilus IAF 3205 and Luctobacillus casei ATCC 7469. Table 1 shows the source of cariogenic streptococci and what test animals were used. TABLE1. THE CARI~GENICs~~~~~ococc~ EXAMINED Species
Source
Test animals
References
Strep. HS-6
Hamster
Conventional
strep. Streu. Strep. Strep.
Hamster Human HulllaD Human
Conventional hamster and rat Gnotobiotic rat Gnotobiotic rat Gnotobiotic rat
E-49 LM-7 GS-5 PK-1
I
I
0
I 4
Cariogenic l
PITZGERALDand KWI%
hamster
wm
streptococcus
PITZGEXALD(1963) G~B~NS et al. (1966) GIBBONSet al. (1966) GIBBONSet 01. (1966)
PK-I
Cell numbers
0 Gly-Pro-NAase A Leu-NAase
I
8
I I2 Incubation
I 16 time,
I
20
I 24
hr
PIG. 1. Time course of the cell numbers, glycyl-prolyl-naphthylamidase (Gly-Pro-NAase) and leucyl-naphthylamidase (km-NAase) during the incubation of cariogenic streptococcus PK- 1.
ARYLAhiINOPEPTIDASE
ACTIVITIES
IN HUMAN
ORAL
BACTERIA
677
The growth culture for each test organism was brain heart infusion broth (Difco) which was inoculated with 5 x lo6 cells/ml and incubated aerobically for 24 hr. Two cultures were made, the second being started 12 hr after the first. For each strain, the growth of the culture was studied by removing aliquots at 0,2,4,6,8 and 12 hr from the first culture and at 12, 16, l&20 and 24 hr from the second culture and making viable plate counts on brain heart infusion agar (Difco) supplemented with 1 per cent glucose and 0.5 per cent yeast extract. Colonies were counted after 48 hr aerobic incubation at 37°C. Enzyme activity was measured using cells removed from the culture at various stages of growth. Ceils were harvested, washed and suspended in O-01 M tris-HCl buffer at pH 7 -0. Arylaminopeptidase activity was measured by using the substrates glycyl-cprolyl&naphthylamide or L-leucyl-@raphthylamide, as reported in our previous paper (OYA et al., 1968). The suspensions of washed bacteria in O-01 M tris-HCl buffer,
i
I
Streptococcus l
mitis
ATCC
9811
Cell numbers
0 Gly-Pro-NAose
1 I 4
A Leu-NAase
I 8
I 12
Incubation
I 16
time,
I 20
I 24
hr
2. Time course of the cell numbers, glycyl-prolyl-naphthybmklase (Gly-ProNAase) and leucyl-naphthylamidase (Leu-NAase) during the incubation of Strep. mitis ATCC 9811.
FIG.
H. OYA, T. NAGATSU, Y. KOBAYASHIAND M. TAKEI
678
pH 7.0 were used as the enzyme preparations. The incubation mixture contained 90 pmole tris-maleate buffer, pH 7.0, 0.45 pmole substrate, an appropriate amount of the enzyme and water to 0.90 ml. Incubation was carried out at 37°C for 30 min. Incubation was stopped by the addition of 0.3 ml of 10 per cent Tween-20 in 1 M acetate buffer, pH 4.2, containing O-45 mg of stabilised diazonium salt Fast Garnet GBC. After 30 min, absorbance at 530 rnp was measured. Enzyme activities are expressed as rnp-moles of /3-naphthylamine liberated/30 min/lOg cells. As shown in Fig. 1 (cariogenic streptococcus PK-1) and Fig. 2 (Strep. mitis ATCC 981 l), enzyme activity either toward glycyl-prolyl-naphthylamide or toward leucyl-naphthylamide was increased simultaneously with the increase in the cell numbers. Figures 1 and 2 show that cariogenic streptococcus PK-1 had lower arylaminopeptidase activities than non-cariogenic streptococcus ATCC 9811. The latter had very high glycyl-prolyl-naphthylamidase activity. The arylaminopeptidase activities of 5 cariogenic streptococci, and 3 non-cariogenie streptococci and 2 lactobacilli are shown in Figs. 3 and 4. As shown in Fig. 3,
HS-6
0
Leu-NAase
q
Gly-
LM-7
Pro-NAase
E-49
PK-I
GS-5
FIG. 3. Glycyl-prolyl-naphthylamidase (Gly-Pro-NAase) and leucyl-naphthylamidase (Leu-NAase) of cariogenic streptococci. Enzyme activities were measured at 8, 12, and 16 hr of the incubation.
ARYLAMrN0PJPnmsE
0
ATCC
I2
9811
0
Leu - NAase
El
Gly-Pro-NAase
I(
I6
ATCC
IN HUMAN ORAL BACTERIA
AcIlvITIEs
9222
fl 8
I2
16
IID
10541
8
I2
I FO 3205
I6
679
6
ATCC
12
7469
FIG. 4. Glycyl-prolyl-naphthykmkiase (Gly-Pro-NAase) and leucyl-naphthylamidase (Leu-NAase) of noncariogenic bacteria (Strep. mitis ATCC 9811, Strep. salivarks ATCC 9222, Strep. faecalis IID 10541, Lactobacillm acidophilus IF0 3205 and Lactobacillus casei ATCC 7419). Enzyme activities were measured at 8, 12, and 16 hr of the incubation.
all the cariogenic streptococci tested had low arylaminopeptidase activities. In contrast, among 3 non-cariogenic streptococci, Strep. mitis ATCC 9811 and Strep. salivarius ATCC 9222 had high arylamidase activity. Glycyl-prolyl-naphthylamidase activity was higher than leucyl-naphthylamidase activity in the streptococci (Fig. 3). Both Luctobacillus acidophilus IF0 3205 and Lacfobacillus casei ATCC 7469 had high arylaminopeptidase activities. Leucyl-naphthylamidase activity was higher than glycyl-prolyl-naphthylamidase activity in the lactobacilli (Fig. 4). These results suggest that in dentine caries the involvement of arylaminopeptidase activity in cariogenic streptococci is not significant, since the bacteria contain only low enzyme activity. However, arylaminopeptidase activity may become of importance for the later development of a dentinal carious cavity, since lactobacilli, which become
680
H. OYA,T. NAGATSU, Y. KOBAYASHI ANDM. TAKEI
predominant in the later stage of carious cavity formation (TAKEI et al., 1968; KOBAYASHI et al., 1969), contain high enzyme activity. It was found that the activities of the extracellular enzymes were low compared with the enzyme activities inside cells. Therefore, the production of extracellular enzymes from the bacteria is probably of little significance. Acknowledgements-The authors wish to thank Dr. S. SAKAKIBARA and Dr. K. TAKADA(Institute for Protein Research, Osaka University, Osaka) for the synthesis of glycyl-L-prolyl+-naphthylamide, and Miss Y. NISHIKAWAand Miss Y. SHIBAHARA for their valuable technical assistance. REFERENCES FITZGERALD,R. J. and KEYES, P. H. 1960. Demonstration of the etiologic role of streptococci in experimental caries in hamster. J. Am. dent. Ass. 61,9-31. FITZGEIULD, R. J. 1963. Gnotobiotic contribution to oral microbiology. J. dent. Res. 42, 548-552. B. 1966.Dental caries and alveolar GIBBONS, R. J., BE-, K. S., KNom, P. and m, bone loss in gnotobiotic rats infected with capsule-forming streptococci of human origin. Archs oralBio1. 11,549-560. Hopsu-HAW,V. K. and GLENNER, G. G. 1966. A new dipeptide naphthylamidase hydrolyzing glycyl-prolyl-j-naphthylamide. Histochemie 7, 197-201. KOBAYASHI, Y., KONDO, E., Fumnr, T. and TAKEI, M. 1969.Fundamental studies on the dynamic movement of oral microbes 10. Lactobacillus in the dental carious foci. Aichi-Gakuin J. Dent. Sci. 7, 27-30. LARMAS,M., M~~KINEN,K. K. and SCHEININ,A. 1968. Histochemical studies on the arylaminopeptidase activity in human carious dentine. Acta odont. stand. 26, 127-137. MKKINEN, K. K. 1966. Studies on oral enzymes I. Fractionation and characterization of aminopeptidases of human saliva. Acta odont. stand. 24,579-6&L NAOATSU,I., NAGATSU,T. and YAMAMOTO,T. 1968.Hydrolysis of amino acid fi-naphthylamides by aminopeptidases in human parotid saliva and human serum. Experientia 24, 347-348. OYA, H. and NAGATSU,T. 1969. Glycyl-L-prolyl-fi-naphthylamidase in human submaxillary gland. J. Jap. Biochem. Sot. 41,597-597. OYA, H., YAMAMOTO, T. and NAGATSU,T. 1968. Presence of leucine aminopeptidase activity in human saliva from the parotid gland and the submaxillary-subliil glands. Archs oral Biol. 12, 941-948. SCHROHENLOMR,R. E., OGLE, J. D. and LOGAN, M. A. 1959. Two tripeptides from an enzymatic digest of collagen. J. biol. Chem. 234, 58-61. TAKEI,M., KOBAYASHI,Y., KONW, E. and IWASAKI,S. 1968. Fundamental studies on the dynamic movement of oral microbes 4. Glucose-utilizing microbes in caries areas. Aichi-Gakuin J. dent. Sci. 5, 306-3 11.
1971. Pergamon Press. Printed in Great Britain.
SHORT COMMUNICATION ARYLAMINOPEPTIDASE ACTIVITIES IN HUMAN CARIOGENIC AND NON-CARIOGENIC ORAL BACTERIA H. OYA*, T. NAGATSU*,Y. KOBAYASHI~ and M. TAKEI? *Department of Biochemistry and tDepartment of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan
M~~KINEN (1966) reported the presence of arylaminopeptidase activity in human whole saliva and in human dental plaque. The presence of various arylaminopeptidases in human duct saliva and in human whole saliva, which hydrolyse amino acid-fi-naphthylamides, has also been reported (OYA, YAMAMOTO and NAGATSU,1968). The hydrolysis of glycyl-prolyl+naphthylamide was found to be relatively higher than those of other amino acid+-naphthylamides (NAGATSU,NAGATSUand YAMAMOTO,1968). The enzyme responsible for the hydrolysis of glycyl-prolyl-naphthylamide, i.e., glycyl-prolyl-naphthylamidase, in human submaxillary gland has been proved to be an enzyme distinct from other arylaminopeptidases (OYA and NAGATSU, 1969). Glycyl-prolyl-naphthylamidase was tist discovered in rat liver by HOPSU-HAVUand GLENNER(1966). Since the amino acid sequence of Gly-Pro-X is present in the collagen molecule (it has been found in hydrolysates from achilles tendon of cattle by SCHROHENLOHER, OGLE and LOGAN 1959), glycyl-prolyl-naphthylamidase might be expected to have an important role in the degradation of collagen in the oral cavity. The high activity of arylaminopeptidases in human whole saliva was attributed to oral bacteria (MKKINEN,1966; OYA et al., 1968). If bacteria in carious plaque contain arylaminopeptidases such as glycyl-prolylnaphthylamidase, destruction of dentine organic matrix by proteolysis might be possible. The presence of arylaminopeptidase activity in human carious dentine was demonstrated histochemically (LARMAS,M~~KINENand SCHEININ,1968). They proposed the hypothesis that decalcification and proteolysis might occur simultaneously, and in the same region. TAKEIet al. (1968) and KOBAYASHI et al. (1969) showed that the predominant species of bacteria in the carious plaque changed from streptococci to lactobacilli during the course of the development of a carious cavity in dentine. Therefore, it seemed important to determine those bacterial species which possess high proteolytic enzyme activities. In order to examine the relations of bacterial arylaminopeptidases to the development of caries, we have examined the enzyme activities in several cariogenic and noncariogenic oral bacteria. 675
676
H. OYA, T. NAGATSU,Y.
KOBAYAWI
AND
M. TAKEI
Test organisms were Streptococcus mitis ATCC 9811, Streptococcus salivarius ATCC 9222, Streptococcus faecalis IID 10541, cariogenic streptococci (strains PK-1, HS-6, CG-5, LM-7, E-49), Lactobacillus acidophilus IAF 3205 and Luctobacillus casei ATCC 7469. Table 1 shows the source of cariogenic streptococci and what test animals were used. TABLE1. THE CARI~GENICs~~~~~ococc~ EXAMINED Species
Source
Test animals
References
Strep. HS-6
Hamster
Conventional
strep. Streu. Strep. Strep.
Hamster Human HulllaD Human
Conventional hamster and rat Gnotobiotic rat Gnotobiotic rat Gnotobiotic rat
E-49 LM-7 GS-5 PK-1
I
I
0
I 4
Cariogenic l
PITZGERALDand KWI%
hamster
wm
streptococcus
PITZGEXALD(1963) G~B~NS et al. (1966) GIBBONSet al. (1966) GIBBONSet 01. (1966)
PK-I
Cell numbers
0 Gly-Pro-NAase A Leu-NAase
I
8
I I2 Incubation
I 16 time,
I
20
I 24
hr
PIG. 1. Time course of the cell numbers, glycyl-prolyl-naphthylamidase (Gly-Pro-NAase) and leucyl-naphthylamidase (km-NAase) during the incubation of cariogenic streptococcus PK- 1.
ARYLAhiINOPEPTIDASE
ACTIVITIES
IN HUMAN
ORAL
BACTERIA
677
The growth culture for each test organism was brain heart infusion broth (Difco) which was inoculated with 5 x lo6 cells/ml and incubated aerobically for 24 hr. Two cultures were made, the second being started 12 hr after the first. For each strain, the growth of the culture was studied by removing aliquots at 0,2,4,6,8 and 12 hr from the first culture and at 12, 16, l&20 and 24 hr from the second culture and making viable plate counts on brain heart infusion agar (Difco) supplemented with 1 per cent glucose and 0.5 per cent yeast extract. Colonies were counted after 48 hr aerobic incubation at 37°C. Enzyme activity was measured using cells removed from the culture at various stages of growth. Ceils were harvested, washed and suspended in O-01 M tris-HCl buffer at pH 7 -0. Arylaminopeptidase activity was measured by using the substrates glycyl-cprolyl&naphthylamide or L-leucyl-@raphthylamide, as reported in our previous paper (OYA et al., 1968). The suspensions of washed bacteria in O-01 M tris-HCl buffer,
i
I
Streptococcus l
mitis
ATCC
9811
Cell numbers
0 Gly-Pro-NAose
1 I 4
A Leu-NAase
I 8
I 12
Incubation
I 16
time,
I 20
I 24
hr
2. Time course of the cell numbers, glycyl-prolyl-naphthybmklase (Gly-ProNAase) and leucyl-naphthylamidase (Leu-NAase) during the incubation of Strep. mitis ATCC 9811.
FIG.
H. OYA, T. NAGATSU, Y. KOBAYASHIAND M. TAKEI
678
pH 7.0 were used as the enzyme preparations. The incubation mixture contained 90 pmole tris-maleate buffer, pH 7.0, 0.45 pmole substrate, an appropriate amount of the enzyme and water to 0.90 ml. Incubation was carried out at 37°C for 30 min. Incubation was stopped by the addition of 0.3 ml of 10 per cent Tween-20 in 1 M acetate buffer, pH 4.2, containing O-45 mg of stabilised diazonium salt Fast Garnet GBC. After 30 min, absorbance at 530 rnp was measured. Enzyme activities are expressed as rnp-moles of /3-naphthylamine liberated/30 min/lOg cells. As shown in Fig. 1 (cariogenic streptococcus PK-1) and Fig. 2 (Strep. mitis ATCC 981 l), enzyme activity either toward glycyl-prolyl-naphthylamide or toward leucyl-naphthylamide was increased simultaneously with the increase in the cell numbers. Figures 1 and 2 show that cariogenic streptococcus PK-1 had lower arylaminopeptidase activities than non-cariogenic streptococcus ATCC 9811. The latter had very high glycyl-prolyl-naphthylamidase activity. The arylaminopeptidase activities of 5 cariogenic streptococci, and 3 non-cariogenie streptococci and 2 lactobacilli are shown in Figs. 3 and 4. As shown in Fig. 3,
HS-6
0
Leu-NAase
q
Gly-
LM-7
Pro-NAase
E-49
PK-I
GS-5
FIG. 3. Glycyl-prolyl-naphthylamidase (Gly-Pro-NAase) and leucyl-naphthylamidase (Leu-NAase) of cariogenic streptococci. Enzyme activities were measured at 8, 12, and 16 hr of the incubation.
ARYLAMrN0PJPnmsE
0
ATCC
I2
9811
0
Leu - NAase
El
Gly-Pro-NAase
I(
I6
ATCC
IN HUMAN ORAL BACTERIA
AcIlvITIEs
9222
fl 8
I2
16
IID
10541
8
I2
I FO 3205
I6
679
6
ATCC
12
7469
FIG. 4. Glycyl-prolyl-naphthykmkiase (Gly-Pro-NAase) and leucyl-naphthylamidase (Leu-NAase) of noncariogenic bacteria (Strep. mitis ATCC 9811, Strep. salivarks ATCC 9222, Strep. faecalis IID 10541, Lactobacillm acidophilus IF0 3205 and Lactobacillus casei ATCC 7419). Enzyme activities were measured at 8, 12, and 16 hr of the incubation.
all the cariogenic streptococci tested had low arylaminopeptidase activities. In contrast, among 3 non-cariogenic streptococci, Strep. mitis ATCC 9811 and Strep. salivarius ATCC 9222 had high arylamidase activity. Glycyl-prolyl-naphthylamidase activity was higher than leucyl-naphthylamidase activity in the streptococci (Fig. 3). Both Luctobacillus acidophilus IF0 3205 and Lacfobacillus casei ATCC 7469 had high arylaminopeptidase activities. Leucyl-naphthylamidase activity was higher than glycyl-prolyl-naphthylamidase activity in the lactobacilli (Fig. 4). These results suggest that in dentine caries the involvement of arylaminopeptidase activity in cariogenic streptococci is not significant, since the bacteria contain only low enzyme activity. However, arylaminopeptidase activity may become of importance for the later development of a dentinal carious cavity, since lactobacilli, which become
680
H. OYA,T. NAGATSU, Y. KOBAYASHI ANDM. TAKEI
predominant in the later stage of carious cavity formation (TAKEI et al., 1968; KOBAYASHI et al., 1969), contain high enzyme activity. It was found that the activities of the extracellular enzymes were low compared with the enzyme activities inside cells. Therefore, the production of extracellular enzymes from the bacteria is probably of little significance. Acknowledgements-The authors wish to thank Dr. S. SAKAKIBARA and Dr. K. TAKADA(Institute for Protein Research, Osaka University, Osaka) for the synthesis of glycyl-L-prolyl+-naphthylamide, and Miss Y. NISHIKAWAand Miss Y. SHIBAHARA for their valuable technical assistance. REFERENCES FITZGERALD,R. J. and KEYES, P. H. 1960. Demonstration of the etiologic role of streptococci in experimental caries in hamster. J. Am. dent. Ass. 61,9-31. FITZGEIULD, R. J. 1963. Gnotobiotic contribution to oral microbiology. J. dent. Res. 42, 548-552. B. 1966.Dental caries and alveolar GIBBONS, R. J., BE-, K. S., KNom, P. and m, bone loss in gnotobiotic rats infected with capsule-forming streptococci of human origin. Archs oralBio1. 11,549-560. Hopsu-HAW,V. K. and GLENNER, G. G. 1966. A new dipeptide naphthylamidase hydrolyzing glycyl-prolyl-j-naphthylamide. Histochemie 7, 197-201. KOBAYASHI, Y., KONDO, E., Fumnr, T. and TAKEI, M. 1969.Fundamental studies on the dynamic movement of oral microbes 10. Lactobacillus in the dental carious foci. Aichi-Gakuin J. Dent. Sci. 7, 27-30. LARMAS,M., M~~KINEN,K. K. and SCHEININ,A. 1968. Histochemical studies on the arylaminopeptidase activity in human carious dentine. Acta odont. stand. 26, 127-137. MKKINEN, K. K. 1966. Studies on oral enzymes I. Fractionation and characterization of aminopeptidases of human saliva. Acta odont. stand. 24,579-6&L NAOATSU,I., NAGATSU,T. and YAMAMOTO,T. 1968.Hydrolysis of amino acid fi-naphthylamides by aminopeptidases in human parotid saliva and human serum. Experientia 24, 347-348. OYA, H. and NAGATSU,T. 1969. Glycyl-L-prolyl-fi-naphthylamidase in human submaxillary gland. J. Jap. Biochem. Sot. 41,597-597. OYA, H., YAMAMOTO, T. and NAGATSU,T. 1968. Presence of leucine aminopeptidase activity in human saliva from the parotid gland and the submaxillary-subliil glands. Archs oral Biol. 12, 941-948. SCHROHENLOMR,R. E., OGLE, J. D. and LOGAN, M. A. 1959. Two tripeptides from an enzymatic digest of collagen. J. biol. Chem. 234, 58-61. TAKEI,M., KOBAYASHI,Y., KONW, E. and IWASAKI,S. 1968. Fundamental studies on the dynamic movement of oral microbes 4. Glucose-utilizing microbes in caries areas. Aichi-Gakuin J. dent. Sci. 5, 306-3 11.