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Comparative studies of putrescine degradation by microorganisms
59
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25 452 COMPARATIVE S T U D I E S OF P U T R E S C I N E D E G R A D A T I O N BY MICROORGANISMS R U T H M I C H A E L S AND K I - H A N
KIM
Department of Chemistry, Wayne State University, Detroit, Mich. (U.S.A.) (Received J u n e 28th, 1965)
SUMMARY
Eighteen organisms were surveyed for both diamine ~-ketoglutarate transaminase activity and diamine oxidase activity. Cell-free extracts and a spectrophotometric method were used. This method was based on the conversion of putrescine to 7-aminobutyraldehyde b y either enzyme. The amount of radioactive glutamic acid formed b y each bacterial extract exhibiting transaminase activity agreed with a stoichiometric formation of 7-aminobutyraldehyde calculated. Both enzymes were demonstrated in eight of the organisms tested. 4 organisms tested showed only transaminase activity and four showed only oxidase activity. In 5 organisms neither enzyme could be demonstrated. A high level of transaminase activity but no oxidase activity was found in both Escherichia coli and Aerobacter aerogenes which are two related species. Aside from this, the presence or absence of either enzyme did not bear a relationship to taxonomic groupings. The regulation of activity of these enzymes m a y depend on growth conditions.
INTRODUCTION
Studies of putrescine metabolism in plants and animals indicate that the degradation of putrescine to 7-aminobutyraldehyde is catalyzed by a diamine oxidase of broad specificity 1. This diamine oxidase has been obtained from both plant and animal sources and has been highly purified by several investigators2, a. Putrescine can also be degraded b y microorganisms. Among the microorganisms which have been reported to degrade putrescine are: Escherichia coli 4, Serratia marcescens 5, Corynebacterium pseudodiphtheriticum 5, Mycobacterium smegmatis 6 and several Pseudomonas species4,5, 7. Suspensions of whole cells and manometric methods were used primarily in these reports and it was assumed that the microbial degradation of putrescine was also catalyzed b y a diamine oxidase. In 1962, KIM AND TCtIEN s, using cell-free extracts of a m u t a n t of E. coli B and a spectrophotometric method based on the coupling reaction of Al-pyrroline with o-aminobenzaldehyde 9, reported that the conversion of putrescine to 7-aminobutyraldehyde was catalyzed b y a diamine e-ketoglutarate transaminase and not b y a diamine oxidase. This enzyme was subsequently purified and some of its properties reported 10. The demonstration of the presence of two enzymes which convert putrescine to Biochim. Biophys. Acta, 115 (1966) 5 9 - 6 4
60
R. MICHAELS, K.-H. KIM
~,-aminobutyraldehyde and the uncertainty of the interpretation of whole cell experiments led us to survey the distribution of these enzymes in various microorganisms. The results are reported in this paper. MATERIALS AND METHODS
a-[5-14C~Ketoglutaric acid (sodium salt, specific activity 6.93 mC/mmole) was purchased from Nuclear Chicago Corporation. Nonradioactive putrescine and o-aminobenzaldehyde were purchased from K and K Laboratories. All other chemicals used were from common commercial sources. C. pseudodiphtheriticum (ATTC 1o7o0 ), A erobacter aerogenes (Enterobacter aerogenes ATTC I3O48 ), Hem@hilus parainfluemae (ATTC 79oi), M. smegmatis (ATTC 10143 ) and Pseudomonas fluorescens (ATTC 3430) were obtained from the American Type Culture Collection. Chromobacterium violaceum strain No. 9 was supplied by Dr. W. A. CORPE and Phytomonas fascians was supplied by Dr. R. MENASSE. Gelasinospora calospora and Sordaria fimicola were received from Dr. J. MANIOTIS.The Pseudomonas species was isolated in our laboratory. All other cultures were obtained from the stock culture collection of the Microbiology Division, Department of Biology, Wayne State University. The Aerobacter, Enterobacter, Chromobacterium, Serratia and Pseudomonas species were grown in a medium containing 2 g putrescine (neutralized with HC1) per liter of distilled water and a mixture of inorganic salts according to KIM1° (P medium). Chromobacterium was also grown on P medium with the addition of 2 g glucose per liter (PG medium). Phytomonas was grown on PG medium with the addition of o.i g yeast extract per liter. Sordaria and Gelasinospora were grown on PG medium containing I g of KNOs and o.I g yeast extract per liter. Mycobacterium was grown on YOUMANS' AND KARLSON'S11 medium with the addition of 2 g putrescine per liter. Hemophilus was grown on Difco Brain Heart Infusion medium supplemented with 5% Difco Bacto Supplement B and 2 g of putrescine per liter. The other organisms were grown on the "adaptation medium" described b y RAZlN et al. 5. The medium contained mineral salts, o.I % yeast extract and 0.3% putrescine. All cultures were grown with aeration at room temperature and were transferred to their respective media IO times. The cells were harvested by centrifugation near the end of the log phase of growth and washed with 0.8% (w/v) saline solution. Cell-free extracts were prepared from freshly harvested or frozen cells by suspending the cells in 4 parts of o.I M Tris-HC1 buffer (pH 8.5) and then treating them for 20 min in a Raytheon io kcyclesfsec sonic oscillator. Cell debris was removed by centrifugation at IO ooo × g for 20 min. The extracts were then dialyzed against eight liters of o.ooi M Tris buffer (pH 8.5) for 24 h with two changes of buffer solution during that period. The extracts were either immediately assayed or frozen and assayed within one week. The pea seedling extract was prepared from eight day old etiolated seedlings and subjected to ammonium sulfate fractionation according to the procedure of HASSE AND SCHMIDT 12.
Diamine ~-ketoglutarate transaminase was assayed according to the procedure of KIM1°. The amounts of ~,-aminobutyraldehyde obtained b y the action of either putrescine transaminase or diamine oxidase were determined b y coupling with Biochim. Biophys. Acta, 1I 5 (1966) 59-64
PUTRESCINE
D E G R A D A T I O N BY MICROORGANISMS
61
o-aminobenzaldehyde and measuring the absorption at 435 m/*. When the transaminase system: was used, the incubation mixture contained in a total volume of 0,8 ml: IOO/~moles of Tris-HC1 buffer (pH 9.o), 5/*moles of putrescine; 5/*moles of a-ketoglutarate; o.I/*mole of pyridoxal phosphate; 2/,moles of o-aminobenzaldehyde and o.2 ml of cell extract.To test for the oxidase system; c~-ketoglutarate was omitted from the reaction mixture. The reaction was stopped after I h by the addition of 0.2 ml of lO% trichloroacetic acid. The absorption at 435 m/, was measured 4 h later. An increase in absorbance obtained with the "transaminase system" beyond that obtained with the oxidase system was taken as partial evidence of the presence of putrescine transaminase. Radioactive glutamic acid was isolated from the reaction mixture containing labeled a-ketoglutarate. The deproteinized reaction mixture was passed through a Dowex-5o W-X4(H+ ) column (0.5 c m x IO cm). IO ml of deionized water were used to remove the unreacted a-ketoglutarate from the column. The glutamic acid formed b y the transaminase was then eluted from the column with successive portions of 5 ml of I N HC1 and 5 ml of 2 N HC1. The two eluates were combined. The radioactivity of an aliquot of the eluate was measured using a Packard liquid scintillation counter. The remainder of the eluate was then evaporated to dryness, suspended in o.I ml of distilled water and chromatographed on W h a t m a n No. I paper in phenolwater solvent (80:20, v/v). The paper strips were scanned using a Vanguard model 880 autoscanner. RESULTS
The organisms surveyed for both diamine a-ketoglutarate transarninase activity and diamine oxidase activity are listed in Table I. The three Pseudomonas species grew well without any prolonged lag period after transfer from nutrient agar slants to a medium containing putrescine as sole carbon and nitrogen source. Serratia and the two Aerobacter species were also transferred to P medium from nutrient agar slants; however, a period of two to four days was required before any appreciable growth could be observed. After successive transfers to fresh P medium, these organisms grew without any noticeable lag. When PG medium was used instead, growth was observed immediately after the first transfer. Chromobacterium grew slowly on P medium and somewhat less slowly on PG medium. The rate of growth did not increase after successive transfers. Phytomonas required the addition of glucose and o.o: % yeast extract. Some organisms did not grow on PG medium to which the afore mentioned quantity of yeast extract had been added. They were, therefore, grown on "adaptation medium" or the other media described in the METrIODSsection. Table I shows that 4 of the :8 species tested exhibited both transaminase and oxidase activity. The oxidase activity was low compared to the transaminase activity. Four organisms exhibited only transaminase activity and one organism exhibited only oxidase activity. Neither enzyme was found in the remaining five organisms tested. Three of the cell-free extracts (Chromobacterium, Phytomonas and p e a seedling) which showed strong oxidase activity, showed not an increase, but ~_ decrease in absorbance when the transaminase system was employed. This disparity was not investigated further, but it is probable that all three organisms possess a Biochim. Biophys. Acta, i x 5 (z966) 5 9 - 6 4
62
R. MICHAELS, K.-H. KIM
TABLE I DISTRIBUTION DIFFERENT
OF
DIAMINE
~-KETOGLUTARATE
TRANSAMINASIg
AND
DIAMINE
OXIDASE
AMONG
SPECIES
Absorbance (435 m/*)
Enzyme(s) present
" Transaminase system"
" Oxidase system"
Transaminase
Oxidase
Baci!lus cereus A erobacter (Enterobacler) aerogenes 21/Iycobacterium smegmatis Pseudomonas aeruginosa Pseudomonas fluorescens Chromobacterium violaceum
o. 145 0.43o o.19o o.15o 0.077
0.035 0.078 0.040 0.030 0.032
+ + + + +
+ + + + +
grown on P medium grown on PG medium Pea seedling
0.455 0.080 0.500 0.500
o.640 0.030 0.730 0.730
probably + + probably + probably +
+ + + +
0.325 o. 120 0.265 0.055
o.oo o.oo o.oo o.oo
+ + + +
o.oo
o.o 4
o.oo
o.oo
o.oo o.oo o.oo o.oo
o.oo o.oo o.oo o.oo
Organism
Group i
Phylomonas fascians
Group 2 A erobacler aerogenes Pseudomonas species Serralia marcescens Staphylococcus aureus
Group 3 Hemophilus parain[tuenzae
+
Group 4 A lkaligenes faecalis Corynebacterium pseudodiphtheriticum Gelasinospora calospora Proteus morganii Sordaria fimicola
t r a n s a m i n a s e in addition to an oxidase as i n d i c a t e d b y t h e f o r m a t i o n of g l u t a m i c acid to be discussed later. T ab l e I I shows the a m o u n t s of g l u t a m i c acid o b t a i n e d from t h e e x t r a c t s b y e m p l o y i n g the " t r a n s a m i n a s e s y s t e m " . These cell-free e x t r a c t s had been e x h a u s t i v e l y dialyzed to r e m o v e cofactors such as D P N and T P N , and the g l u t a m a t e found could n o t be a t t r i b u t e d to the presence of g l u t a m i c d e h y d r o g e n a s e act i v i t y . In these e x p e r i m e n t s , labeled ~ - k e t o g l u t a r a t e was a d d e d to the i n c u b a t i o n m i x t u r e an d t h e l a b el ed g l u t a m a t e f o r m e d b y the t r a n s a m i n a s e was eluted as described in the METHODS. W h e n the c h r o m a t o g r a m s of th e eluates were scanned, only one peak w i t h the same RF as t h a t o b t a i n e d with s t a n d a r d g l u t a m a t e was found. Table I I also shows t h a t the a m o u n t of r a d i o a c t i v e g l u t a m i c acid formed b y each bacterial e x t r a c t agreed with a stoichiometric f o r m a t i o n of y - a m i n o b u t y r a l d e h y d e calculated. A small a m o u n t of label was found in the column eluates of the reaction m i x t u r e s with e x t r a c t s from P r o t e u s and Hemophilus. H o w e v e r , no r a d i o a c t i v e peak on p a p e r c h r o m a t o g r a m s was o b t a i n e d and this r a d i o a c t i v i t y is p r o b a b l y n o t g l u t a m i c acid. Biochim. Biophys. Acta, 115 (1966) 59-64
PUTRESCINE TABLE
63
D E G R A D A T I O N BY M I C R O O R G A N I S M S
II
FORMATION OF GLUTAMIC ACID AND ~]-AMINOBUTYRALDEHYD]~
Organism
t~moles of glutamic acid
lzmoles of T-aminobutyraldehyde
A erobacter aerogenes Bacillus cereus Chromobacterium violaceum
1.o35 0.o6
i.oi 0.05
o.05
0.04
g r o w n on P G m e d i u m
Chromobacterium violaceum grown on P medium
Hemophilus parainfluenzae Mycobacterium smegmatis Pea seedling
Phytomonas fascians Proteus morganii Pseudomonas fluorescens Pseudomonas species Serratia marcescens Staphylococcus aureus
0.05 o.o9 o.o2 o. 14
o.o 7
o.o 3 o.o6
o.o3 o.o6
o. 14
o. 13
0-03
0.03
I t is, therefore, concluded that Proteus has neither enzyme and Hemophilus has only diamine oxidase. Appreciable quantities of labeled glutamic acid were found in Chromobacterium (grown on P medium), pea seedling and Phytomonas eluates, although no increase in absorbance was detected when the "transaminase system" was employed. The presence of glutamic acid implies that transaminase was probably present, in spite of the fact that no increase in absorbance could be observed. DISCUSSION
The presence of diamine transaminase and/or diamine oxidase in the majority of organisms tested was demonstrated b y the use of cell-free extracts. Table I shows that the eighteen organisms tested could be divided into four groups. In Group I, both diamine transminase and diamine oxidase were demonstrated. In Group 2 only transaminase was demonstrated. Group 3 exhibited only oxidase and the organisms in Group 4 exhibited neither enzyme. The bacteria within these four groups do not seem to have m a n y other physiological characteristics in common, nor do they fall into established taxonomic groups. The organisms in Group 4 required either PG medium with the addition of nitrate (Gelasinospora, Sordaria) or "adaptation medium", which contained o.1% yeast extract in addition to putrescine. It seems quite likely that the constituents of the yeast extract also served as carbon and nitrogen sources here and that the organisms in this group did not degrade putrescine in spite of repeated subculture. A high level of transaminase activity was found in both A. aerogenes strains which are known to be closely related to E. coli in which a high level of transaminase had previously been found 8. More organisms exhibited transaminase activity than oxidase activity and eight of the eighteen species tested exhibited both transaminase and oxidase activity. The presence of both enzymes in cell-flee extracts Biochim. Biophys. Acta, 115 (1966) 5 9 - 6 4
64
R. MICJ-IAELS, K.-H. KIM
of these microorganisms is reminiscent of the recent report of HASSE AND SCHMIDT 12 showing the presence of both enzymes in pea seedlings. The regulation of activity of these enzymes is not understood but m a y depend on the conditions of growth. Preliminary experiments with Chromobacterium show that although transaminase activity remained constant, different levels of oxidase activity were found when the organism was grown in media containing only putrescine or glucose and putrescine. It is clear that the influence of various factors such as cultural conditions or age of the cells on these enzymes will have to he examined critically in order to understand the regulation of their synthesis. ACKNOWLEDGEMENTS
We wish to express our appreciation to Dr. T. T. TCHEN for helpful discussions. This work was supported by United States Public Health Service grants (GM 12648 ) and (AM o5384). REFERENCES I E. A. ZELLER, in P. D. BOYER, H. LARDY AND I(. MYRB'/,CK, The Enzymes, Academic Press, New York and London, 1963, p. 313 . 2 P. J. G. MANN, Biochem. J., 59 (x955) 609. 3 H. TABOR,J. Biol. Chem., 188 (1951) 125. 4 E, F. GALLS, Bioehem. J., 36 (1942) 64. 5 S. RAZlN, l. GERY AND U. BACHRACH, Biochem. J., 71 (1959) 551. 6 U. BACHRACH, S. PERSKY AND S. RAZIN, Bioehem. J., 76 (196o) 306. 7 W. B. JAKOBY AND J. FREDERICI~S, J. Biol. Chem., 234 (1959) 2145. 8 K. KIM AND T. T. TCHEN, Bioehem. Biophys. Res. Commun., 9 (1962) 99. 9 13. HOLMSTEDT, L. LANSSON AND R. THAM, Biochim. Biophys. Acta, 48 (1961) 182. io K. KI~, J. Biol. Chem., 239 (1964) 783 . i i G. P. YOUMANS AND A. G. KARLSON, Am. Rev. Tubere., 55 (1947) 529 . 12 K, HASSE AND G. SCHMIDT, Biochem. Z., 337 (1963) 69.
Bioehim. Biophys. Aeta, 115 (1966) 59-64
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25 452 COMPARATIVE S T U D I E S OF P U T R E S C I N E D E G R A D A T I O N BY MICROORGANISMS R U T H M I C H A E L S AND K I - H A N
KIM
Department of Chemistry, Wayne State University, Detroit, Mich. (U.S.A.) (Received J u n e 28th, 1965)
SUMMARY
Eighteen organisms were surveyed for both diamine ~-ketoglutarate transaminase activity and diamine oxidase activity. Cell-free extracts and a spectrophotometric method were used. This method was based on the conversion of putrescine to 7-aminobutyraldehyde b y either enzyme. The amount of radioactive glutamic acid formed b y each bacterial extract exhibiting transaminase activity agreed with a stoichiometric formation of 7-aminobutyraldehyde calculated. Both enzymes were demonstrated in eight of the organisms tested. 4 organisms tested showed only transaminase activity and four showed only oxidase activity. In 5 organisms neither enzyme could be demonstrated. A high level of transaminase activity but no oxidase activity was found in both Escherichia coli and Aerobacter aerogenes which are two related species. Aside from this, the presence or absence of either enzyme did not bear a relationship to taxonomic groupings. The regulation of activity of these enzymes m a y depend on growth conditions.
INTRODUCTION
Studies of putrescine metabolism in plants and animals indicate that the degradation of putrescine to 7-aminobutyraldehyde is catalyzed by a diamine oxidase of broad specificity 1. This diamine oxidase has been obtained from both plant and animal sources and has been highly purified by several investigators2, a. Putrescine can also be degraded b y microorganisms. Among the microorganisms which have been reported to degrade putrescine are: Escherichia coli 4, Serratia marcescens 5, Corynebacterium pseudodiphtheriticum 5, Mycobacterium smegmatis 6 and several Pseudomonas species4,5, 7. Suspensions of whole cells and manometric methods were used primarily in these reports and it was assumed that the microbial degradation of putrescine was also catalyzed b y a diamine oxidase. In 1962, KIM AND TCtIEN s, using cell-free extracts of a m u t a n t of E. coli B and a spectrophotometric method based on the coupling reaction of Al-pyrroline with o-aminobenzaldehyde 9, reported that the conversion of putrescine to 7-aminobutyraldehyde was catalyzed b y a diamine e-ketoglutarate transaminase and not b y a diamine oxidase. This enzyme was subsequently purified and some of its properties reported 10. The demonstration of the presence of two enzymes which convert putrescine to Biochim. Biophys. Acta, 115 (1966) 5 9 - 6 4
60
R. MICHAELS, K.-H. KIM
~,-aminobutyraldehyde and the uncertainty of the interpretation of whole cell experiments led us to survey the distribution of these enzymes in various microorganisms. The results are reported in this paper. MATERIALS AND METHODS
a-[5-14C~Ketoglutaric acid (sodium salt, specific activity 6.93 mC/mmole) was purchased from Nuclear Chicago Corporation. Nonradioactive putrescine and o-aminobenzaldehyde were purchased from K and K Laboratories. All other chemicals used were from common commercial sources. C. pseudodiphtheriticum (ATTC 1o7o0 ), A erobacter aerogenes (Enterobacter aerogenes ATTC I3O48 ), Hem@hilus parainfluemae (ATTC 79oi), M. smegmatis (ATTC 10143 ) and Pseudomonas fluorescens (ATTC 3430) were obtained from the American Type Culture Collection. Chromobacterium violaceum strain No. 9 was supplied by Dr. W. A. CORPE and Phytomonas fascians was supplied by Dr. R. MENASSE. Gelasinospora calospora and Sordaria fimicola were received from Dr. J. MANIOTIS.The Pseudomonas species was isolated in our laboratory. All other cultures were obtained from the stock culture collection of the Microbiology Division, Department of Biology, Wayne State University. The Aerobacter, Enterobacter, Chromobacterium, Serratia and Pseudomonas species were grown in a medium containing 2 g putrescine (neutralized with HC1) per liter of distilled water and a mixture of inorganic salts according to KIM1° (P medium). Chromobacterium was also grown on P medium with the addition of 2 g glucose per liter (PG medium). Phytomonas was grown on PG medium with the addition of o.i g yeast extract per liter. Sordaria and Gelasinospora were grown on PG medium containing I g of KNOs and o.I g yeast extract per liter. Mycobacterium was grown on YOUMANS' AND KARLSON'S11 medium with the addition of 2 g putrescine per liter. Hemophilus was grown on Difco Brain Heart Infusion medium supplemented with 5% Difco Bacto Supplement B and 2 g of putrescine per liter. The other organisms were grown on the "adaptation medium" described b y RAZlN et al. 5. The medium contained mineral salts, o.I % yeast extract and 0.3% putrescine. All cultures were grown with aeration at room temperature and were transferred to their respective media IO times. The cells were harvested by centrifugation near the end of the log phase of growth and washed with 0.8% (w/v) saline solution. Cell-free extracts were prepared from freshly harvested or frozen cells by suspending the cells in 4 parts of o.I M Tris-HC1 buffer (pH 8.5) and then treating them for 20 min in a Raytheon io kcyclesfsec sonic oscillator. Cell debris was removed by centrifugation at IO ooo × g for 20 min. The extracts were then dialyzed against eight liters of o.ooi M Tris buffer (pH 8.5) for 24 h with two changes of buffer solution during that period. The extracts were either immediately assayed or frozen and assayed within one week. The pea seedling extract was prepared from eight day old etiolated seedlings and subjected to ammonium sulfate fractionation according to the procedure of HASSE AND SCHMIDT 12.
Diamine ~-ketoglutarate transaminase was assayed according to the procedure of KIM1°. The amounts of ~,-aminobutyraldehyde obtained b y the action of either putrescine transaminase or diamine oxidase were determined b y coupling with Biochim. Biophys. Acta, 1I 5 (1966) 59-64
PUTRESCINE
D E G R A D A T I O N BY MICROORGANISMS
61
o-aminobenzaldehyde and measuring the absorption at 435 m/*. When the transaminase system: was used, the incubation mixture contained in a total volume of 0,8 ml: IOO/~moles of Tris-HC1 buffer (pH 9.o), 5/*moles of putrescine; 5/*moles of a-ketoglutarate; o.I/*mole of pyridoxal phosphate; 2/,moles of o-aminobenzaldehyde and o.2 ml of cell extract.To test for the oxidase system; c~-ketoglutarate was omitted from the reaction mixture. The reaction was stopped after I h by the addition of 0.2 ml of lO% trichloroacetic acid. The absorption at 435 m/, was measured 4 h later. An increase in absorbance obtained with the "transaminase system" beyond that obtained with the oxidase system was taken as partial evidence of the presence of putrescine transaminase. Radioactive glutamic acid was isolated from the reaction mixture containing labeled a-ketoglutarate. The deproteinized reaction mixture was passed through a Dowex-5o W-X4(H+ ) column (0.5 c m x IO cm). IO ml of deionized water were used to remove the unreacted a-ketoglutarate from the column. The glutamic acid formed b y the transaminase was then eluted from the column with successive portions of 5 ml of I N HC1 and 5 ml of 2 N HC1. The two eluates were combined. The radioactivity of an aliquot of the eluate was measured using a Packard liquid scintillation counter. The remainder of the eluate was then evaporated to dryness, suspended in o.I ml of distilled water and chromatographed on W h a t m a n No. I paper in phenolwater solvent (80:20, v/v). The paper strips were scanned using a Vanguard model 880 autoscanner. RESULTS
The organisms surveyed for both diamine a-ketoglutarate transarninase activity and diamine oxidase activity are listed in Table I. The three Pseudomonas species grew well without any prolonged lag period after transfer from nutrient agar slants to a medium containing putrescine as sole carbon and nitrogen source. Serratia and the two Aerobacter species were also transferred to P medium from nutrient agar slants; however, a period of two to four days was required before any appreciable growth could be observed. After successive transfers to fresh P medium, these organisms grew without any noticeable lag. When PG medium was used instead, growth was observed immediately after the first transfer. Chromobacterium grew slowly on P medium and somewhat less slowly on PG medium. The rate of growth did not increase after successive transfers. Phytomonas required the addition of glucose and o.o: % yeast extract. Some organisms did not grow on PG medium to which the afore mentioned quantity of yeast extract had been added. They were, therefore, grown on "adaptation medium" or the other media described in the METrIODSsection. Table I shows that 4 of the :8 species tested exhibited both transaminase and oxidase activity. The oxidase activity was low compared to the transaminase activity. Four organisms exhibited only transaminase activity and one organism exhibited only oxidase activity. Neither enzyme was found in the remaining five organisms tested. Three of the cell-free extracts (Chromobacterium, Phytomonas and p e a seedling) which showed strong oxidase activity, showed not an increase, but ~_ decrease in absorbance when the transaminase system was employed. This disparity was not investigated further, but it is probable that all three organisms possess a Biochim. Biophys. Acta, i x 5 (z966) 5 9 - 6 4
62
R. MICHAELS, K.-H. KIM
TABLE I DISTRIBUTION DIFFERENT
OF
DIAMINE
~-KETOGLUTARATE
TRANSAMINASIg
AND
DIAMINE
OXIDASE
AMONG
SPECIES
Absorbance (435 m/*)
Enzyme(s) present
" Transaminase system"
" Oxidase system"
Transaminase
Oxidase
Baci!lus cereus A erobacter (Enterobacler) aerogenes 21/Iycobacterium smegmatis Pseudomonas aeruginosa Pseudomonas fluorescens Chromobacterium violaceum
o. 145 0.43o o.19o o.15o 0.077
0.035 0.078 0.040 0.030 0.032
+ + + + +
+ + + + +
grown on P medium grown on PG medium Pea seedling
0.455 0.080 0.500 0.500
o.640 0.030 0.730 0.730
probably + + probably + probably +
+ + + +
0.325 o. 120 0.265 0.055
o.oo o.oo o.oo o.oo
+ + + +
o.oo
o.o 4
o.oo
o.oo
o.oo o.oo o.oo o.oo
o.oo o.oo o.oo o.oo
Organism
Group i
Phylomonas fascians
Group 2 A erobacler aerogenes Pseudomonas species Serralia marcescens Staphylococcus aureus
Group 3 Hemophilus parain[tuenzae
+
Group 4 A lkaligenes faecalis Corynebacterium pseudodiphtheriticum Gelasinospora calospora Proteus morganii Sordaria fimicola
t r a n s a m i n a s e in addition to an oxidase as i n d i c a t e d b y t h e f o r m a t i o n of g l u t a m i c acid to be discussed later. T ab l e I I shows the a m o u n t s of g l u t a m i c acid o b t a i n e d from t h e e x t r a c t s b y e m p l o y i n g the " t r a n s a m i n a s e s y s t e m " . These cell-free e x t r a c t s had been e x h a u s t i v e l y dialyzed to r e m o v e cofactors such as D P N and T P N , and the g l u t a m a t e found could n o t be a t t r i b u t e d to the presence of g l u t a m i c d e h y d r o g e n a s e act i v i t y . In these e x p e r i m e n t s , labeled ~ - k e t o g l u t a r a t e was a d d e d to the i n c u b a t i o n m i x t u r e an d t h e l a b el ed g l u t a m a t e f o r m e d b y the t r a n s a m i n a s e was eluted as described in the METHODS. W h e n the c h r o m a t o g r a m s of th e eluates were scanned, only one peak w i t h the same RF as t h a t o b t a i n e d with s t a n d a r d g l u t a m a t e was found. Table I I also shows t h a t the a m o u n t of r a d i o a c t i v e g l u t a m i c acid formed b y each bacterial e x t r a c t agreed with a stoichiometric f o r m a t i o n of y - a m i n o b u t y r a l d e h y d e calculated. A small a m o u n t of label was found in the column eluates of the reaction m i x t u r e s with e x t r a c t s from P r o t e u s and Hemophilus. H o w e v e r , no r a d i o a c t i v e peak on p a p e r c h r o m a t o g r a m s was o b t a i n e d and this r a d i o a c t i v i t y is p r o b a b l y n o t g l u t a m i c acid. Biochim. Biophys. Acta, 115 (1966) 59-64
PUTRESCINE TABLE
63
D E G R A D A T I O N BY M I C R O O R G A N I S M S
II
FORMATION OF GLUTAMIC ACID AND ~]-AMINOBUTYRALDEHYD]~
Organism
t~moles of glutamic acid
lzmoles of T-aminobutyraldehyde
A erobacter aerogenes Bacillus cereus Chromobacterium violaceum
1.o35 0.o6
i.oi 0.05
o.05
0.04
g r o w n on P G m e d i u m
Chromobacterium violaceum grown on P medium
Hemophilus parainfluenzae Mycobacterium smegmatis Pea seedling
Phytomonas fascians Proteus morganii Pseudomonas fluorescens Pseudomonas species Serratia marcescens Staphylococcus aureus
0.05 o.o9 o.o2 o. 14
o.o 7
o.o 3 o.o6
o.o3 o.o6
o. 14
o. 13
0-03
0.03
I t is, therefore, concluded that Proteus has neither enzyme and Hemophilus has only diamine oxidase. Appreciable quantities of labeled glutamic acid were found in Chromobacterium (grown on P medium), pea seedling and Phytomonas eluates, although no increase in absorbance was detected when the "transaminase system" was employed. The presence of glutamic acid implies that transaminase was probably present, in spite of the fact that no increase in absorbance could be observed. DISCUSSION
The presence of diamine transaminase and/or diamine oxidase in the majority of organisms tested was demonstrated b y the use of cell-free extracts. Table I shows that the eighteen organisms tested could be divided into four groups. In Group I, both diamine transminase and diamine oxidase were demonstrated. In Group 2 only transaminase was demonstrated. Group 3 exhibited only oxidase and the organisms in Group 4 exhibited neither enzyme. The bacteria within these four groups do not seem to have m a n y other physiological characteristics in common, nor do they fall into established taxonomic groups. The organisms in Group 4 required either PG medium with the addition of nitrate (Gelasinospora, Sordaria) or "adaptation medium", which contained o.1% yeast extract in addition to putrescine. It seems quite likely that the constituents of the yeast extract also served as carbon and nitrogen sources here and that the organisms in this group did not degrade putrescine in spite of repeated subculture. A high level of transaminase activity was found in both A. aerogenes strains which are known to be closely related to E. coli in which a high level of transaminase had previously been found 8. More organisms exhibited transaminase activity than oxidase activity and eight of the eighteen species tested exhibited both transaminase and oxidase activity. The presence of both enzymes in cell-flee extracts Biochim. Biophys. Acta, 115 (1966) 5 9 - 6 4
64
R. MICJ-IAELS, K.-H. KIM
of these microorganisms is reminiscent of the recent report of HASSE AND SCHMIDT 12 showing the presence of both enzymes in pea seedlings. The regulation of activity of these enzymes is not understood but m a y depend on the conditions of growth. Preliminary experiments with Chromobacterium show that although transaminase activity remained constant, different levels of oxidase activity were found when the organism was grown in media containing only putrescine or glucose and putrescine. It is clear that the influence of various factors such as cultural conditions or age of the cells on these enzymes will have to he examined critically in order to understand the regulation of their synthesis. ACKNOWLEDGEMENTS
We wish to express our appreciation to Dr. T. T. TCHEN for helpful discussions. This work was supported by United States Public Health Service grants (GM 12648 ) and (AM o5384). REFERENCES I E. A. ZELLER, in P. D. BOYER, H. LARDY AND I(. MYRB'/,CK, The Enzymes, Academic Press, New York and London, 1963, p. 313 . 2 P. J. G. MANN, Biochem. J., 59 (x955) 609. 3 H. TABOR,J. Biol. Chem., 188 (1951) 125. 4 E, F. GALLS, Bioehem. J., 36 (1942) 64. 5 S. RAZlN, l. GERY AND U. BACHRACH, Biochem. J., 71 (1959) 551. 6 U. BACHRACH, S. PERSKY AND S. RAZIN, Bioehem. J., 76 (196o) 306. 7 W. B. JAKOBY AND J. FREDERICI~S, J. Biol. Chem., 234 (1959) 2145. 8 K. KIM AND T. T. TCHEN, Bioehem. Biophys. Res. Commun., 9 (1962) 99. 9 13. HOLMSTEDT, L. LANSSON AND R. THAM, Biochim. Biophys. Acta, 48 (1961) 182. io K. KI~, J. Biol. Chem., 239 (1964) 783 . i i G. P. YOUMANS AND A. G. KARLSON, Am. Rev. Tubere., 55 (1947) 529 . 12 K, HASSE AND G. SCHMIDT, Biochem. Z., 337 (1963) 69.
Bioehim. Biophys. Aeta, 115 (1966) 59-64