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Factors affecting urea inactivation of NADP-specificβ-isocitrate dehydrogenase
722
SHORT COMMUNICATIONS
BBA 63416 Factors affecting urea inactivation of NADP-specific f3-isocitrate dehydrogenase Several electrophoretically distinct forms of NADP-specific isocitrate dehydrogenase (threo-Ds-isocitrate:NADP + oxidoreductase (decarboxylating), EC 1.1.I.42) have been reported to occur in Escherichia coli grown under different culture conditions 1. Since E. coli as well as most other bacterial species 2 contain only the NADPspecific form of this enzyme, it has been suggested 3 that the regulation of isocitrate metabolism may be controlled by multiple forms (isoenzymes) of this key enzyme under different growth conditions. The a- and fl-forms of NADP-specific isocitrate dehydrogenase, which occur in glucose-grown E. coli, have been separated and shown to exhibit marked differences with respect to urea inactivation 3. A similar differential susceptibility to urea has been observed with the L D H - I and LDH-5 isoenzymes of lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) 4-6. Although the phenomenon is not understood, the observation has been the basis of methods for determining lactate dehydrogenase patterns in serum 7,8. The present studies provide an insight into the inactivation of the fl-form of NADP-specific isocitrate dehydrogenase in 2.0 M urea. E. coli was grown aerobically in a simI~le mineral-salts medium containing o.2% glucose as carbon source and cell-free extracts prepared as previously described 9. The fl-isocitrate dehydrogenase was isolated employing DEAE- and CM-Sephadex column chromatography as reported previously ~. For enzyme assays, the change in absorbance at 34 ° hi# was followed using a Cary 14 spectrophotometer. Protein concentrations were determined by measuring the absorbance at 215 and 225 m/~ in a Beckman DU spectrophotometer according to the method described by MURPHY AND KIES 1°. 0. 5 ml of 4.0 M urea freshly prepared in o.oi M phosphate buffer (pH 7.5) was added to 0.5 ml of the same buffer containing fl-isoeitrate dehydrogenase and the solution placed in an ice bath at o °. Immediately after mixing, and at intervals thereafter, aliquots were assayed for enzymatic activity. The results are shown in Fig. I and are expressed as percent of activity in the control remaining at the times indicated. Under these conditions, 60% of the initial enzyme activity was lost after IO rain and only 5°/0 remained after I h. The effectiveness of NADP and dithiothreitol in protecting fl-isocitrate dehydrogenase against inactivation in 2.0 M urea is shown in Fig. 2. A solution of the enzyme in o.oi M phosphate buffer (pH 7.5) containing varying amounts of either NADP, dithiothreitol or a mixture of these was incubated in an ice bath at o ° for 30 min. An equal volume of 4.0 M urea was then added to each tube and incubation continued for I h at o °. Aliquots were then removed and assayed for enzymatic activity. Appropriate controls containing no urea were also included. Both dithiothreitol and NADP alone afford partial protection to the enzyme against inactivation in 2.0 M urea. In the presence of either 3.27 mM NADP or lO5 mM dithiothreitol, the enzyme retained 45-50% of the initial activity after I h at o ° in 2.0 M urea. In the absence of either NADP or dithiothreitol, greater than Biochim. Biophys. Acta, 191 (1969)-722-724
723
SHORT COMMUNICATIONS
80
100 ~-•
•
~"N
X,,, No Urea
60
80 = "G
60 / o
f
Oithiothreitol
40 20
o;
1'o
°~o........,-~ 2.0M Urea ~ °'-----~o,
2'0
3'0
;o
Time ( minutes )
5'o
6o
o~ u
0
2'1 0.654
4'2
6'3
1.31
1.96
8'. 2.62
105 mM DTT 3.27 mM NADP
Fig. I. Inactivation of/5-isocitrate dehydrogenase at o° in 2 M urea. The assay mixture contained (in/~moles) : potassium phosphate buffer (pH 7.5), 15o; MnCI,, 0.5; NADP, 0.5, and DL-isocitrate (potassium salt), 2. 5. The protein concentration was 6.0/~g and the total volume was i.o ml. Fig. 2. Protection of fl-isocitrate dehydrogenase against inactivation in 2 M urea by NADP and dithiothreitol (DTT). Assay conditions the same as described in Fig. i.
9 5 % of t h e initial c a t a l y t i c a c t i v i t y was lost u n d e r t h e same conditions. A t lower concentrations, t h e p r o t e c t i v e effect afforded b y either N A D P or d i t h i o t h r e i t o l decreased. I n t h e presence of b o t h N A D P a n d d i t h i o t h r e i t o l , the e n z y m e could be comp l e t e l y p r o t e c t e d a g a i n s t i n a c t i v a t i o n in 2.0 M urea. As shown in Fig. 2, preincub a t i o n of fi-isocitrate d e h y d r o g e n a s e w i t h b o t h N A D P a n d d i t h i o t h r e i t o l at a concent r a t i o n of each which alone afforded 4 0 - 4 5 % protection, r e s u l t e d in c o m p l e t e prot e c t i o n of the enzyme. A t lower c o n c e n t r a t i o n s also, a m i x t u r e of b o t h N A D P a n d d i t h i o t h r e i t o l was f o u n d to be more effective t h a n either c o m p o u n d alone in affording protection. fl-Isocitrate d e h y d r o g e n a s e in t h e presence of 2.0 M u r e a for I h loses g r e a t e r t h a n 9 0 % of its c a t a l y t i c a c t i v i t y . This is an irreversible i n a c t i v a t i o n in t h a t t h e s u b s e q u e n t r e m o v a l of u r e a b y dialysis or b y d i l u t i o n does n o t restore t h e e n z y m a t i c activity. P r e i n c u b a t i o n of/5-isocitrate d e h y d r o g e n a s e w i t h N A D P , t h e specific cofactor r e q u i r e d for c a t a l y t i c a c t i v i t y , p r o t e c t s t h e enzyme from i n a c t i v a t i o n during subseq u e n t exposure to urea. T h e m a x i m u m p r o t e c t i o n afforded b y N A D P , however, is 4 0 - 4 5 % a n d f u r t h e r increasing t h e c o n c e n t r a t i o n of N A D P does n o t result in a n y g r e a t e r degree of p r o t e c t i o n . This o b s e r v a t i o n suggests t h a t one of t h e sites on t h e n a t i v e p r o t e i n which is affected b y u r e a is i n v o l v e d w i t h N A D P binding. As in t h e case of N A D P , p r e i n c n b a t i o n of fl-isocitrate d e h y d r o g e n a s e w i t h d i t h i o t h r e i t o l r e s u l t e d in a m a x i m u m of 4 0 - 4 5 % p r o t e c t i o n a g a i n s t s u b s e q u e n t u r e a i n a c t i v a t i o n a n d higher c o n c e n t r a t i o n s of d i t h i o t h r e i t o l d i d n o t result in a n y g r e a t e r Biochim. Biophys. Acta, 191 (1969) 722-724
724
SHORT COMMUNICATIONS
degree of protection. This data suggests that in the absence of reducing conditions urea causes a partial disruption of the tertiary structure of the enzyme resulting in the exposure and subsequent oxidation of essential - S H groups. When this occurs in a reducing environment, the oxidation of these - S H groups is prevented and the enzyme maintains 40-45% of the native catalytic activity. If, however, the enzyme is either preincubated with NADP or if the exposure to urea is conducted under reducing conditions, the enzyme is only partially inactivated. Finally, if the native enzyme is preincubated with NADP and subsequently treated with 2.0 M urea under reducing conditions, there is no loss of catalytic activity. This work was supported b y grants from the National Science Foundation (GB-7396) and National Institutes of Health (5 R O I A I 03866-08). One of the authors (H.C.R.) is a Research Career Development Awardee of National Institutes of Health (K3-AI-6928).
Department of Biochemistry, Research Laboratories, Albert Einstein Medical Center, Philadelphia, Pa. I9z4r (U.S.A.) I 2 3 4 5 6 7 8 9 IO
H E N R Y C. R E E V E S * B E T T E A N N ~BREHMEYER
H. C. REEVES, B. A. BREEMEYER AND S. J. AJL, Bacteriol. Proc., (1968) zI 7. T. I~AGLAND, T. I~AWASAKI AND J. LOWENSTEIN, J. Bacteriol., 91 (1966) 236. H. C. REEVES, B. A. BREHMEYER AND S. J. AJL, Science, 162 (1968) 359. D. T. PLUMMER, J. H. WILKINSON AND W. A. WITHYCOMBE, Biochem. J., 89 (1963) 48. I. A. BRODY, Nature, 2Ol (1964) 685. R. RICHTERICH AND A. BURGER, Helv. Physiol. Acta, 21 (1963) 59. A. E. H. EMERY, G. E. MOORES AND V. HUDSON, Clin. Chim. Acta, 19 (1968) 159. S. G. WELSRMAN AND E. C. RIXON, Clin. Chim. Acta, 19 (1968) 121. H. C. REEVES AND S. J. AjL, J. Bacteriol., 84 (1962) 186. J. B. MURPHY AND M. W. KIES, Biochim. Biophys. Acta, 45 (196o) 382.
Received August 7th, 1969 * Present a d d r e s s : D e p a r t m e n t o f B o t a n y a n d Microbiology, Arizona S t a t e U n i v e r s i t y , T e m p e , Ariz. 85281 , U.S.A.
Biochim. Biophys. Acta, 191 (1969) 722-724
BBA 63423
On the effects of cyanide on phenoxazinone synthetase Phenoxazinone synthetase, an enzyme which is believed to be involved in actinomycin biosynthesis, has been purified from Streptomyces antibioticus and studied by KATZ AND WEISSBACH1. These workers were unable to demonstrate a cofactor requirement of the enzyme, but they showed that it was affected by various metal ions and chelating agents. An interesting observation was that Cu ~+ and diethyldithiocarbamate were inhibitory at low levels (I. lO -4 and I . lO -5 M). This suggested to us that phenoxazinone synthetase might be a copper enzyme. The fact that pheBiochim. Biophys. dcta, 191 (1969) 724-726
SHORT COMMUNICATIONS
BBA 63416 Factors affecting urea inactivation of NADP-specific f3-isocitrate dehydrogenase Several electrophoretically distinct forms of NADP-specific isocitrate dehydrogenase (threo-Ds-isocitrate:NADP + oxidoreductase (decarboxylating), EC 1.1.I.42) have been reported to occur in Escherichia coli grown under different culture conditions 1. Since E. coli as well as most other bacterial species 2 contain only the NADPspecific form of this enzyme, it has been suggested 3 that the regulation of isocitrate metabolism may be controlled by multiple forms (isoenzymes) of this key enzyme under different growth conditions. The a- and fl-forms of NADP-specific isocitrate dehydrogenase, which occur in glucose-grown E. coli, have been separated and shown to exhibit marked differences with respect to urea inactivation 3. A similar differential susceptibility to urea has been observed with the L D H - I and LDH-5 isoenzymes of lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) 4-6. Although the phenomenon is not understood, the observation has been the basis of methods for determining lactate dehydrogenase patterns in serum 7,8. The present studies provide an insight into the inactivation of the fl-form of NADP-specific isocitrate dehydrogenase in 2.0 M urea. E. coli was grown aerobically in a simI~le mineral-salts medium containing o.2% glucose as carbon source and cell-free extracts prepared as previously described 9. The fl-isocitrate dehydrogenase was isolated employing DEAE- and CM-Sephadex column chromatography as reported previously ~. For enzyme assays, the change in absorbance at 34 ° hi# was followed using a Cary 14 spectrophotometer. Protein concentrations were determined by measuring the absorbance at 215 and 225 m/~ in a Beckman DU spectrophotometer according to the method described by MURPHY AND KIES 1°. 0. 5 ml of 4.0 M urea freshly prepared in o.oi M phosphate buffer (pH 7.5) was added to 0.5 ml of the same buffer containing fl-isoeitrate dehydrogenase and the solution placed in an ice bath at o °. Immediately after mixing, and at intervals thereafter, aliquots were assayed for enzymatic activity. The results are shown in Fig. I and are expressed as percent of activity in the control remaining at the times indicated. Under these conditions, 60% of the initial enzyme activity was lost after IO rain and only 5°/0 remained after I h. The effectiveness of NADP and dithiothreitol in protecting fl-isocitrate dehydrogenase against inactivation in 2.0 M urea is shown in Fig. 2. A solution of the enzyme in o.oi M phosphate buffer (pH 7.5) containing varying amounts of either NADP, dithiothreitol or a mixture of these was incubated in an ice bath at o ° for 30 min. An equal volume of 4.0 M urea was then added to each tube and incubation continued for I h at o °. Aliquots were then removed and assayed for enzymatic activity. Appropriate controls containing no urea were also included. Both dithiothreitol and NADP alone afford partial protection to the enzyme against inactivation in 2.0 M urea. In the presence of either 3.27 mM NADP or lO5 mM dithiothreitol, the enzyme retained 45-50% of the initial activity after I h at o ° in 2.0 M urea. In the absence of either NADP or dithiothreitol, greater than Biochim. Biophys. Acta, 191 (1969)-722-724
723
SHORT COMMUNICATIONS
80
100 ~-•
•
~"N
X,,, No Urea
60
80 = "G
60 / o
f
Oithiothreitol
40 20
o;
1'o
°~o........,-~ 2.0M Urea ~ °'-----~o,
2'0
3'0
;o
Time ( minutes )
5'o
6o
o~ u
0
2'1 0.654
4'2
6'3
1.31
1.96
8'. 2.62
105 mM DTT 3.27 mM NADP
Fig. I. Inactivation of/5-isocitrate dehydrogenase at o° in 2 M urea. The assay mixture contained (in/~moles) : potassium phosphate buffer (pH 7.5), 15o; MnCI,, 0.5; NADP, 0.5, and DL-isocitrate (potassium salt), 2. 5. The protein concentration was 6.0/~g and the total volume was i.o ml. Fig. 2. Protection of fl-isocitrate dehydrogenase against inactivation in 2 M urea by NADP and dithiothreitol (DTT). Assay conditions the same as described in Fig. i.
9 5 % of t h e initial c a t a l y t i c a c t i v i t y was lost u n d e r t h e same conditions. A t lower concentrations, t h e p r o t e c t i v e effect afforded b y either N A D P or d i t h i o t h r e i t o l decreased. I n t h e presence of b o t h N A D P a n d d i t h i o t h r e i t o l , the e n z y m e could be comp l e t e l y p r o t e c t e d a g a i n s t i n a c t i v a t i o n in 2.0 M urea. As shown in Fig. 2, preincub a t i o n of fi-isocitrate d e h y d r o g e n a s e w i t h b o t h N A D P a n d d i t h i o t h r e i t o l at a concent r a t i o n of each which alone afforded 4 0 - 4 5 % protection, r e s u l t e d in c o m p l e t e prot e c t i o n of the enzyme. A t lower c o n c e n t r a t i o n s also, a m i x t u r e of b o t h N A D P a n d d i t h i o t h r e i t o l was f o u n d to be more effective t h a n either c o m p o u n d alone in affording protection. fl-Isocitrate d e h y d r o g e n a s e in t h e presence of 2.0 M u r e a for I h loses g r e a t e r t h a n 9 0 % of its c a t a l y t i c a c t i v i t y . This is an irreversible i n a c t i v a t i o n in t h a t t h e s u b s e q u e n t r e m o v a l of u r e a b y dialysis or b y d i l u t i o n does n o t restore t h e e n z y m a t i c activity. P r e i n c u b a t i o n of/5-isocitrate d e h y d r o g e n a s e w i t h N A D P , t h e specific cofactor r e q u i r e d for c a t a l y t i c a c t i v i t y , p r o t e c t s t h e enzyme from i n a c t i v a t i o n during subseq u e n t exposure to urea. T h e m a x i m u m p r o t e c t i o n afforded b y N A D P , however, is 4 0 - 4 5 % a n d f u r t h e r increasing t h e c o n c e n t r a t i o n of N A D P does n o t result in a n y g r e a t e r degree of p r o t e c t i o n . This o b s e r v a t i o n suggests t h a t one of t h e sites on t h e n a t i v e p r o t e i n which is affected b y u r e a is i n v o l v e d w i t h N A D P binding. As in t h e case of N A D P , p r e i n c n b a t i o n of fl-isocitrate d e h y d r o g e n a s e w i t h d i t h i o t h r e i t o l r e s u l t e d in a m a x i m u m of 4 0 - 4 5 % p r o t e c t i o n a g a i n s t s u b s e q u e n t u r e a i n a c t i v a t i o n a n d higher c o n c e n t r a t i o n s of d i t h i o t h r e i t o l d i d n o t result in a n y g r e a t e r Biochim. Biophys. Acta, 191 (1969) 722-724
724
SHORT COMMUNICATIONS
degree of protection. This data suggests that in the absence of reducing conditions urea causes a partial disruption of the tertiary structure of the enzyme resulting in the exposure and subsequent oxidation of essential - S H groups. When this occurs in a reducing environment, the oxidation of these - S H groups is prevented and the enzyme maintains 40-45% of the native catalytic activity. If, however, the enzyme is either preincubated with NADP or if the exposure to urea is conducted under reducing conditions, the enzyme is only partially inactivated. Finally, if the native enzyme is preincubated with NADP and subsequently treated with 2.0 M urea under reducing conditions, there is no loss of catalytic activity. This work was supported b y grants from the National Science Foundation (GB-7396) and National Institutes of Health (5 R O I A I 03866-08). One of the authors (H.C.R.) is a Research Career Development Awardee of National Institutes of Health (K3-AI-6928).
Department of Biochemistry, Research Laboratories, Albert Einstein Medical Center, Philadelphia, Pa. I9z4r (U.S.A.) I 2 3 4 5 6 7 8 9 IO
H E N R Y C. R E E V E S * B E T T E A N N ~BREHMEYER
H. C. REEVES, B. A. BREEMEYER AND S. J. AJL, Bacteriol. Proc., (1968) zI 7. T. I~AGLAND, T. I~AWASAKI AND J. LOWENSTEIN, J. Bacteriol., 91 (1966) 236. H. C. REEVES, B. A. BREHMEYER AND S. J. AJL, Science, 162 (1968) 359. D. T. PLUMMER, J. H. WILKINSON AND W. A. WITHYCOMBE, Biochem. J., 89 (1963) 48. I. A. BRODY, Nature, 2Ol (1964) 685. R. RICHTERICH AND A. BURGER, Helv. Physiol. Acta, 21 (1963) 59. A. E. H. EMERY, G. E. MOORES AND V. HUDSON, Clin. Chim. Acta, 19 (1968) 159. S. G. WELSRMAN AND E. C. RIXON, Clin. Chim. Acta, 19 (1968) 121. H. C. REEVES AND S. J. AjL, J. Bacteriol., 84 (1962) 186. J. B. MURPHY AND M. W. KIES, Biochim. Biophys. Acta, 45 (196o) 382.
Received August 7th, 1969 * Present a d d r e s s : D e p a r t m e n t o f B o t a n y a n d Microbiology, Arizona S t a t e U n i v e r s i t y , T e m p e , Ariz. 85281 , U.S.A.
Biochim. Biophys. Acta, 191 (1969) 722-724
BBA 63423
On the effects of cyanide on phenoxazinone synthetase Phenoxazinone synthetase, an enzyme which is believed to be involved in actinomycin biosynthesis, has been purified from Streptomyces antibioticus and studied by KATZ AND WEISSBACH1. These workers were unable to demonstrate a cofactor requirement of the enzyme, but they showed that it was affected by various metal ions and chelating agents. An interesting observation was that Cu ~+ and diethyldithiocarbamate were inhibitory at low levels (I. lO -4 and I . lO -5 M). This suggested to us that phenoxazinone synthetase might be a copper enzyme. The fact that pheBiochim. Biophys. dcta, 191 (1969) 724-726