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Nicotinamide-adenine dinucleotide phosphate in gastric mucosa
SHORT COMMUNICATIONS
645
BBA 43 171
N icotinamide-adenine dinucleotide phosphate in gastric mucosa The hydrogen ions secreted b y gastric mucosa are probably generated b y an oxidation-reduction mechanism in the oxyntic cells 1. This view is supported b y studies of the efficiency of acid secretion in relation to oxygen consumption2, 3. The components of the oxidation-reduction mechanism are not known. K I D D E R , CURRAN AND REHM4 recently demonstrated the existence of extramitochondrial cytochrome c in bullfrog gastric mucosa, and suggested its involvement in the mechanism of acid secretion. The present communication reports the finding of a high level of N A D P H in frog gastric mucosa, high activity of extramitochondrial NADHP-generating enzymes, and appreciable N A D P H : c y t o c h r o m e c oxidoreductase activity. Gastric mucosae were obtained from fresh frogs of the species Discoglossus pictus. The levels of NADP÷ and N A D P H in the mucosae were determined in neutralized acid and alkaline extracts, respectively, b y the method of VILLEE5. In the enzyme assays, the mucosae were homogenized with 9 vols. of ice-cold o.12 M KC1 and the homogenate was centrifuged at 0-4 ° at 20000 x g for 30 rain. The supernatant was used for the assays, n-Glucose-6-phosphate: NADP + oxidoreductase (EC I.I.I.49) was determined b y the method of L6HR AND WALLER6; Ls-isocitrate:NADP+ oxidoreductase (EC 1.1.1.42 ) b y the method of OCHOAT; N A D P H : c y t o c h r o m e c oxidoreductase (EC 1.6.2.3) by the method of H A A S , HORECKER AND HOGNESSs. Protein concentrations were determined by a biuret method 9. The coenzyme and enzyme assays were also carried out on frog liver for the purpose of comparison. TABLE
I
LEVEL OF N A D P + ANn N A D P H The values are means
IN FROG GASTRIC MUCOSA AND LIVER
-~ S . E . o f d e t e r m i n a t i o n s
o n five a n i m a l s . U n i t s r e f e r t o w e t w t .
Tissue
NA DP + (#moles~g)
NA DPH (l~moles/g)
NA DPH/NA Dp+
Gastric mucosa Liver
o.o15 ± o.004 o.059 ± o.006
O.lO 3 ± o . o 1 8 o.163 ± 0.032
lO.6 ± 3.8 2. 7 ~ 0. 4
TABLE
II
ACTIVITY OF IXTADP+-LINKED ENZYMES IN 2 0 0 0 0 × g SUPERNATANT OF HOMOGENATES OF FROG GASTRIC MUCOSA AND LIVER I n i t i a l a c t i v i t i e s w e r e m e a s u r e d a t 25 ° i n t e r m s of /~moles N A D P + r e d u c e d p e r m i l l i n t h e c a s e of t h e N A D P H - g e n e r a t i n g e n z y m e s , a n d i n t e r m s of / z m o l e s c y t o c h r o m e c r e d u c e d p e r m i n i n t h e c a s e of N A D P H : c y t o c h r o m e c o x i d o r e d u c t a s e . V a l u e s a r e m e a n s ± S . E . of d e t e r m i n a t i o n s o n five a n i m a l s . U n i t s r e f e r t o p r o t e i n c o n t e n t of s u p e r n a t a n t .
Enzyme
Gastric mucosa (initial activity/rag protein)
Liver (initial activity/rag protein)
Glucose-6-phosphate: NADP + oxidoreductase Isocitrate :NADP + oxidoreductase NADPH :cytoehrome c oxidoreductase
0 . 2 6 0 ± O.Ol 5 0 . 0 6 6 ± O.Ol 7 0.009 ± o.ooi
0.038 ± 0.007 o.o12 ± 0.002 O.OLO ~- o . o o i
Bioehim. Biophys. Acta, 143 (1967) 6 4 5 - 6 4 6
646
SHORT COMMUNICATIONS
The levels of N A D P + and N A D P H observed in the gastric mucosae and livers are shown in Table I. I t is seen t h a t a high level of N A D P H is m a i n t a i n e d in b o t h tissues. Th e N A D P H / N A D P + ratio is a p p a r e n t l y higher in gastric m u c o s a than in liver (P ab o u t o . I o in the data). This correlates w i t h the finding of higher activities of N A D P H - g e n e r a t i n g e n z y m e s in the mucosae (Table II). Th e a c t i v i t y of N A D P H : c y t o c h r o m e e oxidoreductase was found to be the same in the mucosae and livers (Table II). E x t r a m i t o c h o n d r i a l c y t o c h r o m e c m a y function as an electron acceptor in the generation of h y d r o g e n ions for secretion in gastric mucosa a. Th e present findings suggest the possibility t h a t N A D P H m a y be the source of the reducing equivalents. A l t h o u g h direct evidence remains to be discovered, it is of interest to suggest t h a t the h y d r o g e n ions produced in th e o x i d o r e d u c t i o n b et w een the N A D P H and the cytochrome c are c a p t u r e d for secretion. This work has been s u p p o r te d b y a g r a n t from t h e S m i t h Kline and F r e n c h Foundation.
Department of Physiology, Royal University of Malta, Valletta (Malta)
W. H. BANNISTER SHELAGH MORRISSEY*
E. J. CONWAYAND T. G. BRADY, Nature, 162 (1948) 456. I-[. W. DAVENPORT,Federation Proc., i i (1952) 715 . V~:.t-I. BANNISTER,J. Physiol. London, 177 (1965) 429 . G. "W. KIDDER, P. F. CURRANAND W. S. REHM, Am. J. Physiol., 211 (1966) 513 . C. tL. VILLEE, Biochem. J., 83 (1962) 191. G. W. LOHR AND H. D. ~¢VALLER,in H. BERGMEYER, Methods of Enzymatic Analysis, Academic Press, New York, 1963, p. 744. 7 S. OCHOA,J. Biol. Chem., 174 (1948) 133. 8 J~. HAAS, B. C. HORECKERAND T. R. HOGNESS,J. Biol. Chem., 136 (194o) 747. 9 A. G. GORNALL,J. J. BARDAWILLAND M. M. DAVID,J. Biol. Chem., 177 (I949) 751 i 2 3 4 5 6
R e c e i v e d May I2th, 1967 * Present address: Department of Physiology, Queen Elizabeth College, University of London, Great Britain.
Biochim. Biophys. Acta, 143 (1967) 645-646
645
BBA 43 171
N icotinamide-adenine dinucleotide phosphate in gastric mucosa The hydrogen ions secreted b y gastric mucosa are probably generated b y an oxidation-reduction mechanism in the oxyntic cells 1. This view is supported b y studies of the efficiency of acid secretion in relation to oxygen consumption2, 3. The components of the oxidation-reduction mechanism are not known. K I D D E R , CURRAN AND REHM4 recently demonstrated the existence of extramitochondrial cytochrome c in bullfrog gastric mucosa, and suggested its involvement in the mechanism of acid secretion. The present communication reports the finding of a high level of N A D P H in frog gastric mucosa, high activity of extramitochondrial NADHP-generating enzymes, and appreciable N A D P H : c y t o c h r o m e c oxidoreductase activity. Gastric mucosae were obtained from fresh frogs of the species Discoglossus pictus. The levels of NADP÷ and N A D P H in the mucosae were determined in neutralized acid and alkaline extracts, respectively, b y the method of VILLEE5. In the enzyme assays, the mucosae were homogenized with 9 vols. of ice-cold o.12 M KC1 and the homogenate was centrifuged at 0-4 ° at 20000 x g for 30 rain. The supernatant was used for the assays, n-Glucose-6-phosphate: NADP + oxidoreductase (EC I.I.I.49) was determined b y the method of L6HR AND WALLER6; Ls-isocitrate:NADP+ oxidoreductase (EC 1.1.1.42 ) b y the method of OCHOAT; N A D P H : c y t o c h r o m e c oxidoreductase (EC 1.6.2.3) by the method of H A A S , HORECKER AND HOGNESSs. Protein concentrations were determined by a biuret method 9. The coenzyme and enzyme assays were also carried out on frog liver for the purpose of comparison. TABLE
I
LEVEL OF N A D P + ANn N A D P H The values are means
IN FROG GASTRIC MUCOSA AND LIVER
-~ S . E . o f d e t e r m i n a t i o n s
o n five a n i m a l s . U n i t s r e f e r t o w e t w t .
Tissue
NA DP + (#moles~g)
NA DPH (l~moles/g)
NA DPH/NA Dp+
Gastric mucosa Liver
o.o15 ± o.004 o.059 ± o.006
O.lO 3 ± o . o 1 8 o.163 ± 0.032
lO.6 ± 3.8 2. 7 ~ 0. 4
TABLE
II
ACTIVITY OF IXTADP+-LINKED ENZYMES IN 2 0 0 0 0 × g SUPERNATANT OF HOMOGENATES OF FROG GASTRIC MUCOSA AND LIVER I n i t i a l a c t i v i t i e s w e r e m e a s u r e d a t 25 ° i n t e r m s of /~moles N A D P + r e d u c e d p e r m i l l i n t h e c a s e of t h e N A D P H - g e n e r a t i n g e n z y m e s , a n d i n t e r m s of / z m o l e s c y t o c h r o m e c r e d u c e d p e r m i n i n t h e c a s e of N A D P H : c y t o c h r o m e c o x i d o r e d u c t a s e . V a l u e s a r e m e a n s ± S . E . of d e t e r m i n a t i o n s o n five a n i m a l s . U n i t s r e f e r t o p r o t e i n c o n t e n t of s u p e r n a t a n t .
Enzyme
Gastric mucosa (initial activity/rag protein)
Liver (initial activity/rag protein)
Glucose-6-phosphate: NADP + oxidoreductase Isocitrate :NADP + oxidoreductase NADPH :cytoehrome c oxidoreductase
0 . 2 6 0 ± O.Ol 5 0 . 0 6 6 ± O.Ol 7 0.009 ± o.ooi
0.038 ± 0.007 o.o12 ± 0.002 O.OLO ~- o . o o i
Bioehim. Biophys. Acta, 143 (1967) 6 4 5 - 6 4 6
646
SHORT COMMUNICATIONS
The levels of N A D P + and N A D P H observed in the gastric mucosae and livers are shown in Table I. I t is seen t h a t a high level of N A D P H is m a i n t a i n e d in b o t h tissues. Th e N A D P H / N A D P + ratio is a p p a r e n t l y higher in gastric m u c o s a than in liver (P ab o u t o . I o in the data). This correlates w i t h the finding of higher activities of N A D P H - g e n e r a t i n g e n z y m e s in the mucosae (Table II). Th e a c t i v i t y of N A D P H : c y t o c h r o m e e oxidoreductase was found to be the same in the mucosae and livers (Table II). E x t r a m i t o c h o n d r i a l c y t o c h r o m e c m a y function as an electron acceptor in the generation of h y d r o g e n ions for secretion in gastric mucosa a. Th e present findings suggest the possibility t h a t N A D P H m a y be the source of the reducing equivalents. A l t h o u g h direct evidence remains to be discovered, it is of interest to suggest t h a t the h y d r o g e n ions produced in th e o x i d o r e d u c t i o n b et w een the N A D P H and the cytochrome c are c a p t u r e d for secretion. This work has been s u p p o r te d b y a g r a n t from t h e S m i t h Kline and F r e n c h Foundation.
Department of Physiology, Royal University of Malta, Valletta (Malta)
W. H. BANNISTER SHELAGH MORRISSEY*
E. J. CONWAYAND T. G. BRADY, Nature, 162 (1948) 456. I-[. W. DAVENPORT,Federation Proc., i i (1952) 715 . V~:.t-I. BANNISTER,J. Physiol. London, 177 (1965) 429 . G. "W. KIDDER, P. F. CURRANAND W. S. REHM, Am. J. Physiol., 211 (1966) 513 . C. tL. VILLEE, Biochem. J., 83 (1962) 191. G. W. LOHR AND H. D. ~¢VALLER,in H. BERGMEYER, Methods of Enzymatic Analysis, Academic Press, New York, 1963, p. 744. 7 S. OCHOA,J. Biol. Chem., 174 (1948) 133. 8 J~. HAAS, B. C. HORECKERAND T. R. HOGNESS,J. Biol. Chem., 136 (194o) 747. 9 A. G. GORNALL,J. J. BARDAWILLAND M. M. DAVID,J. Biol. Chem., 177 (I949) 751 i 2 3 4 5 6
R e c e i v e d May I2th, 1967 * Present address: Department of Physiology, Queen Elizabeth College, University of London, Great Britain.
Biochim. Biophys. Acta, 143 (1967) 645-646