81
Biochem. J. (1977) 166, 81-88 Printed in Great Britain
Role of Pyridoxal Phosphate in Mammalian Polyamine Biosynthesis LACK OF REQUIREMENT FOR MAMMALIAN S-ADENOSYLMETHIONINE DECARBOXYLASE ACTIVITY
By ANTHONY E. PEGG Department of Physiology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, U.S.A. (Received 31 December 1976) 1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of
methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine} to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity of ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.
Polyamine biosynthesis in mammalian cells is thought to be controlled by the activities of two key enzymes, L-ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50). Many growth-promoting stimuli have been shown to increase cellular polyamine concentrations, and this increase is brought about by increased activities of these decarboxylases (Morris & Fillingame, 1974; Raina & Janne, 1975; Tabor & Tabor, 1976). Evidence obtained by using specific antisera and making direct measurements of the amount of enzyme protein indicates that these increases in activity are due to increased amounts of enzyme protein (Pegg, 1974; Holtta, 1975; Theoharides & Canellakis, 1976; A. E. Pegg, unpublished work). Although the exact function of polyamines within the cell is not yet well understood, there is compelling evidence that these basic molecules play an essential role in cellular physiology (Tabor & Tabor, 1972, 1976; Raina & Janne, 1975). Studies with inhibitors of polyamine formation have indicated that the enhanced concentrations of polyamines found in growing cells are essential for continued growth and Vol. 166
DNA synthesis (Kay & Pegg, 1973; Otani et al., 1974; Fillingame et al., 1975; Relyea & Rando, 1975; Mamont et al., 1976; Poso & Janne, 1976; WilliamsAshman et al., 1976). It is possible that an important facet of growth retardation by vitamin B-6 deficiency might be due to inability to synthesize polyamines because of decreased availability of an essential cofactor for L-ornithine decarboxylase and/or S-adenosyl-L-methionine decarboxylase. Mammalian L-ornithine decarboxylase has been purified in a number of laboratories (Raina & Janne, 1975; Tabor & Tabor, 1972, 1976), and there is general agreement with the original observations based on studies of the enzyme from rat prostate (Pegg & Williams-Ashman, 1968; Pegg, 1970) that this enzyme has a rather loosely bound pyridoxal phosphate cofactor. There is controversy, however, about the prosthetic group of mammalian Sadenosylmethionine decarboxylase. Early experiments indicated that activity was lost on addition of 4-bromo-3-hydroxybenzyloxyamine (NSD-1055) and other reagents which react with carbonyl groups (Pegg & Williams-Ashman, 1969; Pegg, 1970), but
A. E. PEGG
82 activity could not be restored by later addition of pyridoxal phosphate. Subsequently, two separate groups have claimed that rat liver S-adenosylmethionine decarboxylase could be stimulated by addition of pyridoxal phosphate (Feldman et al., 1972; Sturman & Kremzner, 1974a), but others were unable to obtain such a stimulation (Williams-Ashman et al., 1972; Schmidt & Cantoni, 1973; Pegg, 1974; Hannonen, 1976). The present paper provides data on the effects of vitamin B-6 deficiency on polyamine concentrations and the activities of these decarboxylases. Even under conditions designed to maximize the possibility of exposing S-adenosylmethionine decarboxylase in an apoenzyme form, it was not possible to demonstrate a requirement for pyridoxal phosphate. Further evidence that pyridoxal phosphate is not involved in the action of mammalian S-adenosylmethionine decarboxylase was obtained by studies of the absorption spectrum of the purified enzyme and its inactivation by reagents reacting with carbonyl groups. These studies suggest that the mammalian enzyme may resemble the bacterial equivalent in having a covalently bound cofactor which contains a carbonyl group. However, polyamine concentrations were significantly decreased by giving a vitamin B-6-deficient diet. The possible mechanisms of this decrease are discussed.
Methods S-Adenosylmethionine decarboxylase and ornithine decarboxylase activities were assayed by determination of the release of "4CO2 from carboxyl'4C-labelled substrates as previously described (Pegg & Williams-Ashman, 1969). For the measurement of S-adenosylmethionine decarboxylase activity, the assay medium contained 50 mM-sodium phosphate buffer, pH7.2, 1 mM-dithiothreitol, 0.2mM-S-adenosyl[carboxyl-14C]methionine (2-5mCi/mmol) and up to 2 units of enzyme protein in a total volume of 0.3 ml. For ornithine decarboxylase assay the medium contained 50mM-sodium phosphate buffer, pH7.2, 2.5mM-dithiothreitol, 0.2mM-pyridoxal phosphate, 2mM-DL-[1-14C]ornithine (1-5mCi/mmol) and up to 4 units of enzyme in a total volume of 0.3 ml. A unit of enzyme was defined as that catalysing the decarboxylation of 1 nmol of substrate during a 30min incubation at 37°C. Tissue extracts for the assay of enzyme activity were prepared by homogenization in 3 vol. of 1OmMsodium phosphate (pH7.2)/2.5 mM-dithiothreitol/ 0.1 mM-disodium EDTA. The homogenate was centrifuged for h at 105000g and the supernatant fraction used for assay. The protein present was determined by the method of Lowry et al. (1951), with crystalline bovine serum albumin as a standard.
Highly purified S-adenosylmethionine decarboxylase was purified from the livers of rats treated 24h before death with 80mg of methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine}/kg body wt. The drug was dissolved in 0.9 % (w/v) NaCI at a concentration of 40mg/ml and administered to male rats weighing between 300 and 400g by intraperitoneal injection. This treatment increases the concentration of the S-adenosylmethionine decarboxylase in the liver up to 20-fold (Pegg et al., 1973; H6ltta et al., 1973; Pegg, 1974) and thus provides an enriched starting material for the purification. The enzyme was then purified by precipitation with (NH4)2SO4, affinity chromatography on columns of methylglyoxal bis(guanylhydrazone) linked to Sepharose and chromatography on DEAE-cellulose as previously described (Pegg, 1974). The final fraction was then passed through a column (40cmx2.5cm) of Bio-Gel A 1.5m previously equilibrated with 2.5mM-putrescine/lOmMsodium phosphate (pH7.0)/1 mM-dithiothreitol/ O.1mM-disodium EDTA, and 5ml fractions were collected. The fractions having the highest activity from the column eluate were concentrated to about 1 ml by using a pressurized ultrafiltration cell (Amicon Corp., Lexington, MA, U.S.A.). The final preparation had a specific activity of 5800-6000 units/ mg and was stored at -20°C in small lots to avoid repeated freezing and thawing, which rapidly inactivated the enzyme. In the absence of putrescine activity was lost very rapidly, but, under the conditions described above, activity was lost only at the rate of about 10% per week. Before use, the enzyme was freed from putrescine by passage through a column (1 cmx 20cm) of Sephadex G-10 equilibrated with the buffer required for the particular experiment as described below. The spectrum of the purified enzyme dissolved in 50mM-potassium phosphate, pH7.0, was determined with a Unicam SP.8000 spectrophotometer. For reaction with NaB3H4, 0.2mg of enzyme protein was dissolved in 1 ml of 0.5M-NH4HCO3 and 0.1 ml of 0.1 M-NaOH containing 0.1 M-NaB3H4 (204.8mCi/ mmol) added. After 4h the enzyme was dialysed against 5 x 2 litres of 0.05 M-NH4HCO3 over a 24h period. A small sample was precipitated by the addition of 5% (w/v) HC104 to assay for incorporation of radioactivity into the protein, and the remainder was freeze-dried. A portion of the 3Hlabelled enzyme was mixed with unchanged enzyme in 0.1 M-potassium phosphate/2.5mM-dithiothreitol (pH 6.9) and layered over a linear 5-20% (w/v) sucrose gradient made up in the same buffer solution. The gradient was centrifuged for 48h at 40000rev./ min in the SW 40 rotor of a Beckman model L2-50 ultracentrifuge. Fractions (0.5 ml) were collected and assayed for radioactivity and for S-adenosylmeth-
ionine decarboxylase activity. 1977
PYRIDOXAL PHOSPHATE AND MAMMALIAN POLYAMINE BIOSYNTHESIS Female Wistar-strain rats weighing 40-60 g at the start of the experiment were fed on a pyridoxincdeficient diet (Nutritional Biochemicals Corp., Cleveland, OH, U.S.A.) for 6 weeks. A similar control group of rats received the same diet supplemented with pyridoxine hydrochloride supplied in the drinking water (50,cg/ml). Some of these rats were treated with methylglyoxal bis(guanylhydrazone) as described above. Developing rats were subjected to vitamin B-6 deficiency as described by Dakshinamurti & Stephens (1969). Female rats were placed on the vitamin B-6-deficient diet 1 week after mating and the diet was continued until the suckling rats were killed at 13 days of age. Pyridoxal phosphate concentrations were measured by the ability to re-activate the apoenzyme of bacterial tyrosine decarboxylase (Dakshinamurti & Stephens, 1969; Bayoumi & Smith, 1976). Spermine and spermidine were determined by butanol extraction and separation by paper electrophoresis as previously described (Pegg et al., 1970). Putrescine was determined by the ability to activate yeast S-adenosylmethionine decarboxylase as described by Harik et al. (1973). DL-[1-14C]Ornithine (20-5OmCi/mmol) and S-
adenosyl-L-[carboxyl-'4C]methionine
(25-56mCi/
mmol) were purchased from New England Nuclear Corp., Boston, MA, U.S.A., and diluted to the appropriate specific radioactivity with unlabelled material obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Methylglyoxal bis(guanylhydrazone) was purchased from Aldrich Chemical Co., Milwaukee, WI, U.S.A. Results To obtain a low tissue concentration of pyridoxal phosphate, which might result in the detection of
83
a requirement for this cofactor for mammalian S-adenosylmethionine decarboxylase, pregnant rats were fed on a diet deficient in pyridoxine starting from 1 week after conception and continuing until 12 days after birth. The neonatal rats were therefore deprived of vitamin B-6 both during embryonic development and during suckling. This procedure has been used by others to study the role of pyridoxal phosphate in the development of the nervous system (Dakshinamurti & Stephens, 1969; Bayoumi & Smith, 1972). Suckling rats from mothers fed on the vitamin B-6-deficient diet and from control mothers fed on the same diet supplemented with pyridoxine hydrochloride were killed 13 days after birth, and theactivities of ornithine decarboxylase and S-adenosylmethionine decarboxylase determined in ultracentrifuged extracts from brain, liver and kidney. Results from these assays are shown in Table 1. When assayed in the presence of pyridoxal phosphate, ornithine decarboxylase activity was significantly higher (up to threefold) in extracts from the vitamin-deficient rats, but the reverse was the case when the assays were conducted in the absence of pyridoxal phosphate (Table 1). Thus the stimulation by addition of pyridoxal phosphate was 2-3-fold, depending on the tissue, for extracts from control rats, but was 12-20 fold for rats fed on the vitamin-deficient diet. These results arc similar to those found by others for other pyridoxal phosphatedependent enzymes in extracts from vitamin B-6depleted rats and has been interpreted to indicate an increase in the amount of apoenzyme which may, at least in part, compensate for the decreased concentration of the cofactor (Dakshinamurti & Stephens, 1969; Bayoumi & Smith, 1972). The requirement for pyridoxal phosphate for optimal activity of ornithine decarboxylase in these crude tissue extracts is clear from the data in Table 1.
Table 1. S-Adenosylmethionine decarboxylase and ornithine decarboxylase activities in liver, kidney and brain extracts from pyridoxine-deficient developing rats Developing rats were exposed to pyridoxine deficiency by feeding a vitamin B-6-deficient diet to the mothers as described in the text. Enzyme assays were carried out as described in the Methods section. Pyridoxal phosphate, where present, was added at 0.2mM. Results are shown as means±s.E.M. for five separate determinations. Differences between the averages were tested for statistical significance by Student's t test: *P < 0.01 versus control; tP
Biochem. J. (1977) 166, 81-88 Printed in Great Britain
Role of Pyridoxal Phosphate in Mammalian Polyamine Biosynthesis LACK OF REQUIREMENT FOR MAMMALIAN S-ADENOSYLMETHIONINE DECARBOXYLASE ACTIVITY
By ANTHONY E. PEGG Department of Physiology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, U.S.A. (Received 31 December 1976) 1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of
methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine} to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity of ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.
Polyamine biosynthesis in mammalian cells is thought to be controlled by the activities of two key enzymes, L-ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50). Many growth-promoting stimuli have been shown to increase cellular polyamine concentrations, and this increase is brought about by increased activities of these decarboxylases (Morris & Fillingame, 1974; Raina & Janne, 1975; Tabor & Tabor, 1976). Evidence obtained by using specific antisera and making direct measurements of the amount of enzyme protein indicates that these increases in activity are due to increased amounts of enzyme protein (Pegg, 1974; Holtta, 1975; Theoharides & Canellakis, 1976; A. E. Pegg, unpublished work). Although the exact function of polyamines within the cell is not yet well understood, there is compelling evidence that these basic molecules play an essential role in cellular physiology (Tabor & Tabor, 1972, 1976; Raina & Janne, 1975). Studies with inhibitors of polyamine formation have indicated that the enhanced concentrations of polyamines found in growing cells are essential for continued growth and Vol. 166
DNA synthesis (Kay & Pegg, 1973; Otani et al., 1974; Fillingame et al., 1975; Relyea & Rando, 1975; Mamont et al., 1976; Poso & Janne, 1976; WilliamsAshman et al., 1976). It is possible that an important facet of growth retardation by vitamin B-6 deficiency might be due to inability to synthesize polyamines because of decreased availability of an essential cofactor for L-ornithine decarboxylase and/or S-adenosyl-L-methionine decarboxylase. Mammalian L-ornithine decarboxylase has been purified in a number of laboratories (Raina & Janne, 1975; Tabor & Tabor, 1972, 1976), and there is general agreement with the original observations based on studies of the enzyme from rat prostate (Pegg & Williams-Ashman, 1968; Pegg, 1970) that this enzyme has a rather loosely bound pyridoxal phosphate cofactor. There is controversy, however, about the prosthetic group of mammalian Sadenosylmethionine decarboxylase. Early experiments indicated that activity was lost on addition of 4-bromo-3-hydroxybenzyloxyamine (NSD-1055) and other reagents which react with carbonyl groups (Pegg & Williams-Ashman, 1969; Pegg, 1970), but
A. E. PEGG
82 activity could not be restored by later addition of pyridoxal phosphate. Subsequently, two separate groups have claimed that rat liver S-adenosylmethionine decarboxylase could be stimulated by addition of pyridoxal phosphate (Feldman et al., 1972; Sturman & Kremzner, 1974a), but others were unable to obtain such a stimulation (Williams-Ashman et al., 1972; Schmidt & Cantoni, 1973; Pegg, 1974; Hannonen, 1976). The present paper provides data on the effects of vitamin B-6 deficiency on polyamine concentrations and the activities of these decarboxylases. Even under conditions designed to maximize the possibility of exposing S-adenosylmethionine decarboxylase in an apoenzyme form, it was not possible to demonstrate a requirement for pyridoxal phosphate. Further evidence that pyridoxal phosphate is not involved in the action of mammalian S-adenosylmethionine decarboxylase was obtained by studies of the absorption spectrum of the purified enzyme and its inactivation by reagents reacting with carbonyl groups. These studies suggest that the mammalian enzyme may resemble the bacterial equivalent in having a covalently bound cofactor which contains a carbonyl group. However, polyamine concentrations were significantly decreased by giving a vitamin B-6-deficient diet. The possible mechanisms of this decrease are discussed.
Methods S-Adenosylmethionine decarboxylase and ornithine decarboxylase activities were assayed by determination of the release of "4CO2 from carboxyl'4C-labelled substrates as previously described (Pegg & Williams-Ashman, 1969). For the measurement of S-adenosylmethionine decarboxylase activity, the assay medium contained 50 mM-sodium phosphate buffer, pH7.2, 1 mM-dithiothreitol, 0.2mM-S-adenosyl[carboxyl-14C]methionine (2-5mCi/mmol) and up to 2 units of enzyme protein in a total volume of 0.3 ml. For ornithine decarboxylase assay the medium contained 50mM-sodium phosphate buffer, pH7.2, 2.5mM-dithiothreitol, 0.2mM-pyridoxal phosphate, 2mM-DL-[1-14C]ornithine (1-5mCi/mmol) and up to 4 units of enzyme in a total volume of 0.3 ml. A unit of enzyme was defined as that catalysing the decarboxylation of 1 nmol of substrate during a 30min incubation at 37°C. Tissue extracts for the assay of enzyme activity were prepared by homogenization in 3 vol. of 1OmMsodium phosphate (pH7.2)/2.5 mM-dithiothreitol/ 0.1 mM-disodium EDTA. The homogenate was centrifuged for h at 105000g and the supernatant fraction used for assay. The protein present was determined by the method of Lowry et al. (1951), with crystalline bovine serum albumin as a standard.
Highly purified S-adenosylmethionine decarboxylase was purified from the livers of rats treated 24h before death with 80mg of methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine}/kg body wt. The drug was dissolved in 0.9 % (w/v) NaCI at a concentration of 40mg/ml and administered to male rats weighing between 300 and 400g by intraperitoneal injection. This treatment increases the concentration of the S-adenosylmethionine decarboxylase in the liver up to 20-fold (Pegg et al., 1973; H6ltta et al., 1973; Pegg, 1974) and thus provides an enriched starting material for the purification. The enzyme was then purified by precipitation with (NH4)2SO4, affinity chromatography on columns of methylglyoxal bis(guanylhydrazone) linked to Sepharose and chromatography on DEAE-cellulose as previously described (Pegg, 1974). The final fraction was then passed through a column (40cmx2.5cm) of Bio-Gel A 1.5m previously equilibrated with 2.5mM-putrescine/lOmMsodium phosphate (pH7.0)/1 mM-dithiothreitol/ O.1mM-disodium EDTA, and 5ml fractions were collected. The fractions having the highest activity from the column eluate were concentrated to about 1 ml by using a pressurized ultrafiltration cell (Amicon Corp., Lexington, MA, U.S.A.). The final preparation had a specific activity of 5800-6000 units/ mg and was stored at -20°C in small lots to avoid repeated freezing and thawing, which rapidly inactivated the enzyme. In the absence of putrescine activity was lost very rapidly, but, under the conditions described above, activity was lost only at the rate of about 10% per week. Before use, the enzyme was freed from putrescine by passage through a column (1 cmx 20cm) of Sephadex G-10 equilibrated with the buffer required for the particular experiment as described below. The spectrum of the purified enzyme dissolved in 50mM-potassium phosphate, pH7.0, was determined with a Unicam SP.8000 spectrophotometer. For reaction with NaB3H4, 0.2mg of enzyme protein was dissolved in 1 ml of 0.5M-NH4HCO3 and 0.1 ml of 0.1 M-NaOH containing 0.1 M-NaB3H4 (204.8mCi/ mmol) added. After 4h the enzyme was dialysed against 5 x 2 litres of 0.05 M-NH4HCO3 over a 24h period. A small sample was precipitated by the addition of 5% (w/v) HC104 to assay for incorporation of radioactivity into the protein, and the remainder was freeze-dried. A portion of the 3Hlabelled enzyme was mixed with unchanged enzyme in 0.1 M-potassium phosphate/2.5mM-dithiothreitol (pH 6.9) and layered over a linear 5-20% (w/v) sucrose gradient made up in the same buffer solution. The gradient was centrifuged for 48h at 40000rev./ min in the SW 40 rotor of a Beckman model L2-50 ultracentrifuge. Fractions (0.5 ml) were collected and assayed for radioactivity and for S-adenosylmeth-
ionine decarboxylase activity. 1977
PYRIDOXAL PHOSPHATE AND MAMMALIAN POLYAMINE BIOSYNTHESIS Female Wistar-strain rats weighing 40-60 g at the start of the experiment were fed on a pyridoxincdeficient diet (Nutritional Biochemicals Corp., Cleveland, OH, U.S.A.) for 6 weeks. A similar control group of rats received the same diet supplemented with pyridoxine hydrochloride supplied in the drinking water (50,cg/ml). Some of these rats were treated with methylglyoxal bis(guanylhydrazone) as described above. Developing rats were subjected to vitamin B-6 deficiency as described by Dakshinamurti & Stephens (1969). Female rats were placed on the vitamin B-6-deficient diet 1 week after mating and the diet was continued until the suckling rats were killed at 13 days of age. Pyridoxal phosphate concentrations were measured by the ability to re-activate the apoenzyme of bacterial tyrosine decarboxylase (Dakshinamurti & Stephens, 1969; Bayoumi & Smith, 1976). Spermine and spermidine were determined by butanol extraction and separation by paper electrophoresis as previously described (Pegg et al., 1970). Putrescine was determined by the ability to activate yeast S-adenosylmethionine decarboxylase as described by Harik et al. (1973). DL-[1-14C]Ornithine (20-5OmCi/mmol) and S-
adenosyl-L-[carboxyl-'4C]methionine
(25-56mCi/
mmol) were purchased from New England Nuclear Corp., Boston, MA, U.S.A., and diluted to the appropriate specific radioactivity with unlabelled material obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Methylglyoxal bis(guanylhydrazone) was purchased from Aldrich Chemical Co., Milwaukee, WI, U.S.A. Results To obtain a low tissue concentration of pyridoxal phosphate, which might result in the detection of
83
a requirement for this cofactor for mammalian S-adenosylmethionine decarboxylase, pregnant rats were fed on a diet deficient in pyridoxine starting from 1 week after conception and continuing until 12 days after birth. The neonatal rats were therefore deprived of vitamin B-6 both during embryonic development and during suckling. This procedure has been used by others to study the role of pyridoxal phosphate in the development of the nervous system (Dakshinamurti & Stephens, 1969; Bayoumi & Smith, 1972). Suckling rats from mothers fed on the vitamin B-6-deficient diet and from control mothers fed on the same diet supplemented with pyridoxine hydrochloride were killed 13 days after birth, and theactivities of ornithine decarboxylase and S-adenosylmethionine decarboxylase determined in ultracentrifuged extracts from brain, liver and kidney. Results from these assays are shown in Table 1. When assayed in the presence of pyridoxal phosphate, ornithine decarboxylase activity was significantly higher (up to threefold) in extracts from the vitamin-deficient rats, but the reverse was the case when the assays were conducted in the absence of pyridoxal phosphate (Table 1). Thus the stimulation by addition of pyridoxal phosphate was 2-3-fold, depending on the tissue, for extracts from control rats, but was 12-20 fold for rats fed on the vitamin-deficient diet. These results arc similar to those found by others for other pyridoxal phosphatedependent enzymes in extracts from vitamin B-6depleted rats and has been interpreted to indicate an increase in the amount of apoenzyme which may, at least in part, compensate for the decreased concentration of the cofactor (Dakshinamurti & Stephens, 1969; Bayoumi & Smith, 1972). The requirement for pyridoxal phosphate for optimal activity of ornithine decarboxylase in these crude tissue extracts is clear from the data in Table 1.
Table 1. S-Adenosylmethionine decarboxylase and ornithine decarboxylase activities in liver, kidney and brain extracts from pyridoxine-deficient developing rats Developing rats were exposed to pyridoxine deficiency by feeding a vitamin B-6-deficient diet to the mothers as described in the text. Enzyme assays were carried out as described in the Methods section. Pyridoxal phosphate, where present, was added at 0.2mM. Results are shown as means±s.E.M. for five separate determinations. Differences between the averages were tested for statistical significance by Student's t test: *P < 0.01 versus control; tP
