Studies of the acetyl-CoA-binding site of rat liver spermidine/spermine N1-acetyltransferase.

Rat liver spermidine/spermine N1-acetyltransferase was found to be strongly inhibited by the dyes Cibacron F3GA, Coomassie Brilliant Blue and Congo Red. Inhibition was competitive with respect to acetyl-CoA and Ki values of 0.7 microM and 52 microM were determined for Cibacron F3GA and Coomassie Brilliant Blue respectively. The enzyme was strongly retained by columns of Affi-Gel Blue, which contains Cibacron F3GA linked to agarose. It was not eluted from this adsorbent in the presence of 10 mM-spermidine/0.5 M-NaCl/50 mM-Tris/HCl, pH 7.5, but was released by 1 mM-CoA in 10 mM-spermidine/50 mM-Tris/HCl, pH 7.5. These results are consistent with the presence in the enzyme of a dinucleotide fold that binds acetyl CoA and has a high affinity for Cibacron F3GA. The spermidine/spermine N1-acetyltransferase was irreversibly inactivated by exposure to butane-2,3-dione in sodium borate, pH 7.8, or by exposure to phenylglyoxal or camphorquinone-10-sulphonic acid. All of these reagents are known to interact with arginine residues in proteins under the conditions in which they inactivated the acetyltransferase. Inactivation was prevented by the presence of acetyl-CoA or CoA, but to a lesser extent by 3'-dephospho-CoA and not at all by NAD or adenosine. This protection suggests that an arginine residue at the active site is involved in the binding of the acetyl-CoA substrate. Treatment of the assay mixture but not the spermidine N1-acetyltransferase with alkaline phosphatase prevented the reaction taking place. This suggests that the apparent loss of enzyme activity in response to alkaline phosphatase reported by Matsui, Otani, Kamei & Morisawa [(1982) FEBS Lett. 150, 211-213] is due to dephosphorylation of the acetyl-CoA substrate and that the 3'-phosphate group is essential for activity.     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.     

Phorbol esters stimulate spermidine/spermine N1-acetyltransferase activity in mitogen-stimulated bovine lymphocytes

Phorbol 12-myristate-13-acetate (PMA) is shown to induce spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, in bovine lymphocytes. When PMA and phytohemagglutinin (PHA) were added simultaneously, the enzyme activity was stimulated synergistically. The ability of phorbol esters to stimulate the enzyme activity was consistent with their tumor-promoting ability. Phorbol, which is not a tumor promotor, was incapable of stimulating the enzyme activity. Phorbol diacetate weakly stimulated the activity of the acetylase. Phorbol dibutyrate had a similar stimulatory effect to PMA. These results suggest that the spermidine/spermine N1-acetyltransferase may play an important role in changes in polyamine levels in phorbol ester-treated cells and that the increase in the enzyme activity may have some relationship to the control of cell growth and differentiation by phorbol esters.     

Induction of spermidine/spermine N1-acetyltransferase by methylglyoxal bis(guanylhydrazone)

The anti-tumor agent methylglyoxal bis(guanylhydrazone) was found to be a competitive inhibitor of spermidine/spermine N1-acetyltransferase with a Ki of about 8 microM. Treatment of rats with this drug lead to a very large increase in the total amount of spermidine/spermine N1-acetyltransferase in liver, kidney and spleen. The total increase as measured using a specific antiserum amounted to 700-fold in liver and 100-fold in kidney within 18 h of treatment with 80 mg/kg doses. At least part of this induction was due to a pronounced increase in the half-life of the acetyltransferase which increased from 15 min to more than 12 h. The very large increase in the amount of the enzyme is likely to overwhelm the direct inhibition, and a net increase in the acetylation of polyamines by this enzyme would be expected to occur after treatment with methylglyoxal bis(guanylhydrazone). The acetylated polyamines are known to be rapidly degraded by polyamine oxidase producing putrescine. Direct evidence that a substantial part of the increase in the content of putrescine in the liver of rats treated with methylglyoxal bis(guanylhydrazone) occurs via the induction of this acetylase/oxidase pathway was obtained. These results indicate that methylglyoxal bis(guanylhydrazone) affects cellular polyamine levels not only by means of its inhibitory effect on S-adenosylmethionine decarboxylase and diamine oxidase but also by the induction of spermidine/spermine N1-acetyltransferase. They also raise the possibility that the enormous increase in this enzyme which occurs with higher doses may contribute to the very severe toxicity of methylglyoxal bis(guanylhydrazone).     

Increased activity of spermidine N1-acetyltransferase in the adrenal cortex of rats following administration of exogenous spermidine, carbamylcholine, 2-deoxyglucose and dopamine agonists

The activity of N1-acetyltransferase was increased in the dissected adrenal cortex of the rat following a single administration of spermidine. The activity was maximal 6-8 h after the onset of treatment. The increase in enzyme activity was abolished when the rats were given cycloheximide 2 h after spermidine; this suggests that increased activity results from an augmentation in the synthesis of the enzyme. Adrenocortical spermidine N1-acetyltransferase was also induced by carbamylcholine, 2-deoxyglucose, apomorphine and piribedil, drugs that are known to cause induction of ornithine decarboxylase in that organ. Hypophysectomized rats showed reduced activity of spermidine N1-acetyltransferase when compared to sham-operated controls, and carbamylcholine no longer elicited an increase in enzyme activity in such animals. Adrenocortical spermidine N1-acetyltransferase activity of hypophysectomized rats is induced by corticotropin (ACTH). These results suggest a hormonal control over the activity of the enzyme in the adrenal cortex with ACTH acting as a mediator.     

In vitro reactivation of in vivo ammonium-inactivated glutamine synthetase from Synechocystis sp. PCC 6803.

Glutamine synthetase from Synechocystis sp. strain PCC 6803 is inactivated by ammonium addition to cells growing with nitrate as the nitrogen source. The enzyme can be reactivated in vitro by different methods such as alkaline phosphatase treatment, but not phosphodiesterase, by raising the pH of the crude extract to values higher than 8, by increasing the ionic strength of the cell-free extract, or by preincubation with organic solvents, such as 2-propanol and ethanol. These results suggest that the loss of glutamine synthetase activity promoted by ammonium involves the non-covalent binding of a phosphorylated compound to the enzyme and support previous results that rule out the existence of an adenylylation/deadenylylation system functioning in the regulation of cyanobacterial glutamine synthetase.     

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine activities in rat spleen

Treatment of rats with the glucocorticoid dexamethasone causes an increase in the activity of cytosolic spermidine N1-acetyltransferase both in the spleen and thymus, but not, however, in liver, kidney or lung. The induced spermidine N1-acetyltransferase activity in the spleen catalyses acetylation of spermidine as well as spermine and sym-norspermidine, but not of diamines and histones. The enzyme induction depends on the dose of dexamethasone, and is suppressed by cycloheximide, which suggests that de novo protein synthesis is required for the action of this glucocorticoid. N1-acetylspermidine accumulates in the spleen after dexamethasone treatment, while spermidine progressively decreases and is partly converted into putrescine, the content of which transiently increases. In accordance with previous reports, dexamethasone was found to cause a rapid and large fall in the activity of spleen ornithine decarboxylase which was effected via the appearance of an inhibitor of the enzyme. Glucocorticoids exert large catabolic effects on lymphoid tissues, and further selectively affect the activities of spermidine N1-acetyltransferase and ornithine decarboxylase in the thymus and spleen. These latter selective responses may represent an important early event in lymphoid tissue response to glucocorticoid hormones.     

Occurrence and induction of spermidine-N1-acetyltransferase in Escherichia coli

Spermidine acetylase activity was detected in extracts prepared from $\text{Escherichia coli}$ and there was a marked increase in activity over the early period of growth. This increase reached a maximum 3 h after inoculation and was followed by an increase in ornithine decarboxylase activity. The acetylase was also able to use spermine as a substrate, but not putrescine. With spermidine and acetyl-CoA as substrate, the product formed was exclusively N¹-acetyl-spermidine. This is the first evidence for the occurrence in bacteria of spermidine-N¹-acetyltransferase, an enzyme which has previously been described in mammalian cells. These results suggest that acetylation of spermidine may be involved in the growth of $\text{Escherichia coli}$ and in the regulation of its polyamine content.     

Presence of two trehalose-6-phosphate synthase enzymes in Candida utilis

Abstract Total trehalose-6-phosphate synthase activity decreased in cell extracts from Candida utilis under conditions inducing activation of the regulatory trehalase by protein kinase catalysed phosphorylation. The synthase activity was reactivated by treatment with alkaline phosphatase revealing the presence of an enzyme whose activity is inactivated by reversible phosphorylation. The occurrence in the trehalose-6-phosphate synthase complex of a second synthase enzyme whose activity is not controlled by phosphorylation and dephosphorylation was demonstrated following gel filtration of cell extracts. The activity of the isolated enzymes was differently modified in vitro by the presence of alkaline phosphatase, ATP, glucose or protein kinase.     

The role of Zn(II) in calf intestinal alkaline phosphatase studied by the influence of chelating agents and chemical modification of histidine residues.

Alkaline phosphatase from calf intestine (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is reversibly inhibited at pH 8.0 by incubation with chelating agents. Complete reactivation may be achieved by stoichiometric addition of Zn2+. Atomic absorption spectrometry was used to demonstrate the linear correlation between Zn2+ content and degree of reactivation. The reversibly inhibited enzyme contained 1 Zn2+ per subunit whereas 2 Zn2+ were found in both the reactivated and the native enzyme. At more alkaline pH-values, inactivation by chelating agents becomes irreversible; under such conditions the inactivated alkaline phosphatase still contains 1 Zn2+ per subunit. The conformational changes resulting from the loss of Zn2+ and leading to irreversible inactivation were investigated by optical rotatory dispersion, immunological techniques, and ultraviolet and fluorescence spectroscopy. Azocoupling of the alkaline phosphatase with diazonium-1-H-tetrazole and Zn2+ content measurement of azocoupled enzyme probes indicated that 2 histidine residues per subunit are involved in binding of the catalytically important Zn2+.     

Genetically altered expression of spermidine/spermine N1-acetyltransferase affects fat metabolism in mice via acetyl-CoA

The acetylating enzyme, spermidine/spermine N1-acetyltransferase, participates in polyamine homeostasis by regulating polyamine export and catabolism. Previously, we reported that overexpression of the enzyme in cultured tumor cells and mice activates metabolic flux through the polyamine pathway and depletes the N1-acetyltransferase coenzyme and fatty acid precursor, acetyl-CoA. Here, we investigate this possibility in spermidine/spermine N1-acetyltransferase transgenic mice in which the enzyme is systemically overexpressed and in spermidine/spermine N1-acetyltransferase knock-out mice. Tissues of the former were characterized by increased N1-acetyltransferase activity, a marked elevation in tissue and urinary acetylated polyamines, a compensatory increase in polyamine biosynthetic enzyme activity, and an increase in metabolic flux through the polyamine pathway. These polyamine effects were accompanied by a decrease in white adipose acetyl- and malonyl-CoA pools, a major (20-fold) increase in glucose and palmitate oxidation, and a distinctly lean phenotype. In SSAT-ko mice, the opposite relationship between polyamine and fat metabolism was observed. In the absence of N1-acetylation of polyamines, there was a shift in urinary and tissue polyamines indicative of a decline in metabolic flux. This was accompanied by an increase in white adipose acetyl- and malonyl-CoA pools, a decrease in adipose palmitate and glucose oxidation, and an accumulation of body fat. The latter was further exaggerated under a high fat diet, where knock-out mice gained twice as much weight as wild-type mice. A model is proposed whereby the expression status of spermidine/spermine N1-acetyltransferase alters body fat accumulation by metabolically modulating tissue acetyl- and malonyl-CoA levels, thereby influencing fatty acid biosynthesis and oxidation.     

Stabilization of human serum alkaline phosphatase to histidine-induced heat inactivation by tryptic digestion.

1. Serum alkaline phosphatase [EC 3.1.3.1] was strongly inactivated by histidine during incubation at pH 8.0 and 45degrees; however, tryptic digestion of the serum strongly protected the enzyme against inactivation by histidine. In the absence of histidine, however, neither heat inactivation of the phosphatase nor the effect of trypsin [EC 3.4.21.4] was observed. Factors affecting the alkaline phosphatase inactivation were studied further. 2. The effect of trypsin on the histidine-induced heat inactivation differed considerably according to the tissue source of the enzyme, which suggests a possible method for distinguishing alkaline phosphatase isoenzymes.     

ACETYL-COENZYME A: 1,4-DIAMINOBUTANE N-ACETYLTRANSFERASE: ACTIVITY IN RAT BRAIN DURING DEVELOPMENT, IN EXPERIMENTAL BRAIN TUMOURS AND IN BRAINS OF FISH OF DIFFERENT METABOLIC ACTIVITY

—Acetyl-CoA: 1,4-diaminobutane N-acetyltransferase catalyses the first step of putrescine catabolism in mammalian brain. It may be important in putrescine degradation of other tissues as well. Its specific activity is higher in homogenates of immature than of mature rat brains. A steady decline of putrescine acetylase activity is observed from birth until approx adult levels are reached at day 30. Microsomes and purified nuclei from brains of 2-day-old rats show considerably higher putrescine acetylase activities than the corresponding subcellular organelles from adult brains. Increased putrescine acetylase activities were found in nitroso-ethylurea-induced gliomas, together with a dramatic increase of putrescine concentration. High tissue concentrations of putrescine are, however, not necessarily correlated with enhanced putrescine acetylase activities. In trout brains a linear increase of acetyl-CoA: 1,4-diaminobutane N-acetyltransferase activity was observed together with a decrease of putrescine concentration after adaptation of the animals to increased water temperature.     

The reversible inactivation of pig kidney alkaline phosphatase at low pH

1. Pig kidney alkaline phosphatase is inactivated by treatment with acid at 0°. 2. Inactivated enzyme can be partially reactivated by incubation at 30° in neutral or alkaline buffer. The amount of reactivation that occurs depends on the degree of acid treatment; enzyme that has been inactivated below pH3·3 shows very little reactivation. 3. Studies of the kinetics of reactivation indicate that the process is greatly accelerated by increasing temperature and proceeds by a unimolecular mechanism. The reactivated enzyme has electrophoretic and gel-filtration properties identical with those of non-treated enzyme. 4. The results can be best explained by assuming that a lowering of the pH causes a reversible conformational change of the alkaline phosphatase molecule to a form that is no longer enzymically active but is very susceptible to permanent denaturation by prolonged acid treatment. A reactivation mechanism involving sub-unit recombination seems unlikely.     

Purification and partial characterization of alkaline phosphatase of matrix vesicles from fetal bovine epiphyseal cartilage. Purification by monoclonal antibody affinity chromatography

Alkaline phosphatase of matrix vesicles isolated from fetal bovine epiphyseal cartilage was purified to apparent homogeneity using monoclonal antibody affinity chromatography. The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Tween 20 whereas the major portion of nonspecific protein was removed by this single step. Of various agents tested, 0.6 M 2-amino-2-methyl-1-propanol, pH 10.2, was the most effective in eluting 80-100% of the enzyme initially applied. Both Tween 20 and 2-amino-2-methyl-1-propanol associated with the eluted enzyme were effectively removed by the sequential application of DEAE-cellulose and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 81,000. This molecular weight was nearer reported values for rat liver (Ohkubo, A., Langerman, N., and Kaplan, M. M. (1974) J. Biol Chem. 249, 7174-7180) and porcine kidney (Cathala, G., Brunel, C., Chapplet-Tordo, D., and Lazdunski, M. (1975) J. Biol. Chem. 250, 6040-6045) alkaline phosphatase, than to previously reported values for chicken (Cyboron, G. W., and Wuthier, R. E. (1981) J. Biol. Chem. 256, 7262-7268) and fetal calf (Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1980) Calcif. Tissue Int. 30, 217-225) cartilage matrix vesicle alkaline phosphatase. The purified alkaline phosphatase was activated by micromolar Mg2+. The amino acid composition of cartilage alkaline phosphatase was found to be similar to that previously described for porcine kidney (Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141, 273-282). Double immunoprecipitation data indicated that monoclonal antibody against cartilage alkaline phosphatase cross-reacted with fetal bovine liver or kidney enzyme but failed to react with calf intestinal or rat cartilage enzyme. Thus these observations suggest that alkaline phosphatase of matrix vesicles from calcifying epiphyseal cartilage is a liver-kidney-bone isozyme.     

Purification and characterization of spermidine N1-acetyltransferase from chick duodenum

We have reported that spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in putrescine synthesis in chick duodenum induced by 1 alpha,25-dihydroxycholecalciferol (calcitriol) [Shinki, T., Kadofuku, T., Sato, T. and Suda, T. (1986) J. Biol. Chem. 261, 11712-11716]. In the present study, spermidine N1-acetyltransferase was purified from the duodenal cytosol of calcitriol-treated chicks to homogeneity judged by SDS/polyacrylamide gel electrophoresis. The purified enzyme converted spermidine only to N1-acetyl-spermidine. The apparent molecular mass of the purified spermidine N1-acetyltransferase was found to be 36 kDa by gel filtration on Sephacryl S-200 and 18 kDa by SDS/polyacrylamide gel electrophoresis. When duodenal crude 105,000 x g extracts were directly applied to a Sephacryl S-200 column without prior purification, three peaks with spermidine N1-acetyltransferase activity appeared. The first peak was in the void volume, the second peak was in the fraction corresponding to an apparent molecular mass of 70 kDa, and the third peak was in the fraction corresponding to 36 kDa. These results suggest that spermidine N1-acetyltransferase exists as a dimer of the 18 kDa subunits and is stabilized in (a) form(s) bound to other components or proteins in intact cells.     

Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of approximately 120 kDa that could be solubilized by phospholipase C or Polidocanol.     

Influence of reagents reacting with metal, thiol and amino sites of catalytic activity and l-phenylalanine inhibition of rat intestinal alkaline phosphatase

1. Studies on the inactivation of rat intestinal alkaline phosphatase by several metal-binding agents, namely EDTA, 8-hydroxyquinoline, pyridine-2,6-dicarboxylic acid, αα′-bipyridyl, o-phenanthroline and sodium cyanide, indicated the functional role of a metal, probably zinc, in the catalysis. The metal ligands lowered stereospecific uncompetitive inhibition of the enzyme by l-phenylalanine by an extent that paralleled the decline in enzyme activity. 2. The thiol reagents p-hydroxymercuribenzoate, iodoacetamide and iodine inactivated rat intestinal phosphatase. The enzyme could be protected from inactivation by either cysteine or substrate. The l-phenylalanine inhibition remained unchanged only in the presence of moderately inactivating concentrations of the thiol reagents. 3. Inactivation of the enzyme by the amino-group-blocking reagent, O-methylisourea, provided ample evidence for the participation in the catalysis of the -amino group of lysine. At the same time, l-phenylalanine inhibition remained unaltered even when the enzyme was strongly inactivated. This -amino-group-blocked enzyme exhibited no change in migration in starch gel, in contrast with enzyme treated with acetic anhydride, formaldehyde or succinic anhydride. The Michaelis constant of the enzyme was enhanced by such modifications, but the optimum pH remained the same. 4. d-Phenylalanine acted as a competitive or `co-operative' activator for intestinal alkaline phosphatase after it had been modified by acetylation.     

Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase

The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state. These structures reveal, in particular, that this first mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium fluoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40-fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the first phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (k(cat)), and explains the structural events associated with this artificial evolution.     

Interrelationship between metabolic and genetic regulation of alkaline and acid phosphatases in E. coli cells]

The effect of exogenous orthophosphate and mutations in regulatory genes of alkaline phosphatase on the level of nonspecific acid phosphatase was studied. The level of this enzyme as well as the level of alkaline phosphatase were shown to be regulated by exogenous orthophosphate being derepressed under phosphate starvation. The derepression of acid phosphatase is accompanied by more rapid secretion of enzyme from membranes to soluble fraction. Mutations in all the four regulatory genes decrease the level of enzyme in cells. Genes phoR and phoS, participating in regulation of alkaline phosphatase, are required for the derepression of acid phosphatase under the conditions of phosphate starvation.