The growth promoter spermine interacts specifically with dermatan sulfate regions that are rich inl-iduronic acid and possess antiproliferative activity

We have investigated interactions between spermine, a member of the growth promoting polyamine family, and various glycosaminoglycans. By using gel chromatography and equilibrium dialysis experiments we found that spermine binds tol-iduronic acid-rich dermatan sulfate (K _d, approximately 3.9×10^–4 M) with an affinity similar to that between spermine and DNA. By digesting spermine-dermatan sulfate complexes with chondroitin ABC lyase, the formation of oligosaccharide fragments (tetra-to-decasaccharides) was demonstrated by polyacrylamide gel electrophoresis. Chondroitin sulfate, which is deficient inl-iduronic acid, generates no spermine-protected fragments. Analysis of protected dermatan sulfate oligosaccharides indicates that the majority of thel-iduronic acid residue is non-sulfated and in a periodate-resistant conformation. The oligosaccharides also possess antiproliferative activity.     

Rectification of the Olfactory Cyclic Nucleotide-Gated Channel by Intracellular Polyamines

Polyamine-induced inward rectification of cyclic nucleotide-gated channels was studied in inside-out patches from rat olfactory neurons. The polyamines, spermine, spermidine and putrescine, induced an `instantaneous' voltage-dependent inhibition with K <sub>d</sub> values at 0 mV of 39, 121 μm and 2.7 mm, respectively. Hill coefficients for inhibition were significantly < 1, suggesting an allosteric inhibitory mechanism. The Woodhull model for voltage-dependent block predicted that all 3 polyamines bound to a site 1/3 of the electrical distance through the membrane from the internal side. Instantaneous inhibition was relieved at positive potentials, implying significant polyamine permeation. Spermine also induced exponential current relaxations to a `steady-state' impermeant level. This inhibition was also mediated by a binding site 1/3 of the electrical distance through the pore, but with a K _d of 2.6 mm. Spermine inhibition was explained by postulating two spermine binding sites at a similar depth. Occupation of the first site occurs rapidly and with high affinity, but once a spermine molecule has bound, it inhibits spermine occupation of the second binding site via electrostatic repulsion. This repulsion is overcome at higher membrane potentials, but results in a lower apparent binding affinity for the second spermine molecule. The on-rate constant for the second spermine binding saturated at a low rate (∼200 sec⁻¹ at +120 mV), providing further evidence for an allosteric mechanism. Polyamine-induced inward rectification was significant at physiological concentrations. Received: 17 February 1999/Revised: 27 April 1999     

Polyamine-phospholipid interaction probed by the accessibility of the phospholipidsn-2 ester bond to the action of phospholipase A2

Summary Conditions were used where the action of porcine pancreatic phospholipase A2 on phospholipids can be followed in the absence of added calcium and the catalytic activity is supported by the calcium brought with the nanomolar enzyme. Therefore, alterations in the enzyme velocity resulting from the presence of spermine or spermidine could be specifically studied using 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) and 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphoglycerol (PPHPG) as substrates. Both spermine and spermidine activated the hydrolysis of PPHPG fourfold at polyamine/phospholipid molar ratios of approximately 11 and 121, respectively. Double-reciprocal plots of enzyme activityvs. PPHPG concentration revealed the enhancement to be due to increased apparentV _max while the apparentK _m was slightly increased. In the presence of 4mm CaCl₂ inhibition by polyamines of PPHPG hydrolysis by phospholipase A2 was observed. Using synthetic diamines we could further demonstrate that two primary amino groups are required for the activation. In the absence of exogenous CaCl₂ polyamines inhibited the hydrolysis of PPHPC by phospholipase A2. The presence of 4mm CaCl₂ reversed this inhibition and a twofold activation was observed at 10 m spermine. The results obtained indicate that the activation of PLA2 by spermine and spermidine is produced at the level of the substrate, PPHPG. This implies the formation of complexes of phosphatidylglycerol and polyamines with defined stoichiometries.     

Novel isosteric charge-deficient spermine analogue—1,12-diamino-3,6,9-triazadodecane: synthesis, pK a measurement and biological activity

Ionic interactions are essential for the biological functions of the polyamines spermidine and spermine in mammalian physiology. Here, we describe a simple gram scale method to prepare 1,12-diamino-3,6,9-triazadodecane (SpmTrien), an isosteric charge-deficient spermine analogue. The protonation sites of SpmTrien were determined at pH range of 2.2–11.0 using two-dimensional ¹H-¹⁵N NMR spectroscopy, which proved to be more feasible than conventional methods. The macroscopic pK _a values of SpmTrien (3.3, 6.3, 8.5, 9.5 and 10.3) are significantly lower than those of 1,12-diamino-4,9-diazadodecane (spermine). The effects of SpmTrien and its parent molecule, 1,8-diamino-3,6-diazaoctane (Trien), on cell growth and polyamine metabolism were investigated in DU145 prostate carcinoma cells. SpmTrien downregulated the biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosyl-l-methionine decarboxylase and decreased intracellular polyamine levels, whereas the effects of Trien alone were minor. Interestingly, both SpmTrien and Trien were able to partially overcome growth arrest induced by an ODC inhibitor, α-difluoromethylornithine, indicating that they are able to mimic some functions of the natural polyamines. Thus, SpmTrien is a novel tool to influence polyamine interaction sites at the molecular level and offers a new means to study the contribution of the protonation of spermine amino group(s) in the regulation of polyamine-dependent biological processes.     

Michaelis constant (K m ) of acid phospatase as affected by montmorillonite,illite, and kaolinite clay minerals

The influence of Ca homoionic clay minerals (montmorillonite, illite, and kaolinite) on the activity,K _m , andV _m values of acid phosphatase was examined in model experiments. At each substrate (p-nitrophenyl phosphate) level tested, the addition of increasing amounts of clays (50, 100, and 150 mg, respectively) decreased the activity and increased theK _m value from 1.43×10^–3 m PNP (in the soluble state) to 82.3×10^–3M (montmorillonite), 8.02×10^–3 m (kaolinite), and 7.65×10^–3 m (illite) at the 150 mg level. The maximum enzyme reaction velocity (V _m ) remained nearly constant at different amounts of kaolinite and illite, but increased remarkably with rising quantities of montmorillonite. Apparently, the substrate affinity of sorbed acid phosphatase is significantly lower with montmorillonite than with kaolinite or illite. This may be ascribed to an intensive sorption of both substrate and enzyme to the surface as well as to interlattice sites of montmorillonite.     

Alkaline phosphatase isozymes in the midgut of silkworm: purification of high pH-stable microvillus and labile cytosolic enzymes

Summary Genetically defined alkaline phosphatase (ALP) isozymes from the larval midgut tissues ofBombyx mori were purified and characterized. The membrane-bound form (m-ALP) was solubilized with 1% Triton X-100, then purified by DEAE-Sephacel, Con A-Sepharose 4B and Ultrogel AcA 34 column chromatography. The soluble form (s-ALP) was purified by DEAE-Sephacel, epoxy Toyopearl coupled with phosphonic acid and Ultrogel AcA 34 column chromatography. About 840- and 650-fold purification were achieved for m-ALP and s-ALP, respectively, and both ALPs were homogeneous as judged by poly-acrylamide gel electrophoresis. Both forms were found to be similar (MW=68 000 in gel permeation chromatography, and a single subunit as a monomer in denaturing SDS-polyacrylamide gels with MW=58 000 for m-ALP and MW=61 000 for s-ALP). The pH optima of ALPs were shown to lie at 10.9 (m-ALP) and 9.8 (s-ALP), the former being extremely stable even in pH 10–12 which accords with the physiological milieu inBombyx midgut lumen. The K_m values of the m-ALP and s-ALP forp-nitrophenyl phosphate were 1.99 and 1.49 mM, respectively. Both ALPs had a similar substrate specificity.l-Cysteine strongly inhibited both ALPs, but inhibitory effects ofl-phenylalanine,l-homoarginine andl-leucine were undetectable for s-ALP and very weak for m-ALP. A comparison of enzymatic properties on two ALPs suggested that each isozyme plays different roles.Abbreviations m-ALP membrane-bound alkaline phosphatase - s-ALP soluble alkaline phosphatase     

Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method

Summary The indoxyl—tetranitro BT method for the demonstration of alkaline phosphatase activity has been optimized and its validity for quantitative histochemistry tested. The study has been performed with model films of polyacrylamide gel incorporating homogenate of rat liver and with cryostat sections from the same livers. Addition of polyvinyl alcohol to the incubation medium greatly improved the localization of the final reaction product in cryostat sections. In polyacrylamide films, the formazan production specifically due to alkaline phosphatase was highest when using a medium containing 100mm Tris-HCl buffer, pH 9.0, 0.2–1.0mm substrate, 0.32mm 1-methoxyphenazine methosulphate, 10mm MgCl₂, 5mm sodium azide and 1mm tetranitro BT. For the incubation of cryostat sections in the presence of polyvinyl alcohol, the same medium could be used but the optimum concentrations of substrate and tetranitro BT appeared to be 1–2mm and 5mm respectively. The test minus control reaction was specific for alkaline phosphatase activity and could be inhibited completely with tetramisole. The test minus control reaction was linear with time up to 30 min with model films and up to 15 min with cryostat sections. The formazan production was also linear with the amount of homogenate incorporated in model films and with section thickness up to 18 µm and therefore, the reaction obeyed the Beer—Lambert law. Variation of the substrate concentration yielded aK _M of 0.05mm for aqueous media and aK _M of 0.55mm for polyvinyl alcohol-containing media. The inhibition with tetramisole appeared to be competitive withK _i = 0.07mm for aqueous media andK _i = 0.7mm for polyvinyl alcohol-containing media. These values indicate that the indoxyl—tetranitro BT method is considerably more sensitive than any metal salt or diazonium salt method developed so far. It is concluded that the optimized method described here is a specific, sensitive and valid quantitative histochemical method for the demonstration of alkaline phosphatase activity.     

Effect of l-arginine on metabolism of polyamines in rat’s brain with extrahepatic cholestasis

Cholestatic encephalopathy results from accumulation of unconjugated bilirubin and hydrophobic bile acids in the brain. The aim of this study was to determine disturbances of polyamine metabolism in the brains of rats with experimental extrahepatic cholestasis and the effects of l-arginine administration. Wister rats were divided into groups: I: sham-operated, II: rats treated with l-arginine, III: animals with bile-duct ligation (BDL), and IV: cholestatic-BDL rats treated with l-arginine. Increased plasma γ-glutamyltransferase and alkaline phosphatase activity and increased bile-acids and bilirubin levels in BDL rats were reduced by administration of l-arginine (P < 0.001). Cholestasis increased the brain’s putrescine (P < 0.001) and decreased spermidine and spermine concentration (P < 0.05). The activity of polyamine oxidase was increased (P < 0.001) and diamine oxidase was decreased (P < 0.001) in the brains of BDL rats. Cholestasis increased the activity of arginase (P < 0.05) and decreased the level of citrulline (P < 0.001). Administration of l-arginine in BDL rats prevents metabolic disorders of polyamines and establishs a neuroprotective role in the brain during cholestasis.     

Properties of fructose-1,6-diphosphate phosphatase and fructose-1,6-diphosphate aldolase fromPseudomonas putida

The fructose-1,6-P₂ (FDP) phosphatase, (FDPase) and FDP aldolase fromPseudomonas putida were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. Michaelis-Menten kinetics were observed with, respect to FDP in both FDPase and FDP aldolase. TheK _m for FDP at pH 8.0 was 1.2×10⁻⁵M for FDPase and 3.0×10⁻⁵M for FDP aldolase. The specific activities of these two enzymes (assayed under optimal conditions in cell-free extracts ofP. putida grown ond-fructose), as well as their kinetic properties, are consistent with the suggestion that during growth ond-fructose most, of the FDP generated is converted to fructose-6-P (F-6-P), which is subsequently utilized via the Entner-Doudoroff pathway (EDP).     

Pathways of d-fructose catabolism in species of Pseudomonas

Cell-free extracts of d-fructose grown cells of Pseudomonas putida, P. fluorescens, P. aeruginosa, P. stutzeri, P. mendocina, P. acidovorans and P. maltophila catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose and contained 1-P-fructokinase activity suggesting that in these species fructuse-1-P and fructose-1,6-P₂ were intermediates of d-fructose catabolism. Neither the 1-P-fructokinase nor the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose was present in significant amounts in succinate-grown cells indicating that both activities were inducible. Cell-free extracts also contained activities of fructose-1,6-P₂ aldolase, fructose-1,6-P₂ phosphatase, and P-hexose isomerase which could convert fructose-1,6-P₂ to intermediates of either the Embden-Meyerhof pathway or Entner-Doudoroff pathway. Radiolabeling experiments with 1-¹⁴C-d-fructose suggested that in P. putida, P. aeruginosa, P. stutzeri, and P. acidovorans most of the alanine was made via the Entner-Doudoroff pathway with a minor portion being made via the Embden-meyerhof pathway. An edd ^- mutant of P. putida which lacked a functional Entner-Doudoroff pathway but was able to grow on d-fructose appeared to make alanine solely via the Embden-Meyerhof pathway.Non-Standard Abbreviations cpm counts per min - edd ^- mutant lacking Entner-Doudoroff dehydrase (6-PGA dehydrase) - EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P₂ - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTs PEP: d-fructose phosphotransferase system - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate     

On the turnover of polyamines spermidine and spermine in mouse brain and other organs

The apparent biological half-lives of spermidine and spermine in mouse brain and other organs were determined by measurement of the specific radioactivities of these compounds over long periods of time. The endogenous polyamine pools were labeled by repeated intraperitoneal injections of [1,4-¹⁴C]putrescine·2HCl, [2-¹⁴C]d,l-methionine, [2-³H]l-methionine, andS-adenosyl-[2-³H]l-methionine. Repeated injections were given to ensure labeling of both fast and slow polyamine pools. It was shown that the two parts of the polyamine molecules which derive from ornithine and methionine have significantly different life spans, especially in the brain. Actual turnover rates of polyamines could not be determined because of the active interconversion between spermine and spermidine, and between spermidine and putrescine. The observed reutilization of putrescine originating from spermidine degradation for spermidine biosynthesis, and the analogous reutilization of spermidine in spermine biosynthesis is discussed with respect to its physiological significance and its relationship to cellular organization.     

Developmental changes in mouse brain polyamine metabolism

Mouse brain ornithine decarboxylase (ODC) activity is high at the time of birth, whereas S-adenosyl-l-methionine decarboxylase (SAM-DC) activity is low. ODC activity, and putrescine, spermidine and spermine concentrations decline rapidly during postnatal development to the low level characteristic of mature brains, while SAM-DC activity behaves in the opposite manner. The fluctuations in mouse brain polyamine metabolism are in accord with those found in the rat. The apparentK _m values of ODC and SAM-DC for their substrates decline parallel with the decrease of substrate and product concentrations during ontogeny suggesting substrate and/or product dependent regulation of polyamine synthesis in the developing brain.     

An Effect of Ca2+ on the Intrinsic Cl−-conductance of Rat Kidney Cortex Brush Border Membrane Vesicles

Brush-border membrane vesicles (BBMV) were prepared from superficial rat renal cortex by a divalent²⁺-precipitation technique using either CaCl₂ or MgCl₂. The dependence of the initial [¹⁴C]-d-glucose (or [³H]-l-proline) uptake rate and the extent of the overshoot of d-glucose or l-proline uphill accumulation from solutions containing 100 mm Na⁺ salt, was found to be dependent upon the precipitating divalent cation. With Mg²⁺ precipitation the initial uptake and overshoot accumulation of either d-glucose or l-proline were enhanced compared to BBMV prepared by Ca²⁺ precipitation. When the anion composition of the media was varied (uptake in Cl⁻ media in comparison to gluconate⁻-containing media) it was found that the Cl⁻-dependent component of the initial uptake was markedly depressed with Ca²⁺-prepared BBMV (104.99 ± 33.31 vs. 13.83 ± 1.44 pmoles/sec/mg protein for Mg²⁺ and Ca²⁺ prepared vesicles respectively). When Ca²⁺ was loaded into Mg²⁺ prepared BBMV using a freeze-thaw technique, it was found that the magnitude and Cl⁻ enhancement of d-glucose transport was reduced in a dose-dependent manner. Neomycin, an inhibitor of phospholipase C, had no effect on the reduction of d-glucose uptake by Ca²⁺ in Mg²⁺ prepared vesicles. In contrast, phosphatase inhibitors such as vanadate and fluoride were able to partially reverse the Ca²⁺ inhibition of d-glucose uptake and restore the enhancement due to Cl⁻ media. In addition, inhibitors of protein phosphatase 2B, deltamethrin (50 nm) and trifluoperazine (10 μm), caused partial reversal of Ca²⁺-dependent inhibition of d-glucose uptake. Direct measurement of changes in the bi-ionic (Cl⁻ vs. gluconate⁻) transmembrane electrical potential differences using the cyanine dye, 3,3′-dipropylthiodicarbocyanine iodide DiSC₃-(5) confirmed that Cl⁻ conductance was reduced in Ca²⁺-prepared vesicles. We conclude that a Cl⁻ conductance coexists with Na⁺ cotransport in rat renal BBMV and this may be subject to negative regulation by Ca²⁺ via stimulation of protein phosphatase (PP2B). Received: 14 December 1994/Revised: 27 November 1995     

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles

Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H⁺ gradient (pH_in=6.0, pH_out=8.0)²²Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pH_in=pH_out=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K ₁ =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K⁺-stimulatedp-nitrophenyl phosphatase. In addition²²Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H⁺ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK _M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na⁺/H⁺ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.     

Cellular compartmentation of photosynthetic and photorespiratory enzymes in the heterocystous cyanobacterium Anabaena cylindrica

Activities of enzymes of photosynthesis and photorespiration have been measured in extracts of vegetative cells and heterocysts from the filamentous cyanobacterium Anabaena cylindrica. Phosphoribulokinase, d-ribulose 1,5-bisphosphate carboxylase/oxygenase, phosphoglycollate phosphatase and glycollate dehydrogenase activities were readily measured in vegetative cell extracts, but were undetectable or negligible in heterocyst preparations. The data help to explain why heterocysts are unable to perform photosynthetic CO₂ fixation. They also exemplify the co-ordinate compartmentation of enzymes of photosynthesis and photorespiration which occur in a differentiated phototrophic prokaryote.Abbreviations Ru5P d-ribulose 5-phosphate - RuBP d-ribulose 1,5-bisphosphate - DCPIP 2,6-dichlorophenolindophenol - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulphonate     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum

Summary Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a K_M for putrescine of 2.58×10⁻⁶ m and a V_max of 0.53 nmol per hr per 50 μl serum. Aminoguanidine competitively inhibited the enzyme with a K_I of 1.8×10⁻⁸ m. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The V_max for the ABS putrescine oxidase was 2.10 nmol per hr per 50 μl serum, and the K_M for putrescine, 50.3×10⁻⁶ m. The K₁ of the ABS putrescine oxidase for aminoguanidine was 41×10⁻⁶ m. On the basis of both the K_M and K_I values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56°C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed. This work was supported in part by a grant from the Cystic Fibrosis Foundation.     

Na-dependent <Emphasis Type="SmallCaps">D</Emphasis>-Glucose Transport by Intestinal Brush Border Membrane Vesicles from Gilthead Sea Bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

Brush border membrane vesicles (BBMV) enriched in sucrase, maltase and alkaline phosphatase, and impoverished in Na⁺-K⁺-ATPase, were isolated from proximal and distal intestine of the gilthead sea bream (Sparus aurata) by a MgCl₂ precipitation method. Vesicles were suitable for the study of the characteristics of D-glucose apical transport. Only one D-glucose carrier was found in vesicles from each intestinal segment. In both cases, the D-glucose transport system was sodium-dependent, phlorizin-sensitive, significantly inhibited by D-glucose, D-galactose, α-methyl-D-glucose, 3-O-methyl-D-glucose and 2-deoxy-D-glucose, and showed stereospecificity. Apparent affinity constants of D-glucose transport (K_t) were 0.24 ± 0.03 mM in proximal and 0.18 ± 0.03 mM in distal intestine. Maximal rate of influx (Jmax) was 47.3 ± 2.2 pmols. mg⁻¹ protein for proximal and 27.3 ± 3.6 pmols. mg⁻¹ protein for distal intestine. Specific phlorizin binding and relative abundance of an anti-SGLT1 reactive protein were significantly higher in proximal than in distal BBMV. These results suggest the presence of the same D-glucose transporter along the intestine, with a higher density in the proximal portion. This transporter is compatible with the sodium-dependent D-glucose carrier described for other fish and with the SGLT1 of higher vertebrates.This revised version was published online in June 2005 with a corrected cover date.     

Cytotoxicity of polyamines to Amoeba proteus: Role of polyamine oxidase

It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H₂O₂, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N ¹-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N ¹-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3–20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid ( 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity ( 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.Abbreviations DAO diamine oxidase - DFMO DL--difluoromethylornithine - DP 1-3-diaminopropane - IC₅₀ 50% inhibition concentration - MAO monoamine oxidase - N ¹-ACSP; N ¹-acetylspermine - N¹-ACSPD N ¹-acetylspermidine - N ⁸-ACSPD N ⁸-acetylspermidine - ODC ornithine decarboxylase - PAO(s) polyamine oxidase(s) - PUT putrescine - SP spermine - SPD spermidine     

Interaction of spermine and DNA

The effect of spermine upon the denaturation temperature (T_m) of DNA's of various base compositions has been found to depend upon both the base composition of the DNA and the pH of the solution. Measurement of the hydrogen ion titration curve of spermine as a function of temperature reveals that the net charge of the spermine molecule is undergoing a rapid change with temperature in the range of temperatures at which DNA denatures. Since the value of T_m depends upon base composition, the correlation of the effect of spermine upon T_m with the base composition of the DNA used may be explainable in terms of the changing degree of ionization of spermine. The binding of spermine to native DNA has also been studied by dialysis equilibrium. There is no significant variation either in the number of strongly binding sites or strength of binding with base composition. It is concluded that there is no evidence of correlation between the binding of spermine and the base composition of DNA.     

Rat osseous plate alkaline phosphatase: mechanism of action of manganese ions

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by manganese ions in a similar way as by zinc ions. For concentrations up to 1.0 nm, the enzyme was stimulated by manganese ions, showing site-site interactions (n = 2.2). However, larger concentrations (> 0.1 m) were inhibitory. Manganese ions could play the role of zinc ions stimulating the enzyme synergistically in the presence of magnesium ions (K _d = 7.2 m; V = 1005.5 U mg^–1). Manganese ions could also play the role of magnesium ions, stimulating the enzyme synergistically in the presence of zinc ions (K _d = 2.2 m; V = 1036.7 U mg^–1). However, manganese ions could not substitute for zinc and magnesium at the same time since ion assymetry is necessary for full activity of the enzyme. A steady-state kinetic model for the modulation of enzyme activity by manganese ions is proposed.