Effect of Transjunctional KCl Gradients on the Spermine Inhibition of Connexin40 Gap Junctions
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Dissociation constant
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The effect of spermine (50-400 microM) on the Ca-transporting system of brain mitochondria was studied. In a medium containing Mg2+ and ATP, spermine facilitates the accumulation of Ca2+ by decreasing Km of the uniporter. Spermine inhibits Na-stimulated Ca2+ efflux; this effect is dependent on the ionic strength of the medium--it is decreased when KCl concentration is increased from 20 to 120 mM. Spermine (200 microM) decreases (by 50%) the steady state concentration of Ca2+ maintained by mitochondria. The importance of spermine as a regulator of Ca2+-transport in brain mitochondria is discussed.
Uniporter
Efflux
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Bioorganic chemistry
Plant biochemistry
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The binding of NAD+ and of its fluorescent analogue, nicotinamide 1,N6-ethenoadenine dinucleotide, to glyceraldehyde-3-phosphate dehydrogenase purified from the muscles of young and old rats was studied in detail. Binding of the natural coenzyme was followed both by spectrophotometric titration of the extrinsic absorption band of the enzyme-NAD+ complex and from the degree of quenching of fluorescence of the protein. Binding of the coenzyme analogue was monitored by using the large enhancement in its fluorescence upon forming the complex with the enzyme. Both dinucleotides showed strong negative cooperativity in binding to the enzyme, similar to that displayed in their association with the rabbit muscle enzyme. The enzyme purified from old rats displayed a markedly reduced affinity toward the two dinucleotides, compared with the enzyme isolated from young animals. The various dissociation constants of both dinucleotides from the enzyme from young rats were remarkably similar to the corresponding constants in the rabbit muscle enzyme. The degree of negative cooperativity (i.e., the ratio between the dissociation constants from high- and low-affinity binding sites) in the "young" and "old" enzyme forms was not very different. It was concluded from these results that while modifications in the subunits take place upon aging, the intersubunit interaction is not significantly affected. Increasing concentrations of ATP were found to cause a gradual decrease in the negative cooperativity of NAD+ binding, which completely disappeared in the presence of 5 mM ATP. The observation that all four binding sites of the old enzyme display the same affinity toward NAD+ when the negative cooperativity is removed excludes the possibility that this enzyme form is a mixture of native and inactive species. The different dissociation constants of NAD+ from young and old enzyme forms in the presence of 5 mM ATP also demonstrate the occurrence of age-related modifications in the structure of the individual subunits.
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Glyceraldehyde 3-phosphate dehydrogenase
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Polyamine
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Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell-cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions.
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Preincubation with spermine, of λ T 7 and P 465 phages which were sensitive to oxidized spermine, resulted in a decrease of their susceptibility to the action of oxidized spermine. Phages resistant to oxidized spermine such as T 4 and ϕX 174 became susceptible to this agent after dialysis. The mechanism of phagocidal action of oxidized spermine was examined with 32P-labelled λ phage. Oxidized spermine interfered neither with the absorption of λ phage, nor with the injection of its DNA into the host cells. The injected DNA, however, did not lead to the formation of mature phage. The interaction of oxidized spermine with the DNA of phages T 4 and T 7 was investigated by thermal denaturation studies. DNA treated with oxidized spermine showed the same Tm as untreated DNA. However, the treated DNA was decreased in its hyperchromicity and was renatured to a great extent, even after rapid cooling. These facts are explained by the formation of cross-links which prevents the separation of complementary DNA strands.
Hyperchromicity
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Previous studies have shown that the feeding of putrescine, a biogenic amine and the precursor of the mammalian polyamines, can promote whole-body growth of chicks. The current study was undertaken to determine the effect of spermine, also a biogenic amine and the most cationic of the polyamines, under similar conditions. In Exp. 1, 120 week-old chicks were fed purified crystalline amino acid-based diets containing 0, .2, .4, .6, .8, or 1.0% spermine for 14 d. Spermine proved highly toxic and growth rates were reduced compared with controls when even .2% was fed. In Exp. 2, chicks were fed 0, .0375, .0750, or .1000% spermine. These concentrations proved less toxic than those used in Exp. 1. Supplemental dietary cysteine was then provided at 0, .3, .6, and .9% together with 0, .025, .050, or .400% spermine (Exp. 3) because depletion of cellular glutathione has been suggested as contributing to spermine's toxicity. Even high levels of cysteine supplementation did not overcome spermine's toxicity. Subsequent dietary provision of L-2-oxothiazolidine-4-carboxylic acid (OTC, Exp. 4), a cysteine prodrug, showed that depletion of cellular glutathione was not likely a cause of spermine toxicosis. A trend toward increased weight gain and feed efficiency was observed when low concentrations of spermine were fed. It was concluded, however, that dietary spermine was more toxic to chicks than was previously seen for putrescine, that any growth-promoting effects of dietary spermine are small, and that supplements of dietary cysteine or OTC are unlikely to increase these effects by overcoming spermine toxicosis.
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At the spermine concentration to cover the number of the binding site of spermine 0.016 per nucleotide, the Hill coefficient of the ethidium binding to the calf thymus DNA was 1.7, while the value was 0.38 in the absence of the spermine. On the basis of the data, together with other present data on the viscometric titration of the DNA with spermine and anomalous absorbance-temperature profile at 260nm and viscosity-temperature profile, it can be speculated that allosteric propagation of the conformational transition induced by the binding of the spermine may be involved in the monomolecular collapse of the DNA to a condensed structure.
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Cooperativity
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Dissociation constant
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Spermine is often the most abundant polyamine in human tumors such as breast carcinomas. However, its specific role in tumor biology is still uncertain, since inhibitors of ornithine decarboxylase such as alpha-difluoromethylornithine depress cell growth while leaving spermine content mostly unaffected. We have assessed the specific role of spermine in breast cancer cell growth using N-cyclohexyl-1,3-diaminopropane (C-DAP), a potent spermine synthase inhibitor. In ZR-75-1 cells, C-DAP decreased net cell growth after 14 days by 65% at 50 microm, with an IC50 of about 5 microM, and was about 10 times more potent than N-(n-butyl)-1,3-diaminopropane, another spermine synthase inhibitor. C-DAP acted as a specific inhibitor of spermine biosynthesis, since (a) it depleted spermine content while causing an equal or greater accumulation of spermidine on a molar basis, (b) it rapidly induced S-adenosylmethionine decarboxylase activity and the accumulation of its products due to relief of spermine-dependent inhibition of enzyme expression, and (c) exogenous spermine (1 microM) completely reversed C-DAP-induced growth inhibition. C-DAP and related compounds were accumulated, at least in part, through a mechanism distinct from the polyamine transport system, while also blocking putrescine and spermidine uptake with various potencies. Reversibility of C-DAP-induced growth inhibition by exogenous spermine was progressively lost on prolonged treatment, in association with marked morphological changes. In 4 different human breast cancer cell lines (ZR-75-1, T47-D, MCF-7, and MDA-MB-231), relative growth sensitivity to C-DAP was inversely related to the extent of spermidine accumulation caused by spermine synthase inhibition, suggesting that spermidine overaccumulation can functionally replace spermine. Interestingly, C-DAP strongly potentiated growth inhibition caused by alpha-difluoromethylornithine in all cell lines tested by preventing conversion of residual spermidine to spermine, indicating that spermine synthesis limits alpha-difluoromethylornithine action and that under some critical threshold, spermidine cannot fulfill cellular needs for spermine. Thus, spermine plays specific and important functions in breast tumor growth, and spermine synthase inhibitors could markedly improve the therapeutic effectiveness of existing polyamine depletion strategies, especially in spermine-rich tumors.
Polyamine
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