Role of substrates in Sr2+-induced oscillations of ionic fluxes in rat liver mitochondria

(1) The reason for substrate specificity of Sr²⁺-induced oscillating cation fluxes in isolated rat liver mitochondria was investigated. (2) With succinate as substrate, rotenone prevented oscillation. In this case the mitochondria were only partially able to take up added Sr²⁺ and did not take up any of the released K⁺. Addition of substances decreasing the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio (oxaloacetate or acetoacetate) restored the ability for reuptake of K⁺ and for complete uptake of Sr²⁺ and, therefore, oscillation. (3) Inhibition of substrate-level phosphorylation by arsenite or uncoupling of substrate-level phosphorylation by arsenate in the presence of oligomycin also suppressed the reuptake of cations. This effect of inhibition of substrate-level phosphorylation on oscillation could be circumvented by addition of ATP in the presence of oligomycin. (4) Prevention of the intramitochondrial regeneration of 2-oxoglutarate from acetyl-CoA and oxaloacetate by fluorocitrate or from endogenous glutamate by aminoxyacetate shortened the time during which oscillation with succinate as substrate could be observed. (5) From the key role of substrate level phosphorylation it is concluded that for the reuptake of K⁺ and Sr²⁺ during oscillation, sufficient GTP generation by the succinyl thiokinase (EC 6.2.1.4) reaction is essential. Therefore substrate level phosphorylation seems to be a necessary energy source additional to the respiratory chain. Since the latter process drives the active cation movements, the former may be required for the restoration of a sufficiently low proton conductance of the mitochondrial inner membrane. Oscillation in the absence of exogenous ATP therefore demands 2-oxoglutarate as substrate or the intramitochondrial generation of 2-oxoglutarate for the maintenance of a sufficient GTP production for a longer time.     

The stoichiometry of ion fluxes during Sr2+-induced oscillations in mitochondria

A quantitative study of H⁺, K⁺, Sr²⁺ and succinate fluxes in Sr²⁺-induced oscillatory state of rat liver mitochondria is presented. It was shown that oscillation of succinate content in mitochondria occurs synchronously with oscillations of the cation fluxes. Total charge transferred across the membrane by the registered cations and the succinate-anion is equal to zero. Passive H⁺-influx has been calculated at all stages of the oscillatory cycle. The conclusion is made that electroneutral 2 H⁺/Sr²⁺ exchange is periodically induced in mitochondria. A value of (2 ± 0.2) · 10^-7 mol Sr²⁺/min per mg protein. has been determined for Sr²⁺ by this type of exchange.     

Exploring the antidiabetic and anti-obesity properties of Samanea saman through in vitro and in vivo approaches

In recent years, diabetes and obesity have become a major problem in global health care because of changes in lifestyle, food habits, and age-related metabolic disorders. Diabetes mellitus is one of the most common diseases, affecting millions of people worldwide. Currently, herbal drugs are used to control obesity and diabetes. The present study investigates the anti-obesity, antidiabetic, and antioxidant activities of Samanea saman leaf extract. A methanolic extract of S. saman leaves was prepared by a maceration method. The S. saman leaf extract was studied for its inhibitory effect on glucose utilization using specific in vitro procedures to analyze its antioxidant, anti-obesity, and antidiabetic activities via different assays, such as α-amylase and α-glucosidase inhibition assay, glucose uptake by yeast cells, nonenzymatic glycosylation assay followed by glucose diffusion assay. The outcome of the study showed that the methanolic extract strongly inhibited the pancreatic lipase, α-amylase, and glucosidase activities, compared with the standard drug. The results showed that the extract possessed considerable antioxidant and antidiabetic activities, and further studies are needed to confirm the results using an in vivo model. Thus, it is proposed that S. saman can be used as a therapeutic agent.     

The pattern of glycosyl- and sulfotransferase activities in cancer cell lines: a predictor of individual cancer-associated distinct carbohydrate structures for the structural identification of signature glycans

Carbohydrate chains of cancer glycoprotein antigens contain major outer changes dictated by tissue-specific regulation of glycosyltransferase genes, the availability of sugar nucleotides, and competition between enzymes for acceptor intermediates during glycan elongation. However, it is evident from recent studies with recombinant mucin probes that the final glycosylation profiles of mucin glycoproteins are mainly determined by the cellular repertoire of glycosyltransferases. Hence, we examined various cancer cell lines for the levels of fucosyl-, beta-galactosyl, beta-N-acetylgalactosaminyl-, sialyl-, and sulfotransferase activities that generate the outer ends of the oligosaccharide chains. We have identified glycosyltransferases activities at the levels that would give rise to O-glycan chains as reported by others in breast cancer cell lines, T47D, ZR75-1, MCF-7, and MDA-MB-231. Most breast cancer cells express Gal-3-O-sulfotransferase specific for T-hapten Gal beta1-->3GalNAc alpha-, whereas the enzyme from colon cancer cells exhibits a vast preference for the Gal beta1,4GlcNAc terminal unit in O-glycans. We also studied ovarian cancer cells SW626 and PA-1 and hepatic cancer cells HepG2. Our studies show that alpha1,2-L-fucosyl-T, alpha(2,3) sialyl-T, and 3-O-Sulfo-T capable of acting on the mucin core 2 tetrasaccharide, Gal beta1,4GlcNAc beta1,6(Gal beta1,3)GalNAc alpha-, can also act on the Globo H antigen backbone, Gal beta1,3GalNAc beta1,3Gal alpha-, suggesting the existence of unique carbohydrate moieties in certain cancer-associated glycolipids. Briefly, our study indicates the following: (i) 3'-Sulfo-T-hapten has an apparent relationship to the tumorigenic potential of breast cancer cells; (ii) the 3'-sulfo Lewis(x), the 3-O-sulfo-Globo unit, and the 3-fucosylchitobiose core could be uniquely associated with colon cancer cells; (iii) synthesis of a polylactosamine chain and T-hapten are favorable in ovarian cancer cells due to negligible sialyltransferase activities; and (iv) a 6'-sialyl LacNAc unit and 3'-sialyl T-hapten appear to be prevalent structures in hepatic cancer cell glycans. Thus, it is apparent that different cancer cells are expressing unique glycan epitopes, which could be novel targets for cancer diagnosis and treatment.     

The ADP- and Mg2+-reactive calcium complex of the phosphoenzyme in skeletal sarcoplasmic reticulum Ca2+-ATPase

The effects of ATP on Ca²⁺ binding in the absence of added Mg²⁺ to the purified sarcoplasmic reticulum Ca²⁺-ATPase were studied at pH 7.0 and 0°C. ATP increased the number of Ca²⁺-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca²⁺ binding was 4·10⁵ M^?1, which was not significantly different from that obtained in the absence of ATP. AdoP[CH₂]PP has little effect on the Ca²⁺-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca²⁺ binding. ADP decreased the level of ATP-induced Ca²⁺ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca²⁺ binding exists in the form of an ADP-reactive phosphoenzyme·Ca complex. In addition, the Ca²⁺ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl₂; after the release of Ca²⁺, the phosphoenzyme decayed. These observations suggest that Mg²⁺, added after the ATP-induced Ca²⁺-binding process, may replace the Ca²⁺ on the phosphoenzyme and initiate phosphoenzyme decomposition.     

Inhibition of ruthenium red-induced Ca2+ efflux from liver mitochondria by the antibiotic X-537A

It has been reported (Becker, G.L., Fiskum, G. and Lehninger, A.L. (1980) J. Biol. Chem. 255, 9009-9012) that respiring rat liver mitochondria suspended in KC1 medium containing ATP, Mg2+ and phosphate, maintain a steady state extramitochondrial free Ca2+ concentration of about 0.5 microM. The results reported here show that the addition of the antibiotic X-537A, at concentrations far below those required for ionophorous activity, caused a perturbation in this steady state, lowering the extramitochondrial free Ca2+ concentration by about 0.20 microM. This shift in steady state was clarified by a study of X-537A inhibition of the Ca2+ efflux induced by ruthenium red; a half-maximum effect was observed at approximately 25 nM X-537A. No effect on Ca2+ transport through the influx uniporter was observed. The possibility of a generalized stabilizing action of the antibiotic on the mitochondrial membrane seems to be ruled out by its effectiveness at very low concentrations.     

Quantitative evaluation of respiration induced metabolic oscillations in erythrocytes

The changes in the partial pressures of oxygen and carbon dioxide (P_O2 and P_CO2) during blood circulation alter erythrocyte metabolism, hereby causing flux changes between oxygenated and deoxygenated blood. In the study we have modeled this effect by extending the comprehensive kinetic model by Mulquiney and Kuchel [P.J. Mulquiney, and P.W. Kuchel. Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement, Biochem. J. 1999, 342, 581–596.] with a kinetic model of hemoglobin oxy-/deoxygenation transition based on an oxygen dissociation model developed by Dash and Bassingthwaighte [R. Dash, and J. Bassingthwaighte. Blood HbO₂ and HbCO₂ dissociation curves at varied O₂, CO₂, pH, 2,3-DPG and temperature levels, Ann. Biomed. Eng., 2004, 32(12), 1676–1693.]. The system has been studied during transitions from the arterial to the venous phases by simply forcing P_O2 and P_CO2 to follow the physiological values of venous and arterial blood. The investigations show that the system passively follows a limit cycle driven by the forced oscillations of P_O2 and is thus inadequately described solely by steady state consideration. The metabolic system exhibits a broad distribution of time scales. Relaxations of modes with hemoglobin and Mg²⁺ binding reactions are very fast, while modes involving glycolytic, membrane transport and 2,3-BPG shunt reactions are much slower. Incomplete slow mode relaxations during the 60 s period of the forced transitions cause significant overshoots of important fluxes and metabolite concentrations – notably ATP, 2,3-BPG, and Mg²⁺. The overshoot phenomenon arises in consequence of a periodical forcing and is likely to be widespread in nature – warranting a special consideration for relevant systems.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

Kinetic characterization and substrate requirement for the Ca2+ uptake system in platelet membrane

An ATP-dependent mechanism for Ca²⁺ uptake in human platelet membrane fractions has been identified and characterized. Ca²⁺ uptake into a membrane fraction is shown to be stimulated at low concentrations of ATP and Ca²⁺ and to require magnesium ions. Initial rate kinetics, using Eadie-Scatchard analysis, indicated a single class of calcium uptake sites in the presence of ATP, with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for free [Ca²⁺] of 0.145 μM. Ca²⁺ uptake in the presence of several ATP concentrations demonstrates that ATP binds to at least two sites, representing high and low affinities of 3.21 and 80.1 μM, respectively. The neuroleptic drug fluphenazine inhibited ATP-stimulated calcium uptake ($\text{IC}{}_{50}\text{= 55 μ}\text{M}$), suggesting this ATP-dependent Ca²⁺ uptake system may provide a useful ion-transport model with which to study neuroleptic therapy in humans.     

HgCl2 increases the methemoglobin prooxidant activity. Possible mechanism of Hg2+-induced lipid peroxidation in erythrocytes

In an attempt to elucidate the mechanism of initiation of peroxidation in HgCl2-treated erythrocytes, the effect of HgCl2 on methemoglobin-catalyzed lipid peroxidation was studied. It was found that HgCl2 reinforces the prooxidant action of methemoglobin. This effect seems not to be due to dissociation or degradation of the hemoglobin molecule to heme-containing fragments or iron-containing products of low molecular weight. The results obtained indicate that Hg2+ increases the binding of oxy- and methemoglobin to liposomes. A suggestion is made that the acceleration of methemoglobin-catalyzed peroxidation by HgCl2 is mainly due to increased binding of methemoglobin to liposomes. On the basis of these results and the results obtained previously the possible mechanism of initiation of peroxidation in Hg2+-treated erythrocytes is discussed.     

Possible participation of membrane thiol groups on the mechanism of NAD(P)+-stimulated Ca2+ efflux from mitochondria

The in vitro opioid activities of a series of leucine enkephalin analogs containing a thioamide linkage in place of the peptide bond at various positions of the backbone were determined in mu- and delta-receptor-selective bio- and binding-assays. Thioamide substitution in the 1-2 position resulted in an inactive compound, whereas the same modification in the 2-3 and 4-5 position produced potency enhancement. Most interestingly, the 2-3 modified analog showed a 3 to 5 times higher preference for delta- over mu-receptors than natural leucine enkephalin. These results suggest that subtle backbone modifications can have a profound effect on receptor affinity and selectivity of biologically active peptides.     

On the existence of two populations of mitochondria in a single organ. Respiration, calcium transport and enzyme activities.

Mitochondria isolated from rat heart and kidney cortex by Polytron treatment of the tissues exhibit lower state 3 rates of respiration than mitochondria isolated by Nagarse method. Addition of cytochrome $\text{c}$ to Polytron mitochondria isolated from heart, but not from kidney, increases oxygen uptake to values approaching those of Nagarse-treated preparations. Similar results were observed for Ca²⁺ uptake. Kidney Polytron mitochondria exhibited lower mitochondrial, but higher non-mitochondrial enzyme activities compared to kidney Nagarse mitochondria. Enzyme activities were the same in Polytron and Nagarse mitochondria from heart. The differences between Polytron and Nagarse mitochondria appear to be mainly due to lower cytochrome $\text{c}$ content of Polytron mitochondria from heart and higher contamination of Polytron mitochondria from kidney.     

Product profiles in enzymic and non-enzymic oxidations of the lignin model compound erythro-1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol

The erythro form of the lignin model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol (1) was oxidized with laccase/ABTS, lead(IV) tetraacetate (LTA), lignin peroxidase/H₂O₂, cerium(IV) ammonium nitrate (CAN) and Fenton's reagent. The product profiles obtained with the different oxidants were compared after separation, identification and quantification of the products using HPLC, UV-diode array detector and electrospray ionization mass spectrometry in positive ionization mode. The oxidants generated different product profiles that reflected their different properties. Oxidation with laccase/ABTS resulted almost exclusively in formation of 1-(3,4-dimethoxyphenyl)-3-hydroxy-2-(2-methoxyphenoxy)-1-propanone (2). Oxidation with LTA resulted in more 3,4-dimethoxybenzaldehyde (3) than ketone 2. Lignin peroxidase and CAN gave similar product profiles and aldehyde 3 was the predominant product (only small amounts of ketone 2 were formed). Oxidation with Fenton's reagent resulted in the formation of more aldehyde 3 than ketone 2 but the yields were very low. CAN served as an excellent model for the lignin peroxidase-catalyzed oxidation, while the laccase-mediator system, LTA and Fenton's reagent provided distinctly different product profiles. Erythro-1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol was present among the products obtained on oxidation with LTA, lignin peroxidase, CAN and Fenton's reagent. The differences in redox potential between the oxidants afford an explanation of the diverse product patterns but other factors may also be of importance. The reactions leading to cleavage of the β-ether bond with formation of 1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol (veratrylglycerol) were found to proceed without affecting the configuration at the β-carbon atom.     

The effect of antiestrogens on egg yolk protein synthesis and estrogen-binding to chromatin in the rooster liver

Nafoxidine and CI-628, two well known antiestrogenic compounds, reduce the stimulating effect of estradiol on the estrogen-binding capacity of the liver chromatin from roosters. In vitro both antiestrogens compete with [³H] estradiol for the binding sites on the liver chromatin. They inhibit the estrogen-induced synthesis of egg yolk proteins (vitellogenin) and fail to induce this estrogen-specific protein synthesis by themselves. They show the ability, however, to increase the estrogen-binding sites on the liver chromatin to some extent.     

Catecholamine binding to the α-adrenergic receptors of hamster adipocytes evidence that guanine nucleotides regulate this binding to the α2-receptor subtype

The binding characteristics of the α-component of (?)-[³H]norepinephrine to hamster adipocyte membranes were studied. Binding was rapid, reaching equilibrium in 20 min at 25°C. Dissociation of specific binding by 10 μM phentolamine suggested dissociation from two different sites. The time course of dissociation induced by a 50-fold dilution was unchanged by the addition of norepinephrine, suggesting the absence of cooperative binding sites. [³H]norepinephrine binding was saturable, yielding curvilinear Scatchard plots. Computer modeling of these data further supported the existence of two classes of binding sites, one with high affinity (_D = 23 nM) but low binding capacity (96 fmol/mg protein) and one with low affinity (K_D = 400 nM) but high binding capacity (1000 fmol/mg protein). Adrenergic ligands of competed with [³H]norepinephrine binding in the following order of potency: (?)-norepinephrine>(?)-epinephrine>>(+)-norepinephrine>(?)-isoproterenol. Displacement by the selective α-adrenergic drugs prazosin, clonidine and yohimbine yielded biphasic curves consistent with binding of [³H]norepinephrine to both α₁- (14–22%) and α₂- (78–86%) receptor subtypes. Although Gpp(NH)p failed to alter the binding of [³H]dihydroergocryptine, it severely reduced the binding affinity of (?)-epinephrine, (?)-norepinephrine and the selective α₂-agonist, clonidine. The inhibitory effects of clonidine and of the α-component of (?)-epinephrine on the adrenocorticotropin-stimulated cyclic AMP production in the intact adipocyte were closely correlated with their effects on the binding of both [³H]norepinephrine and [³H]dihydroergocryptine. Conversely, yohimbine but not prazosin markedly antagonised the α-inhibitory effect of norepinephrine on cyclic AMP production. These data led to concluded that [³H]norepinephrine can be successfully used to study the entire α-adrenergic receptor population of hamster fat cells and that the predominant α₂ -receptor subtype exists in two different affinity states for agonists, the proportions of which are modulated by guanine nucleotides.