Salt-containing Media Research Articles

Model development for estimating calcium sulfate dihydrate, hemihydrate, and anhydrite solubilities in multicomponent acid and salt containing aqueous solutions over wide temperature ranges

Thermodynamic solubility of calcium sulfate in multicomponent electrolyte aqueous solutions is of significant importance in many disciplines in science and engineering. A knowledge of environmental chemistry and aqueous solubility of calcium sulfate and several of its hydrates; anhydrite (CaSO4), hemihydrate (CaSO4.0.5H2O), and dihydrate (CaSO4.2H2O); in acid and salt-containing media is of huge practical and theoretical importance. In this study, a solubility calculation model was developed based on coupling of Least Squares Support Vector Machine and Coupled Simulated Annealing (CSA-LSSVM) to model the solubility of various forms of calcium sulfate in aqueous solutions over wide temperature ranges. Comparing the model results to 3079 experimental data gathered from the literature yields a correlation coefficient of 0.9897 and RMSE of 0.0068. The model is able to present accurate solubility results for calcium sulfate dihydrate (CaSO4.2H2O) in aqueous solutions containing H2SO4, ZnSO4, CuSO4, NiSO4, MnSO4, H3PO4, NaCl, CaCl2, MgCl2, KCl, FeCl2, Na2SO4, HNO3, Ca(NO3)2, HCl, AlCl3, Ca(HCO3)2, NaNO3, LiNO3, Li2SO4, MgSO4, Al2(SO4)3, NaClO4, NaOH, KOH at temperatures up to 100 °C, calcium sulfate hemihydrate (CaSO4.0.5H2O) in solutions containing NaCl, CaCl2, MgCl2, KCl, FeCl2, HCl at temperatures up to 125 °C, and calcium sulfate anhydrite (CaSO4) in solutions containing H2SO4, NaCl, CaCl2, MgCl2, Na2SO4, HNO3, HCl, NaNO3, LiNO3, NiSO4, MgSO4, Al2(SO4)3, Mg(NO3)2 at temperatures up to 350 °C. Comparison of the CSA-LSSVM model results to those of Pitzer ion interaction model as well as BR-ANN model in binary, ternary, and multicomponent mixtures reveals the superiority of the CSA-LSSVM model developed in this study in all cases.

Highly stable polyglutamate derivatives/siRNA polyplex efficiently down-relegate survivin expression and augment the efficacy of cisplatin

RNA interfere (RNAi)-based technology holds great promise in cancer treatment. The use of small interfering RNA (siRNA), however, is hampered by its low delivery efficiency in vivo when they are diluted in blood biofluids and in the presence of serum and salt. In this study, we developed the polyglutamate derivative polymer brush, poly(ethyleneglycol) monomethyl ether-b-polyglutamate-g-spermine (mPEG-b-PG-g-spermine, PPGS), which could efficiently deliver survivin-siRNA under ultra-high dilution and in the presence of salt (NaCl 150mM) and serum (10% FBS), most likely due to its PEG-shelled polymer brush structure. On the contrary, aggregation occurred when PEI/siRNA polyplex dispersed in saline and serum-containing media and PEI polyplex dissociated after making a 256-fold dilution. PPGS/si-survivin polyplex exhibited high cellular uptake efficiency and efficiently down-regulated the expression of survivin mRNA in the cisplatin-resistance of non-small cell human lung adenocarcinoma (A549/DDP) cells in the presence of serum. However, either PEI polyplex or Lipofectmine 2000 complex was unstable in serum and salt-containing media and at high dilution rates, which resulted in their dramatical decrease of cellular uptake and gene-silencing efficiency in these conditions. The PPGS/si-survivin polyplex also exhibited synergistic effects of killing the cancer cells by combination treatment with cisplatin. Therefore, the PPGS gene carrier showed great potential in systemic siRNA delivery, and its combination with chemotherapeutic drug is promising in treating drug resistant cancers.

Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress

Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5-MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases.

RsERF1 derived from wild radish (Raphanus sativus) confers salt stress tolerance in Arabidopsis

The change in environmental parameters affects normal growth of plants, eventually reduces agricultural production. Ethylene plays vital roles in plant stress responses, germination, fruit ripening, organ abscission, pathogen response, and senescence. Expression of an ethylene-responsive transcription factor (ERF) was induced in Korean halophyte, Raphanus sativus var. hortensis f. raphanistroides (wild radish) by 200-mM sodium chloride (NaCl). Raphanus sativus ethylene-responsive transcription factor 1 (RsERF1) is also localized to nucleus, similar to other transcription factors. In yeast, RsERF1 showed transcriptional activation property, by expressing the reporter gene. Being a TF, RsERF1 specifically bound to the cis-acting elements, GCC box and DRE/CRT in vitro, to initiate transcription. Homozygous T3 transgenic Arabidopsis, overexpressing RsERF1, showed significant tolerance against salt stress in soil-grown conditions. The tolerance was also marked by an increased germination rate of RsERF1 transgenics in salt-containing media. In RsERF1 overexpression lines, abiotic stress-related genes such as ABF3, ABF4, ADH, Rab18, and SUS1 were upregulated by 200-mM NaCl. ERFs have been studied and proven for their tolerance potential against various abiotic stresses, but RsERF1 belongs to an ERF subgroup called ethylene-responsive transcription factor related to AP2 (ERF-RAP2). Thus, this is a first report for ERF-RAP2 from Korean halophyte cDNA library. We believe that extensive posttranslational modification studies will reveal the role and location of RsERF1 in stress tolerance pathway.

Alfalfa (Medicago sativa L.) clones tolerant to salt stress: in vitro selection

AbstractIn order to quickly and efficiently evaluate the salt tolerance of alfalfa, salinity tests were conducted on Medicago sativa L. var. australis, var. icon, var. loi, and var. gea, under in vitro conditions. Pregerminated seeds of four varieties were subjected to five different NaCl concentrations (0, 50, 100, 150, 200 mM). The influence of saline stress was estimated on the basis of survival percentage, growth parameters, and electrolyte leakage. The seedlings surviving on the medium enriched with salt at the highest concentration were presumed to be tolerant and represented the mother plants for the production of in vitro clones. In the following step, the clones were evaluated in vitro to confirm the salt tolerance. The influence of mild salt stress (75 mM NaCl) on the growth parameters of selected clones was examined. At the end of this trial, the proline accumulation and sodium content in alfalfa shoots were also quantified. The results suggest an increased level of proline promotes salt tolerance. Medicago sativa L. var. icon is highly tolerant in comparison with the other varieties tested. In vitro selection of M. sativa L. varieties on salt-containing media allowed us to obtain clones with increased salinity tolerance.

Osmoadaptative accumulation of N ɛ-acetyl-β-lysine in green sulfur bacteria and Bacillus cereus CECT 148T

The compatible solute N(ɛ)-acetyl-β-lysine (NeABL), thus far considered unique to methanogenic Archaea, has been found to accumulate in several strains of green sulfur bacteria (GSB) and Bacillus cereus CECT 148(T) under salt stress. A similar mixture of compatible solutes including trehalose, α-glutamate, β-glutamate and NeABL has been detected in salt-tolerant GSB strains of different phylogenetic branches. The ability of B. cereus to synthesize this compound was predicted from available genomic data, and nuclear magnetic resonance analyses of cultures grown in salt-containing media indicated that NeABL was present in the solute pools of osmotically challenged cells. The present results describe for the first time in the bacterial domain the use of this compound for osmoadaptation.

Effect of Nicotine on Chorda Tympani Responses to Salty and Sour Stimuli

The effect of nicotine on the benzamil (Bz)-insensitive (transient receptor potential vanilloid-1 variant cation channel, TRPV1t) and the Bz-sensitive (epithelial Na(+) channel, ENaC) salt taste receptors and sour taste was investigated by monitoring intracellular Na(+) and H(+) activity (pH(i)) in polarized fungiform taste receptor cells (TRCs) and the chorda tympani (CT) nerve responses to NaCl, KCl, and HCl. CT responses in Sprague-Dawley rats and both wildtype and TRPV1 knockout (KO) mice were recorded in the presence and absence of agonists [resiniferatoxin (RTX) and elevated temperature] and an antagonist (SB-366791) of TRPV1t, the ENaC blocker (Bz), and varying apical pH (pH(o)). At concentrations <0.015 M, nicotine enhanced and at >0.015 M, it inhibited CT responses to KCl and NaCl. Nicotine produced maximum enhancement in the Bz-insensitive NaCl CT response at pH(o) between 6 and 7. RTX and elevated temperature increased the sensitivity of the CT response to nicotine in salt-containing media, and SB-366791 inhibited these effects. TRPV1 KO mice demonstrated no Bz-insensitive CT response to NaCl and no sensitivity to nicotine, RTX, and elevated temperature. We conclude that nicotine modulates salt responses by direct interaction with TRPV1t. At pH(o) >8, the apical membrane permeability of nicotine was increased significantly, resulting in increase in TRC pH(i) and volume, activation of ENaC, and enhancement of the Bz-sensitive NaCl CT response. At pH(o) >8, nicotine also inhibited the phasic component of the HCl CT response. We conclude that the effects of nicotine on ENaC and the phasic HCl CT response arise from increases in TRC pH(i) and volume.

Ethanol Modulates the VR-1 Variant Amiloride-insensitive Salt Taste Receptor. II. Effect on Chorda Tympani Salt Responses

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by direct measurement of intracellular Na+ activity ([Na+]i) using fluorescence imaging in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. CT responses to KCl and NaCl were recorded in Sprague-Dawley rats, and in wild-type (WT) and vanilloid receptor-1 (VR-1) knockout mice (KO). CT responses were monitored in the presence of Bz, a specific blocker of the epithelial Na+ channel (ENaC). CT responses were also recorded in the presence of agonists (resiniferatoxin and elevated temperature) and antagonists (capsazepine and SB-366791) of VR-1 that similarly modulate the Bz-insensitive VR-1 variant salt taste receptor. In the absence of mineral salts, ethanol induced a transient decrease in TRC volume and elicited only transient phasic CT responses. In the presence of mineral salts, ethanol increased the apical cation flux in TRCs without a change in volume, increased transepithelial electrical resistance across the tongue, and elicited CT responses that were similar to salt responses, consisting of both a phasic component and a sustained tonic component. At concentrations <50%, ethanol enhanced responses to KCl and NaCl, while at ethanol concentrations >50%, those CT responses were inhibited. Resiniferatoxin and elevated temperature increased the sensitivity of the CT response to ethanol in salt-containing media, and SB-366791 inhibited the effect of ethanol, resiniferatoxin, and elevated temperature on the CT responses to mineral salts. VR-1 KO mice demonstrated no Bz-insensitive CT response to NaCl and no sensitivity to ethanol. We conclude that ethanol increases salt taste sensitivity by its direct action on the Bz-insensitive VR-1 variant salt taste receptor.

Structural and functional characterization of AtPTR3, a stress-induced peptide transporter of Arabidopsis

A T-DNA tagged mutant line of Arabidopsis thaliana, produced with a promoter trap vector carrying a promoterless gus (uidA) as a reporter gene, showed GUS induction in response to mechanical wounding. Cloning of the chromosomal DNA flanking the T-DNA revealed that the insert had caused a knockout mutation in a PTR-type peptide transporter gene named At5g46050 in GenBank, here renamed AtPTR3. The gene and the deduced protein were characterized by molecular modelling and bioinformatics. Molecular modelling of the protein with fold recognition identified 12 transmembrane spanning regions and a large loop between the sixth and seventh helices. The structure of AtPTR3 resembled the other PTR-type transporters of plants and transporters in the major facilitator superfamily. Computer analysis of the AtPTR3 promoter suggested its expression in roots, leaves and seeds, complex hormonal regulation and induction by abiotic and biotic stresses. The computer-based hypotheses were tested experimentally by exposing the mutant plants to amino acids and several stress treatments. The AtPTR3 gene was induced by the amino acids histidine, leucine and phenylalanine in cotyledons and lower leaves, whereas a strong induction was obtained in the whole plant upon exposure to salt. Furthermore, the germination frequency of the mutant line was reduced on salt-containing media, suggesting that the AtPTR3 protein is involved in stress tolerance in seeds during germination.

Flocculation kinetics of an ochre suspension in salt(NaCl)-containing aqueous media in the presence of anionic and cationic acrylamide copolymers

The sedimentation kinetics of an ochre suspension in salt (NaCl)-containing aqueous media was studied in the presence of ionogenic (anionic, A, and cationic, C) acrylamide copolymers with high molecular weight (M > 2 × 106) using a VT–0.5 torsion balance. The ionic strength of the dispersion medium varied in the wide range from 0.001 N to 0.4 N. The flocculation proceeded predominantly by a `bridge' mechanism, and the fraction of macromolecules inactive in the acts of floccule formation was significantly higher for C copolymer as compared with A copolymer. A drastic fall in the flocculating activities of A and C copolymers when passing from salt-free to salt-containing media is caused mainly by two following events:

Survival of Escherichia coli during long-term starvation: effects of aeration, NaCl, and the rpoS and osmC gene products

The survival of Escherichia coli was investigated during long-term starvation in rich media. In aerated cultures, E. coli lost the ability to form colonies earlier in NaCl-free Luria broth than in LB medium containing NaCl. Improved survival at low aeration and the sensitivity to hydrogen peroxide in aging cultures indicated a major role for oxidative stress in cell mortality. Mutants in rpoS, lacking the σ s subunit of RNA polymerase, showed altered survival in salt-containing media. However, in the absence of NaCl, although these mutants exhibited a massive loss of viability during the first 2 days, this was followed by a stabilization of the number of survivors. The starved culture contained survivors until at least day 9, long after a wild-type strain had completely lost viability. This peculiar behavior suggests that, in rich media of low osmotic pressure, σ s helps in short-term survival but hampers long-term survival. Mutants in osmC, a member of the rpoS regulon, also exhibited reduced survival and increased sensitivity to oxidative stress. The biochemical function of the envelope protein OsmC remains unknown, but present data indicated that it participates, directly or indirectly, in the defense against oxidative compounds.

The Design of a Pharmaceuttcally Acceptable Liposomal Formulation of Recombinant Interleukin-2 (Ril-2) for Locoregional Anticancer Immunotherapy

Liposomes composed of egg-phosphatidylcholine and egg-phosphati-dylglycerol containing recombinant interleukin-2 (rIL-2) were prepared and characterised for use in locoregional anticancer immunotherapy. During the encapsulation studies it was observed that the protein precipitated. Therefore, the impact of the precipitation phenomenon on the characteristics and in vivo performance of rIL-2 liposomes was studied. Recombinant IL-2 was diluted in various aqueous media and the amount of precipitated protein determined. Also, the in vitro bioactivity, chemical stability, and in vivo antitumor efficacy of liposomes prepared with precipitated rIL-2 or non-precipitated rIL-2 were assessed.Massive protein precipitation (60 up ± 95% of total protein) was observed upon dilution of rIL-2 in salt-containing media, but not upon dilution in 5% glucose or water. Liposomes prepared with precipitated rIL-2 were shown to release 50% of the entrapped rIL-2 over a three-day period at 37° in protein-containing media. Loss of rIL-2 bioactivity and chemical integrity was observed during storage at 4° over a 4-week period. Locoregional administration of precipitated rIL-2 in the guinea pig Line 10 tumor model resulted in significantly more tumor growth inhibition than administration of non-precipitated rIL-2. Liposomes containing non-precipitated rIL-2 were found to elicit similar antitumor effects as precipitated rIL-2. The results point to the importance of proper characterisation of new rIL-2 formulations as the physical properties of formulated rIL-2 may strongly influence its bioactivity.

Variables affecting results of sodium chloride tolerance test for identification of rapidly growing mycobacteria.

The sodium chloride tolerance test is often used in the identification of rapidly growing mycobacteria, particularly for distinguishing between Mycobacterium abscessus and Mycobacterium chelonae. This test, however, is frequently unreliable for the identification of some species. In this study we examined the following variables: medium manufacturer, inoculum concentration, and atmosphere and temperature of incubation. Results show that reliability is improved if the test and control slants are inoculated with an organism suspension spectrophotometrically equal to a 1 McFarland standard. Slants should be incubated at 35 degrees C in ambient air and checked weekly for 4 weeks. Growth on control slants should be critically evaluated to determine the adequacy of the inoculum; colonies should number greater than 50. Salt-containing media should be examined carefully to detect pinpoint or tiny colonies, and colonies should number greater than 50 for a positive reaction. Concurrent use of a citrate slant may be helpful for distinguishing between M. abscessus and M. chelonae. Molecular methodologies are probably the most reliable means for the identification of rapidly growing mycobacteria and should be used, if possible, when unequivocal species identification is of particular importance.

Interaction of Recombinant Interleukin-2 with Liposomal Bilayers

Liposomes have been employed as a delivery system for recombinant interleukin-2 (rIL-2) in cancer immunotherapy. In this study the effects of the rIL-2-bilayer interaction on protein structure were investigated. It was shown that rIL-2 adsorbs to liposomal membranes when added to preformed liposomes. Polarized fluorescence decay studies showed that the single tryptophan in “native” rIL-2 has a relatively large motional freedom, although iodide quenching of this residue’s fluorescence was relatively ineffective. However, adsorption of rIL-2 to liposomes alters this situation dramatically— fluorescence intensity increased 2-fold and the residue became more susceptible to iodide quenching. At the same time, the average fluorescence lifetime of the fluorophore is extended. Interestingly, circular dichroism studies showed that no major conformational changes occurred in rIL-2’s secondary structure upon adsorption. These observations support the hypothesis that intramolecular quenching takes place in the native rIL-2 molecule, which is abrogated upon adsorption to the liposomal membrane, resulting in a higher fluorescence intensity. Fluorescence anisotropy decay experiments indicate that the protein forms self-aggregates under the low-ionic strength conditions used, confirming the earlier observations on the tendency of the protein to precipitate in salt-containing media.

New methods of transplutonium element separation and determination

Some recently developed new methods of separation and determination of transplutonium elements (TPEs) are described. The solvent extraction of TPE III by bidentate organophosphorus reagents from strongly acidic, salt-containing media and their extraction from alkaline solution by various reagents as well as isolation of TPEs in “unusual” oxidation states are discussed. Stabilization of americium as Am IV, Am V, Am VI and berkelium as Bk IV is considered. The separation of actinides and rare earth elements can be carried out by countercurrent chromatography which is based on the retention of the organic phase in a rotating column of a planetary centrifuge under the action of centrifugal forces while the stationary phase is continuously pumped through. It has been shown that TPEs can be successfully separated by this technique. Attention has been paid mainly to consideration of contemporary methods for determination of actinides, special distinction of such methods being low limits of determination, high precision and selectivity. A large number of coulometric methods for determination of americium and berkelium characterized by high precision and selectivity as well as luminescence methods have been developed.

Physiological Adaptation to High Ion Concentrations or Water Deficit by Callus Cultures of Highbush Blueberry, Vaccinium corymbosum

Callus cultures of highbush blueberry were selected for 10 passages on medium supplemented with 50 rnol m-3 NaCl, 50 rnol m-3 KCl, 25 mol m-3 Na2SO4, 25 mol m-3 K2SO4 or 100 mol m-3 mannitol. On all salts, growth of selected callus was greater (200-250%) than that of non-selected callus, and selected callus grew optimally on the type of salt on which it was selected. Conventional (whole plant analysis) and electron probe X-ray microanalysis showed that selected callus accumulated more ions (approximately 1.5-3.0-fold) than non-selected callus on all salts, and there was a positive correlation between vacuolar ion concentration and fresh weight. Growth of NaCl-selected callus but not non-selected callus was greatly enhanced (2.25-fold) in the presence of 100 mol m-3 mannitol, while growth of a mannitol-selected callus line was also enhanced. In callus grown on NaCl or mannitol, slight increases in levels of glycinebetaine, choline and proline were measured. Sucrose, glucose, fructose, sorbitol and malate concentrations significantly increased in callus grown on NaCl or mannitol, and selected callus produced 4-fold more sugars than non-selected callus. The total increases in concentrations of all measured sugars were 210 μmol gFW-1 in NaCl-selected callus grown on 50 mol m-3 NaCl, and 296 μmol gFW-1 in mannitol selected callus grown on 100 rnol m-3 mannitol. The results of this study indicate that adaptation of blueberry callus cultures for optimal growth on salt-containing media is probably due to adaptation to water stress, not tolerance to specific ions. Osmotic adjustment, achieved by ion uptake and production of sugars, appears to be the physiological mechanism of adaptation.

RADIOACTIVE NUCLIDES IN THE ENVIRONMENT

The data on preconcentration of a number of radionuclides (90Sr, 238-240pu, 241Am) from natural waters by coprecipitation, ultrafiltration with watersoluble complex forming polymers, dyalisis and membrane extraction are presented. It has been shown that the precipitation as well as ultrafiltration using polyphosphates, polyalcohols and polyoxines are the most appropriate for the group preconcentration. The selective preconcentration can be performed by ion exchange (137Cs-ferrocyanides), membrane extraction (90Sr crown ethers) and by ultrafiltration (238-240pu polyphosphortungstates). An additional deep purification from macrocomponents or other interfering nucludes, if necessary, can be achieved by extraction-chromatographic methods. The data on analysis of soil, water and biological objects from Chernobyl NPP obtained by abovementioned methods are presented. Among the possible sources of continuous impact of radionuclides in biosphere highly radioactive wastes are the most valuable. The preliminary isolation of long-lived transplutonium elements is very important. In the work for this purpose the solvent extraction of TPE (III) by bidentate organophosphorus reagents from strong acid, salt-containing media, extraction from alkaline solution by variuos alkylpyrocatechols and alkylderivatives of amine-alcohols as well as isolation of TPE in “unusual” oxidation states have been used.

Association of putrescine, spermidine, spermine, and GABA with structural elements of brain cells

The present experiments are the first survey of the association of endogenous and exogenous putrescine, spermidine, and spermine with subcellular structures of rat brain cortex. The differences of distribution in subfractions obtained from salt-free and salt-containing density gradients were studied, with the following results: (1) In contrast with liver preparation, putrescine and the polyamines spermidine and spermine are not distributed in parallel with RNA. (2) In salt-containing media, putrescine and the polyamines were preferentially associated with synaptosomes and with synaptosomal membranes. Significant association with myelin constituents was observed only in salt-free media. (3) Exogenous putrescine and the polyamines were less firmly attached to synaptosomes and to synaptosomal membrane fractions than the endogenous amines. There is good evidence for similar subcellular localizations of putrescine and GABA. Putrescine seems to be entrapped in the nerve endings. (4) Uptake studies with crude mitochondria under conditions of "high-affinity uptake" showed no temperature-sensitive component of polyamine accumulation in synaptosomes, in contrast with GABA, monoacetylputrescine, and ornithine. (5) Polyamines bound to myelin constituents or mitochondria could be displaced by a 200-fold concentration of nonradioactive amines; this was not the case with polyamines bound to synaptosomes. Mg(2+) did not effectively compete with spermine for binding sites at synaptic regions. (6) Electrical stimulation and stimulation by mono- and bivalent cations did not change the concentrations of the polyamines and GABA in guinea pig cortex. (7) There is no evidence for a neurotransmitter role of putrescine, spermidine, or spermine, although these compounds might function as modulators of neurotransmission.

The localization of mitochondrial creatine kinase, and its use for the determination of the sidedness of submitochondrial particles

1. 1. Creatine kinase (EC 2.7.3.2) is extracted from heart mitochondria by suspension in salt-containing media. 2. 2. Digitonin treatment of isolated heart mitochondria causes rupture of the outer membranes and release of adenylate kinase (EC 2.7.4.3), while creatine kinase remains bound to the mitochondrial membranes. Although the outer membranes are ruptured by the action of digitonin, they can not be obtained in reasonable yield by differential centrifugation, indicating that most of the ruptured outer membranes are still connected to the inner membranes, which could be confirmed by electron microscopy. 3. 3. The addition of the detergent Lubrol WX to intact heart mitochondria has no effect upon the activity of the creatine kinase, indicating that this enzyme is not latent. 4. 4. In submitochondrial particles, obtained by ultrasonic treatment of heart mitochondria and removal of intact mitochondria by centrifugation, creatine kinase becomes partially latent. This indicates that in some of the submitochondrial particles the enzyme is localized inside the enveloping inner membrane fragment. Evidence is also presented for the existence of submitochondrial particles with the same orientation of the enveloping membrane as intact mitochondria, as indicated by the latency of aspartate aminotransferase (EC 2.6.1.1), a matrix enzyme that is firmly bound to the inside of the inner membrane. 5. 5. From Paragraphs 2–4 it is concluded that mitochondrial creatine kinase is bound to the outside of the inner mitochondrial membrane, in contrast to mitochondrial adenylate kinase, which is not bound to either of the mitochondrial membranes.

Transport Systems for 1,4-Diaminobutane, Spermidine, and Spermine in Escherichia coli

Uptake processes for 1,4-diaminobutane, spermidine, and spermine have been studied in Escherichia coli. The uptake systems can be divided into two components. One is a rapid adsorption that is not dependent on active metabolic processes, and can be observed at 0° as well as at 37°. The adsorbed amine can be largely removed by washing the bacteria with the nonradioactive amine or with salt-containing media. The amount of the adsorbed amine was less than 1% of the added amine for 1,4-diaminobutane, 3 to 5% for spermidine, and 7 to 15% for spermine. The second is a larger, time-dependent component which depends on metabolic processes, does not occur at 0°, and is markedly decreased by the omission of glucose or by the addition of dinitrophenol. The accumulated amines cannot be removed by washing the bacteria with solutions of the nonradioactive amines; they are released, however, by treatment of the bacteria with toluene or butanol-1. Studies are also reported on the effect of pH, concentration, and inhibitors on these systems.