Assessment study on the high-performance liquid chromatography-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate.

The HPLC-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate (SDS) was assessed with special attention to the behavior of the surfactant. A significant amount of SDS was found to be adsorbed to the hydroxyapatite packed in the column from the starting buffer, 50 mM sodium phosphate buffer, pH 7.0, only when the buffer contained SDS in a concentration at or above its critical micelle concentration. When the phosphate buffer concentration was increased while the SDS concentration was kept at 1 mg/ml, the adsorbed surfactant was desorbed in advance of the release of proteins. Polypeptides derived from proteins could be successfully separated only when the column had been thoroughly equilibrated with the above-mentioned starting buffer solution. When a protein polypeptide complexed with SDS, which had been similarly equilibrated, was applied to the column, an amount of SDS corresponding to 75-90% (w/w) of the surfactant originally bound to the polypeptide was released upon its binding to the hydroxyapatite. On the other hand, porin, an Escherichia coli outer membrane protein, retaining its trimeric native structure in the presence of SDS, released a significantly smaller amount of SDS. When the membrane protein was denatured to give a single polypeptide, it behaved in a manner similar to that of the other protein polypeptides. The mechanism of binding of the protein polypeptides was discussed on the basis of these results. The native and denatured entities of porin could be efficiently separated as the result of the difference in their mode of interaction with the hydroxyapatite.     

Differential refractometric determination of binding of sodium dodecyl sulfate to protein using high-performance gel chromatography

When sodium dodecyl sulfate (SDS) is added to a high-performance gel chromatographic column equilibrated with a buffer solution containing SDS at a level above the critical micelle concentration, the surplus SDS migrates as micelles giving a sharp peak. The presence of an unfolded protein in the sample solution gives a polypeptide peak in advance of the SDS micelle peak. As the result of SDS binding to the polypeptide, the SDS micelle peak is attenuated in comparison to that in the absence of protein. Thus the amount of SDS bound to the polypeptide can be determined accurately and simply from the decrease in the area of the SDS micelle peak. This approach is particularly useful for precise determination of bound SDS, which is pertinent to understanding the state of the protein polypeptide-SDS complex under the conditions of SDS-polyacrylamide gel electrophoresis.     

Physico-chemical studies of micelle formation on sepia cartilage collagen solutions in acetate buffer and its interaction with ionic and nonionic micelles. Hydrodynamic and thermodynamic studies

Sepia cartilage collagen (pepsin-extracted) in acetate buffer (pH = 2.98) forms micelles at a particular concentration below which they do not normally form. The critical micelle concentration (cmc) of the collagen was determined in buffer as well as in SDS, cetyltrimethylammonium bromide (CTAB) and Tween-80 micellar environments at different temperatures. Mutual interaction of collagen micelles with the ionic and nonionic micelles through the formation of the mixed micelle concept has also been found. The cmc of collagen decreased in the presence of SDS and Tween-80 micelles whereas it increased in the presence of CTAB micelles. This clearly suggests that the micelle formation of collagen is facilitated by the presence of SDS and Tween-80 and hindered by CTAB micelles. The various thermodynamic parameters were estimated from viscosity measurements and the transfer of collagen into the micelles of various surfactants and the reverse phenomenon was analyzed. This analysis has also been modelled conceptually as a different phase and the results have supported the above phenomenon. Our thermodynamic results are also able to predict the exact denaturation temperature as well as the structural order of water in the collagen in various environments. The hydrated volumes, Vh, of collagen in the above environments and intrinsic viscosity were also calculated. The low intrinsic viscosity, [eta], of collagen in an SDS environment compared to buffer and other surfactant environments suggested more workable systems in cosmetic and dermatological skin care preparations. The one and two-hydrogen-bonded models of this collagen in various environments have been analyzed. The calculated thermodynamic parameters varied with the concentration of collagen. The change of thermodynamic parameters from coil-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results. Thermodynamic results suggest that the stability of the collagen in the additive environments is in the following order: SDS greater than Tween-80 greater than buffer greater than CTAB.     

In vitro binding of 70S ribosomes to membrane structures of Escherichia coli

It was found that the maximal disattachment of the ribosomes from the membrane structures is observed upon their treatment with 10 mM tris-HCl buffer, pH 7.5, containing 250 mM sucrose, 750 mM KCl, 5 mM magnesium acetate and 1 mM EDTA or puromycin. The most effective attachment of ribosomes to the membrane occurs in 10 mM tris-HCl buffer, pH 7.5, containing 5% sucrose and Mg2+. The increase of Mg2+ concentration in the medium from 0.5 mM up to 1 mM results in a 2-fold increase of the ribosomes bound to the membranes. The concentration of the ribosomal material involved in the reaction is very essential for ribosome binding to the membranes. The amount of ribosomes bound to the membranes increases proportionally to the increase of the ribosome concentration in the reaction mixture.     

Analysis of protein-surfactant interactions--a titration calorimetric and fluorescence spectroscopic investigation of interactions between Humicola insolens cutinase and an anionic surfactant

We have studied interactions of cutinase (HiC) from Humicula insolens and sodium dodecyl sulphate (SDS) by parallel calorimetric and fluorescence investigations of systems in which the concentration of both components was changed systematically. Results from the two methods exhibit a number of synchronous characteristics, when plotted against the total SDS concentration, [SDS]tot. The molecular origin of several of these anomalies was assigned, and five intervals of [SDS]tot in which different modes of interactions dominated were identified. Going from low to high [SDS]tot, these modes were: binding of (a few) SDS to native HiC, formation of oligomeric protein aggregates, denaturation of HiC and adsorption of SDS on denatured protein. For [SDS]tot>3-6 mM (depending on the protein concentration), the adsorption saturated, and no further protein-detergent interaction could be detected. Two particularly conspicuous anomalies in the calorimetric data were ascribed to respectively denaturation and saturation. It was found that [SDS]tot at these points depended linearly on the (total) protein concentration, [HiC]. We suggest that this reflects the balance between bound and free SDS [SDS]tot=[SDS]aq+[HiC] Nb where [SDS]aq and Nb are, respectively, the aqueous ("free") concentration of SDS and the average number of SDS bound per protein. Interpretation of the results along these lines showed that at 22 degrees C and pH 7.0, HiC denatures with approximately 14 bound surfactant molecules at [SDS]aq=1.0 mM. Saturation is characterized by Nb approximately 39 and [SDS]aq=2.2 mM. The latter value is equal to CMC in the (protein free) buffer. These results are discussed with respect to the SDS-binding capacity of HiC and the origin and location of the saturation point.     

Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes

The partition equilibria of sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate between water and bilayer membranes were investigated with isothermal titration calorimetry and spectroscopic methods (light scattering, (31)P-nuclear magnetic resonance) in the temperature range of 28 degrees C to 56 degrees C. The partitioning of the dodecyl sulfate anion (DS(-)) into the bilayer membrane is energetically favored by an exothermic partition enthalpy of Delta H(O)(D) = -6.0 kcal/mol at 28 degrees C. This is in contrast to nonionic detergents where Delta H(O)(D) is usually positive. The partition enthalpy decreases linearly with increasing temperature and the molar heat capacity is Delta C(O)(P) = -50 +/- 3 cal mol(-1) K(-1). The partition isotherm is nonlinear if the bound detergent is plotted versus the free detergent concentration in bulk solution. This is caused by the electrostatic repulsion between the DS(-) ions inserted into the membrane and those free in solution near the membrane surface. The surface concentration of DS(-) immediately above the plane of binding was hence calculated with the Gouy-Chapman theory, and a strictly linear relationship was obtained between the surface concentration and the extent of DS(-) partitioning. The surface partition constant K describes the chemical equilibrium in the absence of electrostatic effects. For the SDS-membrane equilibrium K was found to be 1.2 x 10(4) M(-1) to 6 x 10(4) M(-1) for the various systems and conditions investigated, very similar to data available for nonionic detergents of the same chain length. The membrane-micelle phase diagram was also studied. Complete membrane solubilization requires a ratio of 2.2 mol SDS bound per mole of total lipid at 56 degrees C. The corresponding equilibrium concentration of SDS free in solution is C (sat)(D,F) approximately 1.7 mM and is slightly below the critical micelles concentration (CMC) = 2.1 mM (at 56 degrees C and 0.11 M buffer). Membrane saturation occurs at approximately 0.3 mol SDS per mol lipid and the equilibrium SDS concentration is C (sat)(D,F)approximately equal 2.2 mM +/- 0.6 mM. SDS translocation across the bilayer is slow at ambient temperature but increases at high temperatures.     

Outer membrane proteins of Escherichia coli. I. Effect of preparative conditions on the migration of protein in polyacrylamide gels

Outer membrane protein of Escherichia coli prepared for polyacrylamide gel electrophoresis by solubilization of the membrane in an organic solvent followed by dialysis into sodium dodecyl sulfate (SDS) solution or by solublization of the membrane directly in SDS solution followed by dialysis into a SDS-urea solution and brief heating at 100 °C resulted in a simple polypeptide profile on SDS-containing gels. This polypeptide pattern was characterized by a single major protein band migrating with an apparent molecular weight of about 42,000 daltons which accounted for about 70% of the total protein on the gel. However, if the outer membrane protein is dissolved in SDS solution without urea and heated at 70 °C, major bands are observed in three regions of the gel: A broad band or group of bands near the top of the gel with an apparent molecular weight of much greater than 42,000 daltona (peak A), a second band with the same mobility as the 42,000-dalton band in boiled samples (peak B), and a third, faster-migrating band with an apparent molecular weight of less than 42,000 daltons (peak C).Elution of protein from A or C followed by heating at 100 °C converts this protein to a form migrating with peak B. If the outer-membrane protein is dissolved in SDS solution at 37 °C with no further heating and applied to gels, peak B dissappears completely and A and C increase. These can be partially converted to peak B by urea treatment. Protein from peaks A and C was isolated by chromatography on Sephadex in the presence of SDS, and the intrinsic viscosity of this protein was measured before and after boiling. The intrinsic viscosity of protein from peak A was 35 cc/g both before and after boiling, while the intrinsic viscosity of protein from peak C was 28 cc/g before boiling and 35 cc/g after boiling. These results are best explained by assuming that the protein in peak A represents aggregates of a 42,000-dalton species which are dissociated by boiling or solvent treatment and that the protein in peak C represents a monomeric form of the 42,000-dalton protein which is not fully reacted with SDS and which is converted to the “rigid rod” conformation characteristic of protein-SDS complexes only upon boiling or solvent treatment.     

A low-angle laser light scattering study of the association behavior of a major membrane protein of Rhodospirillum rubrum chromatophore at various concentrations of sodium dodecyl sulfate where polypeptides derived from water-soluble globular proteins are solubilized monomerically

The major membrane protein of Rhodospirillum rubrum chromatophore could be solubilized in the presence of free sodium dodecyl sulfate (SDS) in concentration above 0.8 mM. At this concentration, the protein was highly associated to give a weight-averaged molecular weight as high as one million as determined by the low-angle laser light scattering technique. With the increase of free SDS concentration, the aggregates were progressively dissociated to give a molecular weight of 8300 at the critical micelle concentration of SDS. Three protein polypeptides derived from typical water-soluble globular proteins, bovine serum albumin, ovalbumin and beta-lactoglobulin, were found to be solubilized monomerically even at 0.8 mM free SDS. The results obtained suggest that there is substantial difference in the mode of solubilization between polypeptides derived from intrinsic membrane proteins and those from water-soluble globular proteins.     

SDS-induced conformational changes and inactivation of the bacterial chaperonin GroEL

The inactivation and conformational changes of the bacterial chaperonin GroEL have been studied in SDS solutions with different concentrations. The results show that increasing the SDS concentration caused the intrinsic fluorescence emission intensity to increase and the emission peak to slightly blue-shift, indicating that increasing the SDS concentration can cause the hydrophobic surface to be slightly buried. The changes in the ANS-binding fluorescence with increasing SDS concentration also showed that the GroEL hydrophobic surface decreased. At low SDS concentrations, less than 0.3 mM, the GroEL ATPase activity increased with increasing SDS concentration. Increasing the SDS concentration beyond 0.3 mM caused the GroEL ATPase activity to quickly decrease. At high SDS concentrations, above 0.8 mM, the residual GroEL ATPase activity was less than 10% of the original activity, but the GroEL molecule maintained its native conformation (as indicated by the exposure of buried thiol groups, electrophoresis, and changes of CD spectra). The above results suggest that the conformational changes of the active site result in the inactivation of the ATPase even though the GroEL molecule does not markedly unfold at low SDS concentrations.     

A new method to precipitate myosin V from rat brain soluble fraction

Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.     

Studies on the preparation of rat liver plasma membrane fractions and on their polypeptide patterns

Plasma membrane and bile canalicular membrane fractions were prepared from rat liver using NaHCO3, NaHCO3--CaCl2, and K2HPO4-KH2PO4 buffers (all at pH 7.4). The amount (expressed as milligrams protein per gram liver) of plasma membrane fraction exceeded the amount of bile canalicular membrane fraction using each of these three media; the use of NaHCO3-CaCl2 afforded a substantially higher yield of both types of membranes. The two membrane fractions exhibited complex patterns of polypeptides (greater than 30) on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Several reproducible differences in polypeptide patterns were observable between the two membrane fractions; in particular, components possibly corresponding to the heavy chain of myosin and to action were prominent in the bile canalicular membrane fraction. The effects of incubation in the above three buffers and in Tris--HCl (pH 7.4) on the polypeptide patterns of both types of membrane were studied. Many polypeptides were released from each type of membrane in all of these media. Differential effects on the polypeptide patterns of either type of membrane fraction were observed among the various buffers. In terms of minimizing loss of polypeptides, in general, NaHCO3--CacCl2 appeared to be the best buffer and Tris--HCl the worst buffer. The significance of these results for the preparation and storage of liver cell plasma membrane fractions is briefly discussed.     

The Isolation of Bacterial Polysaccharides with Anti-inflammatory Activity

It was found in the previous paper that wheat gluten polypeptides gave higher molecular weights in SDS-PAGE than in sedimentation equilibrium. To clear the cause of the abnormality of gluten polypeptides in SDS-PAGE, behaviors of the SDS complex of gliadin IV were investigated in comparison with those of the standard proteins. The amount of bound SDS for reduced and alkylated gliadin IV was not different from the value obtained with usual proteins. In accordance with the results of Reynolds et al., the plot of log [η] against log M gave a slope of 1.1, supporting a rodlike structure of the complex. The intrinsic viscosity of the SDS complex of gliadin IV gave a higher value of 0.28 dl/g in comparison with the corresponding value of 0.15 dl/g for the standard proteins. The sedimentation constant was lower in gliadin IV (1.61 S) than in the standard (1.77 S). These facts indicate that the gliadin IV complex has a higher frictional coefficient for its molecular weight of protein, suggesting a more elongated structure than usual. The helix content of the complex of gliadin IV was extremely low (12%). It was suggested that the high proline content of gliadin gives an elongated structure to the SDS complex and this structure causes a low electrophoretic migration mobility and overestimation of molecular weight in SDS-PAGE.     

Conformational changes of α-lactalbumin and its fragment,Phe31-Ile59, induced by sodium dodecyl sulfate

Conformational changes of bovine α-lactalbumin in sodium dodecyl sulfate (SDS) solution were studied with the circular dichroism (CD) method using a dilute phosphate buffer ofpH 7.0 and ionic strength 0.014. The proportions of α-helix and β-structure in α-lactalbumin were 34% and 12%, respectively, in the absence of SDS. In the SDS solution, the helicity increased to 44%, while the β-structure disappeared. In order to verify the structural change from β-structure to α-helix, the moiety, assuming the β-structure in the α-lactalbumin, was isolated by a chymotryptic digestion. The structure of this α-lactalbumin fragment, Phe³¹-Ile⁵⁹, was almost disordered. However, the fragment adopted a considerable amount of α-helical structure in the SDS solution. On the other hand, the tertiary structure of α-lactalbumin, detected by changes of CD in the near-ultraviolet region, began to be disrupted before the secondary structural change in the surfactant solution. Dodecyl sulfate ions of 80 mol were cooperatively bound to α-lactalbumin. Although the removal of the bound dodecyl sulfate ions was tried by the dialysis against the phosphate buffer for 5 days, 4 mol dodecyl sulfates remained per mole of the protein. The remaining amount agreed with the number of stoichiometric binding site, determined by the Scatchard plot, indicating that the stoichiometric binding was so tight.     

The role of magnesium and potassium ions in the molecular mechanism of ribosome assembly: hydrodynamic, conformational, and thermal stability studies of 16 S RNA from Escherichia coli ribosomes

In an attempt to understand the role of magnesium ion in ribosome assembly in vitro, the hydrodynamic shape, conformation, and thermal stability of ribosomal 16 S RNA were studied systematically as a function of Mg2+ concentration by sedimentation velocity, intrinsic viscosity, circular dichroism, and difference ultraviolet absorption spectroscopy. These results were then compared with the corresponding parameters obtained for 16 S RNA under the optimal conditions of reconstitution, i.e., at 37 degrees C, 20 mM Mg2+, an ionic strength equal to 0.37, and pH 7.8 [S. H. Allen, and K.-P. Wong (1978) J. Biol. Chem. 253, 8759-8766]. When the 360 mM KCl required for reconstitution of 30 S ribosomes is added to the medium, only subtle conformational changes are observed, consistent with the destabilization of the conformation, thus making the RNA molecule more "open" and accessible to protein binding. However, when the concentration of Mg2+ is lowered from 20 to 1 mM, the hydrodynamic parameters indicate that the 16 S RNA is partially unfolded, while thermal denaturation studies suggest that the amount of base-stacking and base-pairing is not concomitantly altered. Further removal of the Mg2+ by dialysis against a pH 7.8 buffer containing no Mg2+ results in a drastic decrease of secondary structure and indicates that the Mg2+ is required for maintenance of the pairing, stacking, and stability of the nucleotide bases, in addition to the long range interactions which result in a compact structure. The results suggest that the 20 mM Mg2+ is required for the 16 S RNA molecules to assume the proper secondary and tertiary structure containing the protein-binding sites, while the high K+ concentration (360 mM KCl) is needed for "loosening up" the RNA, making the protein binding sites more accessible to the ribosomal proteins for molecular recognition and binding as well as for the conformational changes that occur during ribosome assembly.     

Structure of micelles formed by monodisperse hexa-(γ-benzyl-L-glutamate) in solution

The angular dependence of light scattering and the concentration dependence of the relative viscosity have been measured in solutions of o-nitrophenylthio-hexa-(γ-benzyl-L -glutamate) ethylamide in ethylene dichloride. Both the reduced intensity of scattered light and the reduced viscosity of the solution suddenly increase above a certain critical concentration, below which both of them remain low and constant. The Debye plot of light scattering indicates that primary micelles having an aggregation number 48 are formed at the critical micelle concentration and that secondary micelles, each consisting of 294 molecules, then appear in increasing amounts with increasing concentration beyond the critical micelle concentration. The secondary micelle is rodlike and has a length of 1170 Å, if it is rigid. An analysis of the reduced viscosity leads to the intrinsic viscosity for the primary micelle, 0.360 dL g^?1, and to that of the secondary micelle, 1.28 dL g^?1. If the secondary micelle is represented by a prolate ellipsoid, it should have an axial ratio of 47. If the polypeptide chains are extended in the micelle, the observed aggregation number and axial ratio of the secondary micelle can well accommodate the intermolecularly hydrogen-bonded in-register β-structure of anti-parallel chains. In the primary micelle, some folded polypeptide chains are involved, and an intermolecularly hydrogen-bonded out-of-register structure would form a rather open network.     

Sodium dodecyl sulfate-mediated transfer of electrophoretically separated DNA-binding proteins

A modified procedure for the transfer of electrophoretically-separated proteins from sodium dodecyl sulfate (SDS)-polyacrylamide gels onto nitrocellulose filters has been developed. During the diffusion mediated transfer, the SDS-protein complexes were maintained and SDS was added to the buffer. This increases the number of polypeptide species bound to the filter thereby giving an accurate replica of the original gel pattern. The immobilization in the gel of certain polypeptides characterized as DNA-binding proteins, which is observed when SDS is eliminated prior to blotting is avoided. The molecules blotted in the presence of SDS remain immunoreactive and able to bind DNA.     

Alterations in Ca2+-binding on plasma membrane after adaptation to salt stress of tobacco cells in suspension

Electrophoresis was used to study effects of salinity on the characteristics of Ca2+ binding to the outer surface of plasma membrane (PM) of protoplasts isolated from two types of tobacco (Nicotiana tabacum L., cv. Bright Yellow) cultured cells that were adapted (tolerant) and unadapted (sensitive) to 50 mM NaCl stress. Electrophoretic analysis of salt-sensitive NaCl-unadapted cells shows that Na+ induced an appreciably higher degree of reduction in the amount of Ca2+ bound to PM compared with K+ with increasing concentration from 0.1 to 30 mM. In salt-tolerant NaCl-adapted cells, however, both Na+ and K+ ions induced almost the same degree of reduction in the amount of Ca2+ bound to PM in the physiological concentration range of Ca2+ in the medium between 2 and 4 mM. These results suggest that, under the physiological conditions, PM of salt-sensitive NaCl-unadapted cells has an appreciable amount of PM-bound Ca2+ that is desorbed much easier by Na+ than K+, whereas PM of salt-tolerant NaCl-adapted cells has the PM-bound Ca2+ that can be equally desorbed by Na+ and K+.     

Interaction of sodium and potassium ions with Na+, K+-ATPase. III. Cooperative effect of ATP and Na+ on complete release of K+ from E2K

The effects of Na+ and ATP on the K+ binding to Na+, K+-ATPase were investigated by the centrifugation method with radioactive K+ in the absence of Mg2+. In the presence of 10 microM 43KCl, 0.6 and 10 mM Na+ decreased the amount of bound K+ to one-half and zero, respectively. On the other hand, 10 microM and 10 mM ATP decreased the amount of K+ to 60 and 25-40%, respectively. When the combined effect of ATP and Na+ was tested, 10 microM ATP decreased the Na+ concentration giving half-maximal inhibition of the K+ binding to one-third, showing synergistic inhibition by both ligands, though increase in ATP concentration seemed to depress the inhibitory effect of Na+. The synergistic inhibition by ATP and Na+ suggests that the release of K+ from E2K is not completed by the binding of ATP alone but is completed by the binding of Na+ in addition to ATP during the cycle of Na+, K+-dependent ATP-hydrolysis as well as ion-transport.     

Dissimilar effects of Na+ and K+ on the promotion of glucosyltransferase secretion in Streptococcus salivarius

A defined growth medium (designated AP11), in which the concentrations of Na+ and K+ could be altered independently of one another, was developed for Streptococcus salivarius ATCC 25975. The addition of 100 mM-Na+ to AP11-medium containing 25 mM-K+ initially reduced the rate of expression of extracellular glucosyltransferase (GTFe). However, once S. salivarius had adaptated to grow in the presence of 100 mM-Na+, the rate of GTFe expression was stimulated. In fact once adapted to the presence of Na+ in the environment the same increase in the rate of enzyme expression was observed in all batch cultures irrespective of the K+ concentration (2-50 mM). At 2 mM-K+ there was no change in the level of saturation of the membrane lipids when the Na+ concentration was increased from 0 mM to 100 mM. Na+-stimulation of GTFe expression was confirmed in non-proliferating cell suspensions at different K+ concentrations. In non-proliferating cell suspensions, GTFe expression outlined a rectangular hyperbola with respect to K+ concentration when the K+ concentration was stepped up from 2 mM. The increase in GTFe synthesis and secretion was transient and was similar to that previously reported by us in Na+-rich medium, though it did not reach the same high levels. The reduced transient stimulation of GTFe expression correlated both with an enrichment in the saturated fatty acids of the membrane lipids of S. salivarius, and with the fact that the degree of saturation was only slightly reduced when the K+ concentration was stepped up from 2 mM to 50 mM. Needless to say, the final octadecenoic to octadecanoic (C18:1/C18:0) fatty acid ratio retained its direct correlation with the transient increase in GTFe production following the step up in K+ concentration, giving rise to an apparent biphasic plot when combined with that previously reported.     

The amounts of adenosine di- and triphosphates bound to H-meromyosin and the adenosinetriphosphatase activity of the H-meromyosin-F-actin-relaxing protein system in the presence and absence of calcium ions. The physiological functions of the two routes of myosin adenosinetriphosphatase in muscle contraction.

The rates of the ATPase [EC 3.6.1.3] reaction of the H-meromyosin-F-actin-relaxing protein system were measured in 2 mM MgCl2, 50mM KC1, and 10mM Tris-HC1 at pH 7.8 and 20 degrees in the presence and absence of 0.05-0.1 mM Ca2+ ions. The concentrations of H-meromyosin (HMM) and the F-actin-relaxing protein (F-A-PR) complex were 3.4 and 3 mg/ml, respectively, and the ATPase reaction was coupled with 4 mg/ml of pyruvate kinase [EC 2.7.1.40] and 1 or 20 mM phosphoenolpyruvate to regenerate ATP. The amount of ADP bound to HMM during the ATPase reaction was determined by measuring the amount of ADP remaining in the reaction mixture. The amount of ATP bound to HMM was determined by subtracting the amount of bound ADP from the total amount of nucleotides bound to HMM, which was measured by a rapid flow-dialysis method. The following results were obtained. 1. The ATPase activity of the HMM-F-A-RP system increased linearly with increase in the amount of ATP added, and was independent of the presence of 0.05 mM Ca2+, when the amount of ATP added was less than 1 mole/mole of HMM. In the presence of 0.05 mM Ca2+, the ATPase activity reached a maximal level when 1.2-1.5 mole of ATP was added per mole of HMM, and maintained this level even at 3 moles of added ATP/mole of HMM. In the presence of 3mM EGTA, the ATPase activity decreased with increase in the amount of ATP added, from 1.5 to 3 moles of ATP/mole of HMM, and reached the level of the HMM ATPase reaction at 3 moles of added ATP/mole of HMM. Similar results were observed when the concentration of HMM was maintained at 3.4 mg/ml and the concentration of the F-A-RP complex was decreased from 3 to 1 or 0.5 mg/ml.