Temperature-sensitive mutant rho-115 rho-RNA binary complexes, and stabilization by substrates and analogues

Summary To determine the molecular basis for the temperature-sensitivity of pure rho RNA-dependent ATPase from Escherichia coli mutant rho-115 cells, we investigated mutant rho binding to [³H] polyC as measured by retention on nitrocellulose filters. Complexes of wild-type rho and polyC incubated at 37°C and 45°C were similarly stable. At 37°C mutant rho-polyC binary complexes were inactivated at a slightly faster rate than complexes with wild-type rho. Upon shift to 45°C the quantity of rho-115 bound to polyC declined immediately, resulting in one-fifth of the quantity of complexes observed at 37°C. Shift back to 37°C restored the level of observed complexes by two-fold. The inclusion of ATP or the analogue - methylene ATP during 45°C incubation resulted in stable mutant rho-polyC complexes. The hydrolysis product ADP was also effective in stabilizing binary complexes at 45°C but this effect was observed with an order of magnitude more ADP than ATP. Adenine, adenosine, AMP or P_i had no stabilizing effect. We conclude that the mutant rho-115 protein exhibits a structural instability as a result of binding RNA. Furthermore ATP confers a wild-type phenotype upon rho-115 protein, probably as a result of conformational change due to binding of this compound. The effect of ATP on the stability of mutant rho-polyC binary complexes supports the model of ATP modulation of rho-RNA interaction proposed by Galluppi and Richardson (1980).     

Reduced superhelicity of plasmid DNA produced by the rho-15 mutation in Escherichia coli

Summary The plasmid pJSF6, a derivative of pBR327, could be maintained at 30° C in strains of Escherichia coli containing the strong rho mutation, rho-15. Plasmids extracted from rho-15 cells were always less negatively supercoiled than plasmids from rho ⁺ cells. Transduction experiments designed to separate the rho gene from possible extragenic suppressors showed that the rho allele consistently determined the degree of plasmid superhelicity. Comparison of the superhelicity of plasmids extracted from the rho-15 and from a gyrB mutant showed that at 30° C the negative supercoiling was reduced by the amounts W _rho=4.0±0.3 and W _gyr=6.0±0.3 turns; the effect of the rho-15 mutation on supercoiling was thus comparable to that of the gyrB mutation. A similar effect of the rho-15 mutation on the superhelicity of pBR329 was observed. The observation that the Rho protein has a role in determining DNA superhelicity (though not necessarily a direct role) provides a new point of view for studying the pleiotropic properties of rho mutants.We dedicate this paper to the cherished memory of Ethel S. Tessman, who died May 10, 1986. She encouraged and advised and stimulated each of us in the development of our careers     

Second-site rho mutation: Genetic linkage and polyC-dependent ATPase

Summary Rho has been purified to homogeneity from Escherichia coli double mutant rho-115 sur-38 cells, and from rho ⁺⁺ and rho-115 cells. The sur-38 mutation suppresses the original rho-115 phenotype. We observe that the polyC-dependent ATPases of these three rho preparations have the same specific activities. However, the ATPase of rho from the double rho-115 sur-38 mutant is extremely heat labile, while that from rho-115 shows a heat lability intermediate between the wild type and the double mutant.Transduction analysis suggests that sur-38 is closely linked to rho-115 in the order ilv-sur-38-rho-115-metE. These data are consistent with the model that the sur-38 mutation affects the structural gene for rho.Contact for offprints     

Rifampicin supersensitivity of rho strains of E. coli, and suppression by sur mutation.

Summary Escherichia coli strains with mutations rho-115, rho-ts15, rho-101 (psu-1) or rho-102 (psu-2) are more sensitive (supersensitive) to rifampicin than isogenic parent strains, as measured by growth rate in broth and colony forming efficiency on solid media with 5, 10, or 20 g of rifampicin per ml. There is no change in sensitivity of rho mutants to the antibiotics penicillin, erythromycin, chloramphenicol, or the detergent desoxycholate. The rho-101 or rho-102 mutations confer rifampicin supersensitivity at 32°C but not 42°C. Mutants of a rho-115 strain that have lost polarity suppression can be isolated by selection for rifampicin resistance. This phenotype, Sur, is not due to reversion of the original rho gene mutation but to a second mutation perhaps in the gene for rho protein or the gene for the subunit of RNA polymerase. One class of Sur mutation, occurring in rho-115 cells isolated as resistant to 20 g of rifampicin per ml, is co-transducible with the marker ilv, and the gene order is rbs-ilv-sur-38. A model suggested by this map position is that the mutations rho-115 and sur-38 define the domain of rho protein which interacts with the subunit of RNA polymerase.     

Synergistic effects of ethanol and temperature on yeast mitochondria

At temperatures lower than 37°C, the ethanol inhibition constant (Ki) for growth or fermentation inrho ⁺ cells of theSaccharomyces cerevisiae strain S288C was always higher (1.1M) than inrho ^– mutants (0.7M). At 37°C these differences disappeared, and both strains were equally inhibited by ethanol (Ki=0.7m). Mitochondrial activity can be inhibited by high ethanol concentration and temperature. In fact, the stronger inhibition by ethanol of therho ⁺ strain at 37°C was due to the fact that, under these conditions, this strain loses the advantage conferred by mitochondrial activity since the induction ofrho ^– cells in the population is very high. This does not result in an increase in the frequency ofrho ^– mutants because of the poor viability of these mutants in conditions of high temperature and ethanol. In consequence, S288C strain becomes as strongly inhibited by ethanol as therho ^– mutant strains. Differences in viability were not related to the fatty acids and ergosterol composition of the strain. In the presence of ethanol, bothrho ⁺ andrho ^– strains modified their lipids in the same way, but these changes did not improve their ethanol tolerance. They were not due to differences in adaptation to ethanol either, since after successive transfers in ethanol, growth () and fermentation () rates in therho ^– mutants were increasingly inhibited with time, whereas in the S288C strain inhibition of and by ethanol remained unaltered. Rather,rho ^– mutants are less viable thanrho ⁺ cells because of the inability of the former to respire. At 37°C the Ki increased to 0.9M ethanol either when mitochondrial from highly ethanol-tolerant wine yeasts were transferred torho ^– mutants of the strain S288C or when the mitochondria of strain S288C were preadapted by growing the strain in glycerol instead of glucose before it was cultivated in ethanol.     

Calcium regulatory proteins and temperature acclimation of actomyosin ATPase from a eurythermal teleost (Carassius auratus L.)

Summary Goldfish (Carassius auratus) were acclimated for 5 months at temperatures of either 2°C or 31°C. Natural actomyosin was prepared from white myotomal muscle and its Mg²⁺Ca²⁺ ATPase activity determined. Temperature acclimation results in adaptations in substrate turnover number and thermodynamic activation parameters of the ATPase. When assayed at 31°C the Mg²⁺Ca²⁺ ATPase of natural actomyosin was 4 times higher in 31°C than 2°C acclimated fish. Arrhenius plots of natural actomyosin ATPase from cold acclimated fish show a break in slope at 15–18°C. In contrast, the temperature dependence of warm acclimated actomyosin was linear. Activation enthalpy (H ) of the ATPase, calculated over the range 0–16°C, was approximately 8,000 cal/mole lower in 2°C than 32°C acclimated fish.In contrast, desensitised actomyosins from which the calcium regulatory proteins have been removed show a linear temperature dependence in the range 0–32°C and have similar properties in 2°C and 31°C acclimated fish. Cross-hybridisation of regulatory proteins (tropomyosin-troponins complex) from cold-acclimated fish to desensitised actomyosin from warm-acclimated fish alters the ATPase towards that of cold-acclimated natural actomyosin and vice versa. The results suggest that the regulatory proteins can influence the kinetics of the ATPase and, furthermore, that they are involved in the acclimation of the actomyosin to different cell temperatures.     

Effect of pH on stability of sarcoplasmic reticulum calcium pump in rabbit heart

The sarcoplasmic reticulum (SR) membranes isolated from rabbit heart were preincubated at pH 6.8 or 7.8 and their Ca²⁺ pump properties were compared at pH 6.8. The ATP-dependent azide insensitive oxalate-stimulated Ca²⁺ uptake was reduced more rapidly from the membranes preincubated at 37°C at pH 7.8 than from those preincubated at pH 6.8. The Ca²⁺–Mg²⁺-ATPase, and the Ca²⁺-dependent formation of 110 kDa acylphosphate were also inhibited by the preincubation at the higher pH. Including 1 mM DTT in the preincubation medium reduced the inactivation. The preincubation at 37°C in the presence or absence of DTT caused membranes to become more leaky as the loss of Ca²⁺ uptake was more rapid than that of ATPase or the acylphosphate formation. The loss of these activities was not accompanied by a breakdown of the protein as monitored in Western blots. It is hypothesized that the SR Ca²⁺ pump inactivation involves a key-SH group and that the lower pH provides a compensatory protective mechanism for the SR during acidosis.     

Evidence implicating a membrane ATPase in the control of passive permeability of excitable cells

The temperature sensitivity of the ATPase enzyme systems in a muscle microsomal preparation from the crayfish, Astacus pallipes, was studied. Preincubation of the enzyme preparation in the range 33–36°C produced a marked inactivation of the ATPases; the Mg⁺⁺-dependent ATPase was very much more sensitive to this treatment than the Na⁺-K⁺-Mg⁺⁺-dependent ATPase. Thus, the Arrhenius μ for the inactivation of the Mg⁺⁺-dependent ATPase produced by eight minute preincubation is > 100 Kcals. These results are compared with the changes that are observed during the heat death of the whole animal, where exposure to 35°C produces a dramatic change in Na⁺ permeability within five minutes. Arrhenius μ for heat death is also > 100 Kcals and operates over the identical critical temperature range. It is suggested that the Mg⁺⁺-dependent ATPase controls passive permeability in these excitable cells and the results also confirm the view that Mg⁺⁺ and Na⁺-K⁺-Mg⁺⁺ ATPases are separate enzymes.     

Mitochondrial resistance to miconazole in Saccharomyces cerevisiae

Summary One mutant of mitochondrial origin resistant to miconazole has been isolated and characterized in S. cerevisiae. The mutation is linked to the locus oli1, the structural gene for subunit 9 of ATPase on mitochondrial DNA. Miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect. Levels of ATP decreased to one-third of the control in the wild type in the presence of miconazole, while they were unaffected in the mutant.Abbreviations MNNG N-methyl-N-nitrosoguanidine - Mic^s/Mic^r phenotypic sensitivity/resistance to miconazole - M ₁ ^R mitochondrial locus conferring miconazole resistance - rho⁺/rho^- grand/cytoplasmic petite - rho^o cytoplasmic petite deleted of all mitochondrial DNA - w⁺ mitochondrial locus conferring polarity of recombination     

Cell inactivation,mutation and DNA strand-break induction by γ-rays at very low temperatures

Cell inactivation, mutation and DNA strand-break induction by -radiation have been investigated at very low temperatures (–78° C, –196° C, and –268° C). InEscherichia coli Y _mel ,lacI ⁺ lacI ^– andSalmonella typhimurium TA102,his ^–his ⁺ dose-modifying factors determined for low radiation doses are similar for both mutation induction and cell inactivation. The sensitivity of repair-deficient strainsE. coli polA ^– andE. coli recA ^– was also reduced at low temperature to a comparable extent. This suggests that the lesions which are responsible for cell inactivation and mutagenesis could be strongly mutually related and/or that different types of lesions which are responsible for cell inactivation and mutation induction in bacteria are reduced at low temperature to the same or similar extent. Likewise, a lower yield of DNA strand breaks in plasmids irradiated at low temperature was observed.     

Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling

Photosynthetic activity, in leaf slices and isolated thylakoids, was examined at 25° C after preincubation of the slices at either 25° C or 4° C at a moderate photon flux density (PFD) of 450 mol·m^–2·s^–1, or at 4° C in the dark. The plants used wereSpinacia oleracea L.,Cucumis sativus L. andNerium oleander L. which was acclimated to growth at 20° C or 45° C. The plants were grown at a PFD of 550 mol·m^–2·s^–1. Photosynthesis, measured as CO₂-dependent O₂ evolution, was not inhibited in leaf slices from any plant after preincubation at 25° C at a moderate PFD or at 4° C in the dark. However, exposure to 4° C at a moderate PFD induced an inhibition of CO₂-dependent O₂ evolution within 1 h inC. sativus, a chilling-sensitive plant, and in 45° C-grownN. oleander. The inhibition in these plants after 5 h reached 80% and 40%, respectively, and was independent of the CO₂ concentration but was reduced at O₂ concentrations of less than 3%. Methyl-viologen-dependent O₂ exchange in leaf slices from these plants was not inhibited. There was no photoxidation of chlorophyll, in isolated thylakoids, or any inhibition of electron transport at photosystem (PS)II, PSI or through both photosystems which would account for the inhibition of photosynthesis. The conditions which inhibit photosynthesis in chilling-sensitive plants do not cause inhibition inS. oleracea, a chilling-insensitive plant, or in 20° C-grownN. oleander. The CO₂-dependent photosynthesis, measured at 5° C, was reduced to about 3% of that recorded at 25° C in chilling-sensitive plants but only to about 30% in the chilling-insensitive plants. Methyl-viologen-dependent O₂ exchange, measured at 5° C, was greater than 25% of the activity at 25° C in all the plants. The results indicate that the mechanism of the chilling-induced inhibition of photosynthesis does not involve damage to PSII. That inhibition of photosynthesis is observed only in the chilling-sensitive plants indicates it is related, in some way, to the disproportionate decrease in photosynthetic activity in these plants at chilling temperatures.Abbreviations Chl chlorophyll - DPIPH reduced form of 2,6-dichlorophenol-indophenol - DMQ 2,5-dimethyl-p-benzoquinone - MV methyl viologen - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density (photon fluence rate) - PSI and PSII photosystem I and II, respectively     

Resistance adaptation of the freshwater crayfish and thermal inactivation of membrane-bound enzymes

Summary Heat death and resistance adaptation of freshwater crayfish are thought to be properties of its muscle membranes. The inactivation at high temperatures of a membrane-bound enzyme, the Ca⁺⁺-stimulated ATPase of crayfish abdominal muscle sarcoplasmic reticulum, and the effect of thermal acclimation of crayfish upon the inactivation kinetics have been investigated. In the absence of KCl, the Ca⁺⁺-stimulated ATPase is irreversibly inactivated with pseudo-first order kinetics at temperatures that cause heat death in the whole animal. 0.1–10.0 mM KCl resulted in slower inactivation, while 100 mM KCl activated the enzyme to 120–180% of its original activity. Enzyme activation by KCl and heat involved a shift in the enzyme concentration/activity curve. Thermal acclimation of crayfish had no significant effect upon the kinetics or Arrhenius activation energy for enzyme inactivation (100.6±10.5 and 92.3±14.6 kcal/mole for preparations from 4°C and 25°C acclimated crayfish).Ca⁺⁺-stimulated ATPase isolated from heat dead crayfish exhibited normal in vitro activity due presumably to the high intracellular K⁺ concentration. Nevertheless, the close correspondence between heat death temperatures and inactivation temperatures for several membrane-bound enzymes of muscle is thought to reflect some perturbation of muscle structure that occurs during heat death.Abbreviations ATP Ademosine 5-Triphosphate - EGTA Ethyleneglycol-bis [-amino-ethyl ether] - N N-tetraacetic acid - Hepes N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic acid - FSR Fragmented sarcoplasmic reticulum - Tris Tris (hydroxymethyl)aminomethane     

Influence of ionic composition,buffering agent,and pH on the histochemical demonstration of myofibrillar actomyosin ATPase

Summary The influence of the composition of the preincubation medium on the histochemical demonstration of myofibrillar actomyosin ATPase, including a variety of carboxylic acid and non-carboxylic acid buffering compounds and neutral salts, was studied. In inorganic salt-free systems the rate of the activation of type I fibers and inactivation of type II fibers was accelerated when the carboxylic acids had longer chain length or multiple carobxyl groups. Of these factors, the number of carboxyl groups was dominant with a 100 mM citrate buffer producing a sharp differentiation between fiber types. In contrast, the time course of the response was exceptionally long in an acetate buffer. The time course of the ATPase reaction was also modified by other buffers at pH 4.60. The most notable were an ascorbate — glycine buffer system which produced little or no deviation from the alkaline preincubation staining pattern after prolonged preincubation and a pyrophosphate system which produced a rapid change. Neutral salts in the preincubation medium accelerated the time course of the inactivation — activation process with the order for the halogen salts of K⁺ being F⁻<Cl⁻<Br⁻<I⁻, which is a progression by molecular weight. The only sequence for cations on the myofibrillar actomyosin ATPase was Li⁺< Na⁺<K⁺. The response to salts was concentration dependent. An interaction existed between buffering compound, type of salt, and pH. These experiments demonstrate that the histochemical differentiation of fiber types by the myofibrillar actomyosin ATPase reaction depends upon a modification of some component(s) of the myofibrillar complex that can be influenced by a number of factors.     

The effects of assay temperature upon the pH optima of enzymes from poikilotherms: A test of the imidazole alphastat hypothesis

Summary A study of the thermal responses of Na-ATPase and NaK-ATPase activities in microsomes prepared from gill tissue of rainbow trout (Salmo gairdneri) revealed further evidence that the two activities are distinct from one another. Arrhenius plots of the NaK-ATPase from sea water-adapted fish and the Na-ATPase from fresh water-adapted fish were linear (Fig. 4) with estimated activation energies of 19.5 and 7.7 kcal/mole, respectively. The Na-ATPase and NaK-ATPase both showed optimum activity at 45°C (Figs. 2 and 3). The Mg-ATPase from fresh water fish showed a distinct temperature optimum at 24°C (Fig. 1) while Mg-ATPase activity from sea water fish was optimum at temperatures of about 15–24 °C (Fig. 3). The Na⁺ dependence of the Na-ATPase and the NaK-ATPase was examined at an assay temperature of 37 °C (Fig. 5) and the results compared with those obtained at 13 °C. No apparent differences were noted for the Na-ATPase, but with the NaK-ATPase both theK _0.5 for Na⁺ and optimum Na⁺ concentration increased at the higher assay temperature. Finally, evidence is presented showing the Na-ATPase to be distinct from Mg-ATPase activity in fresh water trout gill microsomes.Abbreviation HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid     

Revertants from RNase III negative strains of Escherichia coli

Summary E. coli strains carrying the rnc-105 allele do not show any level of RNase III in extracts, grow slower than rnc ⁺ strains at temperatures up to 45°C and fail to grow at 45°C. Revertants which can grow at 45°C were isolated. The vast majority of them still do not grow as fast as rnc ⁺ strains and did not regain RNase III activity. The mutation(s) which caused them are suppressor mutations (physiological suppressors) which do not map in the immediate vicinity of the rnc gene. A few of the revertants regain normal growth, and contain normal levels of RNase III. They do not harbor the rnc-105 allele and therefore are considered to be true revertants. By using purines other than adenine it was possible to isolate rnc ⁺ pur ^- revertants from an rnc ^- pur ^- strain with relative ease. They behaved exactly like the true rnc ⁺ revertants isolated from rnc ^- strains at 45°C.A merodiploid strain which contains the rnc ⁺ gene on an episome behaves exactly like an rnc ⁺ strain with respect to growth and RNA metabolism, eventhough its specific RNase III activity is about 60% of that of an rnc ⁺ strain; thus the level of RNase III is not limiting in the cell.The rnc ^- strains show a characteristic pattern of transitory molecules, related to rRNA, 30S, 25S, p23 and 18S, which are not observed in rnc ⁺ strains. This pattern is unchanged in rnc ^- strains and in the revertants which are still lacking RNase III, regardless of the temperature in which RNA synthesis was examined (30° to 45°C). On the other hand, in the rnc ⁺ strains as well as in the true revertants and the rnc ⁺/rnc ^- merodiploid, the normal pattern of p16 and p23 is observed at all temperatures. These findings suggest that all the effects observed in RNase III^- strains are due to pleiotropic effects of the rnc-105 allele, and that the enzyme RNase III is not essential for the viability of the E. coli cell.     

Inactivation by Na+,K+-ATPase of cytosol glucocorticoid receptors from rat brain and liver

Summary We have examined the effect of Na⁺,K⁺-ATPase on ³H-triamcinolone acetonide binding capacity of cytosol glucocorticoid receptors from rat brain and liver. Preincubation of the brain or liver cytosol with Na⁺,K⁺-ATPase (10 units/ml) at 30 °C resulted in a rapid loss of specific ³H-triamcinolone acetonide binding, with a half-life of approximately 7 min. The ATPase effect could be prevented by the addition of 10^–5 M ouabain, or substantially reduced by the omission of Na⁺,K⁺ or Mg⁺². The cytosol receptor bound with ³H-triamcinolone acetonide was totally resistant to the inactivation by the ATPase. Since there is some evidence that ATP may bind to glucocorticoid receptor, our findings indicate that an ATP-receptor complex may be essential for steroid binding. The effects of the ATPase in the inactivation of the receptor are very similar to those of alkaline phosphatase reported by others. This raises doubts about the proposal based on the phosphatase inactivation that the cytosol glucocorticoid receptor may be phosphorylated.     

Effects of tellurite on growth of Saccharomyces cerevisiae

The effects of potassium tellurite on growth and survival of rho⁺ and rho⁰ Saccharomyces cerevisiae strains were investigated. Both rho⁺ and rho⁰ strains grew on a fermentable carbon source with up to 1.2 mM K₂TeO₃, while rho⁺ yeast cells grown on a non-fermentable carbon source were inhibited at tellurite levels as low as 50 μM suggesting that this metalloid specifically inhibited mitochondrial functions. Growth of rho⁺ yeast cells in the presence of increasing amount of tellurite resulted in dose-dependent blackening of the culture, a phenomenon not observed with rho⁰ cultures. Transmission electron microscopy of S. cerevisiae rho⁺ cells grown in the presence of tellurite showed that blackening was likely due to elemental tellurium (Te⁰) that formed large deposits along the cell wall and small precipitates in both the cytoplasm and mitochondria.     

Polyphenolic Antioxidants Efficiently Protect Urease from Inactivation by Ultrasonic Cavitation

Inactivation of urease (25 nM) in aqueous solutions (pH 5.0–6.0) treated with low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm², 36–56°C) or high-frequency ultrasound (HFUS; 2.64 MHz, 1 W/cm², 36 or 56°C) has been characterized quantitatively, using first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(us), ultrasonic inactivation. Within the range from 1 nM to 10 M, propyl gallate (PG) decreases by approximately threefold the rate of LFUS-induced inactivation of urease (56°C), whereas resorcinol poly-2-disulfide stops this process at 1 nM or higher concentrations. PG completely inhibits HFUS-induced inactivation of urease at 1 nM (36°C) or 10 nM (56°C). At 0.2–1.0 M, human serum albumin (HSA) increases the resistance of urease treated with HFUS to temperature- and cavitation-induced inactivation. Complexes of gallic acid polydisulfide (GAPDS) with HSA (GAPDS–HSA), formed by conjugation of 1.0 nM GAPDS with 0.33 nM HSA, prevent HFUS-induced urease inactivation (56°C).     

Metabolic and energetic aspects of the growth of Bacillus stearothermophilus in glucose-limited and glucose-sufficient chemostat culture

With a glucose-limited chemostat culture of Bacillus stearothermophilus, increasing the incubation temperature progressively from 45°C to 63°C led to a progressive marked increase in the maintenance rates of glucose and oxygen consumption. Hence, at a fixed low dilution rate the yield values with respect to glucose and oxygen decreased substantially with increased temperature. However, the apparent Y _glucose ^max and values did not decrease but actually increased with temperature, being highest at 63°C (i.e., close to the maximum growth temperature). With glucose-sufficient cultures growing at a fixed low dilution rate (0.2 h^–1) and at their optimum temperature (55°C), glucose and oxygen consumption rates invariably were higher than that of a corresponding glucose-limited culture. Cation (K⁺ or Mg²⁺)-limited cultures expressed the highest metabolic rates and with the K⁺ limited culture this rate was found to be very markedly temperature dependent. As the temperature was increased from 45°C to 63°C the rate of glucose consumption increased 1.8-fold, and that of oxygen consumption by 3.7-fold. The culture pH value also exerted a noticeable effect on the metabolic rate of a glucose-limited culture, particularly at the extremes of pH tolerance (5.5 and 8.5, respectively). A K⁺-limited culture was less affected with respect to metabolic rate by the culture pH value though the steady state bacterial concentration, and thus the cellular K⁺ content, changed substantially. These results are discussed in relation to previous findings of the behaviour of this organism in batch culture, and to the behaviour of other thermophilic Bacillus species in chemostat culture.     

Amino group modification of (Na++K+)-ATPase

The effects of three amino group reagents on the activity of (Na⁺+K⁺)-ATPase³ and its component K⁺-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na⁺ of Mg²⁺ present, the enzyme inactivation process follows complicated kinetics. In the presence of K⁺, Rb⁺, or Tl⁺, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg²⁺ (P ₅₀ 6 µM vs. 1.2 mM). Under certain conditions (100 µM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ and K⁺ have no effects, and ATP only a minor effect, on the degree of modification. The K⁺-stimulatedp-nitrophenylphosphatase activity is less inhibited than the (Na⁺+K⁺)-ATPase activity. Half-inhibition of the (Na⁺+K⁺)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na⁺+K⁺)-ATPase. Mg²⁺ has no effect, and ATP has only a slight protecting effect. The K⁺-stimulatedp-nitrophenylphosphatase activity is inhibited in parallel with the (Na⁺+K⁺)-ATPase activity. Half-inactivation of the (Na⁺+K⁺)-ATPase activity is obtained after 20% modification of the amino groups.This article is No. 52 in the series Studies on (Na⁺+K⁺)-Activated ATPase.