Temperature dependence of N-phenyl-1-naphthylamine binding in egg lecithin vesicles

The temperature dependence of the binding of PhNapNH₂ ($\text{N-}\text{phenyl-1-naphthylamine}$) to vesicles of egg phosphatidylcholine has been determined. The Arrhenius plot of the association constant exhibits a discontinuity at 20.9 °C, some 30 °C above the broad phase transition region of the phospholipid. In the temperature range above 20 °C, $\text{ΔH}{}^0\text{= ?6100}\text{cal·mol}{}^{\mathrm{?1}}$ and $\text{ΔS}{}^0\text{= 9.7}\text{e. u.}$; in the temperature range below 20 °C, $\text{ΔH}{}^0\text{= 0}\text{cal · mol}{}^{\mathrm{?1}}$ and $\text{ΔS}{}^0\text{= 30.4}\text{e. u.}$ These values are consistent with the view that there are well ordered lipid-lipid bonds below 20 °C which are significantly less important above this temperature. The order in the temperature range of 5 to 20 °C, though significantly greater than that above 20 °C, is still significantly less than that in the crystalline state.     

Evidence for a role of RNA in eukaryotic chromosome structure. Metabolically stable, small nuclear RNA species are covalently linked to chromosomal DNA in HeLa cells

An investigation of metabolically stable, chromatin-associated RNA in HeLa cells has revealed that three small RNA species, 193, 171 and 127 nucleotides in length, are covalently linked to double-stranded chromosomal DNA through phosphodiester bonds. These DNA-linked RNAs appear to be members of the small nuclear RNA species that have been identified in a wide variety of eukaryotic cells, and they are tentatively identified as species C, D and G′, in the nomenclature system currently employed for HeLa cell small nuclear RNAs. These DNA-linked RNAs do not appear to be involved in priming DNA replication, since they are of relatively high metabolic stability ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 19}$ hours in HeLa cells with a 21·5-hour cell generation time) and since their covalently contiguous DNA stretches are not enriched in newly replicated material. They lack saturated pyrimidine bases (level of detection = 0·15 mol %) and are therefore not “chromosomal RNA”, as defined by its proponents. The covalent linkage of these small RNA species with chromosomal DNA was discovered by virtue of the fact that when highly purified HeLa cell chromatin is dissociated by chaotropic solutes, these RNAs are released in association with small pieces of double-stranded DNA (approx. 475 nucleotide pairs). These DNA-RNA complexes can then be purified by removing the bulk, high molecular weight DNA by ultra-centrifugation. The resulting DNA-RNA complexes are shown to be covalently joined by several criteria, including equilibrium density-gradient centrifugation in either Cs₂SO₄/dimethylsulfoxide or aqueous Cs₂SO₄/formaldehyde after thermal denaturation (90 °C in 50% formamide, which is 55 deg. C above the melting temperature of this DNA), by the chromat ographicbehavior of the complexes on hydroxylapatite before and after thermal denaturation, and by the demonstration of alkali-resistant ribonucleotides flanking the 3′ hydroxyl termini of the DNA, the latter criterion providing evidence for 3′ to 5′ DNA-RNA phosphodiester bonds. Reconstruction experiments involving addition of the purified RNAs to nuclei or chromatin demonstrate that the covalent DNA-RNA linkages do not arise by ligation events during cell fractionation. Further experiments indicate the existence of a dynamic equilibrium of these small nuclear RNA species between chromosomal and nucleoplasmic loci in vivo, and other considerations suggest that this equilibrium may be cell cycle-dependent. The DNA adjacent to these covalently linked RNAs has the same melting temperature as total HeLa chromosomal DNA and its reassociation kinetics reveal the presence of both repeated and non-repeated sequences, implying that the DNA-linked RNAs are widely distributed throughout the HeLa cell genome. It is proposed that these DNA-linked RNAs are involved in the tertiary structure of chromatin, particularly in relation to the cell cycle.     

Thermal analysis of bacteriophage T4.

The process of bacteriophage T4 morphogenesis was studied using a heat leakage scanning calorimeter. Thermograms of defective mutant 49 (am NG727) in permissive and non-permissive cells of $\text{Escherichia coli}$ showed a difference in thermal properties between packaged and non-packaged DNA molecules. $\text{In vivo}$, non-packaged DNA carried out their thermal transition at 85°C, the same temperature as that of T4 DNA melting measured in the standard saline citrate buffer, while the packaged DNA gave a sharper peak at 87°C due to some interaction with the head shell structure. Empty head shells showed a sharp heat absorption peak at 89°C both $\text{in vivo}$ and $\text{in vitro}$, indicating the high degree of cooperativity in their conformational changes.     

A simple linear transform for the Folin-Lowry protein calibration curve to 1.0 mg/ml

The stability of covalently mercurated DNAs during DNA:DNA reassociation, heteroduplex recovery on sulfhydryl-Sepharose, and S₁ nuclease digestion under a variety of solvent and temperature conditions is described. The nonspecific loss of²⁰³Hg from mercurated DNA can be minimized by use of aqueous formamide solvents in reassociation experiments and by minimizing exposure to sulfhydryl reagents and temperatures above 35°C. Single-stranded DNA is shown to be more sensitive to demercuration than is native, duplex DNA.     

The temperature dependence of the redox potential of horse heart cytochrome c in sodium chloride solutions

The E⁰′ values for the conversion of horse heart cytochrome $\text{c}$ from the oxidized to the reduced form as a function of temperature have been measured in 0.10 M NaCl, 0.10 M sodium phosphate, pH 7.0 solutions in H₂O and D₂O. In H₂O, the decrease in the E⁰′ value is linear with increasing temperature up to 42°C. Above this temperature, the decrease is again linear but with a much greater slope. In D₂O solutions, however, this biphasic behavior was not observed but instead a single line was obtained over the temperature range studied (25°C to 50°C). These results are interpreted in terms of the ability of NaCl to cause a destructuring of the bulk H₂O above 42°C but not in the more stable D₂O (Kreishman, Foss, Inoue and Leifer (1976) $\text{Biochemistry}\text{,}\text{15}$, 5431–5435). This decrease in water structure results in a shift in the equilibrium to the larger oxidized form as indicated by the decrease in E⁰′.     

The src gene product (pp60 src) of avian sarcoma virus rapidly induces DNA synthesis and proliferation of calcium-deprived rat cells

A general characteristic of neoplastic cells, but not their non-neoplastic counterparts, is the ability to proliferate in calcium-deficient medium. NRK cells infected with the transformation-defective, temperature-sensitive, ASV mutant, tsLA23, were unable to proliferate in calcium-deficient medium at the non-permissive 40°C, but they very rapidly initiated DNA synthesis (within 1 hour) and resumed proliferation in this medium after being shifted to 36°C, a temperature permissive for the production of active pp60^src and for neoplastic transformation. These observations suggest that activated pp60^src acts near the $\text{G1}\text{S}$ transition point in the cell cycle to bypass or stimulate a calcium-dependent mechanism required for the initiation of DNA synthesis, which enables the cells to display the neoplastic property of proliferating in calcium-deficient medium.     

The beta-galactosidase-catalyzed hydrolysis of o-nitrophenol-beta-D-galactoside at subzero temperatures: evidence for a galactosyl-enzyme intermediate.

The reaction of β-galactosidase ($\text{E. coli}$ K12) with $\text{o}$-nitrophenyl-β-$\text{D}$-galactoside has been investigated over the temperature range +25° to ?30° using 50% aqueous dimethyl sulfoxide as solvent. At temperatures below ?10° turnover becomes very slow and a burst of $\text{o}$-nitrophenol is observed. Such a burst indicates the existence of a galactosyl-enzyme intermediate whose breakdown is rate-limiting and provides a means of determining the active site normality. The Arrhenius plot for turnover is linear in the ?25 to +25° range with E_a = 26 ± 3 kcal/mole. The presence of the 50% DMSO had no effect on K_m but caused a small decrease in K_cat.     

The effects of environmental factors on growth and the chemical and biochemical composition of marine diatoms. I. Light and temperature effects

Temperature and light interact to modify the chemical and biochemical composition of a nitrogen-limited marine diatom, Thalassiosira allenii Takano, grown at a constant dilution rate in continuous culture and under a light:dark cycle.The percent of the total ¹⁴C incorporated into protein, polysaccharide and lipid, the N/C ratio and the C/cell varied with temperature in a markedly non-linear manner. The N/cell was negatively correlated to temperature. The Chl $\text{a}\text{C}$ ratio was positively correlated with temperature under saturating light and non-saturating light for temperatures > 25 °C, but was constant under non-saturating light conditions for temperatures < 25 °C.Productivity index (PI) was negatively correlated to temperature under saturating light conditions, but did not vary under low light. In each case, the variation in PI with temperature was governed by the variation in Chl $\text{a}\text{C}$.The dark carbon loss rate was exponentially related to temperature and independent of light. Variation in the percent of the total ¹⁴C incorporated into protein and polysaccharide, the $\text{N}\text{C}$ ratio and C/cell was primarily due to the effects of N-limitation < 20 °C and primarily due to the effects of temperature > 20 °C. Variation in N/cell was primarily due to the effects of temperature over the entire range of temperature studied. Variation in Chl $\text{a}\text{C}$ was caused by the interaction of temperature and light effects.In most cases, temperature and nutrient effects interacted to govern how a particular parameter varied with temperature while light affected the range of values over which the parameter varied.The percent of the total ¹⁴C incorporated into protein exhibited a significant linear relationship with $\text{N}\text{C}$.The dark carbon loss rate, $\text{N}\text{C}$ ratio and Chl $\text{a}\text{C}$ ratio data were used to test the applicability of a model for the physiological adaptation of unicellular algae. The model, with parameters derived from a non-linear least-squares fit of the dark carbon loss rate data, adequately described the $\text{N}\text{C}$ ratio between 15 and 25 °C at 290 and 137 μE · m^?2 · s^?1, but failed to describe the data at 28 °C and at 48 μE · m^?2 · s^?1. The Chl $\text{a}\text{C}$ ratio was adequately described by the model under all light and temperature conditions.     

Evaluation of the water environments in deoxygenated sickle cells by longitudinal and transverse water proton relaxation rates

The longitudinal and transverse water proton relaxation rates of oxygenated and deoxygenated erythrocytes from both normal adults and individuals with sickle cell disease were measured as a function of temperature at two different frequencies. The simplest model which fits all of the data consists of three different environments for water molecules. The majority of the water (98%) has a correlation time indistinguishable from bulk water (3 × 10^?11 sec). Secondly, there is a small amount of water (1.3–1.5%) present which has a correlation time of 2–4 × 10 ^?9 sec and is apparently independent of the erythrocyte sample studied. Presumably this water is the hydration sphere around the hemoglobin molecules and its correlation time is significantly slower than bulk water. The third environment contains approximately 0.2% of the water present and has a correlation time≥ 10^?7 sec. This third environment is considered tightly bound to the hemoglobin because the water proton correlation time is very similar to the expected rotational correlation time for the hemoglobin molecules. The value of the transverse relaxation rate, f_b(T_2b)^?1, for the tightly bound water fraction decreases in oxy ($\text{S}\text{S}$), deoxy ($\text{A}\text{A}$), and oxy ($\text{A}\text{A}$) erythrocyte samples as the temperature is increased as expected for a rotational correlation time process. In dramatic contrast,f_b (T_2b)^?1 increases almost linearly as the temperature is increased over the whole 4 ° to 37 °C temperature range in samples of deoxy ($\text{S}\text{S}$) erythrocytes. The observation suggests a continual increase in the formation of deoxyhemoglobulin S polymers rather than a sudden transition from a homogeneous solution of deoxyhemoglobin S molecules to a solid gel.     

Calorimetric investigations of the helix-coil conversion of phenylalaninespecific transfer ribonucleic acid

The enthalpy of the helix-coil conversion of phenylalaninespecific transfer ribonucleic acid from brewer's yeast (tRNA^Phe_{brewer's yeast}) has been measured using both an LKB 10700-2 batch miciocalorimeter and an adiabatic differential scanning calorimeter. In the mixing calorimeter the conversion from coil to helix was induced by mixing a tRNA^Phe solution with a solution containing an excess of MgSO₄. We measured the enthalpy of this reaction stepwise in the temperature range from +9 to +60° C. For the enthalpy of folding of tRNA^Phe from coil to helix this method yielded the remarkably high value of ?310 $\text{kcal}\text{mole}$ of tRNA^Phe. With the differential scanning calorimeter in which the helix-coil conversion is simply induced by raising the temperature we found a value of +240 $\text{kcal}\text{mole}$ of tRNA^Phe at a Tm value of 76° C and a value of +200 $\text{kcal}\text{mole}$ of tRNA^Phe at a T_m value of 50° C. A comparison of the apparent van't Hoff enthalpies with the calorimetrically measured enthalpies shows, that the cooperativity of the system increases continually with rising melting temperatures - which are achieved by increasing Mg²⁺ concentrations - reaching a constant value at about 57° C. Above this temperature value the thermodynamic behaviour of the helix-coil conversion of tRNA^Phe may be approximately described by the model of an all-or-none process.     

Phase transitions of phospholipid single-wall vesicles and multilayers. Measurement by vibrational raman spectroscopic frequency differences

Raman spectroscopic frequency differences between selected carbon-carbon stretching modes of lipid hydrocarbon chains were determined as a function of temperature for use in monitoring lipid phase transition behavior and acyl chain disorder in both multilamellar and single-wall vesicles. Transition temperatues detected by this procedure for pure dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine multilayers were observed at $\text{39±1 °}\text{C}$ and $\text{23±1 °}\text{C}$, respectively. Although the phase transition for unilamellar vesicles of dipalmitoyl phosphatidylcholine occurred at nearly the same temperature as the multilayers, the crystal-liquid crystalline transition for the single-shell vesicles appeared to span a slightly broader temperature range, a characteristic consistent with irregularities in the packing arrangement of the hydrocarbon chains. Within the precision of the Raman spectroscopic method, however, the temperature behavior of both the multilamellar and the unilamellar dimyristoyl phosphatidylcholine assemblies appeared nearly identical. The temperature profile for the Raman frequency differences of an excess water sonicate of 25 mol percent cholesterol in dipalmitoyl phosphatidylcholine served as an example of the effect upon lipid phase transition characteristics of a bilayer component intercalated between the acyl chains. For this particular cholesterol-lipid system the phase transition was broadened over a 30 °C temperature range, in contrast to the narrow 5?4 °C range observed for pure multilayer and single-shell vesicle particles.     

Cellular pH and water permeability control in frog urinary bladder a possible action on the water pathway

Mucosal acidification (from pH 8.1 to 6.0) reversibly inhibited the hydroosmotic responses to oxytocin, cyclic AMP and 8-bromo-cyclic AMP in frog urinary bladder. These inhibitory effects were only observed in the presence of a permeant buffer in the apical medium and could also be elicited by CO₂ bubbling, even when the mucosal pH was clamped at 8.1. Acid pH reduced the oxytocin-induced net water flux faster than norepinephrine or oxytocin removal and the difference was especially important at low temperature. The time course of recovery from acid pH inhibition was, at 20°C, similar to that of the hormonal action, but when the medium temperature was reduced to 6–7°C, the recovery from acid pH inhibition paradoxically became faster while the oxytocin action was markedly slowed down ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of changes in net water fluxes (expressed in min): oxytocin addition at 20°C, $\text{6.2 ± 0.9}$; at 6°C, $\text{24 ± 3}$; oxytocin removal at 20°C, $\text{4.7 ± 0.8}$; at 6°C, $\text{22 ± 3}$; pH inhibition at 20°C, $\text{2.6 ± 0.2}$; at 6°C $\text{2.5 ± 0.2}$; recovery from pH 6 at 20°C, $\text{6.5 ± 0.9}$; at 6°C, $\text{2.7 ± 0.3}$). These results can be explained by accepting two main loci sensitive to medium acidification: (1) the cyclase system and (2) an intracellular, temperature-independent, post-cyclic AMP site. The fact that the intramembranous particle aggregates associated with the oxytocin-induced water permeability increase did not disappear after the flow inhibition by acid pH at low temperature suggests that the second effect could be located at the water channel itself.     

Hydrophobic interactions of the apo and holo forms of human cobalamin binding proteins

The interactions of transcobalamin II (TC II), intrinsic factor (IF) and R-type binding protein of cobalamin (Cb1, vitamin B₁₂) with the hydrophobic chromatography matrix Phenyl-Sepharose CL-4B were investigated. IF-Cb1 and R-Cb1 complexes were not adsorbed on Phenyl-Sepharose at room temperature or at 4°C with buffer containing 50 mM sodium phosphate, pH 7.4 containing 150 mM sodium chloride. The TC II-Cb1 complex adsorbed and could be eluted with buffer containing 50% $\text{v}\text{v}$ glycerol. IF without Cb1 adsorbed and was eluted with 50% glycerol at room temperature and 4°C. At room temperature, R binder without Cb1 eluted with buffer, but later than the R-Cb1 complex. At 4°C, R binder completely adsorbed to the matrix. TC II-without Cb1 bound to the matrix at 4°C and room temperature and could not be eluted with glycerol. These results suggest that Cb1 binding proteins can be separated and identified based on their hydrophobic properties. In addition, upon binding Cb1, TC II, IF and R-type binders undergo a conformational change such that the protein-Cb1 complex shows reduced hydrophobicity.     

Raman studies of conformational changes in model membrane systems

Laser Raman spectra of concentrated samples of phosphatidyl choline and phosphatidyl ethanolamine were taken at approximately 10° intervals over a temperature range of 90°–19°C. The spectral region from 30 to 3300 cm^?1 was investigated. Several new spectral features were discovered which are correlated to phospholipid liquid crystalline structure. It is shown that 1) frequency shifts occur in the $\text{PO}{}_2{}^{\mathrm{?}}$ symmetric stretch band which suggest a change in exposure of the PO₂ group to the solvent upon melting, 2) the frequency of the translational hydrocarbon mode around 150 cm^?1 appears to indicate the degree to which the hydrocarbon chain is extended, 3) the methyl and methylene stretch bands at 2890 and 2850 cm^?1 very clearly demonstrate hydrocarbon chain melting, and 4) the 720 cm^?1 band, previously assigned to the symmetric OPO diester stretch, appears to be due instead to the symmetric CN stretch of choline.     

Evidence for a nuclease in Saccharomyces cerevisiae that affects the cell-free translation of natural messenger RNA.

A postpolysomal extract of $\text{Saccharomyces}\text{cerevisiae}$, treated with micrococcal nuclease to remove endogenous mRNAs, translates exogenous natural and synthetic mRNA templates actively and accurately at 20°C. When the temperature of incubation is 30°C or higher, protein synthesis with yeast poly(A)⁺ mRNA is markedly reduced, but synthesis of polyphenyl-alanine with poly (U) is only slightly affected. The protein synthesizing activity of the extract is decreased 50% in 30 minutes at 37°C, while the ability of yeast mRNA to template for protein synthesis is decreased 50% in 5 to 7 minutes when it is incubated with the postpolysomal fraction at 37°C. The release of radioactivity from isotopically-labeled yeast mRNA, into the acid-soluble form, is also much greater at 37°C than at 20°C. Thus, at the elevated temperatures, the loss of mRNA templating activity and RNA hydrolysis occur more rapidly than the loss of activity of the translational apparatus. The evidence suggests that the failure of the extract to catalyze translation at 30°C or higher, as compared to 20°C, is due to a temperature-stimulated nuclease that degrades mRNA.     

Melting point depression of DNA by tetraaklylammonium bromides

Varied concentrations of tetramethyl- (TMAB), tetraethyl- (TEAB), tetrapropyl- (TRAB), tetrabutyl- (TBAB), tetrapentylammonium bromide (TPAB) and ammonium bromide are monitored for effects on DNA sedimentation, viscosity and melting temperature. No changes are observed at 25–30°C for the hydrodynamic properties of DNA when treated with the quaternary ammonium bromide. At higher temperatures, 30–100°C, the tetraalkyl-ammonium bromides reduce the temperature required for conversion of the DNA double-strand helix to the coil (T_m). A form of Debye-Huckel equation ($\text{T}{}_{\mathrm{m}}\text{=a}\text{mu;}\text{2}\text{+ b}$) accurately describes the results. Ionic strength of the quaternary ammonium compounds is represented by μ/2. The potency of tetraalkylammonium bromide in reducing T_m (“a” in the T_m equation) is correlated with the hydrophobicity of the molecule (number of carbon atoms), r = 0.999 and F = 526 (r represents the correlation coefficient and F represents the test value for significance of the equation evaluated by least squares analysis). The T_m of rat liver chromatin is also reduced in a similar fashion by TPAB. The T_m of poly(dG-dC) is not altered by 0.1 M TBAB whereas the T_m of poly(dA-dT) is greatly reduced by the compound.It is thought that the tetraalkylammonium bromide preferentially binds to the coil of DNA during heating and that a preferentially hydrophobic interaction may occur with the adenine-thymine bases.     

Protein rotation and chromophore orientation in reconstituted bacteriorhodopsin vesicles

Bacteriorhodopsin has been reconstituted into lipid vesicles with dipalmitoyl and dimyristoyls phosphatidylcholine. Circular dichroism (CD) measurements show that the proteins are in a monomeric state above the main lipid phase transition temperature (T_c), 41 and 23°C for dipalmitoyl and dimyristoyl phosphatidylcholine, respectively. Below T_c, the CD spectrum is the same as that found for the purple membrane. The latter result implies that the orientation of the chromophore at these temperatures is most likely the same as in the purple membrane ($\text{70° ± 5°}$ from the normal to the membrane plane).Transient dichroism measurements show that below T_c the proteins are immobile, while above this temperature protein rotation around an axis normal to the plane of the membrane is occurring. In addition, from the data the angle of the chromophore for the rotating proteins with respect to the rotational diffusion axis can be calculated. This angle is found to be $\text{30° ± 3°}$ and $\text{29° ± 4°}$ in dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, respectively. This is considerably smaller than the value of $\text{70° ± 5°}$ for the natural biomembrane. A reversible reorientation of the chromophore above and below the respective main T_c transition temperature could explain the change of angle observed provided that all the molecules rotate above T_c.     

Transcriptional activity and chromatin structural changes in a temperature-sensitive mutant of BHK cells blocked in early G1.

AF8, a temperature-sensitive mutant of BHK $\text{21}\text{13}$ cells, has been shown to arrest at the non-permissive temperature in the G1 phase of the cell cycle. When AF8 cells are released from density-dependent arrest of growth by trypsinization and replating at lower density, 60–80% of the cells enter DNA synthesis and divide at the permissive temperature (33 °C), while only 20% enter DNA synthesis at the non-permissive temperature (39.5 °C). The temperature-sensitive block has been localized 4 to 8 h before the onset of DNA synthesis which begins at 12 h after stimulation. Two biochemical events of the prereplicative phase have been temporally related to this temperature-sensitive block. RNA synthesis as measured in isolated nuclei increases initially at both temperatures, then levels off and declines to control levels at 39.5 °C while continuing to increase at 33 °C. Parallel changes are found in circular dichroism spectra and ethidium bromide binding capacity of isolated chromatin. The results suggest that these biochemical changes are involved in the regulation of the prereplicative phase and the subsequent entrance of cells into DNA synthesis.