Dielectric relaxation of DNA solutions. III. Effects of DNA concentration, protein contamination, and mixed solvents

As a continuation of previous papers [Biopolymers (1976) 15 , 879; (1978) 17 , 1508], the low-frequency dielectric relaxation of DNA solutions was studied with a four-electrode cell and the simultaneous two-frequency measurement. Below a critical concentration, the dielectric relaxation time agrees with the rotational relaxation time estimated from the reduced viscosity and is almost independent of DNA concentration C_p, and the dielectric increment is proportional to C_p. The critical concentration is approximately 0.02% of DNA for molecular weight M_r 2 × 10⁶ and 0.2% for M_r 4.5 × 10⁵ in 1 mM NaCl. Dielectric relaxations are compared for samples before and after deproteinization, and the protein contamination is found to have a minor effect on the dipole moment of DNA. The effect of a mixed solvent of water and ethanol on the dielectric relaxation of DNA is well interpreted in terms of changes in viscosity and the dielectric constant of the solvent, assuming that the relaxation arises from rotation of the molecule with a quasi-permanent dipole due to counterion fluctuation.     

Kinetic study of denaturation and subsequent reduction of disulfide bonds of lysozyme by the rapid ultrasonic absorption measurement

A New technique for the rapid measurement of ultrasonic absorption with a sampling interval of 5 msec has been developed and applied to the kinetic study of denaturation and subsequant redution of hen egg-white lysozyme. The lysozyme is denatured by guanidine hydrochloride (GuHCl) orLiBr, and afetr denaturation by GuHCl, its disulfide bonds are reduced by dithiothreitol (DTT). The ultrasonic adsorption coefficient at 9 MHz increases with denaturation but decreases with reduction. The rate constant of denaturation by GuHCl obtained from the rime variance of ultasonic agrees well with that from uv absorption and optical rotation. The time variance if absorption after GuHCl and Dtt have been simultaneously added exhibits two rate constants. Analysis of the constants as functions of regeant concentrations indicates that the intermediates state between native and reduced states is not necessarily the completely denatured state but depends on the concentartions of GuHCl and DTT.     

Pumpkin (Cucurbita sp.) seed globulin I. Purification, characterization, and subunit structure

A heat stable globulin present in the cotyledons of pumpkinseeds was prepared as crystals which were soluble in a dilutesaline solution below pH 4.5 or in a solution with a high ionicstrength at neutral pHs. The protein was nearly homogeneousby ultracentrifuge analysis, and had a molecular weight of about112,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisseparated the globulin into two subunits, and ß,corresponding to molecular weights of about 63,000 and 56,000daltons, respectively. By reduction of disulfide bonds, thetwo subunits were each separated into two polypeptide chainswith molecular weights of around 36,000 and 22,000 daltons,judged by gel electrophoresis. The amino acid composition ofwhole globulin indicated high contents of arginine, glutamicacid and aspartic acid. The total number of half-cystine residuewas nine and only one residue was shown to be free. The subunitstructure of the globulin is discussed. The protein has beenshown to have oxaloacetate decarboxylase activity, and thisfact was confirmed. However, the activity decreased markedlyat pH 4.5 in a fairly short period. It did not require Mn⁺⁺,and the K_m for oxaloacetate was determined to be 4.1 mM. (Received April 9, 1976; )     

Dielectric relaxation of DNA in aqueous solutions.

Using a four-electrode cell and a new electronic system for direct detection of the frequency differences specturm of solution impedance, the complex dielectric constant of calf thymus DNA (M_r = 4 × 10⁶) in aqueous NaCl at 10°C is measured at frequencies ranging from 0.2 Hz to 30 kHz. The DNA concentrations are C_p = 0.01% and 0.05%, and the NaCl concentrations are varied from C_s = 10^?4 M to 10^?3 M. A single relaxation regions is found in this frequency range, the relaxation frequency being 10 Hz at C_p = 0.01% and C_s = 10^?3 M. At C_p = 0.05% it is evidenced that the DNA chains have appreciable intermolecular interactions. The dielectric relaxaton time τ_d at C_p = 0.01% agrees well with the rotational relaxation time estimated from the reduced visocisty on the assumption that the DNA is not representable as a rigid rod but a coiled chain. It is concluded that the dielectric relaxiatioinis ascribed to the rotation of the molecule. Observed values of dielectric increment and other experimental findings are reasonably explained by assuming that the dipole moment of DNA results from the slow counterion fluctuation which has a longer relaxation time than τ_d.     

Kinetics of proton transfer reactions of lysozyme with p-nitrophenol near neutral pH—a study of dynamic properties of Glu 35 and His 15

Kinetics of proton transfer between lysozyme and a pH indicator p-nitrophenol (p-Np) were measured by the temperature-jump method in a pH range of 6.0–7.0. Two well-defined relaxation processes were observed. The fast process (τ ? 15 μsec) was also observed for a lysozyme derivative succinylated at the terminal α-amino group of Lys 1. Therefore, the fast process was found to be attributable to the proton transfer reaction of His 15 with p-Np. The slow process (τ ? 50 μsec) was found to be characteristic of the proton transfer reaction of Glu 35, because it disappeared completely in solution containing a lysozyme derivative having an ester crosslink between the carboxyl group of Glu 35 and indol C-2 of Trp 108. The rate constants for proton transfer from Glu 35 and His 15 to p-Np were found to be 9 × 10⁶/sec/M (±65%, 23°C) and 3 × 10⁸/sec/M (±20%, 25°C), respectively. These data indicate that the proton of the carboxyl group of Glu 35 is kinetically stabilized in lysozyme.     

Fine structures in denaturation curves of bacteriophage lambda DNA. Their relation to the intramolecular heterogeneity in base compositon.

Precise recording of polyphasic optical melting curves was carried out for three kinds of bacteriophage lambda DNA differing in length (lambdac1857s7, lambdacIb2 and lambdacIb2b5). Each of denaturation steps in melting profiles was characterized by two parameters, the melting temperature and the relative size. Any difference in fine structures in melting profiles was not recognized between the intact lambdacI857s7DNA and the DNA fragmented into halves. The change in fine structures in melting profiles caused by the deletions of the b2 and b5 region agreed qualitatively well with the prediction based on the physical and the genetical maps of phage lambda chromosome. The combined results indicate that, first, the well-known linear relationship between melting temperature and G+C content may apply also to each of denaturation steps in polyphasic melting curves due to heterogeneity of nucleotide distribution in a single DNA species, and, second, the effect of molecular ends on melting fine structures can be neglected at moderate salt concentration (0.01 M less than or equal to Na+ less than or equal to 0.2 M) for such a high molecular weight DNA. The heterogeneous distribution of nucleotides was derived for lambdaDNA and for its b2 and b5 regions.     

Purification and properties of avian liver p-hydroxyphenylpyruvate hydroxylase.

Avian liver p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27) was purified to a 1000-fold increase in specific activity over crude supernatant, utilizing a substrate analogue, o-hydroxyphenylpyruvate, to stabilize the enzyme. The preparation was homogeneous with respect to sedimentation with a sedimentation velocity (s20,w) of 5.3 S. The molecular weight of the enzyme was determined to be 97,000 +/- 5,000 by sedimentation equilibrium, and the molecular weight of the subunits was determined to be 49,000 +/- 3,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis revealed heterogeneity of the purified enzyme. The multiple molecular forms were separable by isoelectric focusing, and their isoelectric points ranged from pH 6.8 to 6.0. The amino acid compositions and tryptic peptide maps of the three forms isolated by isoelectric focusing were very similar. The forms of the enzyme had the same relative activity toward p-hydroxyphenylpyruvate and phenylpyruvate. Conditions which are known to accelerate nonenzymic deamidation of proteins caused interconversion of the multiple molecular forms. Iron was the only transition metal found to be associated with the purified enzyme at significant levels. The amount of enzyme-bound iron present in equilibrium-dialyzed samples was equivalent to 1 atom of iron per enzyme subunit. Purification of the enzyme activity correlated with the purification of the enzyme-bound iron. An EPR scan of the purified enzyme gave a signal at g equal 4.33, which is characteristic of ferric iron in a rhombic ligand field.     

Cell types in the adenohypophysis of the Japanese quail and effects of injection of luteinizing hormone-releasing hormone

The cells of the adenohypophysis of the Japanese quail were studied by both light and electron microscopy after exposure to long photoperiods or injection of lutenizing hormone-releasing hormone (LRH). Six cell types were identified in the adenohypophysis by examining alternate thick and thin sections by light and electron microscopy. In the cephalic lobe, there are four types of glandular cells. They are the prolactin cells, ACTH cells, TSH cells, and gonadotropic cells (FSH?). In the caudal lobe, there are two types of cells, STH cells and gonadotropic cells (LH?). After exposure to long daily photoperiods, gonadotropic cells in both lobes were strongly activated. They became larger and accumulated many granules. ACTH cells became vacuolated; granules were sparse. Synthetic LRH injection (10 mug/0.2 ml/day) for 10 days to the non-photostimulated quail stimulated certain numbers of the gonadotropic cells in the both lobes, although the response of the cells was less than that induced by photostimulation. No response was seen in the other cell types.     

Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110.

The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by G?ttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.