A scanning calorimetric study of the thermal denaturation of the lysozyme of phage T4 and the Arg 96----His mutant form thereof

High-sensitivity scanning calorimetry has been employed to study the reversible thermal unfolding of the lysozyme of T4 bacteriophage and of its mutant form Arg 96----His in the pH range 1.80-2.84. The values for t1/2, the temperature of half-denaturation, in degrees Celsius and for the enthalpy of unfolding in kilocalories per mole are given by (standard deviations in parentheses) wild type t1/2 = 9.63 + 14.41 pH (+/- 0.58) delta Hcal = 5.97 + 2.33t (+/- 4.20) mutant form t1/2 = -19.84 + 21.31 pH (+/- 0.51) delta Hcal = -8.58 + 2.66t (+/- 4.48) At any temperature within the range -20 to 60 degrees C, the free energy of unfolding of the mutant form is more negative than that of the wild type by 3-5 kcal mol-1, indicating an apparent destabilization resulting from the arginine to histidine replacement. The ratio of the van't Hoff enthalpy to the calorimetric enthalpy deviates from unity, the value expected for a simple two-state process, by +/- 0.2 depending on the pH. It thus appears that the nature of the unfolding of T4 lysozyme varies with pH in unknown manner. This complication does not invalidate the values reported here for the temperature of half-completion of unfolding, the calorimetric enthalpy, the heat capacity change, or the free energy of unfolding.     

The circadian rhythm of the N-terminus and C-terminus of the atrial natriuretic factor prohormone

Circadian variation in the circulating concentrations of the N-terminal and C-terminal portions of the atrial natriuretic factor prohormone (pro ANF) was evaluated in 8 men, ages 41-47, who have been followed for 19 years with respect to circadian variation in physiological variables including blood pressure and clinical chemistries. The N-terminus of the ANF prohormone contains two peptides consisting of amino acids 1-30 and 31-67 while the C-terminus contains 1 peptide (amino acids 99-126) of this 126 amino acid prohormone which lower blood pressure and have natriuretic properties. To determine if either the N-terminus and/or the C-terminus of the prohormone have a circadian variation in their circulating plasma concentrations these 8 men had blood samples obtained for radiommunoassay every 3 hr during a 24-hr period. Three radiommunoassays which immunologically recognize (1) the whole N-terminus (i.e. amino acids 1-98), (2) the midportion of the N-terminus (amino acids 31-67) and (3) the C-terminus (amino acids 99-126) of the ANF prohormone were utilized. The whole N-terminus, the midportion of the N-terminus which circulates after being proteolytically cleaved from the rest of the N-terminus, and the C-terminus each had a peak circulating concentration between 0400 and 0700 which were significantly (P less than 0.001) higher than their concentrations at any other time throughout the 24-hr period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of the B to Z transition in poly(dGdC)

The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 mM Na cacodylate, 1 mM MgCl₂, pH 6.3, the B to Z transition is centered at 76.4°C, and is characterized by ΔH_cal = 2.02 kcal (mol base pair)^?1 and a cooperative unit of 150 base pairs (bp). The t_m of this transition is independent of both polynucleotide and Mg²⁺ concentrations. A second transition, with ΔH_cal = 2.90 cal (mol bp)^?1, follows the B to Z conversion, the t_m of which is dependent upon both the polynucleotide and the Mg²⁺ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for ψ(–)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126°C, and is characterized by ΔH_cal = 12.4 kcal (mol bp)^?1 and a cooperative unit of 290 bp. In 5 mM MgCl₂, a single transition is seen centered at 75.5°C, characterized by ΔH_cal = 2.82 kcal (mol bp)^?1 and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of ψ(–)-DNA. A transition at 124.9°C is seen under these solution conditions, with ΔH_cal = 10.0 kcal (mol bp)^?1 and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the ψ-(–) aggregate under these solution conditions.     

Multicomponent phase transitions of diacylphosphatidylethanolamine dispersions

The phase transition properties of aqueous suspensions of a series of nonhydrated (not heated above room temperature) and hydrated 1,2 diacylphosphatidylethanolamines (PE's) have been examined by high sensitivity differential scanning calorimetry at scan rates of 0.02-1.0 K min-1. At all scan rates nonhydrated PE's show a single asymmetric transition curve of excess heat capacity as a function of temperature. Multilamellar dispersions of hydrated PE's, however, exhibit transitions with fine structure, which can be fitted as the sum of three two-state component transitions, at scan rates of 0.02-0.1 K min-1, but give only a single asymmetric transition at 1.0 K min-1. At all scan rates the transition(s) of hydrated samples occur at lower temperatures than those of nonhydrated samples. One of the component transitions of hydrated PE's may be analogous to the pretransition that occurs in 1,2 diacylphosphatidylcholines.     

Incidence of Infectious Drug Resistance Among Lactose-Fermenting Bacteria Isolated from Raw and Treated Sewage

Raw and treated sewage samples were examined for antibiotic-resistant, lactose-fermenting bacteria. Approximately 1% of the total lactose-fermenting bacteria were multiply resistant. Of these organisms, 50% were capable of transferring all or part of their resistance to a drug-sensitive recipient. Only 43% of those isolated on media containing a single antibiotic were capable of resistance transfer, whereas 57% of those recovered on multiple antibiotic plates transferred resistance. R factors conferring resistance to chloramphenicol, streptomycin, and tetracycline; streptomycin and tetracycline; and ampicillin, streptomycin, and tetracycline accounted for 22, 19, and 15%, respectively, of those identified. The data indicate a significant level of infectious drug resistance among the intestinal bacteria of the urban population.     

Heat capacity changes for protein-peptide interactions in the ribonuclease S system.

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. We have substituted the wild-type residue Met-13 with six other hydrophobic residues ranging in size from alanine to phenylalanine and have determined the thermodynamic parameters associated with binding of these analogues to S-protein by titration calorimetry in the temperature range 5-25 degrees C. The heat capacity change (delta Cp) associated with binding was obtained from a global analysis of the temperature dependences of the free energies and enthalpies of binding. The delta Cp's were not correlated in any simple fashion with the nonpolar surface area (delta Anp) buried upon binding.     

Studies of the lipid phase transitions of Escherichia coli by high sensitivity differential scanning calorimetry.

High sensitivity adiabatic differential scanning calorimetry was performed on lipids, membrane vesicles, and whole cells of Escherichia coli enriched in particular unsaturated fatty acids by genetic means. Information concerning the shape of the transition is discussed. Transitions with an asymmetric shape reminiscient of a second order transition were observed. Comparison between the lipid transition observed in whole cells, membrane vesicles, and extracted lipids enriched in elaidate reveal some basic similarities. Studies of synthetic lipids were undertaken in an attempt to interpret the shapes of these transitions as a function of the lipid components of the membrane.     

A scanning calorimetric study of the thermally induced unfolding of various forms of tropomyosin

The reversible thermally induced unfolding of various forms of tropomyosin, a two-chain alpha-helical coiled coil, has been studied by high-sensitivity differential scanning calorimetry (DSC). Included in the study are the reduced and oxidized (disulfide cross-linked) forms of alpha alpha- and beta beta-tropomyosin, and the forms of alpha alpha-tropomyosin in which all sulfhydryl groups have been blocked by carboxymethylation or carboxyamidomethylation. Oxidation or blocking of the sulfhydryl groups of tropomyosin strongly affect the thermotropic behavior of the protein in unpredictable ways. The empirical results presented here are in qualitative agreement with those from an earlier DSC study of the oxidized and carboxymethylated forms of alpha alpha-tropomyosin [S.A. Potekhin and P.L. Privalov (1982) Journal of Molecular Biology, Vol. 159, pp. 519-535], but we find that a different decomposition into subtransitions is possible. Comparison of the alpha alpha and beta beta species indicates, in agreement with extant CD studies, that the noncross-linked beta beta species is somewhat less stable than its alpha alpha counterpart, but that cross-linking enhances the stability of the beta beta doubly cross-linked species by a greater amount and does not lead to the small low-temperature transition ("pretransition") seen in the singly cross-linked alpha alpha species.     

Circadian morphometric variation in gastric parietal cell populations of the rat

Summary Gastric parietal cells of rats maintained under standardized conditions and fed ad libitum were examined by electron microscopy at 6 time points of the 24-h day. Morphometric determinations were made on 4 cell characteristics. The volume density of secretory canaliculi was maximal at the mid-dark sampling point and decreased during the light phase; a secondary peak was seen 1 h before the onset of darkness. The surface density of microvesicles and RER fluctuated inversely with the pattern displayed by secretory canaliculi. The number of multivesicular bodies per cytoplasmic area exhibited a single peak, 1 h after the onset of darkness. It was further noted that parietal cells in the necks and bases of glands differed morphologically and that their organelle populations varied at individual circadian rates.