Temperature-dependence of intramolecular coupling of active sites in pyruvate dehydrogenase multienzyme complexes.

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complexes of Escherichia coli, ox heart and Bacillus stearothermophilus was measured at various temperatures. As the temperature was raised, the extent of active-site coupling was found to increase, approaching a maximum near the physiological growth temperature of the organism. Under these conditions, a single pyruvate dehydrogenase (lipoamide) dimer appeared able to cause a rapid (20s) reductive acetylation of probably all 24 polypeptide chains in the dihydrolipoamide acetyltransferase core of the enzyme complex from E. coli at 37 degrees C, and of most if not all of the 60 polypeptide chains in the dihydrolipoamide acetyltransferase cores of the enzymes from ox heart and B. stearothermophilus at 37 degrees C and 60 degrees C respectively. Experiments designed to measure the inter-core and intra-core migration of enzyme subunits suggested that, in the bacterial enzymes at least, this was not a major contributor to active-site coupling.     

Antimicrobial Properties of Natural Phenols and Related Compounds: I. Obtusastyrene

Obtusastyrene (4-cinnamylphenol) displays effective antimicrobial activity in vitro against a variety of gram-positive bacteria, yeasts, and molds. The activity of obtusastyrene is not appreciably affected by pH, and its minimal inhibitory concentrations, 12 to 25 mug/ml for bacteria and 12 to 100 mug/ml for fungi, compare favorably with those of a number of synthetic, phenolic antimicrobial agents.     

Defective Virions in Human Adenovirus Type 12

Purified preparations of human adenovirus type 12 showed two bands when subjected to isopycnic centrifugation in a density gradient of cesium chloride. Their density difference was about 0.003 g/ml, suggesting a small difference in their deoxyribonucleic acid to protein ratio. Virions with a lighter density can kill human KB cells and induce T antigen as efficiently as the heavy virions. However, they appeared incapable to form plaques. Two passages of the heavy infectious virions at low multiplicity of infection did not produce significant amounts of light virions; however, when it was passed at high multiplicity of infection, the light band became visible in a cesium chloride density gradient.     

Purification and Properties of 4-Hydroxy-2-Ketopimelate Aldolase from Acinetobacter

The chemical synthesis of 4-hydroxy-2-ketopimelic acid is described. An aldolase that cleaves this compound to succinic semialdehyde and pyruvate has been purified from Acinetobacter grown at the expense of 4-hydroxyphenylacetic acid. The molecular weight of the enzyme was about 158,000 from sedimentation equilibrium data; other physical determinations gave values in reasonable agreement. The protein was globular and was dissociated in sodium dodecyl sulfate to give a species of molecular weight 25,700. The enzyme attacked both enantiomers of synthetic 4-hydroxy-2-ketopimelate and was stimulated by Mg(2+) and Mn(2+) ions.     

Bacterial Degradation of 4-Hydroxyphenylacetic Acid and Homoprotocatechuic Acid

A species of Acinetobacter and two strains of Pseudomonas putida when grown with 4-hydroxyphenylacetic acid gave cell extracts that converted 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid) into carbon dioxide, pyruvate, and succinate. The sequence of enzyme-catalyzed steps was as follows: ring-fission by a 2,3-dioxygenase, nicotinamide adenine dinucleotide-dependent dehydrogenation, decarboxylation, hydration, aldol fission, and oxidation of succinic semialdehyde. Two new metabolites, 5-carboxymethyl-2-hydroxymuconic acid and 2-hydroxyhepta-2,4-diene-1,7-dioic acid, were isolated from reaction mixtures and a third, 4-hydroxy-2-ketopimelic acid, was shown to be cleaved by extracts to give pyruvate and succinic semialdehyde. Enzymes of this metabolic pathway were present in Acinetobacter grown with 4-hydroxyphenylacetic acid but were effectively absent when 3-hydroxyphenylacetic acid or phenylacetic acid served as sources of carbon.     

Ultraviolet Mutagenesis and Its Repair in an ESCHERICHIA COLI Strain Containing a Nonsense Codon

Ultraviolet mutagenesis and its repair were studied mainly in WU36-10-89, a uvr(-) strain of Escherichia coli containing a UAG mutation in a gene for leucine biosynthesis. Following ultraviolet (UV) irradiation revertants appearing with or without direct photoreactivation (PR) were classified according to the presence and type of suppressor they contained. We find UV mutation production to be quite specific. An analysis of revertants produced by UV indicates they are formed mainly from GC --> AT and that the miscoding is due to a cytosine residue at the site of mutation in a cytosine-thymine (CT) dimer. We propose that the dimer serves as template during some aspects of repair replication and at the time of replication the C in the dimer directs the insertion of A in the complementary strand. We also note that C --> A and T -->G changes caused by a CT dimer occur much less frequently.     

A study of the properties of hybrids of oxyhaemoglobin and deoxyhaemoglobin with two porphyringlobin species

The fluorescence of porphyringlobin is quenched on adding haemoglobin to its solutions. It is suggested that this result indicates the formation of hybrids (comprising a dimer of porphyringlobin and a dimer of haemoglobin) in which quenching occurs by energy transfer from the porphyrin to the haem groups of the protein. From an analysis of fluorescence quenching, dissociation constants were calculated for the hybrids of oxy- and deoxyhaemoglobin with the fast- and slow-moving porphyringlobin species isolated by chromatography on CM-Sephadex (Treffry & Ainsworth, 1974). The values obtained are: deoxyhaemoglobin-fast-moving porphyringlobin, 0.8x10(-9)m; deoxyhaemoglobin-slow-moving porphyringlobin, 5x10(-10)m; oxyhaemoglobin-fast-moving porphyringlobin, 0.8x10(-6)m; oxyhaemoglobin-slow-moving porphyringlobin, 1.2x10(-7)m. The rates of reactions of solutions of haemoglobin and porphyringlobin, containing hybrids, with the thiol reagent 4,4'-dithiodipyridine showed that the thiol groups of the hybrids deoxyhaemoglobin-fast-moving porphyringlobin and oxyhaemoglobin-slow-moving porphyringlobin react more slowly than expected on the basis of composition alone: this result indicates that the deoxy and slow-moving conformations are the more stable, imposing themselves partially on to the fast-moving or oxy dimer of the hybrid. Also the rate of the reaction of CO with deoxyhaemoglobin is decreased when slow-moving porphyringlobin is added to its solutions: this is reflected in a movement of the oxygen equilibrium curve of such a mixture to higher oxygen partial pressures. Similar experiments with deoxyhaemoglobin solutions containing fast-moving porphyringlobin, showed an initial increase in the rate of CO uptake. Correspondingly, the oxygen equilibrium curve of the mixture showed an increased affinity for oxygen. Approximate calculations to determine the oxygen equilibria of the hybrids indicate that a functional dimer retains co-operative characteristics even when the dimer accompanying it within the tetramer has the reacted conformation.     

The Anisotropic Elastic Properties of the Sarcolemma of the Frog Semitendinosus Muscle Fiber

Tension and curvature of the sarcolemmal tube of the frog muscle fiber were measured at different extensions and were used to calculate the anisotropic elastic properties of the sarcolemma. A model was derived to obtain the four parameters of the elasticity matrix of the sarcolemma. Sarcolemmal thickness was taken as 0.1 μm. Over the range of reversible sarcolemmal tube extension, the longitudinal elastic modulus E_L = 6.3 × 10⁷ dyn/cm², the circumferential modulus E_c = 0.88 × 10⁷ dyn/cm², the longitudinal Poisson's ratio σ_L = 1.2, and the circumferential Poisson's ratio σ_c = 0.18. At tubular rest length E_L = 1.2 × 10⁷ dyn/cm². The sarcolemma is less extensible in the longitudinal direction along the fiber axis than in the circumferential direction. It can be extended reversibly to 48% of its rest length, equivalent to extending the intact fiber from a sarcomere length of 3 μm to about 4.5 μm. The sarcolemma does not contribute to intact fiber tension at fiber sarcomere lengths <3 μm, and between 3 and 4 μm its contribution is about 20%. It also exerts a pressure on the myoplasm, which can be calculated by means of the model. The longitudinal elastic modulus of the whole fiber is 1 × 10⁵ dyn/cm² at a sarcomere length of 2.33 μm.