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Dihydroorotase (DHOase) catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The enzyme from Aquifex aeolicus, a hyperthermophilic organism of ancient lineage, was cloned and expressed in Escherichia coli. The purified protein was found to be a 45-kDa monomer containing a single zinc ion. Although there is no other DHOase gene in the A. aeolicus genome, the recombinant protein completely lacked catalytic activity at any temperature tested. However, DHOase formed an active complex with aspartate transcarbamoylase (ATCase) from the same organism. Whereas the k cat of 13.8 ؎ 0.03 s ؊1 was close to the value observed for the mammalian enzyme, the K m for dihydroorotate, 3.03 ؎ 0.05 mM was 433-fold higher. Gel filtration and chemical cross-linking showed that the complex exists as a 240-kDa hexamer (DHO 3 -ATC 3 ) and a 480-kDa duodecamer (DHO 6 -ATC 6 ) probably in rapid equilibrium. Complex formation protects both DHOase and ATCase against thermal degradation at temperatures near 100°C where the organism grows optimally. These results lead to the reclassification of both enzymes: ATCase, previously considered a Class C homotrimer, now falls into Class A, whereas the DHOase is a Class 1B enzyme. CD spectroscopy indicated that association with ATCase does not involve a significant perturbation of the DHOase secondary structure, but the visible absorption spectrum of a Co 2؉ -substituted DHOase is appreciably altered upon complex formation suggesting a change in the electronic environment of the active site. The association of DHOase with ATCase probably serves as a molecular switch that ensures that free, uncomplexed DHOase in the cell remains inactive. At pH 7.4, the equilibrium ratio of carbamoyl aspartate to dihydroorotate is 17 and complex formation may drive the reaction in the biosynthetic direction.

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The remarkable elastic behaviour of the lung entails both continuous tissue stress and a cyclic stress produced by the fluctuating difference between intrathoracic and intra-alveolar pressures throughout the entire span of life. In spite of these potentially disruptive forces, lung structure is preserved by the fibrous connective-tissue proteinscollagen, elastin, and reticulin. These extracellular proteins are characterized by their insolubility, resistance to destruction, and high tensile strength. Collagen and elastin both exhibit elastic behaviour, but great extensibility is a property only of elastin. The most reasonable assumption is that the elastic fibres, therefore, are principally responsible for the elastic behaviour of the lung tissue, but sure proof is lacking.Recently, Carton, Dainauskas, Tews, and Hass (1960) and Wright (1961) have described the nature of the network of elastic tissue in the lung. We sought to describe more fully the roles of collagenous and elastic fibres in the terminal air spaces of the lung, and thus their respective contributions to tissue elasticity and the general elastic performance of the lung. As the study developed, it seemed important to measure the amounts of collagen and elastin in the lung parenchyma and pleura. These findings have been correlated with the age of the subjects and interpreted according to knowledge about tissue elasticity. METHODSThe study was based on 36 human lungs taken from patients who had died of a variety of diseases. No lungs were taken from patients with a history of cough, sputum production, dyspnoea, or chronic pulmonary disease, or from patients whose clinical history was incomplete. Of these 36, 27 lungs were dried in an inflated state by the extraction process using dilute (0-1 N) sodium hydroxide solution (Pierce, Hocott, and Ebert, 1959). Nine more lungs were also dried in the inflated state but were fixed with formalin vapour. The advantage of the alkaline extraction method is that it permits the morphology of the lung 1 Supported in part by grants HTS5333 and HE04031 from the U.S. Public Health Service to be examined and also the quantities of collagen and elastin to be measured.The lungs were cut into thin slices and examined grossly and with a stereomicroscope. Slices of tissue were variously stained (Lillie, 1954) but not embedded. Elastic tissue was stained by the Taenzer-Unna acidorcein method (in absolute ethanol); Van Gieson's picric acid-acid fuchsin mixture was used to stain collagen; and Fraenkel's method of using orcein and indigo-carmine was employed to stain both collagen and elastin in the same slice. All slices were rapidly dehydrated in alcohol and then in ether, and dried in vacuo to prevent collapse. This procedure enabled one to study the structure of the alveoli, alveolar ducts, and respiratory bronchioles as well as to visualize the relationship of the fibres in three dimensions. Preparations extracted with sodium hydroxide provided a more clearly defined fibrous network than did the formalin-vapour preparation...