The growth of ordered two-dimensional sheets of ribosomal particles from salt-alcohol mixtures

A procedure for the in vitro growth of well-ordered two-dimensional sheets from ribosomal particles using salts and salt-alcohol mixtures has been developed. Employing this procedure, ordered two-dimensional sheets of the wild type as well as of mutated 50 S ribosomal subunits from Bacillus stearothermophilus can readily be obtained. These sheets, stained with uranyl acetate or gold-thioglucose, are suitable for three-dimensional image reconstruction. They consist of relatively small unit cells with dimensions of 160 +/- 15 and 365 +/- 20 A. Diffraction patterns of electron micrographs of these sheets contain features to 25 A resolution.     

Cyanide formation from histidine in Chlorella. A general reaction of aromatic amino acids catalyzed by amino acid oxidase systems.

The formation of HCN from D-histidine in Chlorella vulgaris extracts is shown to be due to the combined action of a soluble protein and a particulate component. Either horse-radish peroxidase (EC 1.11.1.7) or a metal ion with redox properties can be substituted for the particulate component. Ions of manganese and vanadium are especially effective, as are o-phenanthroline complexes of iron. Cobalt ions are less active. The D-amino acid oxidase (EC 1.4.3.3) from kidney and the L-amino acid oxidase (EC 1.4.3.2) from snake venom likewise cause HCN production from histidine when supplemented with the particulate preparation from Chlorella or with peroxidase or with a redox metal ion. The stereospecificity of the amino acid oxidase determines which of the two stereoisomers of histidine is active as an HCN precursor. Though histidine is the best substrate for HCN production, other naturally occurring aromatic amino acids (viz. tyrosine, phenylalanine and tryptophan) can also serve as HCN precursors with these enzyme systems. The relative effectiveness of each substrate varies with the amino acid oxidase enzyme and with the supplement. With respect to this latter property, the particulate preparation from Chlorella behaves more like a metal ion than like peroxidase.     

Three-dimensional crystals of ribosomes and their subunits from eu- and archaebacteria

Ordered three-dimensional crystals of 70S ribosomes as well as of 30S and 50S ribosomal subunits from various bacteria (E. coli, Bacillus stearothermophilus, Thermus thermophilus and Halobacterium marismortui) have been grown by vapour diffusion in hanging drops using mono- and polyalcohols. A new compact crystal form of 50S subunits has been obtained, and it is suitable for crystallographic studies at medium resolution. In addition, from one crystal form large crystals could be grown in X-ray capillaries. In all cases the crystals were obtained from functionally active ribosomal particles, and the particles from dissolved crystals retained their integrity and biological activity.     

The growth of ordered two-dimensional sheets of 70 S ribosomes from Bacillus stearothermophilus

Well ordered two-dimensional sheets of intact 70 S ribosomes from Bacillus stearothermophilus have been obtained in vitro using salt-alcohol mixtures. These sheets consist of relatively small unit cells with dimensions of 200 +/- 20 A and 400 +/- 30 A. Diffraction patterns of electron micrographs of these sheets stained with uranyl acetate contain features to 42 A resolution.     

A compact three-dimensional crystal form of the large ribosomal subunit from Bacillus stearothermophilus

A new form of well-ordered three-dimensional crystals of intact 50 S ribosomal subunits from Bacillus stearothermophilus have been obtained. Electron micrographs of positively stained sections of these crystals revealed that the ribosomal particles are packed closely. The cell parameters have been determined. Representative electron micrographs and their computed contoured filtered images are shown.     

Cyanide formation in preparations from Chlorella vulgaris Beijerinck: Effect of sonication and amygdalin addition

Summary A critical evaluation of a method for recovering HCN from cell extracts is presented. Since crude extracts often bind or metabolize HCN extensively, the HCN recovered by distillation at room temperature represents only the difference between production and consumption. Sonication leads to HCN release from the alga, Chlorella vulgaris Beijerinck. Illumination of extracts at high light intensity in oxygen, with added Mn²⁺ and peroxidase, also stimulates HCN production. In both processes, the HCN is probably formed by oxidation of nitrogenous precursors. Chlorella extracts cause formation of HCN from added amygdalin. No evidence was found, however, for the presence of cyanogenic glycosides in the algae.     

Characterization of crystals of small ribosomal subunits

Crystals of intact small ribosomal subunits from Thermus thermophilus have been obtained from functionally active particles. The crystals (P42(1)2, 407 A x 407 A x 171 A) are suitable for X-ray crystallography analysis to 9.9 A using synchrotron radiation at cryotemperature. Crystallographic data from native and a potential heavy-atom derivative have been collected.     

Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo

Summary The NADH-nitrate oxidoreductase of Chlorella vulgaris has an inactive form which has previously been shown to be a cyanide complex of the reduced enzyme. This inactive enzyme can be reactivated by treatment with ferricyanide in vitro. In the present study, the activation state of the enzyme was determined after different prior in vivo programs involving environmental variations. Oxygen, nitrate, light and CO₂ all affect the in vivo inactivation of the enzyme in an interdependent manner. In general, the inactivation is stimulated by O₂ and inhibited by nitrate and CO₂. Light may stimulate or inhibit, depending on conditions. Thus, the effects of CO₂ and nitrate (inhibition of reversible inactivation) are clearly manifested only in the light. In contrast, light stimulates the inactivation in the presence of oxygen and the absence of CO₂ and nitrate. Since the inactivation of the enzyme requires HCN and NADH, and it is improbable that O₂ stimulates NADH formation, it is reasonable to conclude that HCN is formed as the result of an oxidation reaction (which is stimulated by light). The formation of HCN is probably stimulated by Mn²⁺, since the formation of reversibly-inactivated enzyme is impaired in Mn²⁺-deficient cells. The prevention of enzyme inactivation by nitrate in vivo is in keeping with previous in vitro results showing that nitrate prevents inactivation by maintaining the enzyme in the oxidized form. A stimulation of nitrate uptake by CO₂ and light could account for the effect of CO₂ (prevention of inactivation) which is seen mainly in the presence of nitrate and light. Ammonia added in the presence of nitrate has the same effect on the enzyme as removing nitrate (promotion of reversible inactivation). Ammonia added in the absence of nitrate has little extra effect. It is therefore likely that ammonia acts by preventing nitrate uptake. The uncoupler, carbonylcyanide-m-chloro-phenylhydrazone, causes enzyme inactivation because it acts as a good HCN precursor, particularly in the light. Nitrite, arsenate and dinitrophenol cause an enzyme inactivation which can not be reversed by ferricyanide in crude extracts. This suggests that there are at least two different ways in which the enzyme can be inactivated rather rapidly in vivo.     

Studies on the accessibility of nascent non-helical peptide chains on the ribosome

The accessibility of nascent polypeptides with special structural elements to the ribosome was investigated. Poly(C), poly(C, U) and poly(C, A) mRNAs were translated by E. coli ribosomes in vitro. The resulting peptides which were rich in prolines, remained on the ribosomal particles or were released after addition of puromycin. A protease from Aspergillus oryzae hydrolyzed the released peptides rapidly, whereas the degradation of the unreleased ones was only slightly affected. This result shows that the nascent peptides were protected against proteolytic attack by the ribosomal particles. Interestingly, the protease completely degraded the 30S particles whereas the 50S ones remained intact, even after prolonged incubation.