Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites.

The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)     

喀喇昆仑、昆仑山地区植物元素含量的区域分异和数量分析

对喀喇昆仑、昆仑山地区87种植物21个元素含量及区域分异的研究表明,Ca、Cr、Cd、Fe、V含量比高等植物含量偏高,Ph、P的含量偏低。同种植物在不同地点元素含量有差异。盐柴荒漠植物中Na、K、Mg、P含量较高;高山草甸、冰缘植被植物Ba、Ca、Fe、V、Ti含量较高。各植被类型植物元素含量Na/K差异最大,Ca/Mg较小,Fe/Al差异最小。其变异系数分别为153.5、20.5和15.9.%     

Identification and DNA sequence of fixZ, a nifB-like gene from Rhizobium leguminosarum.

Previously, several mutants which nodulated peas but which failed to fix nitrogen were isolated following Tn5 mutagenesis of pRL 1JI, a symbiotic plasmid of Rhizobium leguminosarum. Two of these alleles, fix52::Tn5 and fix137::Tn5 were in a region of pRL 1JI which hybridized to a probe that contained the nifA gene and the amino-terminal region of the nifB gene of Klebsiella pneumoniae. The nitrogen fixation defect of the fix52::Tn5 mutant strain was corrected by a 2.0kb fragment of the corresponding wild-type DNA cloned in a wide host-range plasmid. The DNA sequence of this region revealed an open reading frame corresponding to the gene within which the fix52::Tn5 allele was located. The polypeptide corresponding to this open reading frame had a deduced molecular weight of 39,936 and the gene was termed fixZ. The deduced amino acid sequence of the fixZ gene product contained two clusters of cysteine residues, suggesting that the protein may contain an iron-sulphur cluster. The sequence of the fixZ polypeptide was very similar to the sequence of the K. pneumoniae nifB gene (provided by W. Arnold and A. Pühler) which is required for the synthesis of the FeMo-cofactor of nitrogenase. It was shown that the previously observed hybridization was due to homology between the amino terminal regions of fixZ and nifB. Upstream from fixZ was found another open reading frame whose 5' terminus was not established, but within which was located the fix137::Tn5 allele. This gene was termed fixY. The deduced amino acid sequence of the sequenced part of fixY showed similarity to that of the regulatory nifA gene of K. pneumoniae (provided by W. J. Buikema and F. M. Ausubel). Thus in R. leguminoarum the fix genes that correspond to the nifA and nifB genes are in the same relative orientation as in K. pneumoniae.     

Structural changes in thin filaments of crab striated muscle.

Low angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits in the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between $\text{26}\text{12}$ and $\text{28}\text{13}$. When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to $\text{28}\text{13}$.In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 × 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity distributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments.The muscle in rigor gave meridional and near-meridional reflections at orders of 2 × 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern.     

Karyology and evolution inSesamoides (Resedaceae)

The meiotic behaviour abnormalities, fertility and size of pollen of 6 taxa ofSesamoides have been analysed. Besides diploids (2x), polyploids (4x, 6x, 8x) have been found. The chromosome base number is x = 10, but an origin from x = 5 is suggested.     

Purification of chicken brain choline acetyltransferase

Chicken brain choline acetyltransferase was purified to homogeneity using ammonium sulfate fractionation, followed by chromatography on DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150, immunoabsorption and Sepharose-CoA columns. A purification of 3500-fold was achieved and the final preparation had a specific activity of 2:32 μmol acetylcholine formed per minute per milligram protein. The purified chicken choline acetyltransferase migrated as a single band on polyacrylamide gel electrophoresis in the presence and absence of sodium deodecyl sulfate. The native enzyme, with a molecular weight of 67,000 daltons, consists of two subunits of identical molecular weight. Chicken choline acetyltransferase has a sharp pH optimum of 7.4. It is activated by sodium chloride and potassium chloride but inhibited by cupric ion and N-ethylmaleimide.     

Somatic embryogenesis and plant regeneration in an interspecific hybrid of Oryza

An embryogenic callus was obtained from immature panicle of an interspecific hybrid (Oryza sativa x O. latifolia) F₁. The medium consisted of HE salts supplemented with 2,4-D, NAA (each 2 mg/l), kinetin (3 mg/l), yeast extract (1360 mg/l) and casein hydrolyzate (300 mg/l). The callus was milk-white in colour compact and granulate in texture. Various developmental stage of embryoid, such as globular, heart-shape, scutellum-shape and mature embryoid were observed in an embryogenic callus. Plantlets were successfully regenerated from 1-month-old callus with more than 80% regenerational frequency in each subculture for 12 passages.