不同发育时期追施氮肥对冬小麦旗叶中光合酶活性的影响

小麦开花后,随着旗叶的衰老,旗叶中1,5-二磷酸核酮糖羧化酶（RuBPC）、磷酸烯醇式丙酮酸羧化酶（PEPC）和乙醇酸氧化酶（GO）活性呈下降趋势。随着追施氮肥时期的推迟,光合酶活性呈增加趋势,这意味着氮肥追施时间后移有利于提高小麦光合速率。在旗叶衰老后期,大穗型品种小麦旗叶中光合酶活性略高于多穗型品种小麦。     

不同类型土壤上种植的小麦籽粒中与淀粉合成相关的酶活性变化

测定池栽条件下灰潮土、水稻土、砂姜黑土上种植的强筋小麦‘郑麦9023’籽粒灌浆过程中腺苷二磷酸葡萄糖焦磷酸化酶（AGPP）、尿苷二磷酸葡萄糖焦磷酸化酶（UGPP）、可溶性淀粉合成酶（SSS）、淀粉粒结合淀粉合成酶（GBSS）、淀粉分支酶（SBE）5个与淀粉合成有关的酶活性变化的结果表明,不同类型土壤上种植的小麦籽粒中AGPP、UGPP、SSS、GBSS、SBE活性均呈单峰曲线变化,花后18d,AGPP、UGPP、SSS和SBE活性达到峰值,而GBSS则在花后24d达到峰值。AGPP、SSS、SBE活性峰值表现为灰潮土〉水稻土〉砂姜黑土,UGPP峰值表现为灰潮土〉砂姜黑土〉水稻土,GBSS峰值则表现为水稻土〉灰潮土〉砂姜黑土。     

丙烯对柿果实采后细胞壁物质代谢和几种生理指标的影响

丙烯处理的柿果实,其软化进程显著加快,呼吸速率和乙烯释放高峰提前,且峰值升高,细胞壁各组分的代谢速度加快,柿果实中多聚半乳糖醛酸酶（PG）和纤维素酶（Cx）活性提高,且高峰提前。     

Role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homo-arginine (hR), and the structure-function-stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active alpha-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in deltaH# and decrease in K(m). To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl- binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility.     

Structures of N-acetylornithine transcarbamoylase from Xanthomonas campestris complexed with substrates and substrate analogs imply mechanisms for substrate binding and catalysis

N-acetyl-L-ornithine transcarbamoylase (AOTCase) is a new member of the transcarbamoylase superfamily that is essential for arginine biosynthesis in several eubacteria. We report here crystal structures of the binary complexes of AOTCase with its substrates, carbamoyl phosphate (CP) or N-acetyl-L-ornithine (AORN), and the ternary complex with CP and N-acetyl-L-norvaline. Comparison of these structures demonstrates that the substrate-binding mechanism of this novel transcarbamoylase is different from those of aspartate and ornithine transcarbamoylases, both of which show ordered substrate binding with large domain movements. CP and AORN bind to AOTCase independently, and the main conformational change upon substrate binding is ordering of the 80's loop, with a small domain closure around the active site and little movement of the 240's loop. The structures of the complexes provide insight into the mode of substrate binding and the mechanism of the transcarbamoylation reaction.     

Chemical synthesis of mouse cripto CFC variants

We report for the first time the chemical synthesis of refolded CFC domain of mouse Cripto (mCFC) and of two variants bearing mutations on residues W107 and H104 involved in Alk4 binding. The domains undergo spontaneous and quantitative refolding in about 4 h, yet with very different kinetics. Disulfide linkages have been assessed by enzyme digestion and mass spectrometry analysis of resulting fragments, and the first experimental studies on structural organization have been conducted by circular dichroism spectroscopy under different pH conditions. Upon refolding, the domains considerably change their conformations, although they do not assume canonical structures, and become highly resistant to enzyme degradation. A comparative study of receptor binding shows that the CFC domain can bind Alk4 and confirms the importance of W107 and H104 for receptor recognition.     

A Refined Model for the Structure of Acireductone Dioxygenase from Klebsiella ATCC 8724 Incorporating Residual Dipolar Couplings

Acireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O₂) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni²⁺-containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.     

Uric acid recycling in the shield bug, Parastrachia japonensis (Hemiptera: Parastrachiidae), during diapause

Nymphs of the univoltine shield bug, Parastrachia japonensis grow by feeding on the drupes of their sole food plant, which are available for only 2 weeks a year. The new adults soon enter a reproductive diapause and survive without feeding for at least 10 months up to 2 years. Uric acid was found to be the predominant component among four waste nitrogenous compounds, i.e., uric acid, allantoin, allantoic acid and urea in the body of both nymphs and adults in all stages, and to be predominantly excreted by the nymphs and reproductive adults. However, adults in diapause excreted negligible amounts of these compounds. Erwinia-like bacteria were found exclusively in the cecum of midgut, in which three uricolytic enzymes, i.e., uricase, allantoinase and allantoicase were detected. Ninety % of adults in diapause could survive on water for 9 months, but those given 0.02% rifampicin aqueous solution all died within this period, with significant reduction of the bacteria and uricase activity in the cecum. Rifampicin treatment resulted in a considerable reduction of free amino acids, especially proline in the hemolymph. These results suggest that uric acid is recycled as an amino acid source with the aid of Erwinia-like bacteria, and uricase functions as a key enzyme for this process.     

Identification of the core structure of lysozyme amyloid fibrils by proteolysis

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive beta-sheet structure and a substantial element of non beta-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the beta-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.