Probing steric and hydrophobic effects on enzyme-substrate interactions by protein engineering

Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin. Subtilisin has broad peptidase specificity and contains a large hydrophobic substrate binding cleft. A conserved glycine (Gly(166)), located at the bottom of the substrate binding left, was replaced by 12 nonionic amino acids by the cassette mutagenesis method. Mutant enzymes showed large changes in specificity toward substrates of increasing size and hydrophobicity. In general, the catalytic efficiency (k(cat)/K(m)) toward small hydrophobic substrates was increased (up to 16 times) by hydrophobic substitutions at position 166 in the binding cleft. Exceeding the optimal binding volume of the cleft ( approximately 160 A(3)), by enlarging either the substrate side chain or the side chain at position 166, evoked precipitous drops in catalytic efficiency (k(cat)/K(m)) (up to 5000 times) as a result of steric hindrance.     

Specific inhibition of nuclear RNA polymerase II by alpha-amanitin

alpha-Amanitin, a toxic substance from the mushroom Amanita phalloides, is a potent inhibitor of DNA-dependent RNA polymerase II (the nucleoplasmic form) from sea urchin, rat liver, and calf thymus. This compound exerts no effect on the activity of polymerase I (nucleolar form) or polymerase III (also nucleoplasmic). The inhibition is due to a specific interaction with polymerase II or with a complex of DNA and polymerase II.     

肠道传播的非甲非乙型肝炎病毒(ET-HEV)核酸探针制备及临床诊断研究初报

病毒性肝炎给人类造成的危害是众所周知的。传统的分类法将肝炎病毒分为甲型、乙型、非甲非乙型和丁型四类。近年发现,非甲非乙型病毒(NANBHV)有两种类型:一类经血液传播(致输血后肝炎),另一类经肠道传播,1989年,Choo和Rcycs等分别将二者克隆,并命名为丙肝病毒(HCV)和戊肝病毒(HEV)。因此,新命名法特别是在1989年9月东京会议正式命名法确定后,将肝炎病毒分为HAV、HBV、HCV、HDV、HEV。在世界范围内,HEV肝炎流行多发地见于中国(特别是新疆)、缅甸、墨西哥、     

Lipid films as transducers for detection of antigen-antibody and enzyme-substrate reactions

The transverse electrical impedance of thin lipid films separating two aqueous saline solutions containing a small concentration of antibodies or enzymes decreases markedly and reversibly after immunological and enzymatic reactions involving such protein molecules, which presumably are adsorbed to the lipid-water interface.     

Live-cell imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B

Endoplasmic reticulum-localized protein-tyrosine phosphatase PTP1B terminates growth factor signal transduction by dephosphorylation of receptor tyrosine kinases (RTKs). But how PTP1B allows for RTK signaling in the cytoplasm is unclear. In order to test whether PTP1B activity is spatially regulated, we developed a method based on F?rster resonant energy transfer for imaging enzyme-substrate (ES) intermediates in live cells. We observed the establishment of a steady-state ES gradient across the cell. This gradient exhibited robustness to cell-to-cell variability, growth factor activation, and RTK localization, which demonstrated spatial regulation of PTP1B activity. Such regulation may be important for generating distinct cellular environments that permit RTK signal transduction and that mediate its eventual termination.     

Specific tissue graft rejection in earthworms

Earthworms are capable of destroying antigenic tissues. Autogeneis transplants healed in regularly and remained permanently viable. Xenografts, by contrast were cicatrized but eventually rejected. Intrafamilial transplants survived longer than interfamilial ones. Xenografts and autografts placed in the graft bed were joined to each other but xenografts were later destroyed although autografts were not. Two xenografts from Eisenia to Lumbricus, performed simul taneously, showed survival endpoints similar to a single xenograft. A 5-day interval between first- and second-set grafting led to an accelerated rejection of both transplants. First-set Allolobophora transplants to Lumbricus performed simulataneously with second-set Eisenia grafts were destroyed at a time different from either of the two Eisenia transplants. A single Allolobophora transplant to Lumbricus was rejected at survival tines equivalent to Allolobophora along with two Eisenia transplants.     

Viral RNA subunits in cells transformed by RNA tumor viruses

Single-stranded 35S and 20S viral RNA species are synthesized in virus-producing mouse and rat cells transformed by the murine sarcoma virus. A transformed hamster cell line that does not produce virus synthesizes 35S, but not 20S viral RNA.     

RNA干扰在植物中的应用

RNA干扰是一种进化上保守的机制,存在于植物、动物、真菌等生物中,目前已成为研究热点,在近几年得到了巨大的发展。对RNA干扰技术的发现过程、机制以及特点进行了综述,并对RNA干扰技术在植物上的应用进行了介绍,为相关研究提供信息。     

立体分子伴侣-脂肪酶特异折叠酶

细菌脂肪酶是一类重要的工业用酶,尤其以来源于假单孢杆菌Pseudomonas与伯克霍尔德菌Burkholderia的脂肪酶应用最为广泛。然而,这类脂肪酶折叠过程中存在一个巨大的能障而无法自发正确折叠。研究发现,这类脂肪酶的编码基因所在操纵子中往往存在一个与其折叠相关的基因,编码一种脂肪酶特异折叠酶。该类蛋白为脂肪酶的立体分子伴侣,可与脂肪酶的折叠中间体相互作用,帮助脂肪酶越过折叠过程中的能量障碍,为其获得正确构象提供必要的空间信息。详细阐述了脂肪酶特异折叠酶的发现、分类、作用机制与应用前景。     

Strong interactions in supported-metal catalysts

Many commercially important catalysts consist of small metal particles dispersed on inorganic oxide surfaces. Although in most cases there is no significant interaction between the metal and the support, strong bonding can be demonstrated in a recently discovered class of supported-metal catalysts. These cases typically involve group VIII metals dispersed on transition metal oxides whose surfaces can be reduced to form cations with lower valences. Spectroscopic measurements indicate that an electron is transferred from the cation (such as Ti(3+) or Nb(4+)) to the metal particle. This, in turn, leads to profound changes in the catalytic and chemisorption properties and the morphology of the metal particles.     

Predator-prey interactions in continuous culture

The exploitation of a bacterial food supply by a protozoan predator does not necessarily lead to the extinction of either species. Even a "homogeneous" experimental system contains sufficient heterogeneity (the boundaries) for avoidance strategies to evolve, which allow the populations to presist.