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71.
The stereochemical course of enzymatic hydrolysis by the solublesialidase from Chinese hamster ovary cells, expressed as a recombinantprotein in insect Sf9 cells, was determined using proton nuclearmagnetic resonance spectroscopy. 4-Methyl umbelliferyl-N-acetylneuraminic acid was employed as substrate, and the stereoselectivityof the enzyme catalysis was ascertained by monitoring the H3axial and equatorial protons of the sialic acid product overthe reaction course. At both high (3 U) and low concentrations(1 U) of the enzyme, the alpha anomer of the sialic acid wasclearly observed as the initial reaction product. The correspondingbeta anomer of sialic acid appeared much later in the reaction,arising from mutarotation of the alpha anomer. Similar studieswere also carried out using the Salmonella typhimurium LT 2sialidase, a protein of similar size and substrate specificity.Both enzymes apparently cleave the alpha linked sialoside substratewith retention of configuration. Based on the observations ofa wide variety of other glycohydrolytic enzymes that have showna strong correlation of the stereoselectivity of catalysis withactive site topology (Gebler et al, J. Biol. Chem. 267, 12559–12561,1992), the results obtained here suggest that the microbialand mammalian sialidases have a homologous active site architectureeven though the molecules do not share significant primary sequencesimilarities. sialidase NMR enzyme mechanism Chinese hamster  相似文献   
72.
While globin genes ctt-2β and ctt-9.1 in Chironomus thummi thummi each have a single intron, all of the other insect globin genes reported so far are intronless. We analyzed four globin genes linked to the two intron-bearing genes in C. th. thummi. Three have a single intron at the same position as ctt-2β and ctt-9.1; the fourth is intronless and lies between intron bearing genes. Finally, in addition to its intron, one gene (ctt-13RT) was recently interrupted by retrotransposition. Phylogenetic analyses show that the six genes in C. th. thummi share common ancestry with five globin genes in the distantly related species C. tentans, and that a 5-gene ancestral cluster predates the divergence of the two species. One gene in the ancestral cluster gave rise to ctn-ORFB in C. tentans, and duplicated in C. th. thummi to create ctt-11 and ctt-12. From parsimonious calculations of evolutionary distances since speciation, ctt-11, ctt-12, and ctn-ORFB evolved rapidly, while ctn-ORFE in C. tentans evolved slowly compared to other globin genes in the clusters. While these four globins are under selective pressure, we suggest that most chironomid globin genes were not selected for their unique function. Instead, we propose that high gene copy number itself was selected because conditions favored organisms that could synthesize more hemoglobin. High gene copy number selection to produce more of a useful product may be the basis of forming multigene families, all of whose members initially accumulate neutral substitutions while retaining essential function. Maintenance of a large family of globin genes not only ensured high levels of hemoglobin production, but may have facilitated the extensive divergence of chironomids into as many as 5000 species. Received: 31 December 1996 / Accepted: 16 May 1997  相似文献   
73.
脱落酸对线粒体Na^+—K^+ATPase活性的影响   总被引:3,自引:0,他引:3  
在提取大豆(Glycine m ax)黄化子叶的线粒体时,于清洗和悬浮介质中加入ABA,发现40 μm ol/L(±) ABA 对线粒体膜结合Na+ -K+ ATPase 活性及线粒体吸氧速率有明显的促进作用。ABA 可使16℃与27℃培养的大豆子叶线粒体膜结合Na+ -K+ ATPase Arrhenius图的折点温度分别下降6.3℃和5.9℃,并将该酶的底物动力学曲线由正协同曲线转变为非协同曲线。表明ABA 可降低线粒体的膜相变温度  相似文献   
74.
Abstract From a panel of monoclonal antibodies of dengue viruses, a serotype-specific epitope of dengue virus 1 was screened from a random peptide library displayed on phage. The epitope was the determinant reactive with monoclonal antibody 15F3-1 that was specific to dengue 1. The screening was monitored by a dot blotting procedure, and after three rounds of screening a consensus motif, HRYSWK, was found. This sequence matches the sequence HKYSWK, corresponding to the amino acid residues 885–890 of polyprotein or residues 111–116 of the non-structural protein 1 of dengue virus serotype 1. The linear epitope was confirmed by testing the antigenicity of chemically synthesized 8-branched peptide.  相似文献   
75.
The maternal behaviour of three species of voles was compared using a semi-naturalistic laboratory system. Meadow vole females spent more time out of the nest, and exhibited less maternal behaviour in terms of nursing and brooding, and more non-social behaviour such as locomoting, eating, and drinking, than the females of the other two species. Across all species, maternal behaviour decreased from parturition onward, while non-social behaviour increased. Paternal care was well developed in the prairie vole and pine vole, but was never observed in the meadow vole. During the first postnatal week, male prairie voles showed a tendency to enter the nest when the female left the young unattended. This trend was not apparent in the male pine vole. The physical parameters of pup development, including eye opening and the development of fur, were similar in all three species. Behavioural development, however, was most rapid in the meadow vole, intermediate in the prairie vole, and slowest in the pine vole. These results are compared with those of previous field and laboratory studies, and are discussed with reference to the life-history strategy of each species.  相似文献   
76.
Blood chemistry values were collected over a three-year period from at least 10 colony-born and 24 wild-born apparently normal common marmosets. BUN, SGOT, creatinine, calcium, phosphorus, alkaline phosphatase, protein, albumin, cholesterol, triglycerides, uric and glucose values were determined. A statistical comparison of baseline values was made between wild-born, colony-born, male and female marmosets. Also the same comparison was made between common marmosets and cotton-top tamarins, white lipped tamarins and human subjects.  相似文献   
77.
Summary In the spider Dinopis, retinae of the posterior median eyes synthesise new photoreceptor membrane in bulk at dusk and destroy it at dawn (Blest, 1978). During the dawn period, there is a rapid, anticipatory differentiation of unusual organelles from the rough endoplasmic reticulum (RER) in the intermediate segments of the receptors. This system is classified as GERL. Its products appear to play a role in the autolysis of photoreceptor membrane. Consistent topographical relationship to Golgi bodies has not been determined. Circumscribed regions of RER whorls first reorganise to yield fenestrated profiles; these differentiate further to a number of structures by condensation and loss of ribosomes. Smooth tubular profiles are termed rigid tubules to indicate their probable homology with the rigid lamellae of vertebrate secretory cells. More complex smooth multilocular bodies are also produced. Evidence is discussed which implies that both rigid tubules and multilocular bodies give rise to condensing vacuoles. These, in turn, pinch off coated vesicles assembled as Nebenkerne. Some rigid tubules are transported to the interrhabdomeral cytoplasm of the receptive segments. At late stages of differentiation, rigid tubules, multilocular bodies and Nebenkerne give strong, positive responses to zinc iodide-osmium tetroxide (ZIO) treatment; early stages and both cis and trans Golgi components do not. GERL differentiation is independent of immediate illumination of the retina at dawn. It is suggested to mediate the lysis of membrane degradation products by the production of hydrolases.We thank Professor D.T. Anderson F.R.S. for our use of field facilities at the Crommelin Biological Field Station of Sydney University at Warrah, Pearl Beach, N.S.W., and Andrew and Sally Austin and Sally Stowe for help in the field. We are indebted to Rod Whitty and the Electron Microscopy Unit for advice and support throughout these studies  相似文献   
78.
Extracts of rat anterior and intermediate-posterior pituitary were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for immunoactive ACTH and endorphin. In both lobes the major forms of immunoactive ACTH have apparent molecular weights of 31,000 (31K), 20–21K, 14K, and 4.5K, and the major forms of immunoactive endorphin have apparent molecular weights of 31K (coincident with the peak of immunoactive ACTH), 13K (a βLPH-like peptide), and 3.5K (a β-endorphin-like peptide). However, the quantitative distribution of immunoactivity among the various forms differs greatly between the lobes. Assays using an extreme COOH-terminal ACTH antiserum indicate that the 31K ACTH/endorphin molecule in rat antierior and intermediate pituitary is similar to the pro-ACTH/endorphin molecule from mouse pituitary tumor cells. A radioimmunoassay that is specific for the NH2-terminal non-ACTH, nonendorphin segment (referred to as 16K fragment) of the mouse pro-ACTH/endorphin molecule was used to assay extracts of rat pituitary. In addition to detecting material at 31K and 20–21K, the 16K fragment radioimmunoassay detects significant amounts of cross-reactive material with an apparent molecular weight of 16K in extracts of both lobes. This result also suggests that the structure and processing of the rat 31K ACTH/endorphin molecule is similar to that of mouse tumor cell pro-ACTH/endorphin. Cell suspensions were prepared from the anterior and intermediate lobes of the rat pituitary and maintained in culture for a 24-h period. The isolated cells from both lobes incorporate [3H] phenylalanine into immunoprecipitable ACTH- and endorphin-containing molecules. By sequential immunoprecipitation with ACTH and endorphin antisera, it is possible to demonstrate directly that a single molecule (31K ACTH/endorphin) has antigenic determinants for both ACTH and endorphin. Significant amounts of 31K ACTH/endorphin are released into the culture medium by isolated anterior lobe and intermediate lobe cells. The isolated intermediate lobe cells synthesize and secrete relatively large amounts of a β-endorphin-like molecule; the isolated anterior lobe cells secrete significant amounts of both a βLPH-like molecule and a β-endorphin like molecule. These same quantitative differences between anterior and intermediate lobe tissue were observed in immunoassays of extracts of the separated lobes and probably reflect differences in the processing of the common precursor. The isolated anterior lobe cells can be stimulated to release increased amounts of immunoprecipitable ACTH and endorphin by incubation with a cyclic AMP analog and a phosphodiesterase inhibitor.  相似文献   
79.
The synthesis of ((±)-16-thioketal and 16-keto PGE2 methyl ester ( and ) is herein described.  相似文献   
80.
Embryonic-chick tendon cells were incubated in suspension for 4h with (14)C-labelled amino acids, cell extracts were subjected to gel filtration, and the effluent was examined by rocket immunoelectrophoresis by using antibodies specific for the beta-subunit of chick prolyl hydroxylase. Two peaks of immunoreactive protein were found. The first peak contained 40% of the immunoreactive protein eluted from the column and 100% of the enzyme activity. Polyacrylamide-slab-gel electrophoresis in sodium dodecyl sulphate of an immunoprecipitate of this peak demonstrated that it consisted of the tetrameric form of prolyl hydroxylase, subunit composition alpha(2)beta(2) where alpha and beta are non-identical subunits. Only the alpha-subunits were labelled, indicating that they were synthesized during the 4h labelling period. The beta-subunits were unlabelled, indicating that they had been synthesized before the labelling period. The second peak eluted from the gel-filtration column contained 60% of the immunoreactive protein eluted from the column and was enzymically inactive. Polyacrylamide-slab-gel electrophoresis of an immunoprecipitate of this peak indicated that it consisted of a single labelled polypeptide chain, identified as cross-reacting protein, which was related to, but not identical with, the beta-subunit of prolyl hydroxylase. Pulse-chase experiments were performed on cultured chick tendon cells to demonstrate that alpha-subunits and cross-reacting protein had half-lives of about 60h. The half-life of beta-subunits was considerably longer, and the kinetic pattern was consistent with their being derived from a labelled precursor such as cross-reacting protein. The data presented here indicate that the active tetrameric form of prolyl hydroxylase in cells is assembled from alpha-subunits which are newly synthesized, and from beta-subunits which are derived from cross-reacting protein.  相似文献   
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