交联琼脂糖珠固定化蛋白酶在纯化牛肺Kunitz抑制剂中的应用

本文介绍了珠状交联琼脂糖及以此作为载体,经氯代环氧丙烷活化后与蛋白酶(胰蛋白酶或糜蛋白酶)结合,制成固定化蛋白酶亲和吸附剂,进而用以亲和层析牛肺提取液中的Kunitz抑制剂的方法。纯化出的抑制剂在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带,与参照物Trasytol(商品Kunitz抑制剂)具有相对应的电泳迁移率,其分子量也相符。纯化产品每毫克蛋白的抑制活力相当于16 000胰蛋白酶BAEE单位。纯化效果为90倍,收率约85％。     

Membrane-microfilament interactions in ascites tumor cell microvilli. Identification and isolation of a large microfilament-associated membrane glycoprotein complex

[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with ascites tumor cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.     

Purification of cytochrome cd1 nitrite reductase from Pseudomonas stutzeri JM300 and reconstitution with native and synthetic heme d1.

Cytochrome cd1 nitrite reductase has been purified from Pseudomonas stutzeri strain JM 300. This enzyme appears to be a dimer with a subunit molecular mass of 54 kDa and its isoelectric point is determined to be 5.4. The N terminus of amino acid sequence has strong homology with that of nitrite reductase from P. aeruginosa. The apoprotein of this enzyme has been reconstituted with native and synthetic heme d1. The nitrite reductase activity measured by NO and N2O gas evolution can be restored to 82% of the activity of the original enzyme when the protein was reconstituted with the native heme d1 and to 77% of the activity when reconstituted with the synthetic heme d1. The absorption spectra of both reconstituted enzymes are essentially identical to that of the original nitrite reductase. These results further substantiate the novel dione structure of heme d1 as proposed. The loss of NO2- reducing activity in the absence of heme d1 and its restoration by addition of heme d1 provides further evidence that heme d1 plays a key role in the conversion of NO2- to NO and N2O.     

子莲杂交优势的利用及新品种选育的研究

1981—1987年的试验证实,利用杂交优势是子莲新品种选育的重要途径。作者通过杂交选育和引种,筛选培育出13号(白花建莲×红千叶)和2号(百叶莲×红花建莲)杂交子莲,杂交优势明显而稳定,其产鼠比对照品种——红花建莲增加20%以上,经济效益十分显著。为了推广这两种杂交子莲,作者还提出了子莲良种繁育时防杂的技术措施。     

The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity.

The domain of thrombomodulin that binds to the anion-binding exosite of thrombin was identified by comparing the binding of fragments of thrombomodulin to thrombin with that of Hirugen, a 12-residue peptide of hirudin that is known to bind to the anion-binding exosite of thrombin. Three soluble fragments of thrombomodulin, containing (i) the six repeated growth factor-like domains of thrombomodulin (GF1-6), (ii) one-half of the second through the sixth growth factor-like repeats (GF2.5-6), or (iii) the fifth and sixth such domains (GF5-6), were examined. Hirugen was a competitive inhibitor for either GF1-6 or GF2.5-6 stimulation of thrombin activation of protein C. GF5-6, which binds to thrombin without altering its ability to activate protein C, competed with fluorescein-labeled Hirugen for binding to thrombin. Therefore, all three thrombomodulin fragments, each of which lacked the chondroitin sulfate moiety, competed with Hirugen for binding to thrombin. To determine whether GF5-6 and Hirugen were binding to overlapping sites on thrombin or were interfering allosterically with each other's binding to thrombin, the effects of each thrombomodulin fragment and of Hirugen on the active site conformation of thrombin were compared using two different approaches: fluorescence-detected changes in the structure of the active site and the hydrolysis of chromogenic substrates. The GF5-6 and Hirugen peptides affected these measures of active site conformation very similarly, and hence GF5-6 and Hirugen contact residues on the surface of thrombin that allosterically alter the active site structure to a similar extent. Full-length thrombomodulin and GF1-6 alter the active site structure to comparable extents, but the amidolytic activity of thrombin complexed to thrombomodulin or GF1-6 differs significantly from that of thrombin complexed to GF5-6 or Hirugen. Taken together, these results indicate that the GF5-6 domain of thrombomodulin binds to the anion-binding exosite of thrombin. Furthermore, the binding of GF5-6 to the anion-binding exosite alters thrombin specificity, as evidenced by GF5-6-dependent changes in both the kcat and Km of synthetic substrate hydrolysis by thrombin. The contact sites on thrombin for the GF4 domain and the chondroitin sulfate moiety of thrombomodulin are still unknown.     

链霉菌AC-57及其产生的阿克拉霉素A的研究

During the course of screening for new antitumor antibiotics, a new anthracycline antibiotic--aclacinomycin A was separated from the broth and mycelium of Streptomyces AC-57. The strain AC-57 was isolated from the soil collected in the Shanghai suburbs. According to its culture and physiological characteristics the producer was identified as Str. galilaeus AC-57. The broth and mycelium were extracted and treated with solvents as usual way. The aclacinomycin A was separated by silica-gel column chromatography eluted with chrolo-form-methanol. Aclacinomycin A, its aglycone and sugar components were identified by comparison of their physico-chemical and spectral data (MS, UV, IR, 1H-NMR, and 13C-NMR) with authentic compound, purified from the market sample.     

1H NMR identification of a beta-sheet structure and description of folding topology in putidaredoxin

Putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a [2Fe-2S] cluster, is the physiological reductant of P-450cam, which in turn catalyzes the monohydroxylation of camphor by molecular oxygen. No crystal structure has been obtained for Pdx or for any closely homologous protein. The application of two-dimensional 1H NMR methods to the problem of structure determination in Pdx is reported. A beta-sheet consisting of five short strands and one beta-turn has been identified from distinctive nuclear Overhauser effect patterns. All of the backbone resonances and a majority of the side-chain resonances corresponding to protons in the beta-sheet have been assigned sequence specifically. The sheet contains one parallel and three antiparallel strand orientations. Hydrophobic side chains in the beta-sheet face primarily toward the protein interior, except for a group of three valine side chains that are apparently solvent exposed. The potential significance of this "hydrophobic patch" in terms of biological activity is discussed. The folding topology, as determined by the constraints of the beta-sheet, is compared with that of other [2Fe-2S] proteins for which folding topologies are known.     

Relaxation dynamics of the gel to liquid-crystalline transition of phosphatidylcholine bilayers. Effects of chainlength and vesicle size.

The relaxation kinetics of the gel to liquid-crystalline transition of five phosphatidylcholine (DC14PC to DC18PC) bilayer dispersions have been investigated using volume perturbation calorimetry, a steady-state technique which subjects a sample to sinusoidal changes in volume. Temperature and pressure responses to the volume perturbation are measured to monitor the relaxation to a new equilibrium position. The amplitude demodulation and phase shift of these observables are analyzed with respect to the perturbation frequency to yield relaxation times and amplitudes. In the limit of low perturbation frequency, the temperature and pressure responses are proportional to the equilibrium excess heat capacity and bulk modulus, respectively. At all temperatures, the thermal response data are consistent with a single primary relaxation process of the lipid. The less accurate bulk modulus data exhibit two relaxation times, but it is not clear whether they reflect lipid processes or are characteristic of the instrument. The observed thermal relaxation behavior of all multilamellar vesicles are quantitatively similar. The relaxation times vary from approximately 50 ms to 4 s, with a pronounced maximum at a temperature just greater than Tm, the temperature of the excess heat capacity maximum. Large unilamellar vesicles also exhibit a single relaxation process, but without a pronounced maximum in the relaxation time. Their relaxation time is approximately 80 ms over most of the transition range.