Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis

Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.     

Subcellular distribution of the 1,4-dihydropyridine receptor in rabbit skeletal muscle in situ: an immunofluorescence and immunocolloidal gold- labeling study

The subcellular distribution of the 1,4-dihydropyridine receptor was determined in rabbit skeletal muscle in situ by immunofluorescence and immunoelectron microscopy. Longitudinal and transverse cryosections (5-8 microns) of rabbit gracilis muscle were labeled with monoclonal antibodies specific against either the alpha 1-subunit (170,000-D polypeptide) or the beta-subunit (52,000-D polypeptide) of the 1,4-dihydropyridine receptor by immunofluorescence labeling. In longitudinal sections, specific labeling was present only near the interface between the A- and I-band regions of the sarcomeres. In transverse sections, specific labeling showed a hexagonal staining pattern within each myofiber however, the relative staining intensity of the type II (fast) fibers was judged to be three- to fourfold higher than that of the type I (slow) fibers. Specific immunofluorescence labeling of the sarcolemma was not observed in either longitudinal or transverse sections. These results are consistent with the idea that the alpha 1-subunit and the beta-subunit of the purified 1,4-dihydropyridine receptor are densely distributed in the transverse tubular membrane. Immunoelectron microscopical localization with a monoclonal antibody to the alpha 1-subunit of the 1,4-dihydropyridine receptor showed that the 1,4-dihydropyridine receptor is densely distributed in the transverse tubular membrane. Approximately half of these were distributed in close proximity to the junctional region between the transverse tubules and the terminal cisternae. Specific labeling was also present in discrete foci in the subsarcolemmal region of the myofibers. The size and the nonrandom distribution of these foci in the subsarcolemmal region support the possibility that they correspond to invaginations from the sarcolemma called caveolae. In conclusion, our results demonstrate that the 1,4-dihydropyridine receptor in skeletal muscle is localized to the transverse tubular membrane and discrete foci in the subsarcolemmal region, possibly caveolae but absent from the lateral portion of the sarcolemma.     

Cause for dark, chilling-induced inactivation of photosynthetic oxygen-evolving system in cucumber leaves

Effects on oxygen evolution of the storage of detached cucumber (Cucumis sativus) leaves at 0°C in the dark were investigated with thylakoids and oxygen-evolving photosystem II membranes isolated from stored leaves. The cold and dark treatment of leaves selectively inactivated electron transport on the oxidizing side of photosystem II. Photosystem II membranes isolated from treated leaves were largely depleted of two proteins of 20 and 14 kilodaltons, which correspond to the extrinsic 23- and 17- kilodalton proteins of spinach functioning in oxygen evolution. The manganese content of photosystem II membranes was also markedly reduced by the treatment. Thus, the inactivation of oxygen evolution induced by the dark, chilling treatment is ascribed to solubilization of the 20- and 14-kilodalton proteins and extraction of manganese.     

Increase of poly(L-lysine) uptake but not fluid phase endocytosis in neuraminidase pretreated Madin-Darby canine kidney (MDCK) cells

The uptake of Poly(L-lysine) conjugates in cultured cells has been used as a model for non-specific adsorptive endocytosis of cationic macromolecules. To study the effect of glycocalyx desialylation on the uptake of cationic macromolecules in epithelial cells, Madin-Darby canine kidney (MDCK) cell monolayers were treated with neuraminidase and incubated with Poly(L-lysine) conjugates. Neuraminidase pretreatment of MDCK cells resulted in a 40% increase in the uptake of Poly(L-lysine) whereas trypsin pretreatment did not. Neuraminidase pretreatment did not increase the endocytosis of fluid phase markers in MDCK cells, nor the uptake of Poly(L-lysine) in L929 fibroblasts. These results suggest that the negative charges, rather than the glycoprotein matrices of glycocalyx, play an important role in the control of the uptake of cationic macromolecules in epithelial cells.     

Lectin analysis of common glycoproteins detected on the surface of continuous microvascular endothelium in situ and in culture: identification of sialoglycoproteins

For many years, molecular interactions with vascular endothelium have been studied in vitro on cultured endothelial cells. Yet, it is clear that the different environmental conditions in vivo vs. in vitro may cause phenotypic drift and altered expression of cell surface molecules. In this study, we identify several endothelial surface proteins of similar apparent molecular mass by radioiodination of cultured microvascular cells and by intravascular radioiodination of rat heart endothelium in situ. The radioiodinated surface polypeptides detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (followed by autoradiography) were subjected to lectin affinity chromatography in order to provide an additional screen for identifying common surface glycoproteins and a means for partial characterization of their glycans. With a battery of 18 lectins, seven major (gp140, gp120, gp100, gp85, gp75, gp60, gp47) and 6 minor (gp330, gp300, gp180, gp160, gp150, gp42) glycoproteins were identified on the cultured cells each with a different lectin binding profile. The lectin binding profiles of many endothelial glycoproteins in situ were similar to those of their counterparts in culture. A common set of seven major glycoproteins with the same apparent molecular masses was found in situ as well as in vitro. These common glycoproteins were characterized further using both sialidase digestion and sequential lectin affinity chromatography of cell lysates. Most of the glycoproteins appear to have both complex-type N-linked and O-linked glycans except for gp60 with only O-linked glycans, gp47 with only complex N-linked sugars, and gp42 with only simple N-linked sugars. A subset of sialoglycoproteins (gp140, gp120, gp100, gp60, gp47) was identified. One of them, gp120, is podocalyxin based on immunoprecipitation with specific antiserum and another one, gp60, is a recently identified albumin binding protein on the surface of cultured microvascular endothelial cells. This study shows that gp60 is indeed present on the surface of endothelium in situ and that it is a sialoglycoprotein with typical O-linked glycans. It is apparent that the continuous type of microvascular endothelium can indeed express in culture and in situ a common set of major glycoproteins.     

Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183

Summary A new thermophilic Bacillus strain 3183 (ATCC 49341) was isolated from hot-spring sediments. The organism grew on pullulan as a carbon source and showed optimum pH and temperature at pH 5.5 and 62° C, respectively, for growth. The strain reduced nitrate to nitrite both aerobically and anaerobically. It produced extracellular thermostable pullulanase and saccharidase activities which degraded pullulan and starch into maltotriose, maltose, and glucose. Medium growth conditions for pullulanase production were optimized. The optimum pH and temperature for pullulanase activity were at pH 6.0 and 75° C, respectively. The enzyme was stable at pH 5.5-7.0 and temperature up to 70° C in the absence of substrate. The K _m for pullulan at pH 6.0 and 75° C was 0.4 mg/ml. The pullulanase activity was stimulated and stabilized by Ca²⁺. It was inhibited by ethylenediaminetetraacetate (EDTA), beta and gamma-cyclodextrins but not by alpha-cyclodextrin and reagents that inhibit essential enzyme SH-groups. Offprint requests to: B. C. Saha     

关于Taylor幂法则的统计学讨论

自Greenwood于1920年把负二项分布引做昆虫种群空间格局模型以来,昆虫种群空间格局分析的理论和方法大致经历了两个阶段的发展:五、六十年代以前,是以少数离散型概率分布为主要模型;其后,各种聚集性指标和一些回归公式被提出,显示了比原有     

白细胞介素2受体α链基因转录起始点和5'调控区结合蛋白的初步研究

 借助于5'和3'末端删切后重建的IL-2R a链基因调控区次级克隆,在体外合成有放射性同位素参入的反意义RNA探针与总RNA进行液相杂交,结果表明TPA或PHA分别活化的T细胞在IL-2R a链表达过程中都在不同程度上有选择地利用了调控区内分别为-58(5')和+1(3')位两个转录起始点中3'转录起始点。热休克使PHA活化细胞更明显地利用+1位点。PHA诱导Jurkat细胞表达IL-2RamRNA斑点杂交证实,Jurkat细胞在活化16小时表达IL-2Ra基本达到高峰,至24小时已明显下降。根据这一规律提取PHA诱导活化15小时的Jurkat细胞S100和NE,进行有关结合蛋白的研究,初步结果显示磷酸纤维素柱的KCI洗脱组分中存在着DNA结合蛋白,有关结合蛋白性质的研究正在进行中。     

肥皂草素类核糖核蛋白体失活蛋白的纯化及其性质研究

 利用强阳离子交换柱从Saponaria officinalis L种子中分离出一种肥皂草素(Saporin)成分。它在无细胞体系中显示了较强的抑制蛋白合成的活性,与抗体连接后能特异性杀伤靶细胞。     

家兔早期胚胎细胞发育能力的研究

本文利用胚泡注射法制作嵌合体对家兔交配后96,120和144小时的ICM细胞的发育能力进行了研究。供体胚胎取自青紫兰灰免,受体胚胎取自新西兰白兔,结果表明96和120小时供胚的ICM细胞与96小时受胚胚泡组合后均能参与发育,形成嵌合兔,144小时者未获得嵌合体。由于120小时的ICM细胞发育的2只表型为雄性的嵌合兔,其中1只不育,其性腺和外周血核型表明不育兔为xx/xy性嵌合,性腺中有处于不同发育程度的卵巢和精细管,外周血含xx和xy两种核型。本实验结果首次证明家兔交配后120小时胚泡的ICM细胞仍具有参与嵌合体发育的能力。它不仅能参与体细胞的分化,并具有形成生殖细胞的能力。交配后144小时胚泡的ICM细胞其发育能力似乎已发生了局限。