Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR and computational studies of interactions between remote residues in gangliosides

Conformational preferences of the gangliosides GM1, GM1b, and GD1a have been investigated by using a systematic combination of NMR distance constraints and molecular mechanics calculations. These gangliosides share a common four-sugar core but differ in the number or placement of sialic acid residues attached to the core. Placement of the sialic acid residues is shown to influence the preferred core conformation. The origin of these effects is postulated to be intramolecular interactions of the sialic acid residues with other remote residues. In the case of GM1, hydrogen bonds between the internal sialic acid and an N-acetyl group on GalNAc are suggested. In the case of GD1a, a hydrogen-bonding network between the terminal and internal sialic acids is suggested to play a role.     

Resonance Raman enhancement of phenyl ring vibrational modes in phenyl iron complex of myoglobin.

Resonance Raman spectra are reported for the organometallic phenyl-FeIII complexes of horse heart myoglobin. We observed the resonance enhancement of the ring vibrational modes of the bound phenyl group. They were identified at 642, 996, 1,009, and 1,048 cm-1, which shift to 619, 961, 972, and 1,030 cm-1, respectively, upon phenyl 13C substitution. The lines at 642 and 996 cm-1 are assigned, respectively, as in-plane phenyl ring deformation mode (derived from benzene vibration No. 6a at 606 cm-1) and out-of-plane CH deformation (derived from benzene vibration No. 5 at 995 cm-1). The frequencies of the ring "breathing" modes at 1,009 and 1,048 cm-1 are higher than the corresponding ones in phenylalanine (at 1,004 and 1,033 cm-1) and benzene (at 992 and 1,010 cm-1), indicating that the ring C--C bonds are strengthened (or shortened) when coordinated to the heme iron. The excitation profiles of these phenyl ring modes and a porphyrin ring vibrational mode at 674 cm-1 exhibit peaks near its Soret absorption maximum at 431 nm. This appears to indicate that these phenyl ring modes may be enhanced via resonance with the Soret pi-pi transition. The FeIII--C bond stretching vibration has not been detected with excitation wavelengths in the 406.7-457.9-nm region.     

Large scale purification and immunolocalization of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation

The differentiation of mammalian urothelium culminates in the formation of asymmetrical unit membrane (AUM). Using gradient centrifugation and detergent wash, we purified milligram quantities of AUMs which, interestingly, contained three major proteins (15, 27, and 47 kDa) that appeared to be identical to the three immunoaffinity purified, putatively AUM-associated proteins that we described earlier (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell Biol., 111, 1207-1216). Peptide mapping and immunoblotting established that these three proteins were distinct molecules. Using monospecific antibodies to these three proteins, we showed that they were all restricted to the superficial urothelial cells and were AUM-associated. The 27- and 15-kDa proteins were detected exclusively on the luminal side of mature, apical AUMs. In contrast, epitopes of the 47-kDa protein were detected on both sides of apical AUMs suggesting a transmembranous configuration. These results (i) provide the strongest evidence thus far that AUM contains three major proteins (the 27-kDa uroplakin I, 15-kDa uroplakin II, and 47-kDa uroplakin III) which form an extremely insoluble complex, (ii) suggest that uroplakin II, like uroplakin I (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216), translocates from one side of the membrane to another during AUM maturation, (iii) indicate that uroplakin III may play a different structural role than uroplakins I and II in AUM formation, and (iv) establish the three uroplakins as markers for an advanced stage of urothelial differentiation.     

中华绒螯蟹血淋巴20-羟蜕皮酮诱发蜕皮和卵巢发育的作用

通过放射免疫法测定了中华绒螯蟹蜕皮周期血淋巴20-羟蜕皮酮(20-HE)含量的变化。血淋巴20-HE同卵母细胞发育各个阶段有密切的时相关系:在卵母细胞小生长期血淋巴20-HE持续上升,经青春蜕皮进入卵母细胞大生长期后又迅速降低。不能及时青春蜕皮的青春期前雌蟹,稍后仍出现20-HE下降的趋势。对不同实验条件和生理状态下雌蟹的比较表明,20-HE具有诱发蜕皮的特性。卵母细胞早期生长必需高浓度的血淋巴20-HE。外源注射的20-HE有刺激卵巢增重的作用。     

球形芽孢杆菌Ts—1毒蛋白的分离纯化

Bacillus sphaericus strain Ts-1 is highly insecticidal to larvae of the mosquito. It's insecticidal component is toxic proteins. The toxin was extracted from spore-crystal complexes by disruption in a Sonicator Cell Disruptor Model W-220F followed by treatment with 0.05 mol/L NaOH. Fraction recovered from chromatography of the spore-crystal complexes on column of Sephadex G-200 were assayed against mosquito larvae and the toxic fractions from gel chromatography were subjected to SDS-PAGE. The toxic proteins in B. sphaericus Ts-1 spore-crystal complex migrated in position corresponding to 42kD and 43kD. Bioassay of the two purified proteins prepared by PAGE indicated that they were all toxic to mosquito larvae. Toxic protein was further purified by DEAE-cellulose chromatography. The toxic protein with a molecular weight of 42kD was obtained.     

Developmental regulation of NAD+ kinase in Neurospora crassa

The specific activity of NAD⁺ kinase (ATP:NAD⁺ 2-phosphotransferase, EC 2.7.1.23) from Neurospora crassa shows sharp peaks when the organism enters a new developmental stage of the asexual life cycle: the peaks are observed during hydration and germination of conidia, at the transition from exponential to stationary growth and at the photostimulated conidiation. As stimulation of NAD⁺ kinase activity by light in conidiating mycelium is not sensitive to translation inhibitors, the activiation of pre-existing molecules, rather than induction of protein synthesis de novo may be supposed. Enzyme electrophoresis revealed the presence of four forms of NAD⁺ kinase having different apparent molecular weights (I=333,000; II=306,000; III=229,000 and IV=203,000). Manifestation of the activity of individual forms of NAD⁺ kinase is developmentally controlled: form III is most abundant during vegetative growth, forms I and II prevail in conidia. At the conidial germination the increase of NAD⁺ kinase activity is associated with the activation of form III, whereas during photostimulation of conidiation form II is the most activated one. Therefore, certain molecular forms of the enzyme may be regarded as biochemical markers for different developmental stages of N. crassa.     

Characterization of ubisemiquinone radical in the cytochrome b-c1 segment of the mitochondrial respiratory chain

The ubiquinone protein, QP-C, in reduced ubiquinone-cytochrome c reductase (the b?c₁-III complex) shows a stable ubisemiquinone radical when the enzyme is reduced by succinate in the presence of catalytic amounts of succinate dehydrogenase and QP-S. At room temperature using EPR technique the redox titration of the b?c₁-III complex in the presence of redox dyes or succinate/fumarate couple reveals that the ubisemiquinone radical has a midpoint potential of approximately +67 mV at pH 8.0. Further analysis yields E₁ of +83 mV and E₂ of +51 mV corresponding to ($\text{QH}{}_2\text{QH·}$) and ($\text{QH·}\text{Q}$) or other electronated forms, respectively. The equilibrium radical concentration has been found to be affected both by pH and succinate/fumarate couple. At pH 9.0 the radical shows the maximal amplitude and stability. Below pH 7.0, little radical was detected. The electron spin relaxation behavior of ubisemiquinone radical, as examined by microwave power saturation, indicates that the ubisemiquinone radical of QP-C is somewhat isolated from other paramagnetic centers. The effects of phospholipids, QP-S, and other agents on ubisemiquinone radical formation as well as the enzymatic activity of QP-C have been studied in detail.     

温度对干旱、盐胁迫下两种黄芪属种子萌发和幼苗生长的影响

为探讨温度对干旱、盐胁迫下黄芪属种子萌发和幼苗生长特性的影响,以黄芪属蒙古黄芪和扁茎黄芪2种种子为研究对象,纯净水处理为对照组,NaCl、PEG处理为实验组,设置4个渗透势水平(0、-0.1、-0.3、-0.5 MPa),置于5种不同的温度(10、15、20、25、30 ℃)下,每日观察并记录两种种子萌发和幼苗生长情况。结果表明：旱盐胁迫下蒙古黄芪和扁茎黄芪种子萌发最适宜的温度分别为25和20 ℃左右;蒙古黄芪耐高温不耐低温,而扁茎黄芪恰恰相反;但25和20 ℃均适宜两种幼苗生长,包括胚根、胚轴和子叶的生长。蒙古黄芪各处理组(除未发芽的种子)的平均发芽时间都比扁茎黄芪长;NaCl胁迫程度的增加使得两种种子的最终发芽率降低,但蒙古黄芪的耐盐性高于扁茎黄芪;随着PEG胁迫程度的增加,二者的发芽均受到抑制,甚至会出现完全不萌发,但扁茎黄芪的耐旱性高于蒙古黄芪;在相同的渗透势时,尤其是-0.5 MPa,PEG比NaCl对两种种子的影响大;交互胁迫作用下,随着渗透势的增加两种幼苗的鲜重、干重以及胚根、胚轴、子叶的长和宽变化较大;利用Design Expert软件预测发现：温度25 ℃、NaCl渗透势为-0.1 MPa,温度24 ℃、PEG渗透势为-0.04 MPa的处理是蒙古黄芪种子萌发和幼苗生长达到最优化的组合;而扁茎黄芪最优化的组合则为23 ℃下NaCl渗透势为-0.07 MPa的处理,20 ℃下PEG渗透势为-0.13 MPa的处理。