萱草花粉中微管蛋白生物化学性质

微管（microtubule）是细胞骨架的重要成份,参与囊泡运输、信息传递等多种生命活动。我们从萱草花粉中纯化了植物微管蛋白,对其生物化学及生物物理学部分性质研究表明,纯化的微管蛋白经超离心法测定沉降系数为6．2S,SDS-PAGE分析α,β微管蛋白分子量为56kD、58kD,凝胶扫描分析纯度为93．7％。等电聚焦电泳测定等电点为pI=5．35。光谱学性质研究结果表明,最大紫外吸收峰为280．8nm,荧光光谱研究表明最大激发波长为282nm;此时的最大发射峰为338nm,圆二色光谱分析二级结构表明小螺旋占27．24％,β-折叠占24．48％,无规卷曲为48．28％,呈典型球蛋白特征。     

口腔常见致龋菌荧光光谱特征的初步研究

目的采用三维荧光光谱分析技术对口腔常见致龋菌荧光光谱特征进行初步分析。方法选择口腔常见致龋菌变形链球菌及远缘链球菌,对其进行复苏和培养,运用三维荧光光谱分析技术对其菌液进行荧光学光谱检测。结果变形链球菌及远缘链球菌的三维荧光光谱图相似,均出现2个荧光峰,最佳激发波长分别位于230nm和280nm,最佳发射波长相同,均为340nm。结论成功获得口腔常见致龋菌(变形链球菌及远缘链球菌)的固有荧光三维光谱图,为致龋菌荧光评价技术的应用提供参考。     

嗜碱细菌环状糊精葡糖基转移酶的纯化和性质

嗜碱细菌52—2除去菌体的培养液经硫酸铵沉淀和DEAE－纤维素离子交换柱层析,得到凝胶电泳均一的环状糊精葡糖基转移酶,纯化了11.5倍,酶活力回收为5.7％。用浓度梯度PAGE测分子量为151700。酶反应最适温度为65℃,50℃以下比较稳定。酶反应最适pH为7．0,在6．0～9.0范围内稳定。Zn²⁺、Hg²⁺、Pb²⁺、Al³⁺、Cu²⁺、Ag⁺和Fe²⁺强烈抑制酶活力。紫外光谱在270nm和244nm处分别有最大和最小吸收。荧光光谱的最大激发波长和发射波长分别为283nm和335nm。用NBS、NEM、NAI、DEP和EDC对酶进行了化学修饰,初步推测组氨酸和色氨酸残基可能为酶活力必需基因,羧基与酶活力有一定关系。     

广西产眼镜蛇毒酸性磷脂酶A2的分离纯化及性质研究

目的　从广西产中华眼镜蛇毒 ( N aja naja atra)中分离了一种新的磷脂酶 A2 ( PLA2 ) ,并研究其性质。　方法　磷脂酶 A2 ( PLA2 )的分离纯化采用 CM5 2、CM-Sepharose CL-6 B离子交换柱和 SepharoseCL-6 B凝胶柱 ,磷脂酶 A2 ( PLA2 )的性质采用经典方法进行。　结果　分离后的磷脂酶 A2 经过聚丙烯酰胺凝胶电泳、SDS-聚丙烯酰胺凝胶电泳和 HPLC检测 ,其为单一组分。SDS-PAGE测定它的分子量为1 5 0 0 0± 1 0 0 0。等电聚焦测得它的等电点为 6 .3。它的最适温度为 5 5℃ ,最适 p H值为 8.5。氨基酸成分分析表明该酶由 1 2 6个氨基酸组成 ,以 Asp、Ala、Gly、Cys居多。Fe3 、Zn2 、EDTA对其酶活力起抑制作用 ;K 、Ca2 、去垢剂对其活力起促进作用。荧光光谱分析表明该酶的色氨酸残基、组氨酸残基可能位于分子表面。药理实验表明 :该酶具有抗胰蛋白酶作用、抗凝血作用、间接溶血作用以及对青蛙具有心脏毒性。　结论　广西产中华眼镜蛇毒磷脂酶 A2 与其它来源的 PLA2 同源性高 ,但性质不尽相同。     

酵母羧甲基化及光照甘油醛-3-磷酸脱氢酶在碘化钾溶液中荧光发色团的形成

CM-GAPDH在碘化钾溶液中,NAD~+的存在下,形成发射波长为383nm的荧光物。对照的NAD~+与碘化钾溶液混合不产生荧光物。全位及半位修饰光照酶的内源荧光在碘化钾溶液中的变化与天然酶的有明显不同。两者在碘化钾中都形成383nm的荧光,但全位修饰光照酶形成383nm荧光的最适碘化钾浓度为1.0M;半位修饰的为0.8M。以上结果暗示:383nm荧光物的形成需要GAPDH和NAD~+同时存在,并且与活性部位巯基修饰的多少有关,该荧光物可能位于GAPDH的活性部位。     

酵母羧甲基化及光照甘油醛-3-磷酸脱氢酶在碘化钾溶液中荧光发色团的形成

CM-GAPDH在碘化钾溶液中,NAD~+的存在下,形成发射波长为383nm的荧光物。对照的NAD~+与碘化钾溶液混合不产生荧光物。全位及半位修饰光照酶的内源荧光在碘化钾溶液中的变化与天然酶的有明显不同。两者在碘化钾中都形成383nm的荧光,但全位修饰光照酶形成383nm荧光的最适碘化钾浓度为1.0M;半位修饰的为0.8M。以上结果暗示:383nm荧光物的形成需要GAPDH和NAD~+同时存在,并且与活性部位巯基修饰的多少有关,该荧光物可能位于GAPDH的活性部位。     

不同激发波长的人体血清自体荧光光谱分析

利用分子荧光光度计测定了激发波长分别为457.9 nm,476.5 nm,488.0 nm,501.7 nm和514.5 nm光的血清自体荧光光谱,并对这些光谱的特征和产生机制进行了分析。实验和分析结果表明:在500 nm~750 nm的波长范围内,518 nm和640 nm附近有两个比较明显的荧光峰;590 nm处有一个非常弱的峰。它们可能主要来自于血清中胆红素,核黄素及其衍生物,β-胡萝卜素,锌卟啉及原卟啉IX等的贡献。这些研究结果将为利用氩离子激光器作为光源进行光谱诊断研究选择最佳激发波长提供一些参考。     

嗜热链霉菌过氧化氢酶的纯化及性质研究

嗜热链霉菌(Thermostreptostreptomyces sp.)T485的除去菌体的培养液,经硫酸铵盐析,Sepha- dex G—100、DEAE—Sephadex A-50及羟基磷灰石等柱层析,得到了凝胶电泳均-的过氧化氧酶,纯化了954倍,得率为7％。用浓度梯度PAGE测定分子量为152000,SDS—PAGE测定亚基分子量为57000,凝胶薄层等电点聚焦测定等电点为4．25。过氧化氢酶的反应最适温度为60℃,最适pH为7．0;对H2O2的K为50 mmol·L-1,Vmas值为6．0 mmol·min-1·mg-1。Nall3和Hg2+对酶活力有强烈抑制作用．Ca2+对酶活力有激活作用。测定了波长200—500nm的吸收光谱,在405nm处有明显的吸收峰。根据受NaN3,抑制和吸收光谱性质,推测它为含血红素酶。此外还测定了过氧化氢酶的氨基酸组成。     

植物28kD蛋白的纯化及部分性质研究

经丙酮粉、硫酸铵分级沉淀和阴离子交换层析等方法,首次从玉米花粉细胞质粗提物中纯化了一种具有ＡＴＰａｓｅ活性的可溶性蛋白。ＳＤＳ－ＰＡＧＥ测得分子量为２８ｋＤ,ＩＥＦ－ＰＡＧＥ测得等电点为８．３。免疫印迹鉴定结果表明该酶蛋白与抗牛脑动蛋白或动力蛋白的抗体无免疫交叉反应。最大紫外吸收波长为２７８ｎｍ,并作了ＣＤ谱分析。底物特异性研究表明：ＡＴＰａｓｅ水解活性最高。药理学研究表明：该酶蛋白可被矾酸钠强烈抑制,但对ＮＥＭ基本不敏感,氟化钠可使酶活力丧失５０％左右,寡霉素、硝酸钾及乌本苷对酶活力没有抑制作用．     

测定植物胞内游离钠离子的研究进展

植物耐盐机制的研究一直是植物抗性研究的焦点。近年来,随着生物学不断发展和新荧光标记技术的运用,胞内钠离子测定逐渐应用于植物盐胁迫研究中。该文论述了以下三方面问题:(1)分别介绍了SBFI、Sodium Green和Coro Na Green三种钠离子荧光指示剂:SBFI是一种双激发波长指示剂,其激发波长是340 nm/380 nm,发射波长是500 nm;Sodium Green和Coro Na Green是单波长指示剂,其激发波长分别是507 nm和492nm,发射波长分别是532 nm和516 nm;(2)比较了酯导入、酸导入、电穿孔和显微注射等几种常见荧光指示剂载入胞内方法的优缺点,重点介绍了一种无损伤低温抑制酯酶法:先将荧光指示剂在缓冲液中低温(4℃)处理2 h,随后回到常温(20℃)在不含荧光指示剂的缓冲液中孵育2 h;(3)阐述了胞内离子浓度计算公式,包括单波长测定公式、双波长比率测定公式。     

Biophysical and Pharmacological Characterization of a Dynamin-like Protein from Day-lily ( Hemerocallis fulva,Liliaceae) Pollens

A novel motor protein , dynamin- like protein from day lily, was purified and identified by FPLC . Here we report its biochemical characterization . The molecular weight of the dynamin-like protein is 100 kD on SDS-PAGE. Isoelectric points are about 6. 15 and 6 .80 . The fluorescence emission wave length of dynamin-like protein is 346 nm by excitation at 280 nm . Through fluorescence spectra analysis, ultraviolet absorption spectrum and derivative spectrum, we conclude that it contains tryptophan and tyrosine residues . Pharmacological study indicates that mercapto may play an important role inenzyme activity of dynamin-like protein .     

紫膜的紫外荧光及分子内的能量转移

以可见光为作用光照射天然紫膜,紫膜蛋白被280nm紫外光激发所发射的荧光强度比对照略有降低.比较天然紫膜、漂白紫膜与菌蛋白三者的紫外荧光强度,前两者无显著变化,但菌蛋白的荧光强度比天然紫膜的荧光强度大2-3倍,表明生色团对蛋白质荧光可能有猝灭作用.用280nm波长光照射紫膜的暗适应形式,可使其转变成光适应形式.若有羟胺存在,以紫外光照射也可使紫膜漂白.光漂白的作用光谱,其紫外部分与紫膜蛋白部分的吸收光谱重合得很好.上述实验证明紫膜蛋白部分吸收的能量可以转移到生色团上,即紫膜存在分子内的能量转移     

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.     

Regulation of ADL6 activity by its associated molecular network

Plant dynamin-like proteins consist of a group of high molecular weight GTPase with diverse structural arrangements and cellular localizations. In addition, unlike animal dynamins, there was no evidence for the involvement of any plant dynamin-like protein in clathrin-mediated vesicle trafficking. In this study we demonstrate that ADL6 (Arabidopsis dynamin-like protein 6), due to its domain arrangement, behaves similarly to the animal dynamins. The association of ADL6 with clathrin-coated vesicles was demonstrated by co-fractionation and immunocytochemical studies. ADL6 also interacted via its C-terminus with gamma-adaptin, an adaptor protein of clathrin-coated vesicles. Our results suggest that ADL6 participates in clathrin-mediated vesicle trafficking originating from the Golgi. In addition, our studies demonstrate that ADL6 intrinsic GTPase activity is regulated by its association with acidic phospholipids and an SH3 (Src homology 3)-containing protein.     

Tyrosine and tyrosinate fluorescence of pig intestinal Ca2+-binding protein.

The single tyrosine residue in both pig and cow intestinal Ca2+-binding proteins fluoresces at 303 nm although the crystal structure of the cow protein shows a hydrogen bond between the hydroxy group of the tyrosine and glutamate-38 [Szebenyi & Moffat (1986) J. Biol. Chem. 261, 8761-8777]. The latter interaction suggests that tyrosinate fluorescence should dominate the emission spectra of these proteins. A fluorescence difference spectrum, produced by subtracting the spectrum of free tyrosine from the spectrum of the protein, gives a peak at 334 nm due to ionized tyrosine. That this component of the emission spectrum is not due to a tryptophan-containing contaminant is shown by its elimination when the protein is denatured by guanidine and when glutamate-38 is protonated. We conclude that, in solution, the tyrosine residue in this protein interacts occasionally with glutamate-38 but that a permanent hydrogen bond is not formed.     

Luminescence characteristics of mitochondrial actin-like protein

Investigations comparing tryptophan fluorescence of mitochondrial actin-like protein with that of skeletal and smooth muscle actins have shown that the mitochondrial protein is characterized by a fluorescence spectrum peak at a longer wavelength (337 nm), by a certain broadening of this peak (56 nm) and by a reduction in the lifespan of the excited state of tryptophanyls (2.7 ns). The data obtained suggest the existence of peculiarities in the structure of mitochondrial actin-like protein.     

An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.     

The S100-b Protein: Tyrosine Residues Do Not Exhibit an Abnormal Fluorescence Spectrum

The β subunit of the bovine brain S100-b protein (ββ) lacks tryptophyl residue but contains one tyrosine. Our experiments show that this protein is characterized by a typical tyrosine fluorescence spectrum, with a maximum at 303 nm. Identical fluroescence properties were found for the rat brain S100-b protein. Comparison with the fluorescence spectrum of the bovine brain S100-a’ protein (α'β), which contains a tryptophan residue in the α’ subunit, enables us to demonstrate that the recent report describing an abnormal fluorescence spectrum for the bovine brain S100-b protein may result from a contamination of the S100-b by the S100-α’ protein.     

The fluorescence from the chromophore of the purple membrane protein.

The fluorescence from the purple membrane protein (PM) of Halobacterium halobium and its relation to the primary photochemical events have been studied. The emission spectrum at 77 degrees K has structure, with peaks at 680, 710-715, and 730-735 nm. The excitation spectrum shows a single peak centered at 580 nm. This and a comparison of the fluorescence intensity at 77 degrees K under a variety of conditions with the amounts of the bathoproduct (or K, the only photoproduct seen at this temperature) formed suggest that the source of the fluorescence is the purple membrane itself, not the photoproduct. From the difference in several of their properties, we suggest that the fluorescing state of the pigment is different from the excited state which leads to photoconversion.     

Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.