ISOLATION AND CHARACTERIZATION OF CHROMATIN FROM NEUROSPORA CRASSA

Different preparations of chromatin isolated from mycelia of Neurospora crassa were analyzed for DNA-associated RNA and proteins. The UV absorption spectra, the ultrastructure of chromatin, and the amino acid composition of the acid-extractable proteins were studied. The protein:DNA ratios range from 1.5 to 2.8; the RNA:DNA ratios range from 0.5 to 1.24. UV absorption shows a macimum at 259 mµ and a minimum at 238–239 mµ. The E280/E260 ranges from 0.59 to 0.70. Electron microscopy reveals a fibrous structure with individual fibers of 120–150 A average diameter. Attempts were made to study the protein by polyacrylamide gel electrophoresis and amino acid analysis. The results indicate that Neurospora chromatin does not contain basic proteins comparable to calf thymus histone. The ratios of basic to acidic amino acids range from 0.93 to 1.19. On electrophoresis, no bands are seen whose positions correspond to those of histones. Staining for basic proteins with fast green or eosin Y at pH 8.2 also shows a negative reaction, suggesting the absence of histones.     

Resonance Raman spectra of cytochrome c oxidase. Excitation in the 600-nm region.

The resonance Raman (RR) spectra of oxidized, reduced, and oxidized cyanide-bound cytochrome c oxidase with excitation at several wavelengths in the 600-nm region are presented. No evidence is found for laser-induced photoreduction of the oxidized protein with irradiation at lambda approximately 600 nm at 195 K, in contrast to the predominance of this process upon irradiation in the Soret region at this temperature. The Raman spectra of all three protein species are very similar, and there are no Raman bands which are readily assignable to either cytochrome a or cytochrome a3 exclusively. The Raman spectra of the three protein species do, however, exhibit a number of bands not observed in the RR spectra of other hemoproteins upon exicitation in their visible absorption bands. In particular, strong Raman bands are observed in the low-frequency region of the RR spectra (less than 500 cm-1). The frequencies of these bands are similar to those of the copper-ligand vibrations observed in the RR spectra of type 1 copper proteins upon excitation in the 600-nm absorption band characteristic of these proteins. In cytochrome c oxidase, these bands do not disappear upon reduction of the protein and, therefore, cannot be attributed to copper-ligand vibrations. Thus, all the observed RR bands are associated with the two heme A moieties in the enzyme.     

Identification of peptides containing tryptophan, tyrosine, and phenylalanine using photodiode-array spectrophotometry

The characteristic absorption spectra of aromatic amino acids between 240 and 310 nm were used to identify tryptophan, tyrosine, and phenylalanine-containing peptides. In acidic solution, the absorption spectra of these amino acids exhibit minima or maxima at 255, 270, and 286 nm. Based on these characteristics, the content of the aromatic amino acid in peptide can be estimated. For this study, 2 nmol of tryptic peptides from human apolipoprotein A-1 was separated by high-performance liquid chromatography using a reverse-phase column. The peptide fragments were monitored by a photodiode-array spectrophotometer. This new approach offers a rapid, simple, sensitive, and direct identification of peptides containing aromatic amino acids. Those containing Trp, which may be of interest for DNA sequencing and important in sequence analysis of proteins, can be selectively purified using this technique.     

Emission spectra from copper proteins containing type-3 centres

Aqueous solutions of copper-proteins containing type-3 centres (ceruloplasmin, tyrosinase, haemocyanin), excited within their absorption bands at 325-345 nm, show typical luminescence spectra. The emission bands peak at 415-445 nm and their decay time is no longer than 10 ns. A strong analogous fluorescence is obtained also by excitation of concentrated solutions of carboxylic acids and amino acids, which show again absorption bands around 330 nm. Such a fluorescence, although less intense, is also observed in copper(II) carboxylate solutions. In contrast, no fluorescence has been recorded in solutions of acetic anhydride and of polypeptides (valinomycin, gramicidin D), which do not have free carboxyl groups. We tentatively attribute this novel fluorescence in the investigated copper proteins to interactions between carboxyl groups of amino acids at, or near, the active site.     

曲霉和青霉属内杂种和亲株的蛋白质电泳图谱比较

曲霉属内黑曲霉(Aspergitlus niger)与米曲霉(A.oryzae)具有特征明显不同的可溶性蛋白质电泳图谱,其种间杂种具有双亲的部分或全部电泳带并与黑曲霉相近。来自杂种Ⅰ的多数分离子电泳带与黑曲霉相近,只有一个分离子产生米曲霉的电泳带并具有米曲霉的遗传特性。青霉属内产黄青霉(Penicillium chrysogenum)与展青霉(P.patulum)种间及种内不同菌株间的电泳图谱基本相同,种内或种间杂种具有双亲的电泳带。结果讨论了蛋白质图谱分析的意义。     

Redox-induced conformational changes in myoglobin and hemoglobin: electrochemistry and ultraviolet-visible and Fourier transform infrared difference spectroscopy at surface-modified gold electrodes in an ultra-thin-layer spectroelectrochemical cell.

Using suitable surface-modified electrodes, we have developed an electrochemical system which allows a reversible heterogeneous electron transfer at high (approximately 5 mM) protein concentrations between the electrode and myoglobin or hemoglobin in an optically transparent thin-layer electrochemical (OTTLE) cell. With this cell, which is transparent from 190 to 10,000 nm, we have been able to obtain electrochemically-induced Fourier-transform infrared (FTIR) difference spectra of both proteins. Clean protein difference spectra between the redox states were obtained because of the absence of redox mediators in the protein solution. The reduced-minus-oxidized difference spectra are characteristic for each protein and arise from redox-sensitive heme modes as well as from polypeptide backbone and amino acid side chain conformational changes concomitant with the redox transition. The amplitudes of the difference bands, however, are small as compared to the total amide I absorbance, and correspond to approximately 1% (4%) of the reduced-minus-oxidized difference absorbance in the Soret region of myoglobin (hemoglobin) and to less than 0.1% of the total amide I absorbance. Some of the bands in the 1560-1490-cm-1 spectral regions could be assigned to side-chain vibrational modes of aromatic amino acids. In the conformationally sensitive spectral region between 1680 and 1630 cm-1, bands could be attributed to peptide C = O modes because of their small (2-5 cm-1) shift in 2H2O. A similar assignment could be achieved for amide II modes because of their strong shift in 2H2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

A purple-blue chromoprotein from Goniopora tenuidens belongs to the DsRed subfamily of GFP-like proteins

A number of recently cloned chromoproteins homologous to the green fluorescent protein show a substantial bathochromic shift in absorption spectra. Compared with red fluorescent protein from Discosoma sp. (DsRed), mutants of these so-called far-red proteins exhibit a clear red shift in emission spectra as well. Here we report that a far-red chromoprotein from Goniopora tenuidens (gtCP) contains a chromophore of the same chemical structure as DsRed. Denaturation kinetics of both DsRed and gtCP under acidic conditions indicates that the red form of the chromophore (absorption maximum at 436 nm) converts to the GFP-like form (384 nm) by a one-stage reaction. Upon neutralization, the 436-nm form of gtCP, but not the 384-nm form, renaturates instantly, implying that the former includes a chromophore in its intact state. gtCP represents a single-chain protein and, upon harsh denaturing conditions, shows three major bands in SDS/PAGE, two of which apparently result from hydrolysis of an acylimine C=N bond. Instead of having absorption maxima at 384 nm and 450 nm, which are characteristic for a GFP-like chromophore, fragmented gtCP shows a different spectrum, which presumably corresponds to a 2-keto derivative of imidazolidinone. Mass spectra of the chromophore-containing peptide from gtCP reveal an additional loss of 2 Da relative to the GFP-like chromophore. Tandem mass spectrometry of the chromopeptide shows that an additional bond is dehydrogenated in gtCP at the same position as in DsRed. Altogether, these data suggest that gtCP belongs to the same subfamily as DsRed (in the classification of GFP-like proteins based on the chromophore structure type).     

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Strategies for assignments.

Natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes) of aqueous native proteins yield numerous narrow single carbon resonances of nonprotonated aromatic carbons. Techniques for the assignment of these resonances are presented. Each technique is applied to one or more of the following proteins: ferricytochrome c from horse heart and Candida krusei, ferrocytochrome c and cyanoferricytochrome c from horse heart, lysozyme from hen egg white, cyanoferrimyoglobins from horse and sperm whale skeletal muscle, and carbon monoxide myoglobin from horse. In all of the protein spectra we have examined, methine aromatic carbons give rise to broad bands. Studies of the narrow resonances of nonprotonated aromatic carbons of proteins are facilitated by removal of these broad bands by means of the convolution-difference method, preferably from spectra recorded under conditions of noise-modulated off-resonance proton decoupling. We present a summary of the chemical shift ranges for the various types of nonprotonated aromatic carbons of amino acid residues and hemes of diamagnetic proteins, based on our results for hen egg white lysozyme, horse heart ferrocytochrome c, horse carbon monoxide myoglobin, and carbon monoxide hemoglobins from various species...     

MICROSPECTROPHOTOMETRY AND THE PHOTORECEPTOR OF PHYCOMYCES I

By applying microspectrophotometry to the sporangiophore of Phycomyces blakesleeanus wild-type and the albino car-10(-) type II, absorption spectra were obtained for 1- to 5-day cultures. Spectra in the growing-zone of the wild-type during Stage IVb, taken from 0.1 to 3 mm below the base of the sporangium, show two distinctly different spectra: one is more characteristic of a carotene, the other of a flavin. Combined, these absorption spectra reproduce closely the action spectrum. For the albino car-10(-), which is deficient in carotenes, only the spectrum characteristic of lumichrome or a reduced flavin was found. A c-type cytochrome was isolated from both strains which, if coupled with a flavin, could permit a photoreversible oxidation-reduction system. Birefringent crystals were observed to be aligned in the growing zone in which the photoreceptor is believed to lie. Micro-spectrophotometry of these crystals shows absorption peaks similar to those of riboflavin crystals.     

河南小麦梭条斑花叶病病原鉴定、病毒提纯及其特性

经鉴定,我国河南邓县冬小麦上发生的一种病毒病的病原,是小麦梭条斑花叶病毒。 对病毒的分离纯化进行了研究,提出了一个简单可靠的病毒纯化程序,获得了较为理想的提纯病毒制品,提纯病毒具有典型的、核酸含量约为5～6％的线状病毒紫外吸收曲线,病毒粒体宽约12nm,长度分布从200～2000nm以上,平均粒体长度约为1000nm,在300nm、600nm及1000nm处有3个分布高峰,用提纯病毒免疫兔子获得效价为1/1024的抗血清,提纯病毒的SDS-聚丙烯酰胺凝胶电泳可检测到多条蛋白带,分子量从36.6kd至28.6kd,其中36.6kd的蛋白为病毒的外壳蛋白,其余的为外壳蛋白的降解产物。     

Tissue specificity of nuclear acidic proteins isolated from bovine brain and adrenal medulla.

Nuclear acidic proteins from bovine brain and adrenal medulla demonstrated dissimilar electophoretic and chemical profiles. The amino acid analysis of the acidic nuclear protein fraction isolated from these tissues revealed some variation in the ratio of acidic to basic amino acids. The estimation of free carboxylic acid groups confirmed the more acidic nature of the brain proteins. In comparing the acrylamide gels either visually or optically, several electrophoretically specific bands were apparent. Although the total number of protein bands from each tissue was approximately the same, the adrenal medulla contained a larger proportion of the more positively charged proteins. These observations are interpreted to indicate that the nuclear acidic protein from brain and adrenal medulla may show functional variation.     

The interaction of Ca2+-binding proteins with the carbocyanine dye stains-all

The interaction of the carbocyanine dye Stains-all with the Ca²⁺-binding proteins calmodulin, troponin C, and parvalbumin has been monitored by means of absorption spectra and CD. In the absence of Ca²⁺, complexes with Stains-all of all three proteins exhibit at high dye: protein mole ratios an intense J absorption band at 600–650 nm, which is associated with a characteristic CD spectrum. In the cases of calmodulin and troponin C, the J-band is progressively lost as the dye: protein ratio decreases and is replaced by bands of the γ and β types at 450–550 nm, which likewise give rise to characteristic CD spectra. For parvalbumin, only the J-band is observed; its intensity is undiminished at the lowest dye: protein ratios examined. In the presence of excess Ca²⁺ the J-band is lost for all three proteins. For calmodulin and troponin C it is replaced by σ- and β-bands; in the case of parvalbumin the bound dye is released. A tentative model has been proposed to account for these observations.     

Purification of bacteriorhodopsin and characterization of mature and partially processed forms

Bacteriorhodopsin (BR) essentially free of native lipids has been prepared in a highly stable state. Purple membrane was solubilized in Triton X-100 and BR was purified by size exclusion chromatography using 3-[cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonic acid (CHAPSO) detergent at pH 5. Molar ratios of phospholipid/BR ranged from 0.4 to 0.05 corresponding to 94-98% phospholipid removal. Purified BR has an absorbance ratio (A280nm/A548nm) of 1.5-1.6 in the dark-adapted state which is the highest purified BR/protein ratio reported to date. The purified BR in CHAPSO shows maximum stability in the pH range 5.0-5.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of native purple membrane and solubilized BR from most Halobacterium halobium JW-3 cultures show 3 higher molecular weight bands in addition to BR. Immunological staining and amino acid sequencing indicates that these additional proteins are partially processed forms of the BR precursor protein. The BR preprotein contains 13 additional amino acids on the NH2 terminus which are removed by post-translational processing in at least four steps. Isoelectric focusing separated most delipidated and non-delipidated BR samples into 8 bands. Incomplete BR post-translational processing BR is thought to be largely responsible for the multiplicity of isoelectric BR species. The principal components have pI values of 5.20 and 5.24 and both have absorption maxima at 550 nm, characteristic of detergent-solubilized BR. BR in Triton X-100 or nonylglucoside, delipidated BR in CHAPSO, and BR in intact purple membrane all have a dark-adapted ratio of 13-cis to all-trans-retinal of 1.9:1.     

Biochemical composition of stigmatic exudate of Eichhornia crassipes (Mart.) solms

The stigmatic exudate of Eichhornia crassipes (Mart.) Solms is moderately soluble in water, but is readily soluble in 80% ethanol. Soluble sugars, fructose, sucrose, total phenol, hydroxy phenol, free amino acids, soluble proteins and free fatty acids were found in the exudate. The exudate is devoid of organic acids and alkaloids, while proteins, soluble sugars, free fatty acids and free amino acids were the principal constituents. The stigma possesses detectable quantities of these compounds and shows a progressive increase in the amount of these substances up to the stage just prior to anthesis. The ultraviolet absorption profile of the stigmatic exudate in ethanol shows characteristic absorption peaks and bathochromatic shifts on addition of NaOH. The peaks probably correspond to those of simple phenolic compounds.     

Conformational isomers of insect odorant-binding proteins.

We have identified and cloned the cDNAs encoding odorant-binding proteins (OBPs) from the large black chafer, Holotrichia parallela, and the yellowish elongate chafer, Heptophylla picea. Each species possess two OBPs, the proteins migrating faster in native gels (OBP1) showed high amino acid identity (>88%) to previously identified pheromone-binding proteins (PBPs) from scarab beetles. HparOBP1 and HpicOBP1 have 116 amino acids and six highly conserved cysteine residues. In contrast to OBP1 that gave a single band, both HparOBP2 and HpicOBP2 separated each into two bands in native gels (15%). The N-terminal amino acid sequences for the two bands from each species were indistinguishable, and they had the same molecular masses. Although we sequenced several clones from each species, they all encode only one protein for each species, indicating they are different conformational isomers of the same protein. HparOBP2 and HpicOBP2 have 133 amino acids and cysteine residues are conserved in proteins of the same family.     

ELEGTROPHORETIC VARIATION IN PROTEINS AND ENZYMES OF THE TUMOR-FORMING HYBRID NICOTIANA GLAUCA X N. LANGSDORFFII AND ITS PARENT SPECIES

Leaf and tumor extracts of the genetically tumor-conditioned amphiploid Nicotiana glauca X N. langsdorffii, as well as leaf extracts from the parent species and a nontumorous mutant of the amphiploid, were separated on acrylamide gel columns by the method of disc electrophoresis. Gels were stained for general proteins with amido black and specifically for esterases, peroxidases and leucine amino peptidase. The results show characteristic protein and enzyme patterns for leaves of each of the parental species and the amphiploid hybrids. The amphiploids show some bands which are comparable to bands of either one or both of the parental species, while other bands do not have their equivalents in the parental species. Leaf tissue of the tumorous and nontumorous amphiploids were found to differ by a few protein bands, at least two for esterases and at least one for peroxidases. Extracts from tumor tissue show very different patterns from those of the leaves of the same genotype.     

五种鳗鲡的含肉率及肌肉营养成分分析

研究利用营养测试方法对日本鳗鲡、欧洲鳗鲡、美洲鳗鲡、花鳗鲡和太平洋双色鳗鲡共5种养殖鳗鲡的含肉率及肌肉营养成分进行了分析比较。结果表明: 5种鳗鲡含肉率61.77%69.22%, 日本鳗鲡和太平洋双色鳗鲡显著高于欧洲鳗鲡和花鳗鲡 (P0.05); 水分含量为62.34%71.80%, 粗蛋白含量为11.31% 18.47%, 脂肪含量为8.62% 24.48%, 灰分含量为0.92%1.06%; 均含有18种氨基酸, 其中包括8种人体必须氨基酸, 总氨基酸含量存在差异, 鲜味氨基酸含量占37.43%38.77%, 必需氨基酸指数(EAAI)为65.2574.77, 其构成比例符合FAO/ WHO的标准, 色氨酸、异亮氨基酸和缬氨酸等氨基酸为限制性氨基酸; 富含磷、钾、铁和锌等多种矿物元素, 日本鳗鲡和花鳗鲡含量最高; 均含有16种脂肪酸, 其中饱和脂肪酸(SFA) 7种, 不饱和脂肪酸(UFA)9种; 脂肪酸中多不饱和脂肪酸(PUFA)和二十二碳六烯酸(DHA)含量较高, 分别占总量的41.92%48.27%和6.63%16.87%。研究结果表明: 5种鳗鲡的肌肉为高蛋白、鲜味氨基酸与必需氨基酸含量高的优质蛋白源; 富含磷、钾、铁、锌等矿物元素, 可作为补充人体矿物质营养的膳食来源; 脂肪酸以不饱和脂肪酸为主, 多不饱和脂肪酸和DHA比值高。因此, 5种鳗鲡具有较高的营养价值且有益人体健康, 均是优良的水产养殖种类。       

杨尺蠖核型多角体病毒蛋白质的特性

本文研究了杨尺蠖核型多角体病毒(AciNPV)蛋白质特性,用反复调等电点法,从AciNPV的多角体蛋白中分离到A,B两种蛋白,Sepharose 6B柱层析和超离心沉降分析表明,两者均为一个峰纯,沉降系数(S20w)分别为4.9和8.9,对A蛋白进行了16种氨基酸组成分析,Glu、Asp和Leu含量高于其它氨基酸,不含Cys,用SDS-聚丙烯酰胺凝胶电泳和免疫双扩散法分析表明,用两倍体积饱和硫酸铵沉淀法制备的多角体蛋白,与提纯的多角体A、B蛋白之间无差异,多角体蛋白结构多肽由6种组成,分子量在12500—54000道尔顿范围内,其中32000是多角体蛋白的主要多肽,病毒粒子结构多肽和核衣壳蛋白分别由19和7个多肽组成,分子量范围分别在89000～13000和34000～13000之间,间接试验结果表明,在AciNPV中有碱性蛋白酶活性存在。     

黄鳝血清和体表粘液蛋白的比较研究

本文用反相高效液相色谱法测定了黄鳝血清和体表粘液蛋白的氨基酸种类和含量,比较了二者的氨基酸组成变化。结果表明：二者都含有17种氨基酸,血清的氨基酸总量为397．11mg／l00ml,体表粘液蛋白的氨基酸总量为259．29mg／l00m1,血清与粘液蛋白中氨基酸含量差异最大的是蛋氨酸、半胱氨酸。应用SDS—PAGE分析和比较了血清和体表粘液蛋白的分子量大小及特有区带效,黄鳝血清与体表粘液蛋白分子量相同的蛋白区带数为2条,其分子量大小分别为19．5kDa、96．0kDa。其中血清的特有蛋白带为18条,体表粘液的特有蛋白带为8条。此外,对二者的相关性和体表粘液特异性的免疫机制作了探讨。     

A structural investigation of the central chlorophyll a binding sites in the minor photosystem II antenna protein, Lhcb4

Mutant proteins from light-harvesting complexes of higher plants may be obtained by expressing modified apoproteins in Escherichia coli, and reconstituting them in the presence of chlorophyll and carotenoid cofactors. This method has allowed, in particular, the engineering of mutant LHCs in which each of the residues coordinating the central Mg atoms of the chlorophylls was replaced by noncoordinating amino acids [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10056-10061]. The availability of these mutants is of particular importance for determining the precise position of absorption bands for the different chlorophyll molecules, as well as the sequence of energy transfer events that occur within LHC complexes, provided that the structural impact of each mutation is precisely evaluated. Using resonance Raman spectroscopy, we have characterized the pigment-protein interactions in the minor photosystem II antenna protein, Lhcb4 (CP29), in which each of three of the four central chlorophyll a molecules has been removed by such mutations. By comparing the spectra of these mutants with those of the wild-type protein, the state of interaction of the carbonyl group, the coordination state of the central magnesium ion, and the dielectric constant (polarity) of the immediate environment in the binding pocket of the chlorophyll a molecule were defined for each cofactor binding site. In addition, the structural impact of the absence of one chlorophyll a molecule and the quality of protein folding were evaluated for each of these mutated polypeptides.