辛德毕斯病毒装配及其6K蛋白与中间纤维的关系

用温和的选择性抽提方法与整装细胞电镜技术,DGD包埋-去包埋剂电镜技术相结合,对辛德毕斯病毒的装配与宿主细胞中间纤维的关系进行了探讨。电镜观察清晰地显示了病毒“装配中心”被中间纤维所网络,病毒的装配过程显然是以中间纤维为支架;正在装配的核壳体与已装配的核壳体紧密结合在中间纤维丝上。根据电镜照片分析,核壳体可能是沿中间纤维由装配中心向外扩散。应用人工合成6K蛋白所得抗体进行胶体金标记,证明辛德毕斯病毒非结构性6K蛋白也与中间纤维紧密结合。     

Site-specific endonuclease CauB31 from Chloroflexus aurantiacus B3

A sequence-specific endonuclease CauB3I has been isolated from cell extracts of Chloroflexus aurantiacus and partially purified by chromatography on heparin-sepharose; the yield was 3000 units per 1 g of cells. The final preparation is free of non-specific nucleases. It is shown that endonuclease CauB3I recognizes 5' T decreases CCGGA 3' sequence in double-stranded DNA and cleaves it as shown by an arrow. Methylation of adenine in the recognition sequence makes it resistant to CauB3I.     

Human spleen histone H2B. Isolation and amino acid sequence.

The amino acid sequences of human histones have been investigated for studies of histone evolution. The whole histone was prepared from human spleen and was separated into 3 fractions, H4+H3+H2A, H2B, and H1, by our technique of CM-cellulose chromatography. The H2B fraction was further purified by Bio-Gel P-60 chromatography. For sequence determination, the H2B molecule was first split into 4 major fragments I to IV, by limited chymotryptic digestion at pH 5.0 and 15 degrees C, followed by Sephadex G-50 chromatography. Fragments I and III were then digested with trypsin, yielding 18 and 16 peptides, respectively, on column and paper chromatographies. Sequence analyses of these tryptic peptides, as well as chymotryptic fragments II and IV, showed no differences from the corresponding parts of calf thymus H2B sequence, making it possible to locate fragments I to IV at residues 1--40, 41--42, 43--121 and 122--125 of the total sequence. The only new findings were microheterogeneities at residues 39 (75% valine and 25% isoleucine) and 124 (70% serine and 30% alanine). The sequence of the most basic cluster at residues 27--24, -Lys-Lys-Arg-Lys-Arg-Ser-Arg-Lys-, was confirmed with a peptide obtained from fragment I by staphylococcal protease digestion. Thus, it is concluded that the H2B sequence of lower mammals was conserved during the evolutionary process leading to man.     

Modification methylase M.Sau3239I from Streptomyces aureofaciens 3239

By chromatography on phosphocellulose and Heparin-Sepharose the modification methylase M.Sau3239I was detected and partly purified from cells of Streptomyces aureofaciens 3239. Methylation by this enzyme protects DNA from cleavage by the restriction endonuclease R.Sau3239I. The enzyme catalyzes methylation of adenine to N-6-methyladenine in the 5'-CTCGmAG-3' recognition sequence.     

Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures

Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. In mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography.     

Cloning of phosphatase I gene from a psychrophile, Shewanella sp., and some properties of the recombinant enzyme

Psychrophilic phosphatase I from Shewanella sp. is a cold enzyme that was found as a novel protein-tyrosine-phosphatase (PTPase, EC 3. 1.3.48) with a histidine as its catalytic residue [Tsuruta and Aizono (1999) J. Biochem. 125, 690-695]. Here, we determined the nucleotide sequence of a DNA fragment (2,004 bp) containing the phosphatase I gene by cloning with polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence, of the enzyme contained a conserved region of protein-serine/threonine-phosphatase (PPase). The 38.5 kDa-recombinant protein expressed in Escherichia coli was purified to homogeneity by glutathione-Sepharose 4B column chromatography, treatment with endoproteinase and Mono-Q column chromatography. The recombinant enzyme had a specific activity of 49.4 units and, like native psychrophilic phosphatase I, exhibited high catalytic activity at low temperature and PTPase activity.     

BME 361 I--a new site-specific deoxyribonuclease type II from Bacillus megaterium 361]

Bme 361 I, a new site-specific type II deoxyribonuclease, was purified from Bacillus megaterium 361 by chromatography on phosphocellulose P 11 and hydroxylapatite. The enzyme recognizes and cleaves the nucleotide sequence 5'-GG decreases CC-3' in double-strand DNA. Thus it is a true isoschizomer of deoxyribonucleases Hae III and BspR I.     

Identification of angiotensin I in a cyclostome, Lampetra fluviatilis

Angiotensin I (ANG I) was isolated from incubates of plasma and kidney extracts of the river lamprey, Lampetra fluviatilis, using eel vasopressor activity as an assay during purification. Its sequence was Asn-Arg-Val-Tyr-Val-His-Pro-Phe-Thr-Leu as determined by the sequence analysis and mass spectrometry. The sequence was confirmed by identity of the elution profile with the synthetic peptide in two different reverse-phase columns of high-performance liquid chromatography. Lamprey ANG I produced dorsal-aortic pressor responses in L. fluviatilis but the rise was very small in comparison to that produced by angiotensin II. Angiotensin III produced an even bigger increase. It was not possible to demonstrate a difference in response to Asn(1) (lamprey) ANG I and Asp(1) (human) ANG I. The present study directly demonstrated the presence and biological activity of the renin-angiotensin system in the most primitive extant vertebrates, the cyclostomes. Thus the renin-angiotensin system is a phylogenetically old hormonal system that is present throughout the vertebrates.     

Covalent structure of collagen: amino acid sequence of alpha 1 (III)-CB5 from type III collagen of human liver

Type III collagen was prepared from human liver by limited pepsin digestion, differential salt precipitation, and carboxymethylcellulose chromatography. Ten distinct peptides were obtained by cyanogen bromide digestion. The peptide alpha 1 (III)-CB5 was further purified by carboxymethylcellulose chromatography, and its amino acid sequence was determined. Automatic Edman degradation of intact alpha 1 (III)-CB5, tryptic and thermolytic peptides, and hydroxylamine-derived fragments was used to establish the total sequence. The mammalian collagenase site contained in the alpha 1 (III)-CB5 sequence was ascertained by digestion of native type III collagen with purified rheumatoid synovial collagenase. Collagenase cleavage occurred at a single Gly--Ile bond, one triplet before the corresponding specific cleavage site of type I collagen. The present work brings the known sequence of human liver type III collagen to include alpha 1 (III)-CB3-7-6-1-8-10-2-4-5. These correspond to the homologous region of alpha 1 (I)-CB0-1-2-4-5-8-3-7 residues 11--804.     

Further purification and characterization of newly synthesized anionic glycoconjugates secreted by cultured UMR-106 cells: evidence that the major anionic glycoconjugate secreted by these cells is similar to bone sialoprotein II.

Following incubation of UMR-106 cells for 48 h in the presence of [3H]glucosamine and [35S]sulfate, the newly synthesized anionic glycoconjugates were isolated from the culture medium by cetylpyridinium chloride/ethanol precipitation and further separated by DEAE-Sephacel chromatography into two radiolabelled fractions, a major component, UM I, and a minor component, UM II. UM I appeared to be homogeneous as shown by Sepharose CL-4B chromatography under dissociative conditions, and SDS-polyacrylamide gel electrophoresis. It showed a molecular mass of approximately 93 kDa on 4-15% gels. UM I was partially degraded by brief treatment with trypsin, releasing a small, terminal peptide that contained 47.6% of 35S but no 3H. Treatment of UM I with neuraminidase and 0.1 N H2SO4 (1 h at 80 degrees C), respectively, released 27% 3H and 38.4% 3H plus 41% 35S, suggesting the presence of a significant number of sialic acid residues, as shown by Sephadex G-50 chromatography of the digests. Amino acid analysis showed that the UM I glycoconjugate was rich in acidic amino acids (12.6% aspartic acid and 21.2% glutamic acid residues) and its N-terminal sequence was Phe-Ser-Met-Lys-Asn-Phe-, which is identical to the published N-terminal amino acid sequence of rat bone sialoprotein II. Keratanase treatment of UM I released 26% of the incorporated radioactivity, suggesting the presence of keratan sulfate chains. UM II contained a chondroitinase ABC-sensitive proteoglycan.     

Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center

Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate- (PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140 000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70 000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [32P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group.     

Aplysia californica neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and peptide I

The R3-R14 neurons of the marine mollusc Aplysia are neuroendocrine cells that express a gene encoding peptides I, II and histidine-rich basic peptide (HRBP), a myoactive peptide that excites Aplysia heart and enhances gut motility in vitro. Peptide II has been chemically characterized (35), but the complete primary structures of peptide I and HRBP have not been established by amino acid sequence analysis. HRBP, peptide I, and the prohormone (proHRBP) were therefore purified from acid extracts of Aplysia californica neural tissue using sequential gel filtration and reverse-phase high-performance liquid chromatography and chemically characterized. Amino acid sequence analysis demonstrated that HRBP was a 43-residue peptide whose sequence was: less than Glu-Val-Ala-Gln-Met-His-Val-Trp-Arg-Ala-Val-Asn-His-Asp-Arg-Asn-His-Gly- Thr-Gly - Ser-Gly-Arg-His-Gly-Arg-Phe-Leu-Ile-Arg-Asn-Arg-Tyr-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOH. Compositional and sequence analyses of peptide I and proHRBP demonstrated that peptide I was a 26-residue peptide with the following sequence: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala- Leu-Glu-Ser-Val-Leu-Thr-Asp-Phe-Lys-Asp-COOH. These results demonstrated that the pro-HRBP sequence predicted by nucleotide sequence analysis of a cDNA clone (24) was in fact synthesized in R3-R14 neurons. Hydrophilicity and hydrophobicity profiles of preproHRBP, combined with charge distribution profiles and predictive secondary structural analysis, showed that cleavage at dibasic sequences was strongly associated with peaks of hydrophilicity in alpha-helical regions of the preprohormone.     

The rat kidney acylase I, characterization and molecular cloning. Differences with other acylases I.

The soluble acylase I from rat kidney was purified to homogeneity using a five-step procedure. As the resulting protein was found to have a relative molecular mass of 125 kDa based on size-exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunits. The amino-acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding sequence of porcine kidney acylase I. However, as N-furyl-acryloyl-L-methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA coding for the rat kidney acylase I was therefore isolated and found to contain a 1224-bp open reading frame encoding a protein consisting of 408 amino-acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino-acid sequence showed 93.6% and 87.2% identity with that of the human liver and porcine kidney, respectively.     

Amino acid sequence of cytotoxin IIa isolated from the venom of the Indian cobra (Naja naja)

A cytotoxic basic polypeptide, designated as cytotoxin IIa, was purified to homogeneous state from the venom of the Indian cobra (Naja naja) by a combination of gel filtration on Sephadex G-50, CM-cellulose chromatography, and fast protein liquid chromatography. Cytotoxin IIa is a single polypeptide consisting of 60 amino acid residues with four intramolecular disulfide linkages. The toxin showed high cytotoxicity toward Yoshida sarcoma and ascites hepatoma cells as did cytotoxins I and II isolated from the same venom. Analysis of the amino acid sequence revealed that cytotoxin I, IIa, and II are highly homologous in their primary structures and that cytotoxin IIa differs from cytotoxin I only in having Phe 25 and Val 52 in place of Tyr 25 and Glu 52 residues.     

Isolation and determination of the amino acid sequence of chicken calcitonin I from chicken ultimobranchial glands

A radioimmunoassay for chicken calcitonin in chicken ultimobranchial glands was established utilizing a rabbit antiserum against eel calcitonin. This assay method, which is about 100 times as sensitive as the usual bioassay for hypocalcemic activity, was used for monitoring chicken calcitonin during its purification. The immunoreactivity in chicken ultimobranchial extract was separated by SP-Sephadex C-25 chromatography into two fractions. Chicken calcitonin I, which was occurred in the major immunoreactive fraction, was further purified to homogeneity as shown by reverse phase HPLC. In the end, 39 nmol of chicken calcitonin I was obtained from 3,384 chickens following a 12,000-fold purification. The complete amino acid sequence of purified chicken calcitonin I was determined to be H-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Ly s-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH2 and confirmed by synthesis. The specific biological activity of chicken calcitonin I (4,500 MRCU/mg) was identical to that of eel calcitonin, which has the highest specific biological activity among the calcitonins so far isolated. Chicken calcitonin I resembled the calcitonins from the ultimobranchial glands both of salmon and eel in sequence, biological activity, and immunological property.     

链霉菌C-3662产生的纤溶活性蛋白酶的纯化与理化性质

 从链霉菌 C- 3662发酵上清液中 ,通过硫酸铵沉淀 ,CM- Sepharose Fast Flow和 Phenyl-Sepharose Fast Flow等层析色谱 ,分离纯化得到了具有纤溶活性的蛋白酶 CGW- 3,反向 HPLC鉴定纯度为 90 % ;每立升发酵上清液可得到 8mg纯品 ,活性回收率 46% ,CGW- 3为一单肽链蛋白 ,分子量 2 2 72 1 ,对丝氨酸蛋白酶抑制剂 PMSF敏感 ,对 EDTA不敏感 ;其 N端 1 5个氨基酸的顺序为 VVGGTRAAQGEFPFM,与微生物来源的胰蛋白酶类丝氨酸蛋白酶有较高的同源性 . CGW- 3的等电点 p I9.0 ,纤溶活性的最适 p H为 7.5～ 8.0 ,对温度比较敏感 .CGW- 3不仅具有直接降解纤维蛋白作用 ,而且能够激活纤溶酶原     

The complete sequence of the acidic subunit from Mojave toxin determined by Edman degradation and mass spectrometry

Mojave toxin, a heterodimeric, neurotoxic phospholipase complex from Crotalus scutulatus scutulatus, is one of a group of closely related rattlesnake toxins for which much structural information is still lacking. The complete amino-acid sequence of the acidic subunit from Mojave toxin was determined. The three individual peptide chains, derived from the acidic subunit by reductive alkylation, were separated by high-performance liquid chromatography. Fragmentations of the A and B chains were done using specific proteinases and the resulting peptide mixtures were fractionated by reverse-phase high-performance liquid chromatography. Sequence analyses on the intact chains and the fragments from digests were done by automated Edman degradation, carboxypeptidase Y degradation and triple-quadrupole and tandem-quadrupole Fourier-transform mass spectrometry. The sequence for each acidic subunit chain is very similar to the corresponding chain from the related neurotoxin complex, crotoxin, and overall the sequence is similar to the sequences of group I and II phospholipases A2. The N-terminus of the B chain is blocked by pyroglutamic acid. The existence of two distinct and closely related C chains was established. It is unlikely that the small sequence difference can account for the isoforms that are present in purified Mojave toxin and in unfractionated venom.     

化学合成虎纹捕鸟蛛毒素-I基因的克隆和表达

本文报道了全化学合成虎纹捕鸟蛛毒素－Ⅰ基因在大肠杆菌中的表达,表达产物为Ｎ－端是谷胱甘肽硫转移酶的融合蛋白．经ＧＳＨ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化,凝血酶酶解融合蛋白,得到重组ＨＷＴＸ－Ⅰ（ｒＨＷＴＸ－Ⅰ）．质谱和氨基酸顺序分析均表明ｒＨＷＴＸ－Ⅰ系正确表达产物．还原复性的ｒＨＷＴＸ－Ⅰ表现出与天然ＨＷＴＸ－Ⅰ生物学活性的一致性．     

Bme585 I [5'-CCCGC(4/6)-3'], a new isoschizomer of restriction endonuclease Fau I, isolated from a strain of Bacillus mesentericus

Bme585 I is a new member of the restriction endonuclease type IIS family. It was partially purified from the heterothrophic, mesophilic bacterial strain Bacillus mesentericus 585 by ammonium sulphate precipitation and phosphocellulose column chromatography. Bme585 I is a monomeric protein with a molecular mass of 62 kD. The enzyme is active over a broad pH range from 7.0 to 8.8, has a temperature optimum of 37 degrees C and tolerance of NaCl in reaction buffer from 0 to 400 mM. Bme585 I recognizes the asymmetric sequence 5'-CCCGC(4/6)-3' and is therefore an isoschizomer of restriction endonuclease Fau I.     

The sequence around the phosphorylation site of the porcine heart type phosphorylase isoenzyme

1. A tetradecapeptide containing the phosphorylation site was obtained from 32P-labelled pig heart phosphorylase a isoenzyme by alpha-chymotryptic digestion. 2. The peptide was purified by Mono S cation-exchange chromatography and reversed-phase HPLC. 3. The phosphorylated residue was identified as Ser and the sequence was determined: T D G E R R K Q I S V R G L. 4. The sequence was compared to the known sequences of muscle and liver type isophosphorylases and the structural consequences of the amino acid residue exchanges were predicted.