荔枝胚蛋白质的提取方法

以不同体积的Tris-HCl(0.1mol／L,pH8．8)为提取液,结合不同含量(以胚鲜重计)的PVP40,对怀枝、黑叶和桂味等荔枝(Lithi chinensis)品种的胚蛋白质进行提取。结果表明,提取液体积为胚鲜重的5倍(ml g-1 FW),并加入15％的 PVP40时,提取蛋白质的效果最好,可用于荔枝胚可溶性蛋白质含量的测定;胚乳蛋白质的提取则以等体积的提取液(内含2％的PVP40)为佳。加入10% PVP40的胚蛋白提取液可直接进行SDS-PAGE电泳,用10倍于蛋白质提取液体积的乙醇沉淀胚和胚乳的蛋白提取液,可得到最佳的SDS-PAGE电泳效果。     

Purification and Some Properties of Plant Endonuclease from Scallion Bulbs

A plant endonuclease with 3′-nucleotidase activity was purified from scallion bulbs to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 38,000 by SDS-PAGE and 40,000 by Sephadex G-100 gel filtration. The enzyme rapidly hydrolyzed yeast RNA and denatured calf thymus DNA to acid-soluble substances, and hydrolyzed the plasmid pBR322 to yield small DNA fragments at low enzyme concentrations. These four activities were eliminated by treatments with EDTA and tetraethylenepentamine. The enzyme had maximum activity at pH 8.5-9.0 for 3′-AMP, 3′-GMP, and 3′-UMP, at pH 6.5 for 3′-CMP and yeast RNA, and at pH 6.0 for denatured calf thymus DNA and pBR322. During digestion of yeast RNA by the enzyme at pH 6.5, 5′-GMP was released most rapidly, followed by 5′-UMP, 5′-AMP, and 5′-CMP. These properties were different from those of endonucleases isolated from other sources such as mung bean sprouts and wheat seedlings.     

Cathepsin B, the lysosomal thiol proteinase of calf liver

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal-mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4.0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7.4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4.5 and at pH6.8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.     

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.     

Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro. I. Radiolabelling of membrane and secretory proteins

A method for the in vitro perfusion of isolated guinea-pig mammary tissue is described that allows the radiolabelling of secretory and membrane proteins. Glands were depleted of methionine, labelled with [35S]methionine for 5 min and perfused with medium containing an excess of unlabelled methionine for varying times. The structural integrity of the alveoli in the perfused glands appeared well maintained. Epithelial polarity was preserved and junctional complexes were evident. About 20% of the methionine provided in the medium was extracted by glands of 10 g wet weight under the labelling conditions employed. With chase periods from 15 to 40 min, 50-70% of the methionine was incorporated into trichloroacetic-acid (TCA)-precipitable material. The principal radiolabelled proteins recovered from the tissue fractions had Mrs and isoelectric points similar to the major secretory proteins (i.e. caseins and alpha-lactalbumin) of guinea-pig milk. Autoradiography of tissue sections at the resolution of the light microscope showed that secretory proteins were transported from sites of synthesis within secretory cells to the alveolar lumina after 45 min. These highly labelled secretory proteins could be almost completely removed from microsomal fractions by treatment with sodium carbonate solutions. Proteins with Mrs from 30 000 to 200 000 were detected in the washed membranes by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. These labelled membrane-associated proteins persisted in the microsomal membrane fraction after chase periods from 7.5 to 40 min.     

Prostaglandin-E2-9-ketoreductase in rabbit kidney.

The 100,000 xg supernatant of rabbit kidney contains a prostaglandin-E2-9-ketoreductase which has an obligatory requirement for NADPH. This enzyme is localised in the renal cortex and is able to quantitatively convert PGE2 to PGF2alpha. A broad pH profile was evident with an optimum at pH 7 with 5. Kinetic studies indicated a Km of 3 with 2 x 10-4M PGE2. The isoelectric point was at pH 5 with 65 and the molecular weight, as estimated by gel filtration, was 21,800. These values differ from those obtained with enzyme from monkey brain tissue and suggest a tissue specificity of PGE2-9-ketoreductase. By combining isoelectric focussing techniques with sephadex filtration considerable purification of the renal enzyme was achieved.     

Purification and some properties of an alkaline proteinase from rat skeletal muscle.

1. Rat skeletal muscle was homogenized in 0.05M-Tris/HCl, pH 8.5, containing 1M-KCl. Myofibrillar proteins were precipitated by addition of (NH4)2SO4 (33% saturation). 2. The alkaline proteolytic activity that was precipitated with the myofibrillar proteins was solubilized with trypsin (conjugated to Sepharose) and further purified by affinity chromatography, ion-exchange chromatography and gel filtration. 3. The purified enzyme migrates as a single band in polyacrylamide-disc electrophoresis, and has optimum hydrolytic activity with azocasein and [14C]haemoglobin as substrates at pH 9.4 and 9.6 respectively. Its apparent molecular weight, as determined by gel filtration on Sephadex G-75, is 30800. 4. The purified alkaline proteinase is strongly inhibited by equimolar amounts of soya-bean trypsin inhibitor and ovomucoid, whereas di-isopropyl phosphorofluoidate and alpha-toluenesulphonyl fluoride have no effect. On the other hand N-ethylmaleimide and p-chloromercuribenzoate have inhibitory effects on the enzyme activity. 5. Bivalent metal ions (Fe2+, Co2+, Zn2+, Mg2+, Mn2+) diminish the proteolytic activity, at 1mM concentrations. Ca2+ ions and the metal-ion-chelating agent EDTA are without effect on enzyme activity. 6. The enzyme is part of the alkaline proteolytic activity that appears to be associated with myofibrillar proteins.     

Purification and partial characterization of Oenococcus oeni exoprotease

The exoprotease from Oenococcus oeni produced in stress conditions was purified to homogeneity in two steps, a 14-fold increase of specific activity and a 44% recovery of proteinase activity. The molecular mass was estimated to be 33.1 kDa by gel filtration and 17 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggest that the enzyme is a dimer consisting of two identical subunits. Optimal conditions for activity on grape juice were 25 degrees C and a pH of 4.5. Incubation at 70 degrees C, 15 min, destroyed proteolytic activity. The SDS-PAGE profile shows that the enzyme was able to degrade the grape juice proteins at a significantly high rate. The activity at low pH and pepstatin A inhibition indicate that this enzyme is an aspartic protease. The protease activity increases at acidic pH suggesting that it could be involved in the wine elaboration.     

Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.     

水稻花药总蛋白质双向电泳方法的改良与优化(简报)

双向电泳作为蛋白质组学核心技术之一,目前已广泛地应用在植物领域,并且成功应用于水稻代谢和调节等方面的研究。谢锦云等利用溶液法提取温敏核不育水稻花药总蛋白质,利用pH3-10线性胶条分离,经银染显色后检测到约1,000个     

一种食源性溶栓酶的分离纯化与部分酶学性质的研究

目的对以豆渣为原料,接种纳豆菌的发酵物分离纯化和酶学性质研究。方法采用生理盐水浸提、(NH4)2SO4分级沉淀、Sephadex G-100凝胶层析等纯化步骤,得到层析纯的食源性溶栓酶-纳豆激酶,结果经聚丙烯酰胺凝胶电泳显示为二个组分。酶学性质研究表明,以酪蛋白为底物时,最适反应温度为60℃,最适反应pH为8.0,在pH7~9溶液中,37℃以下基本稳定。体外溶栓作用表明,纳豆激酶溶解纤维蛋白的方式主要是直接溶解,而不是纤溶酶原激活剂。     

细胞核单一种类非组蛋白的提取

本文报道了一种提取单一种类染色质非组蛋白的简化方法。在获得核内酚溶性非组蛋白后进行双向电泳。切下胶内点蛋白于缓冲液浸泡抽提,离心得到上清,继用丙酮低温沉淀蛋白。     

Biochemical and histochemical characteristics of proteins homologous to calf lens membrane proteins with high calcium-binding capacity

Proteins with high affinity and capacity for calcium are present in the membranes of calf lens fiber and epithelial cells. They can be extracted from these membranes by means of EDTA or EGTA. The tissue specificity and localization of these 30-38 kD EDTA-extractable proteins (EEP) have been examined. Antibodies raised against calf lens fiber EEP specifically form immune complexes with distinct proteins of 30-38 kD in a great variety of non-lenticular tissues. By indirect immunofluorescence microscopy using anti-EEP antiserum, the EEP-like proteins could be detected in fibroblasts, retinal Müller cells, endothelial cells and some types of epithelial cells. Only covering epithelia (cornea, glomerulus) contained significant amounts of these proteins, irrespective of the shape of the cells. EEP-like proteins were absent in secreting epithelial cells of liver, kidney tubules and pancreas. In addition, they were not detected in muscle, nerve and fat cells, erythrocytes and lymphocytes. The localization and the number of EEP-like proteins varied among different cell types. In fibroblasts, containing only two EEP-like proteins (molecular weight (MW) 33.0 and 31.5 kD in calf tissue), predominantly the nucleus was stained. In vitro studies with permeabilized cultured fibroblasts from several species have shown that the nuclear staining was built up of bright spots around unstained nucleoli. In epithelial and endothelial cells of calf tissue, however, most fluorescent label was found in the plasma membranes. Immunoblotting experiments revealed the presence in these cell types of at least five EEP-like proteins, including a 33.0 and 31.5 kD component. The difference in staining pattern between these cells and fibroblasts might thus indicate that the nature or the localization of some of the EEP-like proteins is cell type-specific. Because of their extractability from various tissue membrane fractions by means of EDTA or EGTA it is suggested that at least part of the EEP-like proteins is bound to membrane structures via calcium. This characteristic feature, together with the MW values and the cross-reactivity with anti-EEP antiserum indicate that these proteins and the lens membrane proteins with high calcium-binding capacity share a very high degree of homology and may even be identical.     

Properties of acid phosphatase from scutella of germinating maize seeds

One acid phosphatase (optimum pH at 5.4) was purified from maize scutellum after 96 hr of germination. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulfate (SDS). The enzyme has a MW of 65 000 ± 4000 as determined by Sephadex G-200 gel filtration and SDS-PAGE. The enzyme contained 16% neutral sugars, and cations are not required for activity. The purified enzyme was not inactivated by DTNB at pH 8. The hydrolysis of glucose-6-phosphate in the presence of 4 mM fluoride and 4 mm EDTA, at pH 6.7 (optimum pH), seems to be catalysed by this acid phosphatase.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Purification and some properties of membrane-bound phospholipase B from Torulaspora delbrueckii

Membrane-bound phospholipase B was purified to a homogeneous state from Torulaspora delbrueckii cell homogenate. Cell homogenate was extracted with Triton X-100, and the enzyme was precipitated with acetone. The acetone powder was washed repeatedly with Tris-HCl buffer (pH 8.0) until no phospholipae B activity was detected in the soluble fraction. The enzyme was extracted with Triton X-100 from the final residue and purified about 1,390-fold by sequential chromatofocusing, Sepharose 6B, and DEAE-Sephadex A-50 column chromatography. The final preparation showed a single broad protein band on SDS-polyacrylamide gel electrophoresis when stained with silver stain reagent and PAS-reagent. The molecular weight of phospholipase B was about 390,000 and 140,000-190,000 as estimated by gel filtration on Sepharose 6B and SDS-polyacrylamide gel electrophoresis, respectively, suggesting that phospholipase B is an oligomeric protein. The isoelectric point was at pH 4.5. Phospholipase B has two pH optima, one acidic (pH 2.5-3.0) and the other alkaline (pH 7.2-8.0). At acidic pH the phospholipase B activity was greatly increased in the presence of divalent metal ions, although metal ions are not a factor for enzyme activity. On the other hand, at alkaline pH the enzyme required Ca2+ or Mn2+ for activity. The pH- and thermal-stabilities at both pHs were similar. The phospholipase B hydrolyzed all diacylphospholipids tested at acidic pH, but hydrolyzed only phosphatidylcholine at alkaline pH. The hydrolysis rates of lysophospholipids were much higher (about 10-fold) than those of diacylphospholipids at both pHs.     

Membrane-bound choline acetyltransferase from human brain: Purification and properties

Choline acetyltransferase (ChAT; EC 2.3.1.6) was separated from human caudate/putamen into three fractions by successive extractions into apotassium phosphate buffer, a high salt (NaCl) buffer and a buffer containing 0.6% Triton X-100. The Triton-X-solubilized fraction is the membrane-bound ChAT (mChAT) and represents about 40% of the total ChAT. After centrifugation, mChAT was precipitated by ammonium sulfate at 35–65% saturation. The crude enzyme preparation was fractionated in turn on a DEAE-Sepharose, a hydroxylapatite and a phosphocellulose columns. Finally, mChAT was applied to a CoA-Sepharose column equilibrated with buffer containing 100 mM choline chloride and was specifically eluted with buffer containing acetyl-CoA. The presence of both substrates greatly stabilized the enzyme and ChAT was recovered almost quantitatively. The final preparation of mChAT has a specific activity of 37.2 mol of acetylcholine synthesized per min-mg protein. The purified mChAT has a pH optimum of 8.3. It migrated as two bands on SDS-PAGE with molecular weights of 67,000 and 62,000 daltons, respectively. Immunoblot autoradiography showed that an antiserum prepared previously against soluble ChAT also cross-reacted with both bands of mChAT, indicating that both forms of this enzyme are related. Furthermore, as previously reported for soluble ChAT, Fab-Sepharose chromatography could be used for the purification of mChAT and this preparation also resolved into two bands of 10% SDS gel.Special Issue dedicated to Prof. Eduardo De Robertis.     

Improved Method for Prufication of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase from Bovine Cerebral White Matter

Abstract: An improved procedure of the solubilization and purification of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) from bovine cerebral white matter is reported. To remove easily extractable protein, the tissue was homogenised in 10 vol. of 0.5 M-ammonium acetate containing 10 mM-Tris. HCI, pH 6.9, at 4°C and centrifuged at 105,000 g for 60 min. The precipitate was extracted with 10 vol. of 0.5% Triton X-100 containing 10 mM-Tris. HCI, pH 6.9, and centrifuged, By this extraction, over 70% soluble protein could be removed in the supernatant and most CNPase activity was kept in the precipitate. The precipitate was extracted with 10 vol. of 1% Triton X-100 and 1 M-ammonium acetate mixture containing 10 mM-Tris.HCI, pH 8.2, and centrifuged at 105,000 g for 60 min. The extract contained 54% of CNPase and the specific activity was fivefold that of the original homogenate. Subsequently, the extractions were carried out with 2% Triton X-100-2 M-ammonium acetate and 4% Triton X-100-4 M-ammonium acetate at pH 8.2. The recovery of CNPase was found to be nearly 90% from the original homogenate, without loss of enzyme activity during extraction, while much CNPase activity was lost when guanidinium chloride was used as the extraction medium. Using the Triton X-100-ammonium acetate extract, several column chromatography techniques were applied to purify the enzyme. In the first step, Phenyl-Sepharose CL-4B column chromatography was performed by eluting with a double-linear gradient of ammonium acetate and Triton X-100. In the second step, the fraction containing CNPase after Phenyl-Sepharose CL-4B column chromatography was applied to a Sepharose 6B column and the enzyme was eluted with 1% Triton X-100- I M-ammonium acetate, pH 8.2. The peak containing CNPase was applied to CM-Sepharose CL-6B column chromatography in the final step. The enzyme was eluted with a linear gradient of KCI. In this step, CNPase eluted as a sharp peak and the specific activity was approximately 2300 pmol 2′-AMP formed/min/mg protein. The recovery of CNPase from the original homogenate was about 50%. By the isoelectrofocusing technique, the pI of CNPase was found to be 8.6. When Reisfeld polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis were carried out on the purified CNPase, only one protein band, corresponding to CNPase activity, was detected. Its molecular weight was estimated to be approximately 51,000 as the active enzyme form. K, value of the purified enzyme for 2′,3′-CAMP calculated from a Lineweaver-Burk plot was 3.13 mM.     

Simultaneous staining of proteins during polyacrylamide gel electrophoresis in acidic gels by countermigration of Coomassie brilliant blue R-250

A method for the simultaneous staining of proteins during polyacrylamide gel electrophoresis with Coomassie brilliant blue R-250 at pH 2.5 is described. Calf thymus whole histone and cytochrome c were stained by this method and the results obtained were similar to that obtained by staining after electrophoresis.     

Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, beta-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, beta-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.