Purification of a glucose/mannose specific lectin,isoform 1, from seeds of Cratylia mollis mart. (Camaratu Bean)

A quantitatively main molecular form ofCratylia mollis lectin, isoform 1 (iso 1) was purified by affinity chromatography on Sephadex G-75, followed by ion exchange chromatography on CM-cellulose. Another lectin form was identified in the latter step. Iso 1 is specific for glucose/mannose, with a main subunit of 31 kDa mol wt; the native protein is basic (pI 8.5-8.6) and the constituent polypeptides had a pI range of 5.15–7.75. An antibody to the protein was raised in a rabbit, and the conjugate was active in an immunosorbent assay.     

Hyperalkaline and thermostable phosphatase inThermus thermophilus

The phosphatases existing in the extreme thermophilic bacteriumThermus thermophilus have been studied. Utilizing ion exchange, hydrophobic, pseudoaffinity, and affinity chromatography, a number of distinct phosphatase activities were identified. At least four phosphatases, with optimum pH ranging between 5.0 and 11.5, were assayed withp-nitrophenylphosphate, and two with optimum pH between 7.0 and 11.0, with³²P-casein as substrate. The authors have focused on the hyperalkaline phosphatase and have tried to purify and characterize it. This hyperalkaline phosphatase reaches a maximal level at the stationary phase of the growth, and is co-purified with alkaline phosphatase with optimum pH of 10.2. The enzymes present a relative mol wt of 65 and 58 kDa, respectively, as judged by SDS-PAGE and Sephadex G-150 column, and possess similar properties, indicating that they are isoforms. These enzymes barely function in the presence of tartrate, and are inhibited by EDTA, pyrophosphate, and molybdate. Among the metals tested, Hg²⁺ appeared as the strongest inhibitor of the hyperalkaline phosphatase. The two enzymes are thermostable and, upon treatment at 90°C for 10 min, 75% of their activity remains. The physiological role and function of these phosphatases need further investigation.     

Preparative alkaline urea gradient PAGE: application to purification of extraordinarily-stable disulfide-linked homodimer of human growth hormone

The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.     

Recent progress in the electrochromatography of proteins

We have constructed a column for the application of an electrical field to a flowing packed bed of Chromatographic media (electrochromatography), and we have studied the effect the electrical field on the elution of bovine serum albumin, β-lactoglobulin A and B, α-lactalbumin, and myoglobin from packed beds of Sephadex beads. The elution behavior of the model proteins on Sephadex with a high degree of crosslinking (Sephadex G-25) and with a lower amount of crosslinking (Sephadex G-75) was measured. We confirmed that proteins exhibited an unexplained electrically driven retention on the column packed with G-75. The electrically driven retention effect was greatly reduced or absent in columns packed with the more highly crosslinked G-25. The potential of electrochromatography for high-resolution separations was shown by the partial separation of β-lactoglobulin A and B by using short columns with the electric field polarity such that electrophoresis opposed convective flow in the column. Certain commerical equipment, instruments or materials are identified in this article to specify adequately the experimental procedure. Such identification does not imply recommendation by NIST, nor does it imply that the materials or equipment are necessarily the best available for the purpose.     

Chemical Studies of Formosan Cobra (Naja Naja Atra) Venom Part IV. Estimation of molecular weights of some purified protein fractions by sephadex gel filtration

Molecular weights of four purified fractions, Fractions VIII (cobraneurotoxin), IX (5′-nucleotidase), XI (cardiotoxin) and XII (cardiotoxin) of Formosan cobra venom were estimated by gel filtration on Sephadex G-100 and G-75 columns.     

Die multikomponenten Formen von Cellulase (EC 3.2.1.4) ausAspergillus oryzae

The enzyme cellulase fromAspergillus oryzae was resolved into four multiple forms, using anion exchange chromatography on DEAE Sephadex A-50 and gel filtration on Sephadex G-75. The stages of fractionation were followed by electrophoresis on cellulose acetate strips. These enzyme forms are characterized by different enzyme activities and isoelectric points.

Herrn Univ.-Prof. Dr.Hans Tuppy zum 65. Geburtstag herzlichst gewidmet.     

银诱导大鼠肝脏金属硫蛋白的提纯与鉴定

Ｗｉｓｔａｒ公鼠经腹腔注射ＡｇＮＯ３后可诱导肝脏合成ＭＴ。经匀浆、乙醇沉降、Ｓｅｐｈａｄｅｘ－７５、ＤＥＡＥ－５２两次柱层析，可得到两个亚型。原子吸收测定结果表明：该蛋白分别含７份Ａｇ、２份Ｚｎ和２份Ｃｕ，具有与Ｃｄ５Ｚｎ２－ＭＴ并不相同的二级、三级结构。进一步研究表明，蛋白中伴随Ｃｕ和Ｚｎ的含量与所用诱发剂的种类、数量均有关，且Ｃｕ和Ｚｎ（通过ＭＴ）具有某种微妙的联系。     

The Surfactin and Lichenysin Isoforms Produced by Bacillus licheniformis HSN 221

Surfactin and lichenysin are typical members of the lipopeptide family. Four surfactin and two lichenysin isoforms were isolated by Pre RP-HPLC from the cell-free broth of Bacillus licheniformis HSN 221 cultivated in YPD medium. The molecular weight of each isoform was determined by ESI-MS. The peptide part of each isoform was elucidated by Q-Tof MS/MS combined with HPLC, and the fatty acid part was analyzed by GC/MS. It showed that the four surfactin isoforms have the primary structures of anteiso(iso)C₁₃, isoC₁₄, anteiso(iso)C₁₅, anteiso(iso)C₁₅-surfactin-methyl ester, and the two lichenysin isoforms have those of nC₁₄ and anteiso(iso)C₁₅, respectively.     

Effect of Bacillus circulans D1 thermostable xylanase on biobleaching of eucalyptus kraft pulp

The alkalophilic Bacillus circulans D1 was isolated from decayed wood. It produced high levels of extracellular cellulase-free xylanase. The enzyme was thermally stable up to 60°C, with an optimal hydrolysis temperature of 70°C. It was stable over a wide pH range (5.5—10.5), with an optimum pH at 5.5 and 80% of its activity at pH 9.0. This cellulase-free xylanase preparation was used to biobleach kraft pulp. Enzymatic treatment of kraft pulp decreased chlorine dioxide use by 23 and 37% to obtain the same kappa number (κ number) and brightness, respectively. Separation on Sephadex G-50 isolated three fractions with xylanase activity with distinct molecular weights.     

Mg2+与胆汁蛋白质相互作用研究

胆汁是一中性溶液,在胆汁固形物中,胆汁酸盐、胆固醇、磷脂、蛋白质、胆红素分别占总质量的65%、3%、20%、5%及1%。蛋白质是胆汁固形物中第四多成分,在胆汁的生理功能的发挥和胆结石的形成中起到重要的作用。研究表明,胆汁蛋白质的主要成分是糖蛋白,主要由胆囊粘膜上皮细胞产生     

Gel filtration of protected peptides on sephadex G-50 in hexamethylphosphoramide containing 5% water.

Gel permeation chromatography on G-50 and G-75 Sephadex gels, using 5% water in hexamethylphosphoramide (phosphoric trisdimethylamide) has been applied to the purification of large protected peptides which are outside the molecular weight range of the Sephadex LH-20-dimethylformamide system.     

大鼠肾脏汞结合金属硫蛋白的联用技术表征研究

通过凝胶色谱(SEC)及反相液相色谱(RPC)分别与电感耦合等离子体质谱(ICP-MS)和电喷雾电离质谱(ESI-MS)联用的技术，对灌喂HgCl₂大鼠的肾组织中诱导的多金属结合金属硫蛋白(MT)的结构进行表征分析。通过SEC-ICP-MS曲线上的金属信号及紫外检测的MT光谱吸收特征，可确认组织提取液中MT在色谱曲线上的位置。与对照组相比，灌喂HgCl₂大鼠肾中Hg-MT的诱导量显著增加，与此同时，Cu-MT在染毒组的诱导量也相应增加，说明MT在染汞大鼠肾脏中起着对汞的解毒和对铜的调节作用。MT样品提取物经葡聚糖凝胶(G-75)柱分离纯化并用透析法脱盐后，用细内径反相色谱柱分离，进行梯度洗脱，并与ESI-MS联用，可获得总离子流色谱图及与色谱峰对应的质谱图，通过对质谱信号的解析及参考脱金属MT构型的有关报道，对汞诱导大鼠肾组织MT的不同亚型和次亚型分子结构进行推断。结果证明，在灌喂HgCl₂大鼠的肾组织中存在着一系列汞结合的MT形态。     

Parameteruntersuchung zur Alkalitrennung auf Sephadex-S?ulen

Zusammenfassung Das Prinzip der Alkalitrennung auf Dextrangelen wurde bereits früher in dieser Zeitschrift beschrieben [18]; Vorzüge und damalige Grenzen dieser neuen Möglichkeit der Alkalitrennung waren erlÄutert.Nun wird die Anwendung von 75 Vol.-% Methanol als günstigstes Elutionsmittel für Alkalitrennungen auf Sephadex-SÄulen begründet. Als geeignetstes Gelmaterial erweist sich Sephadex G -25 fine. Folgende Parameter, die die Alkalitrennung auf Sephadex-G-25-SÄulen in 75 Vol.-% Methanol beeinflussen, werden systematisch untersucht, um durch optimale Parameterkombination zu möglichst günstigen Trennbedingungen zu gelangen: Korngrö\e und -gestalt, pH-Wert, Durchflu\geschwindigkeit, Aufgabekonzentration, Temperatur.

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Summary The principle of alkali separations on Dextran gels was described earlier in this journal [18] and a limited scope of this new separation technique was given.The application of 75% (v/v) methanol as best eluant for alkali separations on Sephadex columns is elucidated. Sephadex G-25 fine proves to be the most suitable gel material for this purpose. The following parameters are of great influence on the alkali separation on Sephadex G-25 columns with 75% (v/v) methanol as eluant: particle shape and size, pH, flow rate, alkali concentration, temperature. They are investigated systematically to find the most favourable conditions for the alkali separation by optimal parameter combination.

     

Effect of Insulin-Dependent Cytoplasmic Regulator on Murine Melanoma KML Cell Proliferation

Insulin-dependent cytoplasmic regulator (IDR), the activity of which correlates with the insulin level in blood, was isolated from rat liver and fractionated on a Sephadex G-25 column. Radioautography established that IDR stimulates by 30% proliferation of murine melanoma KML cells in culture. This is indicated by ³ H-thymidine incorporation into the cell nucleus. It is proposed that insulin stimulates proliferation of the melanoma KML cell cultures through an intracellular intermediate, IDR     

Purification and properties of three cellobiases from Aspergillus niger A20

Three cellobiases, here called cellobiase A, B, and C, from the culture filtrate of Aspergillus niger A20, were purified by precipitation with ammonium sulphate, gel filtration through Sephadex G-75, and column chromatography of DEAE-cellulose. The purified enzymes were homogeneous on polyacrylamide disk electrophoresis. The mol wt of the purified enzymes were estimated by SDS-gelelectrophoresis to be 88,000, 80,000, and 71,000 for cellobiases A, B, and C, respectively. The enzymes were active at pH 4.5 and 55–60°C. The pattern of their aminoacid compositions showed high contents of aspartic acid, glutamic acid, threonine, serine, and glycine. The apparent K_m values for cellobiose were 0.9, 1.63, and 1.0 mM for cellobiases A, B, and C, respectively. Calcium ions stimulated cellobiases B and C, and Co²⁺ and Mg²⁺ ions stimulated cell obiase A. The purified enzymes hydrolyzed cellobiose and aryl-β-d-glucosides, but they had no action on sucrose, maltose, and cellulose. The three cellobiases catalyzed transglycosylate reaction, and the major product formed from cellobiose was tetramer of glucose.     

Isolation, Purification, and Properties of α-Amylase from Bacillus subtilis-7A

Cultivation ofBacillus subtilis-7A on waste from alcohol production yielded an active extracellular enzyme -amylase with MW 75 kDa. The enzyme was isolated from the culture medium by 60% saturated ammonium sulfate and purified until homogeneous by gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. The optimum temperatures for the complex and purified enzyme are 30 and 50°C, respectively. The optimum activity for both preparations occurred at pH 6.5. The substrate specificity of the isolated preparations was studied.     

金属硫蛋白异构体及亚型异构体的液相色谱分离与质谱鉴别

建立了金属硫蛋白(MT)异构体及亚型异构体的色谱分离与质谱鉴别方法。将金属硫蛋白混合物通过弱阴离子DEAE Sephadex A-25离子交换柱,结合离线电感耦合等离子体质谱(ICP-MS)对锌诱导金属硫蛋白的两个异构体MT-1和MT-2进行分离和检测;利用Sephadex G-25凝胶排阻色谱柱对得到的两个金属硫蛋白异构体进行脱盐;探索脱盐后的金属硫蛋白异构体在不同色谱条件下的C18反相色谱柱上的保留行为,进而实现各个亚型异构体的分离;通过在线电喷雾质谱检测实现了对金属硫蛋白各个亚型异构体的鉴别。结果表明,通过优化色谱条件,由离子交换色谱及凝胶排阻色谱得到的金属硫蛋白各亚型异构体在酸性条件下均得到了良好的分离,质谱检测结果与前人的文献报道结果一致。该方法可使金属硫蛋白各异构体均达到最佳的分离效果。     

An investigation of the venom of Renard's viperVipera ursini renardi. III. Isolation of phospholipases A2

Two phospholipases A₂ with molecular weights of 15,600 and 13,200 and pI values of 7.57 and 6.73, respectively, have been isolated from viper venom in the pure state with the aid of gel filtration on Sephadex G-75 followed by ion-exchange chromatography on CM-cellulose and SE-Sephadex C-25.Institute of Biochemistry, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 389–392, May–June, 1980.     

Bacitracin production by a new strain ofBacillus subtilis

A new strain ofBacillus subtilis C 126 was isolated from sugar cane fermentation and produced an antibiotic that inhibited the growth ofMicrococcus flavus. The production of the antibiotic in culture medium followed to extraction withn-butanol, thin layer chromatography, and microbiological tests indicated that a polypeptide antibiotic was produced. The fraction obtained by Sephadex G-25 column and analyzed by HPLC indicated that bacitracin complex was produced.     

Purification and characterization of two xylanases from alkalophilic and thermophilic Bacillus licheniformis 77-2

The alkalophilic bacteria Bacillus licheniformis 77-2 produces significant quantities of thermostable cellulase-free xylanases. The crude xylanase was purified to apparent homogeneity by gel filtration (G-75) and ionic exchange chromatography (carboxymethyl sephadex, Q sepharose, and Mono Q), resulting in the isolation of two xylanases. The molecular masses of the enzymes were estimated to be 17 kDa (X-I) and 40 kDa (X-II), as determined by SDS-PAGE. The K _m and V _max values were 1.8 mg/mL and 7.05 U/mg protein (X-I), and 1.05 mg/mL and 9.1 U/mg protein (X-II). The xylanases demonstrated optimum activity at pH 7.0 and 8.0–10.0 for xylanase X-I and X-II, respectively, and, retained more than 75% of hydrolytic activity up to pH 11.0. The purified enzymes were most active at 70 and 75°C for X-I and X-II, respectively, and, retained more than 90% of hydrolytic activity after 1 h of heating at 50°C and 60°C for X-I and X-II, respectively. The predominant products of xylan hydrolysates indicated that these enzymes were endoxylanases.