α1-Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing

1. Improved resolution of mixtures of alpha(1)-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14-0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36-0.72 pH unit. After treatment with 0.025m-H(2)SO(4) at 80 degrees C for 1h, which removed all the sialic acid, the increase was 0.40-0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.     

α1 acute-phase globulins of rats. Isolation and some properties

1. A method is described for the isolation of certain of the alpha(1)-globulins of rat plasma that are known to increase in concentration after tissue damage (acute-phase globulins). 2. Although apparently homogeneous when examined by disc electrophoresis at pH9, these proteins could be subdivided further by isoelectric fractionation. 3. Treatment with neuraminidase removed approx. 60% of the sialic acid originally present in these proteins and gave almost completely homogeneous material of decreased mobility when examined by disc electrophoresis in polyacrylamide gel. When subjected to immunoelectrophoresis this material gave a single arc. 4. The homogeneity of the isolated materials was examined by ultracentrifugation. The single peak thus found is consistent with molecular weights of 45000-46000. 5. The isolated materials were shown to be glycoproteins containing approx. 15% of carbohydrate, and to have isoelectric points in the range pH4.4-4.8.     

Electrophoretic determination of sulfhydryl groups and its application to complex protein samples, in vitro protein synthesis mixtures, and cross-linked proteins

Without prior fractionation, the number of sulfhydryl groups of individual polypeptides in a protein mixture can be determined, provided their molecular weights and approximate isoelectric points are known. Urea-denatured protein samples are reacted with iodoacetamide and iodoacetate in a modified version of Creighton's procedure. After separation by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and isoelectric focusing, the number of sulfhydryl groups is determined by counting the protein bands which have additional negative charges. This method requires little material and provides an additional parameter, besides the molecular weight and isoelectric point, for the identification and characterization of a protein. The sensitivity may be enhanced for nonradioactive proteins by using 14C-labeled iodoacetamide and iodoacetate. The procedure has been applied to prokaryotic in vitro protein synthesis mixtures, bacterial membrane protein, and trypsin-cleaved or chemically cross-linked subunits of the F1 ATPase from Escherichia coli.     

Isolation and partial characterization of the major outer membrane protein of Chromatium vinosum.

The 42,000 major outer membrane protein of Chromatium vinosum was purified by a combination on ion-exchange chromatography, gel filtration, and isoelectric focusing. Upon isoelectric focusing, the final material produced four major hands. Three of the four bands were isolated and analyzed for similarity or differences. Protease peptide maps and cyanogen bromide maps of the three isoelectric species were identical. When the isolated isoelectric species were refocused, each produced multiple isoelectric species, suggesting that the procedure used was generating the multiple charged species. Protease treatment of the isolated outer membrane produced a 31,000 fragment from the 42,000 protein. This fragment was isolated by preparative sodium sulfate-polyacrylamide gel electrophoresis. Although the amino acid compositions of the 42,000 protein and its 31,000 trypsin fragment were different, their polarity index was the same (45%). The amino-terminal sequences of the 42,000 protein and 31,000 trypsin fragment were identical, and it concluded that the amino-terminal was buried in the membrane.     

Biochemical purification of monkey and human LDH isoenzyme 5 suitable as immunization antigen

A method consisting in an ion-exchange chromatography on DEAE-Sephadex A50 and an affinity chromatography on Oxamate-hexyl-Sepharose for the purification of monkey and human LDH isoenzyme 5 is presented. Analytical isoelectric focusing reveals minor variants in the monkey LDH isoenzyme 5. This material has been used to produce an antiserum from rabbits which recognizes all the human LDH isoenzymes containing the M subunit, without cross-reacting with LDH1.     

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material.The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic cross-point (pH 4.1) was observed for the fraction enriched in inside-out grana thylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

Anomalous behaviour of yeast isocitrate dehydrogenase during isoelectric focusing

Isoelectric focusing of yeast isocitrate dehydrogenase apparently reveals a number of ;isoenzymes'. These have isoelectric points near pH5.5 in crude material, but during purification the mean isoelectric point progressively rises to pH7.0 and the band pattern changes. The shift in isoelectric point during purification is apparently genuine, since it is also manifested in the electrophoretic and chromatographic properties of the enzyme. The multiple forms, however, are an artifact, generated by exposure of the enzyme to Ampholine, since their activities vary with the protein/Ampholine ratio and they cannot be observed in any system from which Ampholine is excluded. There are no detectable isoenzymes of yeast isocitrate dehydrogenase.     

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition.

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material. The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic crosspoint (pH 4.1) was observed for the fraction enriched in inside-out granathylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

Characterization of a soluble factor that specifically suppresses the in vitro generation of cells cytotoxic for syngeneic tumor cells in mice.

A tumor-specific soluble factor found in extracts of thymocytes from mice bearing small P815 tumors has been demonstrated. This factor is capable of significantly suppressing the in vitro generation of syngeneic cells cytotoxic for P815 targets if it is added to culture vessels within the first 30 hr of culture. The suppressive factor eluted from Sephadex G-100 after hemoglobin and was estimated to have a m.w. in the range of 40 to 60,000. Preparative isoelectric focusing of thymic extracts established that the suppressive material has an isoelectric point in the range of pH 4.6 to 4.9. The suppressive activity of extracts could be removed by passage of the material through immunoadsorbent columns prepared from membrane proteins of P815 cells but not by analogous columns prepared by using L1210 membrane proteins. The suppressive material was not removed by its passage through immunoadsorbent columns containing anti-mouse immunoglobulin.     

全柱成像毛细管等电聚焦电泳测定艾塞那肽等电点

目的:采用新一代全柱成像毛细管等电聚焦电泳技术(CIEF-WCID)测定艾塞那肽等电点。方法:采用互补性金属氧化物半导体成像技术对样品等电聚焦过程进行实时记录,根据适宜的marker计算得到艾塞那肽的等电点,并对方法的准确度与重复性进行考察。结果:测得艾塞那肽等电点为5.46,与凝胶电泳结果基本一致,相对标准偏差为0.11%。CIEF-WCID方法快速准确,相对误差小于2.5%,重复性良好。结论:CIEF-WCID作为一种新的技术手段可用于艾塞那肽等电点的分析,方法快速、准确、重复性好,可为多肽的质量控制提供一种可靠的分析方法。     

Isolation of a granulocyte colony inhibitory factor derived from human polymorphonuclear neutrophils

An inhibitory factor obtained from mature human granulocytes which suppresses granulocyte and monocyte-macrophage colony formation by an action on the endogenous colony stimulating factor-producing cells has been partially purified and characterized. The methods for purification consisted of a combination of ultracentrifugation, DEAE-Sephadex chromatography, SDS polyacrylamide gel electrophoresis and isoelectric focusing. The material had a molecular weight range of 102--128 000 and an isoelectric point between pH 6.2 and 6.4. The inhibitory factor was found to be heat stable and glycoprotein in nature.     

Studies on heterogeneity of the lysine-rich histone from ox pancreas

1. Histone H1 from ox pancrease has been isolated by preparative electrophoresis at pH 2.7 in polyacrylamide slab gel, using the fraction F1 of Oliver et al. (1972, Biochem. J., 129, 349--353) as starting material. 2. The isolated histone H1 showed higher heterogeneity on isoelectric focusing than on polyacrylamide electrophoresis in long gel. The isoelectric points of the main subfractions of histone H1 were at pH 8.0--8.4.     

PURIFICATION OF GONAD-STIMULATING SUBSTANCE OBTAINED FROM RADIAL NERVES OF THE STARFISH, ASTERIAS AMURENSIS

Purification of starfish gonad-stimulating substance (GSS), which induces shedding of gametes and oocyte maturation, was carried out using lyophilized radial nerves of Asterias amurensis as source material. In the first series of experiments, 1.3 mg of the purified GSS, which induced spawning at a concentration of 0.0096 μg/ml, was isolated from acetone powder of lyophilized radial nerves of 7,360 starfish through several steps of purification procedures consisting of gel-filtrations on Sephadex G–50 and G–25 columns of various sizes and ion-exchange chromatography on DEAE-Sephadex columns by gradient as well as step-wise elution. With this sample, the molecular weight and amino acid composition of GSS were estimated. Another series of experiments, conducted on a similar amount of material with purification procedures which were essentially the same as those of the first series except for employing 2 steps of partition chromatography instead of extensive gel-filtration, gave about 0.1 mg of purified sample which served as material for studies of the amino acid composition and electrophoretic properties of GSS.
The molecular weight of Asterias GSS was found to be about 2,100, as determined with the sedimentation equilibrium method. GSS seemed to consist of the following 22 amino acid residues: aspartic acid (2), threonine (1), serine (6), glutamic acid (1), proline (1), glycine (4), alanine (2), valine (1), isoleucine (1), leucine (1), histidine (1), and ornithine (1). The isoelectric point of GSS was found to be at about pH 4.5 as determined by the isoelectric focusing method.     

红肉蜜柚汁胞蛋白质双向电泳体系优化研究

为建立适合红肉蜜柚发育过程中汁胞蛋白质组学的研究体系,以着色初始时期的红肉蜜柚汁胞为试材,摸索最适合的蛋白质双向电泳体系参数。结果表明,酚提取法比TCA-丙酮法更适合于红肉蜜柚汁胞总蛋白质的提取,提取得率高,双向电泳图谱清晰。采取18 cm、线性pH 3～10 和18 cm、线性pH 4～7 相结合的分析策略,可获得更为完整的蛋白质组信息,其第一向等电聚焦最佳参数（总伏小时数）分别为30 000 Vhs和43 000 Vhs。     

Partial purification and characterization of the glucagon receptor

Specific labeling of liver plasma membrane glucagon receptors has been achieved by the photoincorporation of a 125I-labeled photoderivative of glucagon, NE-4-azidophenylamidinoglucagon. Identification of glucagon receptors was facilitated by irradiating membranes in the presence of excess unlabeled glucagon. Isoelectric focusing of radioiodinated membrane proteins revealed one major band of glucagon displaceable material which had an isoelectric point of 5.85. When this material was isolated and run on SDS-polyacrylamide gels a major labeled band of Mr55000 was obtained which had properties consistent with those of the glucagon receptor. These studies indicate that a purification of the glucagon receptor of greater than 700-fold can be attained through the use of isoelectric focusing and SDS-polyacrylamide electrophoresis.     

Isoelectric point determination of human and camel beta-endorphin, alpha-endorphin and enkephalins

The isoelectric point of the camel and the human β-endorphin, of the α-endorphin and the enkephalins were determined by analytical isoelectric focusing on 1 mm thin polyacrylamide gel slab. The difficulty of staining peptides as short as β-endorphin or smaller was overcomed using a modification of Bibring and Baxandall's or Faupel and Von Arx's staining method. The camel β-endorphin gives two bands having isoelectric point of 10.3 and 10.4, the human β-endorphin focus at pH 9.9, while α-endorphin, leu and met-enkephalin at pH 5.9, 5.5 and 5.45 respectively. The staining method described coupled with the isoelectric focusing seems to be fit for discriminating β-endorphin in a crude rat pituitary extract.     

表面活性剂对谷氨酸结晶的影响之研究

本文介绍了表面活性剂对等电点提取谷氨酸的收得率的影响。通过正交试验确定提取过程的最佳条件。试验结果表明,它可使等电点法收得率提高10～20%。     

Isoelectric focusing of multiple forms of DNase in thin layers of polyacrylamide gel and detection of enzymatic activity with a zymogram method following separation

Multiple forms of bovine pancreatic DNase (DNases A, B, C, and D) are separated by isoelectric focusing in thin layers of polyacrylamide gel with a carrier ampholyte in the pH range 4–6. The isoelectric points of DNases A, B, C, and D are 5.22, 4.96, 5.06, and 4.78, respectively. A zymogram method for detecting DNase activity as bands in the gel following isoelectric focusing is described. The method detects microgram amounts of DNase and has only one step. It can be used with the parified cazyme as well as with crude extracts of tissues containing DNase. By this method, two major components of DNase in ovine pancreas and at least three in malted barley as well as two previously unideatified forms of DNase in bovine pancreas with isoelectric points of 5.12 and 5.48 (DNases E and F) are observed.     

Antibodies to guinea pig lymphokines. VII. Reactivity with products of lymphoid and nonlymphoid cells.

It was shown previously, that an antiserum directed against highly purified fractions of migration inhibitory factor inhibits delayed hypersensitivity reactions in vivo and in vitro. Using radiolabeling techniques we determined that the anti-lymphokine serum reacted primarily with three lymphocyte activation products (m.w. 60,000, 45,000, and 30,000) all of which had a similar isoelectric point of 5.2. The cellular origin of this material was investigated. It was found that activated B cells, B leukemia cells (L2C), and growing fibroblasts produced material of a similar m.w. as analyzed on SDS-PAGE. No cross-reaction was found with radiolabeled products of activated murine and human lymph node cells and of SV 40-infected African green monkey kidney cells. The isoelectric point of the reactive material from B cells, leukemia cells, and fibroblasts was determined at 5.2. In addition to material with pI 5.2, lymph node cells also produced material with pI 3.5 to 4.5, which focused at pH 5.0 to 5.4. After neuraminidase treatment macrophage migration inhibitory activity in fibroblast culture supernatants could be absorbed specifically to insolubilized anti-lymphokine antibody. These findings suggests that lymphoid and nonlymphoid cells are capable of producing molecules whose physicochemical and functional properties appear to be identical.     

Comparison of isoelectric focusing in Sephadex vs immobiline flatbeds for the recovery of follicle regulatory protein from porcine follicular fluid

We describe and compare the use of isoelectric focusing (IEF) in a granulated Sephadex matrix and in polyacrylamide immobilized pH gradients to separate an aromatase inhibitor (follicle regulatory protein: FRP) in preparative amounts from porcine follicular fluid (PFF). The starting material for IEF was derived from pFF after passage through agarose immobilized textile dye Orange A (0.5 KC1 eluent). Before IEF, some Orange A bound (OAB) material was further purified on a FPLC employing a Mono-Q anion exchange column. Previous use of chromatofocusing indicated that aromatase inhibitory activity is largely concentrated in OAB fractions with a pI in the ranges of pH approximately 4.5 and approximately 6.5. The current study revises these findings to provide a more precise measure of the isoelectric points in question to pH 4.73 +/- 0.05 and pH 6.41 +/- 0.06. The use of Sephadex was limited by gradient instability and the selection of pH ranges available. IEF using immobilized pH gradients had several advantages over Sephadex: 1) broader selection of gradients from 0.1 to 7.0 pH units; greater resolving power, and enhanced stability. The principal disadvantage of the immobiline system was the recovery of focused material from the gel matrix. The use of isoelectric focusing with immobilized pH gradients on a preparative scale to purify FRP from OAB resulted in a greater than 50% recovery with a substantial increase in specific activity (from ID50 approximately 300 micrograms/ml to 20 ng/ml).