Regulation of Chitinase Production by the 5'-Untranslated Region of the ybfM in Serratia marcescens 2170

Glycine-rich protein (GRP), a cell wall protein, was extracted with hot water from the aleurone layer of soybean seeds. GRP was purified by adsorption on DEAE-Sephadex, Sephadex G-100 gel chromatography, and anion exchange HPLC. The estimated molecular size of GRP was approximately 30 kDa and GRP contained 59% glycine and 15% serine. The N-terminal amino acid sequence was a novel Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-.     

Purification and Characterization of a Glycine-rich Polypeptide Tightly Bound to Cell Walls from Soybean Aleurone Layers

A part of cell walls in soybean aleurone layers remained undigested after pectinase and cellulase treatments, and the features of the undigested cell walls were similar to those of Casparian strips. Glycine-rich polypeptides (GRPP) were extracted with 0.4 n NaOH from the undigested cell walls, Casparian strip-like tissues. Approximately 6.5–kDa GRPP obtained by gel-permeation chromatography from the extract was purified by anion-exchange HPLC and reverse-phase HPLC. The major amino acids of GRPP were glycine (69%) and serine (13%). The n-terminal amino acid sequence of GRPP was the same polyglycine as 30 kDa glycine-rich protein (GRP). GRPP would participate in adhesion between neighboring cells in aleurone layers because of tight binding to the cell wall.     

Alpha-enolase is restricted to basal cells of stratified squamous epithelium.

We have developed a monoclonal antibody against a 50-kDa protein that binds preferentially to basal cells in the limbus of rat, rabbit, and human corneas (J. D. Zieske, G. Bukusoglu, and M. A. Yankauckas, Invest. Ophthalmol. Visual Sci. 33, 143-152, 1992). Here we report on the purification and identification of the antigen. The 50-kDa antigen was purified from rabbit limbal and corneal epithelium using HPLC methodology including anion exchange (DEAE) followed by reverse-phase (C18) chromatography. The purified 50-kDa protein was then digested with endoproteinase Lys-C, and a reproducible profile comprising approximately 20 peptides was observed by reverse-phase HPLC of the digest. Sequence analysis of five peptides ranging in length from 4 to 20 residues revealed that the 50-kDa protein was alpha-enolase, a glycolytic enzyme. Overall, 57 amino acids were identified with a 95% sequence homology. Localization of alpha-enolase in rat epithelium by immunofluorescence microscopy demonstrated that simple epithelium contained low or undetectable levels of the enzyme. Stratified squamous epithelium, however, showed high levels of alpha-enolase, which was localized specifically to cells of the basal layer. Epidermal, corneal limbal, oral mucosal, vaginal, and laryngeal epithelium all showed cytoplasmic binding specific to the basal cells. These data indicate that the glycolytic enzyme alpha-enolase is preferentially localized in the basal cell layer of stratified squamous epithelium and suggest that glycolytic activity is concentrated in these cells. The localization pattern suggests that a major change in metabolism occurs as cells leave the mitotically active basal cell layer and migrate toward terminal differentiation in the suprabasal cell layers.     

Theromin, a novel leech thrombin inhibitor

We purified the most potent thrombin inhibitor described to date from the rhynchobdellid leech Theromyzon tessulatum. Designated theromin, it was purified to apparent homogeneity by gel permeation and anion exchange chromatography followed by two reverse-phase steps of high performance liquid chromatography. The primary sequence of theromin (a homodimer of 67 amino acid residues including 16 cysteine residues) was determined by a combination of reduction and s-beta-pyridylethylation, Edman degradation, trypsin enzymatic digestion, and matrix-assisted laser desorption mass spectrometry measurement. Theromin exhibits no sequence homology with any other thrombin inhibitors. Furthermore, theromin significantly diminishes, in a dose-dependent manner, the level of human granulocyte and monocyte activation induced by lipopolysaccharides. In summary, this potent thrombin inhibitor promises to have high biomedical significance.     

Purification and characterization of a new member of the S-100 protein family from human placenta.

A novel Ca(2+)-binding protein which is termed S-100P was purified from human placenta with a hydrophobic column followed by an anion exchange column and reverse phase high performance liquid chromatography (HPLC). Molecular mass of the protein was 10 kDa according to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Using immunoblotting technique, anti-human calcyclin antibodies did not bind to the S-100P. Isoelectric point of S-100P was pI = 4.6. S-100P did not formed disulfide-linked dimer. Calcium binding ability was proved by UV difference spectrometry, urea/alkaline gel electrophoresis, and 45Ca overlay technique. A ninety amino acid sequence of S-100P was determined. It is 49% identical with human S-100 beta, 38% with human calcyclin, and 37% with human cystic fibrosis antigen.     

啤酒酵母生产的重组水蛭素的纯化及脱色

对啤酒酵母生产的重组水蛭素变异体1(rHV1)进行多步骤的纯化。首先将分泌到培养上清液中的水蛭素进行硫酸铵沉淀和SephadexG-50凝胶过滤,再用Q-SepharoseHP阴离子交换层析分离,最后用HPLCSP-5PW阳离子交换柱脱色及HPLCC8柱反相层析。真空干燥后得到的水蛭素蛋白经SPS-PAGE、N端氨基酸序列分析、抗凝血酶活力分析鉴定,证明已获得高纯度的重组水蛭素HV1制剂,为利用基因工程方法生产重组水蛭素的规模化生产及临床应用提供了依据     

N-terminal sequence analysis of human placental protein 14, purified in high yield from decidual cytosol

Human placental protein 14 (PP14) has been purified in high yield from first trimester decidual cytosol. High-performance liquid chromatography on anion exchange, gel filtration and reverse-phase chromatography were used. The protein obtained is approximately 97% pure with an overall recovery of about 50% from the original tissue extract. The first 24 amino acids of the N-terminal were found to be Met-Asp-Ile-Pro-Gln-Thr-Lys-Gln-Asp-Leu-Glu-Leu-Pro-Lys-Leu-Ala-Gly-Thr-Glu-His - Glu-Met-Ala-Met. PP14 has been characterized in this study to be a dimeric glycoprotein of Mr 60,000, with homologous subunits having an Mr of 28,000.     

Characterization of a pathogenesis-related class 10 protein (PR-10) from Astragalus mongholicus with ribonuclease activity.

A pathogenesis-related (PR) class 10 protein (designated AmPR-10) was first isolated from the Chinese medicinal material Astragalus mongholicus using a combination of affinity chromatography on Zn-chelate Agarose 4B, ion exchange chromatography on QAE Sephadex A-25 and gel filtration on Sephadex G50. The purified AmPR-10 showed a single band with a molecular mass of 17.2kDa in SDS-PAGE. The molecular mass of intact AmPR-10 was determined to be 32.8kDa by gel filtration. Thus, AmPR-10 is a dimeric protein composed of two identical subunits. AmPR-10 was a glycoprotein detected by periodic acid-Schiff (PAS) staining and its neutral carbohydrate content was 13.7%. The carbohydrate was mainly composed of 73.0% (w/w) arabinose, 15.0% (w/w) glucose and 4.8% (w/w) fructose on the basis of high-performance anion exchange chromatography (HPAEC) analysis. Its N-terminal sequence of 15 amino acid residues was determined as GVISFNEETISTVAP, and showed significant sequence homology to some pathogenesis-related (PR) class 10 proteins. This sequence had 80% identity with the PR-10 protein LlPR10.1C from Lupinus luteus (yellow lupine) followed by 73.3% identity with the PR-10 protein PR10.2 from Medicago sativa (alfalfa), suggesting it is a new member of PR-10 proteins. AmPR-10 exhibited ribonuclease (RNase) activity as do some other PR-10 proteins. The optimal pH and temperature for RNase activity were pH 6.0 and 60 degrees C, respectively. The RNase activity was stable within pH 5.0-11.0. It was stable up to 60 degrees C at pH 6.0. The purification and characterization of AmPR-10 in this investigation furnish additional data to the relatively scanty literature pertaining to Astragali radix proteins.     

The primary structure of glycoprotein III from bovine adrenal medullary chromaffin granules. Sequence similarity with human serum protein-40,40 and rat Sertoli cell glycoprotein.

Glycoprotein III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secretory vesicles of the adrenal medulla, by chromatography using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatography (HPLC). Characterization of this glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase HPLC, amino acid analysis and partial NH2-terminal sequence analysis indicated that GPIII was a disulfide-linked heterodimer with 37-kDa subunits. Analysis of in vitro translation products of adrenal medullary poly(A)+ RNA by immunoprecipitation using an anti-GpIII serum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that both subunits are synthesized from a single precursor. Partial NH2-terminal sequence analysis allowed construction of oligonucleotides which were used as primers for a polymerase chain reaction to generate a GpIII-specific DNA probe. This probe was used to isolate a cDNA clone encoding the GpIII precursor from a bovine adrenal medullary cDNA library. The predicted amino acid sequence of GpIII has greater than 80% similarity to human serum protein-40,40, a protein implicated in the complement system, and to a major secretory product of Sertoli cells, glycoprotein 2, which is thought to play a role in spermatogenesis. Northern blot analysis confirmed that RNA encoding GpIII is also abundant in liver, testis, and brain.     

A 60-kDa phosphorylated protein from fetal human bone

A phosphorylated protein was isolated and purified from fetal human bone. Fetal and adult human bones were decalcified with EDTA, and the extract from the fetal bone was fractionated using Q-Sepharose anion exchange chromatography. The fraction containing Ser(P) was purified by Sephacryl S-200 molecular sieving and C4 reverse-phase HPLC. The purified protein had a molecular weight of 60000 on SDS-PAGE, where the protein was stained with Rhodamine-B. The amino acid composition of this protein was different from any other reported phosphorylated proteins in human bone. However, this phosphorylated protein was difficult to detect in the adult bone extract on SDS-PAGE.     

Purification and Chemical Characterization of Peptide G1, an Invertebrate Neuropeptide That Stimulates Cyclic GMP Metabolism

Bioassay analysis of extracts of the major neurosecretory structures of the American lobster have revealed several different agents with stimulatory effects on the cyclic GMP metabolism of various lobster tissues. The most potent of these is a peptide extracted from the sinus gland, a neurohemal organ found in the animal's eyestalk. This molecule, called peptide G1 (for its effects on cyclic GMP metabolism), can increase the cyclic GMP content of every lobster tissue tested, sometimes by as much as 200-fold. In this article, we describe the purification and some of the chemical properties of peptide G1. Purification was accomplished by sequential anion exchange and reverse-phase HPLC. The purified peptide is a large, extremely hydrophobic molecule. Its apparent molecular mass on a reducing sodium dodecyl sulfate-containing gel is 6.4 kDa, and its calculated molecular mass (based on an amino acid analysis of the purified material) is 8.2 kDa. Amino acid analysis reveals a high proportion of leucine and valine residues. The amino terminus of the molecule is not susceptible to Edman degradation, but sequencing studies were successfully carried out on tryptic fragments. Based on the estimated size of the molecule, these studies provide approximately 60% of the total sequence. No homologies with any previously sequenced peptide were observed, but biochemical similarities to as yet unsequenced peptides found in extracts of sinus glands from other crustaceans (hyperglycemic hormone and moult-inhibiting hormone) are described.     

Characterisation of bombesin-like immunoreactivity in human fetal lung

A sensitive radioimmunoassay for bombesin-like immunoreactivity (BLI) was developed and utilised in conjunction with G50 gel chromatography and reverse-phase HPLC, to study the content and molecular characteristics of bombesin-like peptides in acid extracts of human fetal lung. The antiserum, (B5), is directed towards the C-terminal region of the bombesin molecule and cross-reacts 70% with synthetic porcine GRP and the synthetic GRP fragment, GRP (14-27). Specimens of lung were collected from fetuses of gestational ages 15-22 weeks, following prostaglandin termination of pregnancy. The tissue was extracted into 0.1 N HCl at 90 degrees C. The mean BLI content was 50.2 pg/mg wet weight of tissue (range 15.5-136 pg/mg; n = 13). No correlation between gestational age and BLI content could be established. G50 gel chromatography of acid extracts, under dissociating conditions, revealed two peaks of BLI, one in the position of synthetic porcine GRP and the second, constituting greater than 90% of the immunoreactivity, eluting with synthetic amphibian bombesin. Reverse-phase ODS silica HPLC of this major G50 peak, utilising a methanol/trifluoroacetic acid gradient, indicated that this peptide was similar to the GRP C-terminal fragment, GRP (14-27). We have therefore (1) confirmed the presence and heterogeneity of BLI in human fetal lung, and (2) shown, for the first time, that the majority of this BLI more closely resembles a fragment of GRP than amphibian bombesin itself.     

A cytocidal tissue kallikrein isolated from mouse submandibular glands

A cytocidal factor against mouse thymocytes was purified from the submandibular glands of female BALB/c mice using Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. SDS-PAGE and amino acid sequence analysis revealed that the cytocidal factor was mouse glandular kallikrein (mGK)-6. mGK-6 showed an optimal enzyme activity at pH 10 and a cytocidal activity against thymocytes in a dose-dependent manner.     

一种新的烙铁头蛇毒肌肉毒素

用分子筛和快速蛋白质液相色谱从烙铁头（ＴＲｒｉｍｅｒｅｓｕｒｕｓ ｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒中分离了一个新的碱性肌肉毒素,命名为ＴＭＰＢ。它的分子量为１６０００,等电点为９．２．用蛋白质序列仪测定了其Ｎ端２４个氨基酸残基,ＴＭＰＢ与其他两个从同种蛇毒中分离到的碱性磷酯酶Ａ２的同源性分别为４１．７％和５４．２％《     

Purification and NH2-terminal sequence of a plasmacytoma growth factor derived from the murine macrophage cell line P388D1

Plasmacytoma growth factor (PCT-GF), a putative macrophage-derived lymphokine essential for the in vitro viability and proliferation of early generation plasmacytomas, was purified from conditioned medium of the murine macrophage cell line P388D1. The purification of PCT-GF was accomplished by a batch concentration on trimethylsilyl-controlled pore glass beads, followed by: gel filtration chromatography; hydrophobic interaction HPLC; and reverse-phase HPLC. SDS-PAGE analysis of the purified PCT-GF revealed a single band of Mr 23,000. The amino terminal sequence of PCT-GF was established as NH2-Pro-Thr-Ser-Gln-Val-Arg-Arg-Gly-Asp-Phe-Thr-Glu-Asp-Thr-Thr-Pro-Asn- Arg-Pro-Val-Tyr-Thr. No significant homology was found between this sequence and proteins in the National Biomedical Research Foundation database, suggesting that PCT-GF is a new lymphokine unrelated to previously described growth and differentiation factors.     

Purification and characterization of recombinant human interleukin-1 beta produced in Escherichia coli

Recombinant human interleukin-1 beta (rIL-1 beta) produced in Escherichia coli was purified to homogeneity by a combination of mass ion exchange column chromatography, ion exchange and gel filtration high performance liquid chromatography. The purified rIL-1 beta had a molecular weight of 18 kD on SDS-polyacrylamide gel electrophoresis and an isoelectric point of 6.9 on analytical isoelectric focusing. These values were almost same as those of natural interleukin-1 beta. The amino acid composition and amino acid sequence of the amino terminal region were consistent with those deduced from the cDNA sequence. In addition, the primary structure was confirmed by peptide mapping with lysyl-endopeptidase on reverse phase HPLC. Besides rIL-1 beta with amino terminal Ala, two molecular species, [Met0] rIL-1 beta and [desAla1] rIL-1 beta, were also obtained. Biological and physicochemical properties of the three species of rIL-1 beta were compared.     

大肠杆菌表达重组人生长激素的纯化

目的 :获得具有生物学活性的重组人生长激素 (rhGH)。方法 :PBV -GH/DH5α菌体经超声破菌、反复洗涤后获得包涵体。将包涵体变性、复性 ,用硫酸铵盐析 ,离子交换层析和凝胶层析进行纯化。产物经SDS -PAGE、HPLC、N末端 15个氨基酸序列检测验证。结果 :终产物rhGH纯度达 98.2 % ,比活性大于 3.0IU/mg。分子量为 2 2kDa ,N末端氨基酸序列与DNA序列推导的氨基酸序列完全一致。结论 :从自构建的PBV -GH/DH5α工程菌中获得高纯度、高活性重组人生长激素。其纯化工艺为中试生产提供可靠依据。     

Macrophage migration inhibitory factor (MIF) produced by a human T cell hybridoma clone

A human T cell hybridoma clone, F5, producing high levels of macrophage migration inhibitory factor (MIF) was established by the emetine-actinomycin D selection method. This clone produced two species of MIF which were separated on a Phenyl Sepharose column. We purified MIF-2 (the more hydrophobic species of the two) to homogeneity from the conditioned medium of stimulated F5 cells by a series of steps that included hydrophobic chromatography, ion-exchange chromatography. Ricinus communis lectin affinity chromatography, and high-performance liquid chromatography on anion exchange and reverse-phase columns. Purified MIF was digested with endoproteinase Lys-C and Asp-N. The amino acid sequences of the generated peptides were determined. No sequence similarity with any other protein was found. The molecular weight of MIF-2 was estimated to be 45 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitates with anti-peptide antibodies. These results show that F5MIF-2 is a novel cytokine.     

Purification of guinea pig tumor necrosis factor (TNF): comparison of its antiproliferative and differentiative activities for myeloid leukemic cell lines with those of recombinant human TNF

Recently, we suggested that the effect of differentiation inducing factor (D-factor) which is found in the supernatant of macrophages, and induced the differentiation of a mouse myeloid leukemic cell line, M1, into macrophage-like cells, may be a result of the cooperative effects of tumor necrosis factor (TNF) and interleukin 1 (IL-1). In this study, we purified guinea pig (G.P.) TNF secreted from peritoneal macrophages and compared the antiproliferative and differentiative effects of the G.P. TNF with those of recombinant human TNF (rHuTNF). The purification scheme consisted of ultrafiltration, gel filtration-high performance liquid chromatography (HPLC), DEAE-HPLC, and reverse-phase HPLC. The cytotoxic activity of the purified substance was approximately 1.5 x 10(8) U/mg. The isoelectric point was 5.2. The molecular weight was 40 to 45 kDa as estimated by gel filtration and 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequence was determined to be Ser-Ala-Ser-Gln-Asn-Asp . . . . Approximately 76 or 71% homology between G.P. TNF and mouse or human TNF exists in the NH2-terminal 21 residues. The purified G.P. TNF and rHuTNF demonstrated D-factor activity only in the presence of recombinant human IL-1 alpha in M1 cells. We also determined the effect of TNF on two human myeloid leukemic cell lines (THP-1 and U937). The purified G.P. TNF and rHuTNF inhibited the growth of U937 cells, but did not induce their differentiation. In THP-1 cells, TNF slightly inhibited the growth and induced differentiation. In mouse cell lines G.P. TNF was more effective than rHuTNF for differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate.

A factor of low M(r) with growth-promoting effects on rabbit embryos was extracted and purified from commercial bovine serum albumin (BSA). This embryotrophic factor was extracted from BSA dissolved in formic acid by membrane filtration (membrane cutoff of M(r) 10,000) and then freeze-drying of the filtrate. The extract was purified successively by chromatography on G-10 Sephadex, QAE-Sephadex A-25 anion exchange and high-performance liquid chromatography (HPLC) reverse-phase columns. Mass spectrometry of the active reverse-phase material indicated that the major component in this material had an M(r) of 192. The embryotrophic factor in the low M(r) extract of BSA was shown to be citrate, because: (i) the mass spectra of the active reverse-phase material and citrate were identical, (ii) the activity was eluted at the identical position to citrate on an analytical HPLC anion-exchange column, (iii) the original BSA sample was shown by enzyme assay to be heavily contaminated by citrate and (iv) citrate stimulated cell proliferation and expansion of blastocysts.