Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity

A novel monocyte-derived neutrophil-activating peptide (MONAP) produced by lipopolysaccharide- and phorbol myristate acetate-stimulated human peripheral blood monocytes was purified by sequential ion exchange-high performance liquid chromatography (HPLC), size exclusion HPLC, and reversed phase HPLC. Biologic activities of the purified cytokine were monitored by either an enzyme release assay or a chemotaxis assay, using peripheral human neutrophils. Purified MONAP was found to be homogeneous, giving a single peak on size-exclusion HPLC, reversed-phase HPLC, as well as a single 10-kDa band on silver-stained polyacrylamide gels. Purified MONAP stimulate human neutrophil chemotaxis at an estimated molarity of 5 x 10(-11) M. Half-maximal enzyme release of cytochalasin B pretreated neutrophils occurred at 2 to 3 x 10(-10) M, whereas superoxide anion production elicited by various concentrations of MONAP was found to be low. Isolated human peripheral monocytes, as well as human eosinophils, showed no chemotactic response to MONAP, indicating neutrophil specificity. MONAP activity was separated from thymocyte-stimulating activity by reversed-phase HPLC, indicating nonidentity with interleukin (IL)-1. This was further supported by heat resistance of MONAP, which is in contrast to the heat sensitivity of IL-1. In addition, IL-1 obtained as a by-product during isolation of MONAP did not stimulate human neutrophil chemotaxis.     

Purification and partial biologic characterization of a human lymphocyte-derived peptide with potent neutrophil-stimulating activity

A novel neutrophil-activating peptide is detected in supernatants from mitogen-stimulated human T lymphocyte preparations. This chemotaxin was purified to apparent homogeneity by sequential Gel G-75 permeation chromatography, wide pore reversed phase (RP-8) HPLC, size exclusion HPLC, and reversed phase (RP-18) HPLC. Additional characterization of this lymphocyte-derived neutrophil-activating peptide (LYNAP) resulted in a single peak upon reversed phase HPLC and size exclusion HPLC. SDS-PAGE under nonreducing conditions revealed a single line at 10 kDa. LYNAP stimulated neutrophil chemotaxis (ED50 of 3 +/- 3 ng/ml), chemokinesis (ED50 of 2 +/- 2 ng/ml), and caused degranulation of cytochalasin B pretreated human neutrophils (ED50 of 20 ng/ml). In purified human monocytes, chemotactic responses to LYNAP at doses up to 100 ng/ml were absent, indicating nonidentity with a lymphocyte-derived monocyte chemotactic factor previously described by other workers. LYNAP shows biochemical and biologic similarities to a recently detected monocyte-derived neutrophil-activating peptide (MONAP). Moreover, desensitization experiments revealed cross-deactivation between LYNAP and MONAP, not, however, between these two chemotactic peptides and other well characterized polymorphonuclear leukocyte chemotaxins, e.g., C5a, FMLP, leukotriene B4, or platelet-activating factor. This finding points toward structure identity or homology of both chemotaxins, MONAP and LYNAP.     

Partial purification of hatching activity for Globodera rostochiensis from potato root diffusate

The potential of several chromatographic methods for isolating hatching factors for potato cyst nematodes from potato root diffusate was investigated using a bioassay based on emergence of juveniles from cysts. Gel filtration provided an overall estimate of molecular weight of 437 Da for the hatching activity and ion exchange chromatography indicated that at least 60% of the recovered activity was anionic in nature. Material less polar than the hatching activity could be removed by passing potato root diffusate through a reversed-phase Sep-Pak C₁₈ cartridge and the elutant showed 83.3 ± 4.4% (mean of 32 cysts) of the initial hatching activity. High performance liquid chromatography (HPLC) with a reversed phase, C₁₈ column and gradient elution (0–80% CH₃OH in water) confirmed that much of the hatching activity was polar and that it was not retained by this method of separation. A weak anion exchange resin achieved slight retention of much of the hatching activity and an ion pairing reagent lowered the polarity sufficiently to allow some retention in subsequent reversed phase HPLC on a C_IS column. Both ion exchange and ion pairing HPLC suggested that hatching activity was not chromatographed as a single compound and indicated that fractions able to influence the nucleolus of the nucleus within the dorsal pharyngeal gland cell did not always show hatching activity.     

Purification of a mammalian 5-lipoxygenase from rat basophilic leukemia cells

5-Lipoxygenase (5-lipox) has been purified to homogeneity from the 20,000 xg supernatant of sonicated rat basophilic leukemia (RBL-1) cells using a 4-step procedure. Purification was achieved primarily through the use of anion-exchange HPLC on two different media. Using the supernatant from 1 X 10(9) cells, approximately 33 micrograms of the enzyme can be routinely isolated with an estimated net yield of 5-10%. Purified 5-lipox consists of a single Mr 73,000 band on SDS gels (reduced or unreduced). When the purified enzyme was incubated with radiolabeled arachidonic acid and products analyzed by both straight phase and reversed phase HPLC, 5-hydroperoxyeicosatetraenoic acid (5-HPETE) was the only enzymatic product detected. The purified enzyme exhibits the same characteristic lag phase and premature cessation of reaction as does the 5-lipox activity seen in crude cell homogenates.     

Purification of a mammalian 5-lipoxygenase from rat basophilic leukemia cells

5-Lipoxygenase (5-lipox) has been purified to homogeneity from the 20,000 xg supernatant of sonicated rat basophilic leukemia (RBL-1) cells using a 4-step procedure. Purification was achieved primarily through the use of anion-exchange HPLC on two different media. Using the supernatant from 1 × 10 ⁹ cells, approximately 33 μg of the enzyme can be routinely isolated with an estimated net yield of 5–10%. Purified 5-lipox consists of a single Mr 73,000 band on SDS gels (reduced or unreduced). When the purified enzyme was incubated with radiolabeled arachidonic acid and products analyzed by both straight phase and reversed phase HPLC, 5-hydroperoxyeicosate-traenoic acid (5-HPETE) was the only enzymatic product detected. The purified enzyme exhibits the same characteristic lag phase and premature cessation of reaction as does the 5-lipox activity seen in crude cell homogenates.     

A new member of the AKH/RPCH family that stimulates locomotory activity in the firebug, Pyrrhocoris apterus (Heteroptera)

A new member of the AKH/RPCH family was isolated and identified from the corpora cardiaca of the firebug Pyrrhocoris apterus. The peptide was isolated in a single step by reversed phase HPLC and the structure deduced from the multiple MS (MS(N)) electrospray mass spectra and amino acid analysis as that of an octapeptide with the sequence pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-NH(2): this sequence was confirmed by synthesis. The synthetic peptide induced lipid mobilisation and stimulated locomotory activity in macropterous females. This peptide, designated as Pyrrhocoris apterus adipokinetic hormone (Pya-AKH), is the first identified adipokinetic hormone described in a representative species of the suborder Heteroptera.     

Synthesis and structural characterization of charybdotoxin, a potent peptidyl inhibitor of the high conductance Ca2(+)-activated K+ channel.

Charybdotoxin (ChTX), a potent inhibitor of the high conductance Ca2(+)-activated K+ channel (PK,Ca) is a highly basic peptide isolated from venom of the scorpion Leiurus quinquestriatus hebraeus, whose primary structure has been determined (Gimenez-Gallego, G., Navia, M. A., Reuben, J. P., Katz, G. M., Kaczorowski, G. J., and Garcia, M. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3329-3333). The synthesis of this peptide using continuous flow solid phase fluorenylmethyloxycarbonyl-pentafluorophenyl ester methodology has now been achieved. The 1-37-amino acid hexasulfhydryl peptide oxidizes readily to give the tricyclic disulfide structure in good yield. This folded synthetic material is identical to native toxin based on three criteria: co-migration with ChTX on reversed phase high performance liquid chromatography (HPLC); competitive inhibition of 125I-labeled monoiodotyrosine charybdotoxin binding to bovine aortic sarcolemmal membrane vesicles with a Ki (10 pM) identical to that of native toxin; blockade of PK,Ca activity in excised outside-out patches from bovine aortic smooth muscle with the potency and inhibitory properties characteristic of ChTX (i.e. appearance of silent periods interdispersed with normal bursts of channel activity in single channel recordings). Selective enzymatic digestion of native or synthetic ChTX by simultaneous exposure to chymotrypsin and trypsin yields identical reversed phase HPLC profiles. Analysis of the sequence and amino acid composition of the resulting fragments defines a disulfide bond arrangement (Cys7-Cys28, Cys13-Cys33, Cys17-Cys35) which differs from that previously suggested. This configuration predicts a highly folded tertiary structure for ChTX which, together with observations from electrophysiological and binding experiments, suggests a possible mechanism by which ChTX interacts with PK,Ca to block channel function.     

A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatography

Unquestionably, the purification of polypeptides by chromatographic methods is a considerable bottleneck in their preparation. Peptides synthesised by solid phase synthesis typically contain chromatographically similar impurities that complicate purification by reversed phase high performance liquid chromatography (HPLC) techniques. We report on the application of a slow gradient HPLC protocol that allows, in a single chromatographic step, the purification of hundreds of milligrammes of material. This technique was applied to an extensive collection of synthetic polypeptides some incorporating non‐proteinogenic functionality. In all cases examined, the peptides were not only obtained in high purity peptides but were also recovered in multi‐milligramme amounts. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Reaction System of Haemophilus,Superhelical DNA-relaxing Enzyme is Useful for Screening of DNA-binding Substance

Several substances with antioxidant activity were isolated from the mixture of dehydroascorbic acid and tryptophan when reacted together in ethanol. One of the main antioxidant products was obtained in crystalline form from an n-butanol extract of the reaction mixture by means of Sephadex column chromatography followed by HPLC with a reversed phase column. ¹H- and ¹³C-NMR of the product and its acetate showed its structure as a condensate of dehydroascorbic acid and tryptophan with each single molecule involving a C-spiro structure. By a POV test the activity of this substance was about two-thirds of that of BHA on a molar basis, and the activity of the reaction mixture is greatly attributable to this substance.     

1H NMR characterization of two crambin species

Crambin displays amino acid heterogeneity at positions 22 (Pro or Ser) and 25 (Leu or Ile). Using reversed phase HPLC the crambin mixture can be resolved into two protein fractions. It is shown by amino acid analysis and NMR spectroscopy that these fractions represent single proteins (Ser-22/Ile-25 and Pro-22/ Leu-25 species). A first characterization of the ¹H-NMR spectra of these species is presented.     

Separation and quantification of alkylphosphocholines by reversed phase high performance liquid chromatography

Alkylphosphocholines represent a new class of drugs with remarkable antineoplastic and antiprotozoal activity. For instance, hexadecylphosphocholine has been approved for the topical treatment of skin metastasis. In addition, it was successfully studied in India for the treatment of leishmaniasis. Different phase-I and phase-II-trials resulted in cure rates of more than 97%. To optimize antitumor or antiprotozoal activity, we have prepared alkylphosphocholines differing in chain length and unsaturation. For the qualitative and quantitative analysis of these longer chain analogues, we have used isocratic high performance liquid chromatography. The separation of the alkylphosphocholines with different chain lengths in this reversed phase HPLC system was achieved on a YMC-TMS column with a mobile phase consisting of methanol-water (85:15; v/v) at a flow rate of 1.0 ml/min. Furthermore the cis-/trans-isomers such as oleylphosphocholine and elaidylphosphocholine were clearly separated on a YMC-C8 column with a methanol-water mixture (80:20; v/v) as mobile phase. In the described reversed phase HPLC systems simple refractive index detection and UV detection allow the sensitive and quantitative determination of alkylphosphocholines. These methods are very important for reproducible identification and quantitative determination of saturated and mono-unsaturated alkylphosphocholines with alkyl residues containing up to 25 carbon atoms.     

Secretion of novel and homologous neutrophil-activating peptides by LPS-stimulated human endothelial cells

Human umbilical vein endothelial cells in culture produce two chemotactic polypeptides when stimulated with LPS. The chemotactic factors could be purified to apparent homogeneity by HPLC techniques and were identified as 7.5-kDa and 15-kDa polypeptides by SDS-PAGE under nonreducing conditions. Both factors are potent chemotaxins for human neutrophils demonstrating half-maximal chemotaxis at 2 ng/ml and g ng/ml, respectively. In addition both peptides elicited release of azurophilic granule constituents when neutrophils were pretreated with cytochalasin B. Cross-desensitization experiments by using human neutrophils revealed cross-reactivities between both chemotaxins, not, however, with C5a or FMLP, indicating that both endothelial cell-derived neutrophil activating peptides (ENAP) are homologous. In addition, the 7.5-kDa factor (beta-ENAP) proved to be the quantitatively dominating and more potent chemotaxin as compared to the 15 kDa factor (alpha-ENAP). beta-ENAP shows biochemical and biologic similarities to monocyte- and lymphocyte-derived neutrophil-activating peptides MONAP and LYNAP, which recently were purified and sequenced.     

测序级胰蛋白酶的制备工艺与质量检测简

目的：制备高纯度、酶解效率高、酶切位点专一的测序级胰蛋白酶,应用于蛋白组学研究的蛋白质鉴定与分析中。方法：取实验室自制的猪胰蛋白酶粗酶,经硼氢化钠、甲醛还原甲基化修饰抑制胰蛋白酶自水解,采用高效液相色谱仪15RPC反相柱纯化,收集对应的甲基化胰蛋白酶峰组分,冷冻干燥;甲基化修饰的胰蛋白酶进一步经甲苯磺酰苯丙氨酰氯甲酮（TPCK）修饰,以抑制糜蛋白酶等非特异性酶切活性,并经反相色谱柱再纯化,获得终产物即质谱测序级胰蛋白酶;自制的测序级胰蛋白酶经SDS-PAGE、HPLC反相色谱分析、酶比活力测定,并应用于胶内蛋白质酶切质谱鉴定氨基酸序列等,检测其纯度、酶水解效率及酶切位点特异性。结果：自制甲基化TPCK修饰的测序级胰蛋白酶纯度大于95％,酶比活力为200U/mgP（TAME）以上,质谱分析酶切特异性好;且酶的制备工艺流程稳定,可应用于测序级胰蛋白酶产品的生产与开发中。结论：制备的测序级胰蛋白酶纯度高、酶解效率优、酶切特异性强,可广泛应用于实验室中蛋白质和肽段测序鉴定、HPLC肽段谱图分析等蛋白组学研究分析中。     

Identification and characterization of a novel CCK-like immunoreactive peptide in pig CNS

Antibodies directed against the C-terminus of cholecystokinin octapeptide (CCK8) and caerulein were used to study immunoreactive peptides in pig brain. One antibody, a mouse monoclonal raised to caerulein (c.MAb), reacts equally with heptadecapeptide gastrin (G17), CCK8 and caerulein, the other raised to CCK8 (L48) shows 10 times lower immunoreactivity with caerulein compared with G17 and CCK8. Extracts were purified by adsorption to alginic acid, gel filtration chromatography and reversed phase HPLC. In addition to material with the expected properties of CCK33, 39 and 58 a novel peptide was identified that reacted 10 times better with c.MAb compared with L48. This material emerged in a similar position to CCK58 on Sephadex G50 but had a greater retention time on reversed phase HPLC. It had CCK-like bioactivity and digestion with trypsin gave a fragment showing a pattern of immunoreactivity similar to that of the parent compound. This pattern of activity is distinct from other known mammalian CCKs; the material may represent an addition to the gastrin-CCK family in mammals.     

Insulin-stimulating peptide from tryptic digest of bovine serum albumin

Insulin-stimulating peptide was isolated from a tryptic digest of bovine serum albumin by gel permeation, SP Sephadex column chromatography, reversed phase HPLC and cation-exchange HPLC. This peptide, with a molecular weight of about 8,400, had no insulin-like activity by itself, but enhanced fatty acid synthesis from glucose in rat adipose tissue explants in the presence of suboptimal concentrations of insulin. It also stimulated the effect of insulin on CO2 production from glucose in rat adipocytes, without affecting insulin binding. These stimulations were dose-dependent and were observed at concentrations of more than 2 X 10(-7) M peptide only in the presence of a suboptimal concentration of insulin.     

Separation by high-performance liquid chromatography of alpha- and beta-amino acids: application to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase

Several pairs of alpha- and beta-amino acids were well separated as the ortho-phthalaldehyde derivatives by reversed phase HPLC. These included alpha- and beta-lysine, leucine, tyrosine, serine, aminoisobutyric acid, and beta-hydroxyvaline and its positional isomer. The separation was applicable to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase. However, for unknown reasons, no leucine 2,3-aminomutase activity could be found in rat liver homogenate.     

Synthesis of phosphorothioate oligonucleotide-peptide conjugates by solid phase fragment condensation

Phosphorothioate oligonucleotide-peptide conjugates were synthesized by solid phase fragment condensation (SPFC). Arginine rich peptides could be successfully conjugated in 2.8-13.4% isolated yields. All the products were fully characterized by reversed phase HPLC and MALDI-TOF-MS to give satisfactory results.     

真核细胞基因工程产物人生长激素的纯化研究

应用反相高效液相色谱法（ＲＰ－ＨＰＬＣ）对真核细胞基因表达产物人生长激素进行了纯化研究,发现在７１．６７％的乙晴浓度下人生长激素可有效地与其它杂蛋白分离,乙晴与人生长激素的短时间接触不影响其放射免疫活性．     

Polystyrene/Divinylbenzene Based Monolithic and Encapsulated Capillary Columns for the Analysis of Nucleic Acids by High‐Performance Liquid Chromatography‐Electrospray Ionisation Mass Spectrometry

Monolithic capillary columns are prepared by copolymerization of styrene and divinylbenzene, encapsulated capillary columns by immobilizing silica particles with different pore sizes inside a 200 μm i.d. fused silica capillary by encapsulation of the derivatized silica sorbent in a poly(styrene/divinylbenzene) (PS/DVB) matrix. Both allow the rapid and highly efficient separation of single‐ and double‐stranded DNA by ion‐pair reversed‐phase high‐performance liquid chromatography (IP‐RP‐HPLC). The high resolving power of monolithic and encapsulated capillary columns can be utilized for mutation screening in polymerase chain reaction (PCR) amplified polymorphic loci by denaturing HPLC (DHPLC). Recognition of mutations is based on the separation of homo‐ and heteroduplex species by IP‐RP‐HPLC under denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Separations can be readily hyphenated to electrospray ionization‐mass spectrometry.     

取代P物质类似物的合成及生物活性

使用固相法合成含有不同D-型氨基酸取代的八个P物质类似物。纯化采用凝胶过滤,硅胶柱层或反相高效液相色谱。化合物的SP样作用及拮抗SP样作用在离体豚鼠迴肠纵行肌上进行了试验。所有化合物都显示了不同程度的拮抗作用。其中化合物A-58[D-Arg~1,D-Phe~5,D-Trp~(7,9),Leu~(11)]SP拮抗活性最高。它拮抗SP样作用的剂量比率(EC_(50)之比)在10~(-6)M时为24,在3×10~(-6)M时为52.2。